RESUMO
Microbial rhodopsins are a large family of photoreceptive membrane proteins with diverse light-regulated functions. While the most ubiquitous microbial rhodopsins are light-driven outward proton (H+) pumps, new subfamilies of microbial rhodopsins transporting H+ inwardly, i.e., light-driven inward H+ pumps, have been discovered recently. Although structural and spectroscopic studies provide insights into their ion transport mechanisms, the minimum key element(s) that determine the direction of H+ transport have not yet been clarified. Here, we conducted the first functional conversion study by substituting key amino acids in a natural outward H+-pumping rhodopsin (PspR) with those in inward H+-pumping rhodopsins. Consequently, an artificial inward H+ pump was constructed by mutating only three residues of PspR. This result indicates that these residues govern the key processes that discriminate between outward and inward H+ pumps. Spectroscopic studies revealed the presence of an inward H+-accepting residue in the H+ transport pathway and direct H+ uptake from the extracellular solvent. This finding of the simple element for determining H+ transport would provide a new basis for understanding the concept of ion transport not only by microbial rhodopsins but also by other ion-pumping proteins.
Assuntos
Bombas de Próton , Rodopsina , Bombas de Próton/química , Rodopsina/química , Rodopsinas Microbianas/metabolismo , Transporte de Íons , Bombas de Íon/metabolismo , Prótons , LuzRESUMO
BACKGROUND: Despite the need for specific weaning strategies in neurological patients, evidence is generally insufficient or lacking. We aimed to describe the evolution over time of weaning and extubation practices in patients with acute brain injury compared with patients who are mechanically ventilated (MV) due to other reasons. METHODS: We performed a secondary analysis of three prospective, observational, multicenter international studies conducted in 2004, 2010, and 2016 in adults who had need of invasive MV for more than 12 h. We collected data on baseline characteristics, variables related to management ventilator settings, and complications while patients were ventilated or until day 28. RESULTS: Among the 20,929 patients enrolled, we included 12,618 (60%) who started the weaning from MV, of whom 1722 (14%) were patients with acute brain injury. In the acutely brain-injured cohort, 538 patients (31%) did not undergo planned extubation, defined as the need for a tracheostomy without an attempt of extubation, accidental extubation, and death. Among the 1184 planned extubated patients with acute brain injury, 202 required reintubation (17%). Patients with acute brain injury had a higher odds for unplanned extubation (odds ratio [OR] 1.35, confidence interval for 95% [CI 95%] 1.19-1.54; p < 0.001), a higher odds of failure after the first attempt of weaning (spontaneous breathing trial or gradual reduction of ventilatory support; OR 1.14 [CI 95% 1.01-1.30; p = 0.03]), and a higher odds for reintubation (OR 1.41 [CI 95% 1.20-1.66; p < 0.001]) than patients without brain injury. Patients with hemorrhagic stroke had the highest odds for unplanned extubation (OR 1.47 [CI 95% 1.22-1.77; p < 0.001]), of failed extubation after the first attempt of weaning (OR 1.28 [CI 95% 1.06-1.55; p = 0.009]), and for reintubation (OR 1.49 [CI 95% 1.17-1.88; p < 0.001]). In relation to weaning evolution over time in patients with acute brain injury, the risk for unplanned extubation showed a downward trend; the risk for reintubation was not associated to time; and there was a significant increase in the percentage of patients who underwent extubation after the first attempt of weaning from MV. CONCLUSIONS: Patients with acute brain injury, compared with patients without brain injury, present higher odds of undergoing unplanned extubated after weaning was started, lower odds of being extubated after the first attempt, and a higher risk of reintubation.
