RESUMO
The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to â¼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines.
Assuntos
Escherichia coli/química , Escherichia coli/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Microscopia Crioeletrônica , Espectrometria de Massas , Modelos Moleculares , Multimerização Proteica , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismoRESUMO
Cryo-electron tomography (cryo-ET) enables observation of macromolecular complexes in their native, spatially contextualized cellular environment. Cryo-ET processing software to visualize such complexes at nanometer resolution via iterative alignment and averaging are well developed but rely upon assumptions of structural homogeneity among the complexes of interest. Recently developed tools allow for some assessment of structural diversity but have limited capacity to represent highly heterogeneous structures, including those undergoing continuous conformational changes. Here we extend the highly expressive cryoDRGN (Deep Reconstructing Generative Networks) deep learning architecture, originally created for single-particle cryo-electron microscopy analysis, to cryo-ET. Our new tool, tomoDRGN, learns a continuous low-dimensional representation of structural heterogeneity in cryo-ET datasets while also learning to reconstruct heterogeneous structural ensembles supported by the underlying data. Using simulated and experimental data, we describe and benchmark architectural choices within tomoDRGN that are uniquely necessitated and enabled by cryo-ET. We additionally illustrate tomoDRGN's efficacy in analyzing diverse datasets, using it to reveal high-level organization of human immunodeficiency virus (HIV) capsid complexes assembled in virus-like particles and to resolve extensive structural heterogeneity among ribosomes imaged in situ.
Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Processamento de Imagem Assistida por Computador , Software , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Humanos , Aprendizado Profundo , HIV-1RESUMO
Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in bacteria and eukaryotic organelles of bacterial origin. In Escherichia coli and many other proteobacteria, the SspB adaptor assists ClpXP in degrading ssrA-tagged polypeptides produced as a consequence of tmRNA-mediated ribosome rescue. By tethering these incomplete ssrA-tagged proteins to ClpXP, SspB facilitates their efficient degradation at low substrate concentrations. How this process occurs structurally is unknown. Here, we present a cryo-EM structure of the SspB adaptor bound to a GFP-ssrA substrate and to ClpXP. This structure provides evidence for simultaneous contacts of SspB and ClpX with the ssrA tag within the tethering complex, allowing direct substrate handoff concomitant with the initiation of substrate translocation. Furthermore, our structure reveals that binding of the substrate·adaptor complex induces unexpected conformational changes within the spiral structure of the AAA+ ClpX hexamer and its interaction with the ClpP tetradecamer.
Assuntos
Proteínas de Transporte , Proteínas de Escherichia coli , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Adenosina Trifosfatases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Especificidade por SubstratoRESUMO
Cryo-electron microscopy (cryo-EM) single-particle analysis has proven powerful in determining the structures of rigid macromolecules. However, many imaged protein complexes exhibit conformational and compositional heterogeneity that poses a major challenge to existing three-dimensional reconstruction methods. Here, we present cryoDRGN, an algorithm that leverages the representation power of deep neural networks to directly reconstruct continuous distributions of 3D density maps and map per-particle heterogeneity of single-particle cryo-EM datasets. Using cryoDRGN, we uncovered residual heterogeneity in high-resolution datasets of the 80S ribosome and the RAG complex, revealed a new structural state of the assembling 50S ribosome, and visualized large-scale continuous motions of a spliceosome complex. CryoDRGN contains interactive tools to visualize a dataset's distribution of per-particle variability, generate density maps for exploratory analysis, extract particle subsets for use with other tools and generate trajectories to visualize molecular motions. CryoDRGN is open-source software freely available at http://cryodrgn.csail.mit.edu .
