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1.
Cell ; 171(7): 1520-1531.e13, 2017 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-29153832

RESUMO

Pectin, an integral component of the plant cell wall, is a recalcitrant substrate against enzymatic challenges by most animals. In characterizing the source of a leaf beetle's (Cassida rubiginosa) pectin-degrading phenotype, we demonstrate its dependency on an extracellular bacterium housed in specialized organs connected to the foregut. Despite possessing the smallest genome (0.27 Mb) of any organism not subsisting within a host cell, the symbiont nonetheless retained a functional pectinolytic metabolism targeting the polysaccharide's two most abundant classes: homogalacturonan and rhamnogalacturonan I. Comparative transcriptomics revealed pectinase expression to be enriched in the symbiotic organs, consistent with enzymatic buildup in these structures following immunostaining with pectinase-targeting antibodies. Symbiont elimination results in a drastically reduced host survivorship and a diminished capacity to degrade pectin. Collectively, our findings highlight symbiosis as a strategy for an herbivore to metabolize one of nature's most complex polysaccharides and a universal component of plant tissues.


Assuntos
Besouros/microbiologia , Enterobacteriaceae/genética , Genoma Bacteriano , Animais , Besouros/fisiologia , Enterobacteriaceae/classificação , Enterobacteriaceae/enzimologia , Enterobacteriaceae/fisiologia , Tamanho do Genoma , Pectinas/metabolismo , Simbiose
2.
Proc Natl Acad Sci U S A ; 119(30): e2120339119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35857873

RESUMO

During translation initiation, eIF4G1 dynamically interacts with eIF4E and eIF1. While the role of eIF4E-eIF4G1 is well established, the regulatory functions of eIF4G1-eIF1 are poorly understood. Here, we report the identification of the eIF4G1-eIF1 inhibitors i14G1-10 and i14G1-12. i14G1s directly bind eIF4G1 and inhibit translation in vitro and in the cell, and their effects on translation are dependent on eIF4G1 levels. Translatome analyses revealed that i14G1s mimic eIF1 and eIF4G1 perturbations on the stringency of start codon selection and the opposing roles of eIF1-eIF4G1 in scanning-dependent and scanning-independent short 5' untranslated region (UTR) translation. Remarkably, i14G1s activate ER/unfolded protein response (UPR) stress-response genes via enhanced ribosome loading, elevated 5'UTR translation at near-cognate AUGs, and unexpected concomitant up-regulation of coding-region translation. These effects are, at least in part, independent of eIF2α-phosphorylation. Interestingly, eIF4G1-eIF1 interaction itself is negatively regulated by ER stress and mTOR inhibition. Thus, i14G1s uncover an unknown mechanism of ER/UPR translational stress response and are valuable research tools and potential drugs against diseases exhibiting dysregulated translation.


Assuntos
Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos , Fator de Iniciação Eucariótico 4G , Fatores de Iniciação em Eucariotos , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Resposta a Proteínas não Dobradas , Animais , Códon de Iniciação , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/antagonistas & inibidores , Fator de Iniciação Eucariótico 4G/metabolismo , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Biossíntese de Proteínas , Resposta a Proteínas não Dobradas/genética
3.
Nano Lett ; 2024 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-39319575

RESUMO

Electrophysiological recordings from brain cells are performed routinely using implanted electrodes, but they traditionally require a wired connection to the outside of the brain. A completely passive, wireless device that does not require on-board power for active transmission but that still facilitates remote detection could open the door for mass-scale direct recording of action potentials and transform the way we acquire brain signals. We present a nanofabricated coil that forms a neuroelectromagnetic junction, yielding a highly enhanced magnetic field transduction of electrophysiology. We show that this micrometer-scale device enables remote magnetic detection of neuronal fields from the center of the coil using room temperature superconducting quantum interference device (SQUID) microscopy. Further, time-locked stimulation in conjunction with magnetometry demonstrates thresholding behavior that affirms the viability of the technology for detection with no requirement for wires or on-board power. This strategy may permit unprecedented detection of electrophysiology using magnetoencephalography and magnetic resonance imaging.