Assuntos
Lesões Encefálicas , Desmame do Respirador , Adulto , Humanos , Estudos Prospectivos , Extubação , Intubação Intratraqueal , Lesões Encefálicas/terapia , Respiração ArtificialRESUMO
OBJECTIVES: To describe the changes in ventilator management over time in patients with neurologic disease at ICU admission and to estimate factors associated with 28-day hospital mortality. DESIGN: Secondary analysis of three prospective, observational, multicenter studies. SETTING: Cohort studies conducted in 2004, 2010, and 2016. PATIENTS: Adult patients who received mechanical ventilation for more than 12 hours. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among the 20,929 patients enrolled, we included 4,152 (20%) mechanically ventilated patients due to different neurologic diseases. Hemorrhagic stroke and brain trauma were the most common pathologies associated with the need for mechanical ventilation. Although volume-cycled ventilation remained the preferred ventilation mode, there was a significant (p < 0.001) increment in the use of pressure support ventilation. The proportion of patients receiving a protective lung ventilation strategy was increased over time: 47% in 2004, 63% in 2010, and 65% in 2016 (p < 0.001), as well as the duration of protective ventilation strategies: 406 days per 1,000 mechanical ventilation days in 2004, 523 days per 1,000 mechanical ventilation days in 2010, and 585 days per 1,000 mechanical ventilation days in 2016 (p < 0.001). There were no differences in the length of stay in the ICU, mortality in the ICU, and mortality in hospital from 2004 to 2016. Independent risk factors for 28-day mortality were age greater than 75 years, Simplified Acute Physiology Score II greater than 50, the occurrence of organ dysfunction within first 48 hours after brain injury, and specific neurologic diseases such as hemorrhagic stroke, ischemic stroke, and brain trauma. CONCLUSIONS: More lung-protective ventilatory strategies have been implemented over years in neurologic patients with no effect on pulmonary complications or on survival. We found several prognostic factors on mortality such as advanced age, the severity of the disease, organ dysfunctions, and the etiology of neurologic disease.
Assuntos
Unidades de Terapia Intensiva/estatística & dados numéricos , Doenças do Sistema Nervoso/mortalidade , Doenças do Sistema Nervoso/terapia , Respiração Artificial/métodos , Respiração Artificial/tendências , Adulto , Fatores Etários , Idoso , Lesões Encefálicas Traumáticas/mortalidade , Lesões Encefálicas Traumáticas/terapia , Feminino , Acidente Vascular Cerebral Hemorrágico/mortalidade , Acidente Vascular Cerebral Hemorrágico/terapia , Mortalidade Hospitalar/tendências , Humanos , AVC Isquêmico/mortalidade , AVC Isquêmico/terapia , Tempo de Internação/tendências , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Ventilação não Invasiva/tendências , Estudos Observacionais como Assunto , Estudos Prospectivos , Fatores de Risco , Escore Fisiológico Agudo Simplificado , Traqueotomia/estatística & dados numéricos , Traqueotomia/tendências , Desmame do Respirador/tendênciasRESUMO
This article introduces Web-ARM, a specialized tool, online available, designed to build quantum mechanical/molecular mechanical models of rhodopsins, a widely spread family of light-responsive proteins. Web-ARM allows the rapidly building of models of rhodopsins with a documented quality and the prediction of trends in UV-vis absorption maximum wavelengths, based on their excitation energies computed at the CASPT2//CASSCF/Amber level of theory. Web-ARM builds upon the recently reported, python-based a-ARM protocol [J. Chem. Theory Comput., 2019, 15, 3134-3152] and, as such, necessitates only a crystallographic structure or a comparative model in PDB format and a very basic knowledge of the studied rhodopsin system. The user-friendly web interface uses such input to generate congruous, gas-phase models of rhodopsins and, if requested, their mutants. We present two possible applications of Web-ARM, which showcase how the interface can be employed to assist both research and educational activities in fields at the interface between chemistry and biology. The first application shows how, through Web-ARM, research projects (e.g., rhodopsin and rhodopsin mutant screening) can be carried out in significantly less time with respect to using the required computational photochemistry tools via a command line. The second application documents the use of Web-ARM in a real-life educational/training activity, through a hands-on experience illustrating the concepts of rhodopsin color tuning.