Assuntos
Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/química , Redes Neurais de Computação , Estrutura MolecularRESUMO
Sulfur (S) deficiency is becoming more common in agro-ecosystems worldwide due to factors such as agronomic practices, high biomass production, reduced sulfur emissions, and the use of non-sulfur fertilizers. This review explores the natural occurrence and commercial exploitation of sulfur pools in nature, the mineralization and immobilization of sulfur, the physiological role of sulfur in plants, and its deficiency symptoms. Additionally, the organic and inorganic forms of sulfur in soil, their transformations, and the process of microbiological oxidation of sulfur are discussed. The review also addresses the diversity of sulfur-oxidizing bacteria (SOB) and the various biochemical mechanisms involved in their role in plant productivity and soil reclamation. The measurement of S oxidation rate in soil and the variables that influence the process are also examined. Typically, the rate of oxidation of added elemental S is around 40%-51%, which is available for plant uptake. These characteristics of SOB demonstrate their potential as bioinoculants for increasing plant growth, indicating their use as biofertilizers for sustainable crop production in agro-ecosystems.
Assuntos
Bactérias , Ecossistema , Bactérias/genética , Plantas/microbiologia , Oxirredução , SoloRESUMO
The biomedical aspiration of psychiatry has fundamentally reoriented clinical practice since the DSM-III in 1980 and reverberated in the public sphere. Over time, lay public understanding of the causes of mental suffering has increasingly endorsed biological conceptions. In this paper, I explore the sources from which a neurobiological model for mental suffering reaches ordinary people, and investigate its rhetorical appeal, personal appropriation, and consequences. Drawing on interviews and other data, I show that these sources-physicians, popular media, and advertising-share common ontological and moral assumptions. These assumptions, in turn, influence how people take up neurobiological explanation to account for their suffering, and how, paradoxically, they join it to their projects of self-determination. I conclude by considering how, from a phenomenological perspective, a neurobiological account fails to enhance self-knowledge or determination but leads to a hermeneutic dead end.
Assuntos
Neurobiologia , Psiquiatria , Hermenêutica , Humanos , Princípios Morais , AutoimagemRESUMO
Background. Despite favorable outcomes of laparoscopic surgery in high-income countries, its implementation in low- and middle-income countries (LMICs) is challenging given a shortage of consumable supplies, high cost, and risk of power outages. To overcome these barriers, we designed a mechanical retractor that provides vertical tension on the anterior abdominal wall. Methods. The retractor design is anatomically and mathematically optimized to provide exposure similar to traditional gas-based insufflation methods. Anatomical data from computed tomography scans were used to define retractor size. The retractor is constructed of biocompatible stainless steel rods and paired with a table-mounted lifting system to provide 5 degrees of freedom. Structural integrity was assessed through finite element analysis (FEA) and load testing. Functional testing was performed in a laparotomy model. Results. A user guide based on patient height and weight was created to customize retractor size, and 4 retractor sizes were constructed. FEA data using a 13.6 kg mass (15 mm Hg pneumoperitoneum) show a maximum of 30 mm displacement with no permanent deformation. Physical load testing with applied weight from 0 to 13.6 kg shows a maximum of 60 mm displacement, again without permanent deformation. Retraction achieved a 57% larger field of view compared to an unretracted state in a laparotomy model. Conclusions. The KeyLoop retractor maintains structural integrity, is easily sterilized, and can be readily manufactured, making it a viable alternative to traditional insufflation methods. For surgeons and patients in LMICs, the KeyLoop provides a means to increase access to laparoscopic surgery.