4.
J Endovasc Ther ; : 15266028241258659, 2024 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-38880997

RESUMO

PURPOSE: The aim of this study was to present the covered endovascular reconstruction of the iliac artery bifurcation (CERIB) technique as a revascularization option for aorto-iliac occlusive disease (AIOD) involving the iliac artery bifurcation. METHODS: This was a retrospective single-center study including all patients undergoing a CERIB procedure from January 2021 until December 2022. Covered endovascular reconstruction of the iliac artery bifurcation procedures were performed percutaneously with bilateral femoral access, excepting cases where simultaneous open femoral artery reconstruction was required due to the extension of peripheral arterial occlusive disease. Standard patient, procedural, and follow-up (FU) data including survival and arterial reinterventions were gathered and analyzed. RESULTS: A total of 13 patients were included (11/13 male, median age=70 [60-76] years). Additional open and/or endovascular procedures included endarterectomy of the femoral bifurcation (n=6), rotational atherectomy (n=2), and 1 axillary artery cutdown for upper limb access (n=1). Median operating time was 2.9 hours (1.5-4.9 hours); median radiation duration and dose were 28.4 (14.3-63.3 minutes) and 4090.6 (384.4-9430.1 cGray/cm2), respectively. The stent grafts used for CERIB were BeGraft peripheral n=31, BeGraft Aortic n=4 and BeGraft peripheral plus n=1 (Bentley InnoMed GmbH, Hechingen, Germany). In 2 patients with prior stenting of the common iliac artery (CIA), only the internal and external iliac arteries (IIA and EIA) were treated in this study, and in 2 cases, additional uncovered stents were required for relining. Technical success was 100%. During a midterm FU (median 18 [4-31] months), 2 patients died from cardiovascular reasons and 1 patient with prior iliac stenting required reinterventions for recurring occlusion. CONCLUSION: Covered endovascular reconstruction of the iliac artery bifurcation is a straightforward option for treating AIOD involving the iliac artery bifurcation that allows preservation of internal iliac artery perfusion and shows good early and midterm results. Prior iliac artery stenting may be a risk factor for early occlusion after CERIB. CLINICAL IMPACT: Aorto-iliac occlusive disease with involvement of the internal iliac artery is encountered regularly in vascular surgical practice, but the internal iliac artery is often not included in the revascularization strategy. The present article will increase the awareness for the relevance of including the internal iliac artery in revascularization strategies and presents CERIB as another method to be added to the armamentarium of (endo-)vascular surgeons and interventionalists.

5.
Nucleic Acids Res ; 49(9): 5159-5176, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33893802

RESUMO

The eIF4E are a family of initiation factors that bind the mRNA 5' cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active during stress and that it selects specific mRNA populations based on 5' TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have 'moon-lighting' functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote 'stress resistance' and adaptation.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Biossíntese de Proteínas , Estresse Fisiológico/genética , Animais , Células Cultivadas , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Camundongos , Naftiridinas/farmacologia , Capuzes de RNA/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores
6.
Proc Natl Acad Sci U S A ; 117(47): 29487-29494, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33148806

RESUMO

We report paleomagnetic data showing that an intraoceanic Trans-Tethyan subduction zone existed south of the Eurasian continent and north of the Indian subcontinent until at least Paleocene time. This system was active between 66 and 62 Ma at a paleolatitude of 8.1 ± 5.6 °N, placing it 600-2,300 km south of the contemporaneous Eurasian margin. The first ophiolite obductions onto the northern Indian margin also occurred at this time, demonstrating that collision was a multistage process involving at least two subduction systems. Collisional events began with collision of India and the Trans-Tethyan subduction zone in Late Cretaceous to Early Paleocene time, followed by the collision of India (plus Trans-Tethyan ophiolites) with Eurasia in mid-Eocene time. These data constrain the total postcollisional convergence across the India-Eurasia convergent zone to 1,350-2,150 km and limit the north-south extent of northwestern Greater India to <900 km. These results have broad implications for how collisional processes may affect plate reconfigurations, global climate, and biodiversity.