Assuntos
Teoria Quântica , Rodopsina , Internet , Modelos MolecularesRESUMO
Ethanol-induced behavioral sensitization (EIBS) is thought to play a key role in addiction. However, whether EIBS is linked to an increase in the motivation to self-administerethanol in an operant paradigm has never been demonstrated, and thus, the motivational sensitization theory (increase in drug wanting) has not been yet confirmed. We investigated using the operant ethanol self-administrationparadigm if the motivation to self-administerethanol (breakpoint) is increased in female mice prone to develop EIBS. Outbred female Swiss mice were treated once a day with 2.5-g ethanol per kilogram during 10 days and challenged with the same dose of ethanol 7 days later. EIBS-pronegroup was characterized by a significant increase in locomotion between the challenge day and day 1. When the difference was not significant, mice were considered as the "EIBS-resistant"group. Mice were then trained to nose poke for a 20% ethanol solution reinforcer under a FR1 and then a FR-2schedule of reinforcement. Motivation was assessed more directly with a progressive ratio schedule. Our results show that there is a positive correlation between EIBS and both the level of intake and motivation. Interestingly, acquisition of ethanol self-administrationwas faster in sensitized mice that also display a quick and long-lastingincrease in ethanol intake together with a lack of effect of alcohol challenge on c-Fosexpression restricted to the dorsolateral striatum. These results further support that EIBS vulnerability is crucial in the development of addictive behaviors and suggest a potential link with habit learning processes.
Assuntos
Consumo de Bebidas Alcoólicas/psicologia , Etanol/administração & dosagem , Motivação/efeitos dos fármacos , Animais , Condicionamento Operante , Corpo Estriado/efeitos dos fármacos , Etanol/farmacologia , Feminino , Locomoção , Atividade Motora/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Reforço Psicológico , AutoadministraçãoRESUMO
The engineering of microbial rhodopsins with enhanced fluorescence is of great importance in the expanding field of optogenetics. Here we report the discovery of two mutants (W76S/Y179F and L83Q) of a sensory rhodopsin from the cyanobacterium Anabaena PCC7120 with opposite fluorescence behavior. In fact, while W76S/Y179F displays, with respect to the wild-type protein, a nearly 10-fold increase in red-light emission, the second is not emissive. Thus, the W76S/Y179F, L83Q pair offers an unprecedented opportunity for the investigation of fluorescence enhancement in microbial rhodopsins, which is pursued by combining transient absorption spectroscopy and multiconfigurational quantum chemistry. The results of such an investigation point to an isomerization-blocking electronic effect as the direct cause of instantaneous (subpicosecond) fluorescence enhancement.
Assuntos
Anabaena/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Engenharia de Proteínas , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Transporte de Elétrons , Modelos Moleculares , Proteínas Mutantes/genética , Conformação Proteica , Rodopsinas Microbianas/genética , Espectrometria de FluorescênciaRESUMO
The symbiotic unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) is one of the most abundant and widespread nitrogen (N2 )-fixing cyanobacteria in the ocean. Although it remains uncultivated, multiple sublineages have been detected based on partial nitrogenase (nifH) gene sequences, including the four most commonly detected sublineages UCYN-A1, UCYN-A2, UCYN-A3 and UCYN-A4. However, very little is known about UCYN-A3 beyond the nifH sequences from nifH gene diversity surveys. In this study, single cell sorting, DNA sequencing, qPCR and CARD-FISH assays revealed discrepancies involving the identification of sublineages, which led to new information on the diversity of the UCYN-A symbiosis. 16S rRNA and nifH gene sequencing on single sorted cells allowed us to identify the 16S rRNA gene of the uncharacterized UCYN-A3 sublineage. We designed new CARD-FISH probes that allowed us to distinguish and observe UCYN-A2 in a coastal location (SIO Pier; San Diego) and UCYN-A3 in an open ocean location (Station ALOHA; Hawaii). Moreover, we reconstructed about 13% of the UCYN-A3 genome from Tara Oceans metagenomic data. Finally, our findings unveil the UCYN-A3 symbiosis in open ocean waters suggesting that the different UCYN-A sublineages are distributed along different size fractions of the plankton defined by the cell-size ranges of their prymnesiophyte hosts.