Assuntos
Parede Abdominal , Insuflação , Laparoscopia , Cirurgiões , Parede Abdominal/cirurgia , Humanos , Laparoscopia/métodos , LaparotomiaRESUMO
OBJECTIVE: To report the findings and long-term outcome of 76 sport horses with meniscal injury. STUDY DESIGN: Retrospective case series. ANIMALS: Seventy-six horses with 93 meniscal injuries in 85 stifles. METHODS: Medical records of sport horses diagnosed with meniscal injury during arthroscopy were reviewed. Owner follow up was obtained via telephone interview ≥1.5 years postoperatively. Preoperative and intraoperative findings, and postoperative treatments, were analyzed for potential association with return to athletic performance. RESULTS: The medial meniscus was involved in 82.8% of cases, with grade 1 injuries diagnosed in 76.3% of menisci. Overall, 85.5% of horses returned to athletic performance, with 40% returning to their previous level. The grade of meniscal injury was associated with long-term outcome (P = .023). The presence of preoperative radiographic abnormalities (P = .259) or additional joint pathology (P = 1.00) was not associated with long-term outcomes. Fifty-nine stifles were treated with an orthobiologic: autologous conditioned serum, platelet-rich plasma, or marrow-derived mesenchymal stem cells. There was no association between the use of any orthobiologic and long-term outcome (P = .394). CONCLUSION: This is the first report on long-term outcome of sport horses with meniscal injuries following arthroscopic surgery. Overall, the long-term prognosis was fair, with 40% of horses returning to their previous level of use. Severity of the meniscal injury was a prognostic indicator for return to work. The presence of radiographic abnormalities or additional joint pathology, or the use of orthobiologics, was not associated with long-term outcome. CLINICAL SIGNIFICANCE: These findings can help in prognostication for sport horses with meniscal injuries.
Assuntos
Doenças dos Cavalos , Menisco , Lesões do Menisco Tibial , Animais , Artroscopia/veterinária , Doenças dos Cavalos/patologia , Doenças dos Cavalos/cirurgia , Cavalos , Meniscos Tibiais/cirurgia , Estudos Retrospectivos , Joelho de Quadrúpedes/patologia , Lesões do Menisco Tibial/cirurgia , Lesões do Menisco Tibial/veterináriaRESUMO
Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Homeostase , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Sequência de Bases , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Conformação de Ácido Nucleico , Ligação Proteica , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores/genética , Subunidades Ribossômicas Menores/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/ultraestruturaRESUMO
Appropriate imaging is imperative in evaluating children with a primary hepatic malignancy such as hepatoblastoma or hepatocellular carcinoma. For use in the adult patient population, the American College of Radiology created the Liver Imaging Reporting and Data System (LI-RADS) to provide consistent terminology and to improve imaging interpretation. At present, no similar consensus exists to guide imaging and interpretation of pediatric patients at risk for developing a liver neoplasm or how best to evaluate a pediatric patient with a known liver neoplasm. Therefore, a new Pediatric Working Group within American College of Radiology LI-RADS was created to provide consensus for imaging recommendations and interpretation of pediatric liver neoplasms. The article was drafted based on the most up-to-date existing information as interpreted by imaging experts comprising the Pediatric LI-RADS Working Group. Guidance is provided regarding appropriate imaging modalities and protocols, as well as imaging interpretation and reporting, with the goals to improve imaging quality, to decrease image interpretation errors, to enhance communication with referrers, and to advance patient care. An expanded version of this document that includes broader background information on pediatric hepatocellular carcinoma and rationale for recommendations can be found in Appendix E1 (online).
Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Hepatoblastoma/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Biópsia , Criança , Pré-Escolar , Consenso , Humanos , Lactente , Imageamento por Ressonância Magnética , Guias de Prática Clínica como Assunto , Sistemas de Informação em Radiologia/organização & administração , Tomografia Computadorizada por Raios XRESUMO
We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes. We applied these methods to determine the structures at near-atomic resolution of the influenza hemagglutinin trimer, which adopts a highly preferred specimen orientation, and of ribosomal biogenesis intermediates, which adopt moderately preferred orientations.
Assuntos
Microscopia Crioeletrônica/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/ultraestrutura , Aumento da Imagem/métodos , Imageamento Tridimensional/métodos , Imagem Molecular/métodos , Manejo de Espécimes/métodos , Algoritmos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
One of the more common and important challenges in the imaging of children is minimizing image degradation caused by motion. This is especially important in MRI, which is often preferable in the pediatric population due to better tissue characterization and lack of ionizing radiation. However, due to the length of time needed for most examinations, MRI is among the most sensitive to disruption by patient motion. Traditionally, deep conscious sedation or general anesthesia was the most common method of reducing motion in children who are unable or unwilling to follow direction. As the drawbacks and risks of anesthesia in children become more known and accepted, the development and optimization of means of mitigating motion and anxiety in children without the use of sedation or anesthesia becomes more urgent. In this article we describe the risks of sedation in the pediatric population and explore current methods of reducing both patient anxiety and imaging degradation from motion in the unsedated, free-breathing child. Level of Evidence: 5 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2019;50:365-376.