7.
Appl Environ Microbiol ; 88(9): e0254921, 2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35420439

RESUMO

Multicellular eukaryotes often host multiple microbial symbionts that may cooperate or compete for host resources, such as space and nutrients. Here, we studied the abundances and localization of four bacterial symbionts, Rickettsia, Wolbachia, Sodalis, and Arsenophonus, in the parasitic wasp Spalangia cameroni. Using quantitative PCR (qPCR), we measured the symbionts' titers in wasps that harbor different combinations of these symbionts. We found that the titer of each symbiont decreased as the number of symbiont species in the community increased. Symbionts' titers were higher in females than in males. Rickettsia was the most abundant symbiont in all the communities, followed by Sodalis and Wolbachia. The titers of these three symbionts were positively correlated in some of the colonies. Fluorescence in situ hybridization was in line with the qPCR results: Rickettsia, Wolbachia, and Sodalis were observed in high densities in multiple organs, including brain, muscles, gut, Malpighian tubules, fat body, ovaries, and testes, while Arsenophonus was localized to fewer organs and in lower densities. Sodalis and Arsenophonus were observed in ovarian follicle cells but not within oocytes or laid eggs. This study highlights the connection between symbionts' abundance and localization. We discuss the possible connections between our findings to symbiont transmission success. IMPORTANCE Many insects carry intracellular bacterial symbionts (bacteria that reside within the cells of the insect). When multiple symbiont species cohabit in a host, they may compete or cooperate for space, nutrients, and transmission, and the nature of such interactions would be reflected in the abundance of each symbiont species. Given the widespread occurrence of coinfections with maternally transmitted symbionts in insects, it is important to learn more about how they interact, where they are localized, and how these two aspects affect their co-occurrence within individual insects. Here, we studied the abundance and the localization of four symbionts, Rickettsia, Wolbachia, Sodalis, and Arsenophonus, that cohabit the parasitic wasp Spalangia cameroni. We found that symbionts' titers differed between symbiotic communities. These results were corroborated by microscopy, which shows differential localization patterns. We discuss the findings in the contexts of community ecology, possible symbiont-symbiont interactions, and host control mechanisms that may shape the symbiotic community structure.


Assuntos
Gammaproteobacteria , Rickettsia , Vespas , Wolbachia , Animais , Enterobacteriaceae/genética , Feminino , Hibridização in Situ Fluorescente , Masculino , Rickettsia/genética , Simbiose/fisiologia , Vespas/microbiologia , Wolbachia/fisiologia
8.
Nucleic Acids Res ; 48(3): 1043-1055, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31598688

RESUMO

In recent years translation elongation has emerged as an important contributor to the regulation of gene expression. There are multiple quality control checkpoints along the way of producing mature proteins and targeting them to the right cellular compartment, or associating them correctly with their partners. Ribosomes pause to allow co-translational protein folding, protein targeting or protein interactions, and the pausing is dictated by a combination of the mRNA sequence and structure, the tRNA availability and the nascent peptide. However, ribosome pausing can also lead to ribosome collisions and co-translational degradation of both mRNA and nascent chain. Understanding how the translating ribosome tunes the different maturation steps that nascent proteins must undergo, what the timing of these maturation events is, and how degradation can be avoided when pausing is needed, is now possible by the emergence of methods to follow ribosome dynamics in vivo. This review summarizes some of the recent studies that have advanced our knowledge about co-translational events using the power of ribosome profiling, and some of the questions that have emerged from these studies.