Assuntos
Cianobactérias/isolamento & purificação , Cianobactérias/metabolismo , Fixação de Nitrogênio , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianobactérias/classificação , Cianobactérias/genética , DNA Bacteriano/genética , Haptófitas/microbiologia , Haptófitas/fisiologia , Havaí , Nitrogenase/genética , Nitrogenase/metabolismo , Oceanos e Mares , Filogenia , RNA Ribossômico 16S/genética , Água do Mar/microbiologia , SimbioseRESUMO
BACKGROUND: Intensive care medicine is a relatively young discipline that has rapidly grown into a full-fledged medical subspecialty. Intensivists are responsible for managing an ever-increasing number of patients with complex, life-threatening diseases. Several factors may influence their performance, including age, training, experience, workload, and socioeconomic context. The aim of this study was to examine individual- and work-related aspects of the Latin American intensivist workforce, mainly with academic appointments, which might influence the quality of care provided. In consequence, we conducted a cross-sectional study of intensivists at public and private academic and nonacademic Latin American intensive care units (ICUs) through a web-based electronic survey submitted by email. Questions about personal aspects, work-related topics, and general clinical workflow were incorporated. RESULTS: Our study comprised 735 survey respondents (53% return rate) with the following country-specific breakdown: Brazil (29%); Argentina (19%); Chile (17%); Uruguay (12%); Ecuador (9%); Mexico (7%); Colombia (5%); and Bolivia, Peru, Guatemala, and Paraguay combined (2%). Latin American intensivists were predominantly male (68%) young adults (median age, 40 [IQR, 35-48] years) with a median clinical ICU experience of 10 (IQR, 5-20) years. The median weekly workload was 60 (IQR, 47-70) h. ICU formal training was between 2 and 4 years. Only 63% of academic ICUs performed multidisciplinary rounds. Most intensivists (85%) reported adequate conditions to manage patients with septic shock in their units. Unsatisfactory conditions were attributed to insufficient technology (11%), laboratory support (5%), imaging resources (5%), and drug shortages (5%). Seventy percent of intensivists participated in research, and 54% read scientific studies regularly, whereas 32% read no more than one scientific study per month. Research grants and pharmaceutical sponsorship are unusual funding sources in Latin America. Although Latin American intensivists are mostly unsatisfied with their income (81%), only a minority (27%) considered changing to another specialty before retirement. CONCLUSIONS: Latin American intensivists constitute a predominantly young adult workforce, mostly formally trained, have a high workload, and most are interested in research. They are under important limitations owing to resource constraints and overt dissatisfaction. Latin America may be representative of other world areas with similar challenges for intensivists. Specific initiatives aimed at addressing these situations need to be devised to improve the quality of critical care delivery in Latin America.