Assuntos
Artefatos , Imageamento por Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/psicologia , Ansiedade/prevenção & controle , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Movimento (Física) , RespiraçãoRESUMO
OBJECTIVE. Diagnostic reference levels were developed as guidance for radiation dose in medical imaging and, by inference, diagnostic quality. The objective of this work was to expand the concept of diagnostic reference levels to explicitly include noise of CT examinations to simultaneously target both dose and quality through corresponding reference values. MATERIALS AND METHODS. The study consisted of 2851 adult CT examinations performed with scanners from two manufacturers and two clinical protocols: abdominopelvic CT with IV contrast administration and chest CT without IV contrast administration. An institutional informatics system was used to automatically extract protocol type, patient diameter, volume CT dose index, and noise magnitude from images. The data were divided into five reference patient size ranges. Noise reference level, noise reference range, dose reference level, and dose reference range were defined for each size range. RESULTS. The data exhibited strong dependence between dose and patient size, weak dependence between noise and patient size, and different trends for different manufacturers with differing strategies for tube current modulation. The results suggest size-based reference intervals and levels for noise and dose (e.g., noise reference level and noise reference range of 11.5-12.9 HU and 11.0-14.0 HU for chest CT and 10.1-12.1 HU and 9.4-13.7 HU for abdominopelvic CT examinations) that can be targeted to improve clinical performance consistency. CONCLUSION. New reference levels and ranges, which simultaneously consider image noise and radiation dose information across wide patient populations, were defined and determined for two clinical protocols. The methods of new quantitative constraints may provide unique and useful information about the goal of managing the variability of image quality and dose in clinical CT examinations.
Assuntos
Ruído , Doses de Radiação , Tomografia Computadorizada por Raios X/normas , Adulto , Tamanho Corporal , Meios de Contraste , Humanos , Radiografia Abdominal/normas , Radiografia Torácica/normas , Valores de ReferênciaRESUMO
Aneurysmal subarachnoid hemorrhage has a high mortality rate and, for those who survive this devastating injury, can lead to lifelong impairment. Clinical trials have demonstrated that cerebral vasospasm of larger extraparenchymal vessels is not the sole contributor to neurological outcome. Recently, the focus of intense investigation has turned to mechanisms of early brain injury that may play a larger role in outcome, including neuroinflammation and microvascular dysfunction. Extravasated blood after aneurysm rupture results in a robust inflammatory response characterized by activation of microglia, upregulation of cellular adhesion molecules, recruitment of peripheral immune cells, as well as impaired neurovascular coupling, disruption of the blood-brain barrier, and imbalances in endogenous vasodilators and vasoconstrictors. Each of these phenomena is either directly or indirectly associated with neuronal death and brain injury. Here, we review recent studies investigating these various mechanisms in experimental models of subarachnoid hemorrhage with special emphasis on neuroinflammation and its effect on microvascular dysfunction. We discuss the various therapeutic targets that have risen from these mechanistic studies and suggest the utility of a multi-targeted approach to preventing delayed injury and improving outcome after subarachnoid hemorrhage.