Assuntos
Elongação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Regulação da Expressão Gênica , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Estabilidade de RNA , RNA Mensageiro/metabolismo
9.
Proc Natl Acad Sci U S A ; 116(2): 407-412, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30598434

RESUMO

Zircon crystals from the Jack Hills, Western Australia, are one of the few surviving mineralogical records of Earth's first 500 million years and have been proposed to contain a paleomagnetic record of the Hadean geodynamo. A prerequisite for the preservation of Hadean magnetization is the presence of primary magnetic inclusions within pristine igneous zircon. To date no images of the magnetic recorders within ancient zircon have been presented. Here we use high-resolution transmission electron microscopy to demonstrate that all observed inclusions are secondary features formed via two distinct mechanisms. Magnetite is produced via a pipe-diffusion mechanism whereby iron diffuses into radiation-damaged zircon along the cores of dislocations and is precipitated inside nanopores and also during low-temperature recrystallization of radiation-damaged zircon in the presence of an aqueous fluid. Although these magnetites can be recognized as secondary using transmission electron microscopy, they otherwise occur in regions that are indistinguishable from pristine igneous zircon and carry remanent magnetization that postdates the crystallization age by at least several hundred million years. Without microscopic evidence ruling out secondary magnetite, the paleomagnetic case for a Hadean-Eoarchean geodynamo cannot yet been made.

10.
Int J Mol Sci ; 23(21)2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36362157

RESUMO

The first step in translation initiation consists in the recruitment of the small ribosome onto the mRNA. This preinitiation complex (PIC) loads via interactions with eIF4F that has assembled on the 5' cap. It then scans the 5' TL (transcript leader) to locate a start site. The molecular architecture of the PIC-mRNA complex over the cap is beginning to be resolved. As part of this, we have been examining the role of the 5' TL length. We observed in vivo initiation events on AUG codons positioned within 3 nts of the 5' cap and robust initiation in vitro at start sites immediately downstream of the 5' end. Ribosomal toe-printing confirmed the positioning of these codons within the P site, indicating that the ribosome reads from the +1 position. To explore differences in the eIF4E-5' cap interaction in the context of long versus short TL, we followed the fate of the eIF4E-cap interaction using a novel solid phase in vitro expression assay. We observed that ribosome recruitment onto a short TL disrupts the eIF4E-cap contact releasing all the mRNA from the solid phase, whereas with a long the mRNA distributes between both phases. These results are discussed in the context of current recruitment models.


Assuntos
Fator de Iniciação 4E em Eucariotos , Ribossomos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Ribossomos/genética , Ribossomos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Biossíntese de Proteínas , Capuzes de RNA/metabolismo
11.
Appl Environ Microbiol ; 87(12): e0021221, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-33863703

RESUMO

A pervasive pest of stored leguminous products, the bean beetle Callosobruchus maculatus (Coleoptera: Chrysomelidae) associates with a simple bacterial community during adulthood. Despite its economic importance, little is known about the compositional stability, heritability, localization, and metabolic potential of the bacterial symbionts of C. maculatus. In this study, we applied community profiling using 16S rRNA gene sequencing to reveal a highly conserved bacterial assembly shared between larvae and adults. Dominated by Firmicutes and Proteobacteria, this community is localized extracellularly along the epithelial lining of the bean beetle's digestive tract. Our analysis revealed that only one species, Staphylococcus gallinarum (phylum Firmicutes), is shared across all developmental stages. Isolation and whole-genome sequencing of S. gallinarum from the beetle gut yielded a circular chromosome (2.8 Mb) and one plasmid (45 kb). The strain encodes complete biosynthetic pathways for the production of B vitamins and amino acids, including tyrosine, which is increasingly recognized as an important symbiont-supplemented precursor for cuticle biosynthesis in beetles. A carbohydrate-active enzyme search revealed that the genome codes for a number of digestive enzymes, reflecting the nutritional ecology of C. maculatus. The ontogenic conservation of the gut microbiota in the bean beetle, featuring a "core" community composed of S. gallinarum, may be indicative of an adaptive role for the host. In clarifying symbiont localization and metabolic potential, we further our understanding and study of a costly pest of stored products. IMPORTANCE From supplementing essential nutrients to detoxifying plant secondary metabolites and insecticides, bacterial symbionts are a key source of adaptations for herbivorous insect pests. Despite the pervasiveness and geographical range of the bean beetle Callosobruchus maculatus, the role of microbial symbioses in its natural history remains understudied. Here, we demonstrate that the bean beetle harbors a simple gut bacterial community that is stable throughout development. This community localizes along the insect's digestive tract and is largely dominated by Staphylococcus gallinarum. In elucidating symbiont metabolic potential, we highlight its possible adaptive significance for a widespread agricultural pest.