Assuntos
Cuidados Críticos/tendências , Países em Desenvolvimento/estatística & dados numéricos , Centros Médicos Acadêmicos/organização & administração , Centros Médicos Acadêmicos/estatística & dados numéricos , Adulto , Distribuição de Qui-Quadrado , Estudos Transversais , Feminino , Humanos , América Latina , Masculino , Pessoa de Meia-Idade , Estatísticas não Paramétricas , Inquéritos e Questionários , Recursos HumanosRESUMO
Prochlorococcus is responsible for a significant part of CO2 fixation in the ocean. Although it was long considered an autotrophic cyanobacterium, the uptake of organic compounds has been reported, assuming they were sources of limited biogenic elements. We have shown in laboratory experiments that Prochlorococcus can take up glucose. However, the mechanisms of glucose uptake and its occurrence in the ocean have not been shown. Here, we report that the gene Pro1404 confers capability for glucose uptake in Prochlorococcus marinus SS120. We used a cyanobacterium unable to take up glucose to engineer strains that express the Pro1404 gene. These recombinant strains were capable of specific glucose uptake over a wide range of glucose concentrations, showing multiphasic transport kinetics. The Ks constant of the high affinity phase was in the nanomolar range, consistent with the average concentration of glucose in the ocean. Furthermore, we were able to observe glucose uptake by Prochlorococcus in the central Atlantic Ocean, where glucose concentrations were 0.5-2.7 nM. Our results suggest that Prochlorococcus are primary producers capable of tuning their metabolism to energetically benefit from environmental conditions, taking up not only organic compounds with key limiting elements in the ocean, but also molecules devoid of such elements, like glucose.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose/metabolismo , Oceanos e Mares , Prochlorococcus/metabolismo , Microbiologia da Água , Proteínas de Bactérias/genética , Transporte Biológico Ativo/fisiologia , Proteínas Facilitadoras de Transporte de Glucose/genética , Prochlorococcus/genéticaAssuntos
Doenças do Íleo/etiologia , Obstrução Intestinal/etiologia , Doença Inflamatória Pélvica/complicações , Abscesso/complicações , Abscesso/diagnóstico , Abscesso/terapia , Doença Aguda , Feminino , Humanos , Doenças do Íleo/diagnóstico , Doenças do Íleo/cirurgia , Obstrução Intestinal/diagnóstico , Obstrução Intestinal/terapia , Laparoscopia , Cistos Ovarianos/diagnóstico por imagem , Cistos Ovarianos/terapia , Doenças Ovarianas/complicações , Doenças Ovarianas/diagnóstico , Doenças Ovarianas/terapia , Doença Inflamatória Pélvica/diagnóstico , Doença Inflamatória Pélvica/terapia , Salpingite/diagnóstico , Salpingite/cirurgia , Aderências Teciduais/diagnóstico , Aderências Teciduais/etiologia , Aderências Teciduais/cirurgia , Descarga Vaginal/tratamento farmacológico , Descarga Vaginal/microbiologia , Descarga Vaginal/parasitologia , Adulto JovemRESUMO
Cyanobacteria evolved the oxygenic photosynthesis to generate organic matter from CO2 and sunlight, and they were responsible for the production of oxygen in the Earth's atmosphere. This made them a model for photosynthetic organisms, since they are easier to study than higher plants. Early studies suggested that only a minority among cyanobacteria might assimilate organic compounds, being considered mostly autotrophic for decades. However, compelling evidence from marine and freshwater cyanobacteria, including toxic strains, in the laboratory and in the field, has been obtained in the last decades: by using physiological and omics approaches, mixotrophy has been found to be a more widespread feature than initially believed. Furthermore, dominant clades of marine cyanobacteria can take up organic compounds, and mixotrophy is critical for their survival in deep waters with very low light. Hence, mixotrophy seems to be an essential trait in the metabolism of most cyanobacteria, which can be exploited for biotechnological purposes.
Assuntos
Cianobactérias , Cianobactérias/genética , Cianobactérias/metabolismo , Fotossíntese/fisiologia , Atmosfera , Oxigênio/metabolismoRESUMO
Microbial rhodopsins are photoreceptive transmembrane proteins that transport ions or regulate other intracellular biological processes. Recent genomic and metagenomic analyses found many microbial rhodopsins with unique sequences distinct from known ones. Functional characterization of these new types of microbial rhodopsins is expected to expand our understanding of their physiological roles. Here, we found microbial rhodopsins having a DSE motif in the third transmembrane helix from members of the Actinobacteria. Although the expressed proteins exhibited blue-green light absorption, either no or extremely small outward H+ pump activity was observed. The turnover rate of the photocycle reaction of the purified proteins was extremely slow compared to typical H+ pumps, suggesting these rhodopsins would work as photosensors or H+ pumps whose activities are enhanced by an unknown regulatory system in the hosts. The discovery of this rhodopsin group with the unique motif and functionality expands our understanding of the biological role of microbial rhodopsins.