Assuntos
Barreira Hematoencefálica/fisiopatologia , Citocinas/imunologia , Leucócitos/imunologia , Microglia/imunologia , Microvasos/fisiopatologia , Hemorragia Subaracnóidea/fisiopatologia , Trombose/fisiopatologia , Animais , Moléculas de Adesão Celular , Modelos Animais de Doenças , Humanos , Inflamação/imunologia , Acoplamento Neurovascular/fisiologia , Hemorragia Subaracnóidea/imunologia , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
The threshold for conscious perception of stimuli within the environment varies from individual to individual. Functional neuroimaging studies have suggested that insular cortex activity is positively correlated with perceptual awareness. However, few studies have tested the relationship between awareness and structural variability in the insula. The purpose of this study was to examine structural differences in brain morphology related to perceptual awareness of fearful faces. This study hypothesized that there would be a positive correlation between insular grey matter volume and scores on the forced-choice awareness check task. The forced-choice awareness check task was designed to assess awareness for the presence and location of backward masked fearful and neutral faces, masked with neutral faces. The participants responded by indicating the side on which the masked fearful face appeared, or whether there were two neutral faces. The task included a total of 60 trials. T1-weighted magnetic resonance images were collected to measure grey matter volumes. Individuals that were more aware of backward masked fearful faces had greater grey matter volume in the insula, middle cingulate, anterior temporal pole, ventral striatum, and hippocampus.
Assuntos
Conscientização , Córtex Cerebral/diagnóstico por imagem , Expressão Facial , Reconhecimento Facial , Medo , Substância Cinzenta/diagnóstico por imagem , Adulto , Conscientização/fisiologia , Córtex Cerebral/anatomia & histologia , Reconhecimento Facial/fisiologia , Feminino , Substância Cinzenta/anatomia & histologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Tamanho do Órgão , Adulto JovemRESUMO
OBJECTIVE: To determine the location of the deep and superficial caudal epigastric arteries in relation to 3 midline positions and the relationship between the location of these arteries, body circumference, and body condition score. STUDY DESIGN: Descriptive anatomical study. SAMPLE POPULATION: Nine horses, aged 1-28 years (mean 10.61 ± 8.89 SD). METHODS: Body condition score and body circumference were measured prior to euthanasia. Angiographic studies of the deep and superficial caudal epigastric arteries were performed on resected abdominal walls. The distances between the deep and the superficial caudal epigastric arteries and 3 midline positions were measured. Correlations among these distances, body circumference, and body condition score were analyzed. RESULTS: The location of the deep caudal epigastric artery correlated with body circumference and body condition score at the umbilicus (r = 0.53 and 0.68, respectively), midpoint landmark (r = 0.79 and 0.83, respectively), and prepubic tendon attachment (r = 0.69 and 0.78, respectively). The course of this artery could be estimated by multiplying body circumference by 0.04 ± 0.02 at the umbilicus, 0.07 ± 0.01 at the midpoint landmark, and 0.03 ± 0.015 at the prepubic tendon attachment. The course of the superficial caudal epigastric artery did not correlate with anatomic landmarks. CONCLUSION: The course of the deep caudal epigastric artery could be estimated at 3 midline landmarks on the basis of body circumference and body condition score in equine cadavers. CLINICAL SIGNIFICANCE: Predicting the course of the caudal epigastric arteries in the equine abdomen based on correlation among location, body circumference, and body condition score may prevent iatrogenic damage during creation of laparoscopic portals.
Assuntos
Artérias Epigástricas/anatomia & histologia , Cavalos/anatomia & histologia , Animais , Composição Corporal , Cadáver , Artérias Epigástricas/diagnóstico por imagem , Feminino , HumanosRESUMO
Aims: To determine if a software algorithm can use an individualized distance-morphology difference model, built from three initial pacemaps, to prospectively locate the exit site (ES) of ventricular arrhythmias (VA). Methods and results: Consecutive patients undergoing ablation of VA from a single centre were recruited. During mapping, three initial pacing points were collected in the chamber of interest and the navigation algorithm applied to predict the ES, which was corroborated by conventional mapping techniques. Thirty-two patients underwent ES prediction over 35 procedures. Structural heart disease was present in 16 (7 ischaemic cardiomyopathy, 9 non-ischaemic cardiomyopathy), median ejection fraction 45% [Interquartile range (IQR) 26]. The remainder had normal hearts. The navigation algorithm was applied to 46 VA (24 left ventricle, 11 right ventricular outflow tract, 5 left ventricular outflow tract, 4 right ventricle, 2 epicardial) and successfully located the site of best pacemap match in 45 within a median area of 196.5 mm2 (IQR 161.3, range 46.6-1288.2 mm2). Conclusions: In a diverse population of patients with and without structural heart disease, the ES of VA can be accurately and reliably identified to within a clinically useful target area using a simple software navigation algorithm based on pacemapping.