Assuntos
Besouros/microbiologia , Microbioma Gastrointestinal/genética , Genoma Bacteriano , Staphylococcus/genética , Simbiose , Animais , Feminino , Genômica , Larva/microbiologia , Masculino , Óvulo/microbiologia , Staphylococcus/isolamento & purificação
12.
Microb Ecol ; 80(3): 718-728, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32488484

RESUMO

Many insect species harbor facultative microbial symbionts that affect their biology in diverse ways. Here, we studied the effects, interactions, and localization of two bacterial symbionts-Wolbachia and Rickettsia-in the parasitoid Spalangia endius. We crossed between four S. endius colonies-Wolbachia only (W), Rickettsia only (R), both (WR), and none (aposymbiotic, APS) (16 possible crosses) and found that Wolbachia induces incomplete cytoplasmic incompatibility (CI), both when the males are W or WR. Rickettsia did not cause reproductive manipulations and did not rescue the Wolbachia-induced CI. However, when R females were crossed with W or WR males, significantly less offspring were produced compared with that of control crosses. In non-CI crosses, the presence of Wolbachia in males caused a significant reduction in offspring numbers. Females' developmental time was significantly prolonged in the R colony, with adults starting to emerge one day later than the other colonies. Other fitness parameters did not differ significantly between the colonies. Using fluorescence in situ hybridization microscopy in females, we found that Wolbachia is localized alongside Rickettsia inside oocytes, follicle cells, and nurse cells in the ovaries. However, Rickettsia is distributed also in muscle cells all over the body, in ganglia, and even in the brain.


Assuntos
Interações Hospedeiro-Parasita , Rickettsia/fisiologia , Simbiose , Vespas/microbiologia , Wolbachia/fisiologia , Animais , Feminino , Moscas Domésticas/parasitologia , Masculino , Reprodução , Vespas/fisiologia
13.
Environ Sci Technol ; 54(12): 7354-7365, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32379434

RESUMO

Methanogenic archaea have been shown to reduce iron from ferric [Fe(III)] to ferrous [Fe(II)] state, but minerals that form during iron reduction by different methanogens remain to be characterized. Here, we show that zerovalent iron (ZVI) minerals, ferrite [α-Fe(0)] and austenite [γ-Fe(0)], appear in the X-ray diffraction spectra minutes after the addition of ferrihydrite to the cultures of a methanogenic archaeon, Methanosarcina barkeri (M. barkeri). M. barkeri cells and redox-active, nonenzymatic soluble organic compounds in organic-rich spent culture supernatants can promote the formation of ZVI; the latter compounds also likely stabilize ZVI. Methanogenic microbes that inhabit organic- and Fe(III)-rich anaerobic environments may similarly reduce Fe(III) to Fe(II) and ZVI, with implications for the preservation of paleomagnetic signals during sediment diagenesis and potential applications in the protection of iron metals against corrosion and in the green synthesis of ZVI.


Assuntos
Compostos Férricos , Methanosarcina barkeri , Ferro , Minerais , Oxirredução
14.
Eur Respir J ; 52(5)2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30166321

RESUMO

Despite the beneficial effects of pirfenidone in treating idiopathic pulmonary fibrosis (IPF), it remains unclear if lung fibroblasts (FB) are the main therapeutic target.To resolve this question, we employed a comparative transcriptomic approach and analysed lung homogenates (LH) and FB derived from IPF patients treated with or without pirfenidone.In FB, pirfenidone therapy predominantly affected growth and cell division pathways, indicating a major cellular metabolic shift. In LH samples, pirfenidone treatment was mostly associated with inflammation-related processes. In FB and LH, regulated genes were over-represented in the Gene Ontology node "extracellular matrix". We identified lower expression of cell migration-inducing and hyaluronan-binding protein (CEMIP) in both LH and FB from pirfenidone-treated IPF patients. Plasma levels of CEMIP were elevated in IPF patients compared to healthy controls and decreased after 7 months of pirfenidone treatment. CEMIP expression in FB was downregulated in a glioma-associated oncogene homologue-dependent manner and CEMIP silencing in IPF FB reduced collagen production and attenuated cell proliferation and migration.Cumulatively, our approach indicates that pirfenidone exerts beneficial effects via its action on multiple pathways in both FB and other pulmonary cells, through its ability to control extracellular matrix architecture and inflammatory reactions.


Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/genética , Proteínas/metabolismo , Piridonas/uso terapêutico , Adulto , Idoso , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Hialuronoglucosaminidase , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Transcriptoma
15.
Proc Natl Acad Sci U S A ; 110(21): 8453-8, 2013 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-23650386

RESUMO

The lifetime of the ancient lunar core dynamo has implications for its power source and the mechanism of field generation. Here, we report analyses of two 3.56-Gy-old mare basalts demonstrating that they were magnetized in a stable and surprisingly intense dynamo magnetic field of at least ~13 µT. These data extend the known lifetime of the lunar dynamo by ~160 My and indicate that the field was likely continuously active until well after the final large basin-forming impact. This likely excludes impact-driven changes in rotation rate as the source of the dynamo at this time in lunar history. Rather, our results require a persistent power source like precession of the lunar mantle or a compositional convection dynamo.

16.
Anal Biochem ; 484: 72-4, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25963893

RESUMO

Eukaryotic messenger RNAs (mRNAs) are generally enriched using oligo(dT) selection. However, a significant fraction of mRNAs contain either short or no poly(A). Our technique permits the isolation of mRNAs via their unique biochemical feature, the 5' cap. It involves RNA extraction, blocking of the 3' ribose cis-diol by cordycepin, oxidation of the 5' cis-diol of the CAP to a dialdehyde, coupling to a biotinylated linker, and enrichment on a streptavidin affinity matrix. We demonstrate that it efficiently pulls out a synthetic capped and non-polyadenylated transcript used to spike total cell RNA as well as endogenous histone 3c mRNA reported to be poly(A) negative.


Assuntos
Eucariotos , Polinucleotídeo Adenililtransferase/metabolismo , Capuzes de RNA/química , Capuzes de RNA/metabolismo
17.
Cell Rep ; 43(5): 114126, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38630588

RESUMO

Scanning and initiation are critical steps in translation. Here, we utilized translation complex profiling (TCP-seq) to investigate 48S organization and eIF4G1-eIF1 inhibition impact. We provide global views of scanning and leaky scanning, uncovering a central role of eIF4G1-eIF1 in their regulation. We confirm AUG context importance, with non-leaky genes featuring a Kozak context and cytosine at positions -1 and +5. Capturing 48S complexes associated with eIF1, eIF4G1, eIF3, and eIF2 through selective TCP-seq revealed that the eIF3-scanning ribosome is highly vulnerable to eIF4G1-eIF1 inhibition, and eIF1 tends to dissociate upon AUG recognition. Initiation-site footprint analysis revealed a class spanning -12 to +18/19 from the AUG, representing the entire 48S and enriched with eIF2, eIF1, and eIF4G1, indicative of early initiation. Another eIF3-dependent class extends up to +26 and exhibits reduced eIF2 and eIF4G1 association, suggesting a late/alternative initiation complex. Our analysis provides an overview of scanning, initiation, and evidence for conformational rearrangements in vivo.


Assuntos
Ribossomos , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Iniciação Traducional da Cadeia Peptídica , Humanos , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
18.
ISME J ; 18(1)2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38874172