Assuntos
Algoritmos , Técnicas Eletrofisiológicas Cardíacas/métodos , Software , Taquicardia Ventricular/fisiopatologia , Complexos Ventriculares Prematuros/fisiopatologia , Adulto , Idoso , Displasia Arritmogênica Ventricular Direita/complicações , Cardiomiopatias/complicações , Cardiomiopatia Dilatada/complicações , Ablação por Cateter , Cicatriz/complicações , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/complicações , Miocardite/complicações , Estudo de Prova de Conceito , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/cirurgia , Complexos Ventriculares Prematuros/etiologia , Complexos Ventriculares Prematuros/cirurgiaRESUMO
Our understanding regarding the function of YjeQ (also called RsgA), RbfA, RimM and Era in ribosome biogenesis has been derived in part from the study of immature 30S particles that accumulate in null strains lacking one of these factors. However, their mechanistic details are still unknown. Here, we demonstrate that these immature particles are not dead-end products of assembly, but progress into mature 30S subunits. Mass spectrometry analysis revealed that in vivo the occupancy level of these factors in these immature 30S particles is below 10% and that the concentration of factors does not increase when immature particles accumulate in cells. We measured by microscale thermophoresis that YjeQ and Era binds to the mature 30S subunit with high affinity. However, the binding affinity of these factors to the immature particles and of RimM and RbfA to mature or immature particles was weak, suggesting that binding is not occurring at physiological concentrations. These results suggest that in the absence of these factors, the immature particles evolve into a thermodynamically stable intermediate that exhibits low affinity for the assembly factors. These results imply that the true substrates of YjeQ, RbfA, RimM and Era are immature particles that precede the ribosomal particles accumulating in the knockouts strains.
Assuntos
Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Modelos Biológicos , Complexos Multiproteicos , Mutação , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5-6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit.
Assuntos
Proteínas de Bactérias/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/ultraestrutura , Cinética , Espectrometria de Massas , Conformação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/ultraestruturaRESUMO
Oral Candida albicans has been detected in children with early childhood caries (ECC) and has demonstrated cariogenic traits in animal models of the disease. Conversely, other studies found no positive correlation between C. albicans and caries experience in children, while suggesting it may have protective effects as a commensal organism. Thus, this study aimed to examine whether oral C. albicans is associated with ECC. Seven electronic databases were searched. The data from eligible studies were extracted, and the risk of bias was evaluated. A fixed effects model (Mantel-Haenszel estimate) was used for meta-analysis, and the summary effect measure was calculated by odds ratio (OR) and 95% confidence interval (CI). Fifteen cross-sectional studies were included for the qualitative assessment and 9 studies for meta-analysis. Twelve studies revealed higher oral C. albicans prevalence in ECC children than in caries-free children, while 2 studies indicated an equivalent prevalence. A pooled estimate, with OR = 6.51 and 95% CI = 4.94-8.57, indicated a significantly higher ECC experience in children with oral C. albicans than those without C. albicans (p < 0.01). The odds of experiencing ECC in children with C. albicans versus children without C. albicans were 5.26 for salivary, 6.69 for plaque, and 6.3 for oral swab samples. This systematic review indicates that children with oral C. albicans have >5 times higher odds of having ECC compared to those without C. albicans. Further prospective cohort studies are needed to determine whether C. albicans could be a risk factor for ECC, and whether it is dependent on different sample sources (saliva/plaque).