RESUMO

Insects engage in manifold interactions with bacteria that can shift along the parasitism-mutualism continuum. However, only a small number of bacterial taxa managed to successfully colonize a wide diversity of insects, by evolving mechanisms for host-cell entry, immune evasion, germline tropism, reproductive manipulation, and/or by providing benefits to the host that stabilize the symbiotic association. Here, we report on the discovery of an Enterobacterales endosymbiont (Symbiodolus, type species Symbiodolus clandestinus) that is widespread across at least six insect orders and occurs at high prevalence within host populations. Fluorescence in situ hybridization in several Coleopteran and one Dipteran species revealed Symbiodolus' intracellular presence in all host life stages and across tissues, with a high abundance in female ovaries, indicating transovarial vertical transmission. Symbiont genome sequencing across 16 host taxa revealed a high degree of functional conservation in the eroding and transposon-rich genomes. All sequenced Symbiodolus genomes encode for multiple secretion systems, alongside effectors and toxin-antitoxin systems, which likely facilitate host-cell entry and interactions with the host. However, Symbiodolus-infected insects show no obvious signs of disease, and biosynthetic pathways for several amino acids and cofactors encoded by the bacterial genomes suggest that the symbionts may also be able to provide benefits to the hosts. A lack of host-symbiont cospeciation provides evidence for occasional horizontal transmission, so Symbiodolus' success is likely based on a mixed transmission mode. Our findings uncover a hitherto undescribed and widespread insect endosymbiont that may present valuable opportunities to unravel the molecular underpinnings of symbiosis establishment and maintenance.


Assuntos
Enterobacteriaceae , Insetos , Simbiose , Animais , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/classificação , Feminino , Insetos/microbiologia , Hibridização in Situ Fluorescente , Genoma Bacteriano , Filogenia
19.
ISME Commun ; 4(1): ycae005, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38439943

RESUMO

Insects typically acquire their beneficial microbes early in development. Endosymbionts housed intracellularly are commonly integrated during oogenesis or embryogenesis, whereas extracellular microbes are only known to be acquired after hatching by immature instars such as larvae or nymphs. Here, however, we report on an extracellular symbiont that colonizes its host during embryo development. Tortoise beetles (Chrysomelidae: Cassidinae) host their digestive bacterial symbiont Stammera extracellularly within foregut symbiotic organs and in ovary-associated glands to ensure its vertical transmission. We outline the initial stages of symbiont colonization and observe that although the foregut symbiotic organs develop 3 days prior to larval emergence, they remain empty until the final 24 h of embryo development. Infection by Stammera occurs during that timeframe and prior to hatching. By experimentally manipulating symbiont availability to embryos in the egg, we describe a 12-h developmental window governing colonization by Stammera. Symbiotic organs form normally in aposymbiotic larvae, demonstrating that these Stammera-bearing structures develop autonomously. In adults, the foregut symbiotic organs are already colonized following metamorphosis and host a stable Stammera population to facilitate folivory. The ovary-associated glands, however, initially lack Stammera. Symbiont abundance subsequently increases within these transmission organs, thereby ensuring sufficient titers at the onset of oviposition ~29 days following metamorphosis. Collectively, our findings reveal that Stammera colonization precedes larval emergence, where its proliferation is eventually decoupled in adult beetles to match the nutritional and reproductive requirements of its host.

20.
R Soc Open Sci ; 11(3): 231514, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38481984

RESUMO

Coral reefs, hubs of global biodiversity, are among the world's most imperilled habitats. Healthy coral reefs are characterized by distinctive soundscapes; these environments are rich with sounds produced by fishes and marine invertebrates. Emerging evidence suggests these sounds can be used as orientation and settlement cues for larvae of reef animals. On degraded reefs, these cues may be reduced or absent, impeding the success of larval settlement, which is an essential process for the maintenance and replenishment of reef populations. Here, in a field-based study, we evaluated the effects of enriching the soundscape of a degraded coral reef to increase coral settlement rates. Porites astreoides larvae were exposed to reef sounds using a custom solar-powered acoustic playback system. Porites astreoides settled at significantly higher rates at the acoustically enriched sites, averaging 1.7 times (up to maximum of seven times) more settlement compared with control reef sites without acoustic enrichment. Settlement rates decreased with distance from the speaker but remained higher than control levels at least 30 m from the sound source. These results reveal that acoustic enrichment can facilitate coral larval settlement at reasonable distances, offering a promising new method for scientists, managers and restoration practitioners to rebuild coral reefs.

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