RESUMO
Decades of research on marine N2 fixation focused on Trichodesmium, which are generally free-living cyanobacteria, but in recent years the endosymbiotic cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) has received increasing attention. However, few studies have shed light on the influence of the host versus the habitat on UCYN-A N2 fixation and overall metabolism. Here we compared transcriptomes from natural populations of UCYN-A from oligotrophic open-ocean versus nutrient-rich coastal waters, using a microarray that targets the full genomes of UCYN-A1 and UCYN-A2 and known genes for UCYN-A3. We found that UCYN-A2, usually regarded as adapted to coastal environments, was transcriptionally very active in the open ocean and appeared to be less impacted by habitat change than UCYN-A1. Moreover, for genes with 24 h periodic expression we observed strong but inverse correlations among UCYN-A1, A2, and A3 to oxygen and chlorophyll, which suggests distinct host-symbiont relationships. Across habitats and sublineages, genes for N2 fixation and energy production had high transcript levels, and, intriguingly, were among the minority of genes that kept the same schedule of diel expression. This might indicate different regulatory mechanisms for genes that are critical to the symbiosis for the exchange of nitrogen for carbon from the host. Our results underscore the importance of N2 fixation in UCYN-A symbioses across habitats, with consequences for community interactions and global biogeochemical cycles.
Assuntos
Cianobactérias , Água do Mar , Água do Mar/microbiologia , Transcriptoma , Cianobactérias/genética , Cianobactérias/metabolismo , Fixação de Nitrogênio/genética , Oceanos e Mares , Nitrogênio/metabolismoRESUMO
Transcranial direct current stimulation (tDCS) is an emerging noninvasive brain neuromodulation technique aimed at relieving symptoms associated with psychiatric disorders, including addiction. The goal of the present study was to better identify which phase of alcohol-related behavior (hedonic effect, behavioral sensitization, self-administration, or motivation to obtain the drug) might be modulated by repeated anodal tDCS over the frontal cortex (0.2 mA, 20 min, twice a day for 5 consecutive days), using female mice as a model. Our data showed that tDCS did not modulate the hedonic effects of ethanol as assessed by a conditioned place preference test (CPP) or the expression of ethanol-induced behavioral sensitization. Interestingly, tDCS robustly reduced reacquisition of ethanol consumption (50% decrease) following extinction of self-administration in an operant paradigm. Furthermore, tDCS significantly decreased motivation to drink ethanol on a progressive ratio schedule (30% decrease). Taken together, our results show a dissociation between the effects of tDCS on "liking" (hedonic aspect; no effect in the CPP) and "wanting" (motivation; decreased consumption on a progressive ratio schedule). Our tDCS procedure in rodents will allow us to better understand its mechanisms of action in order to accelerate its use as a complementary and innovative tool to help alcohol-dependent patients maintain abstinence or reduce ethanol intake.
Assuntos
Consumo de Bebidas Alcoólicas/prevenção & controle , Comportamento Animal , Comportamento de Procura de Droga , Etanol/administração & dosagem , Motivação , Estimulação Transcraniana por Corrente Contínua , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/psicologia , Animais , Condicionamento Operante , Etanol/toxicidade , Extinção Psicológica , Feminino , Camundongos , Modelos Animais , AutoadministraçãoRESUMO
The marine cyanobacterium Prochlorococcus can utilize glucose as a source of carbon. However, the relative importance of inorganic and organic carbon assimilation and the timing of glucose assimilation are still poorly understood in these numerically dominant cyanobacteria. Here, we investigated whole microbial community and group-specific primary production and glucose assimilation using incubations with radioisotopes combined with flow cytometry cell sorting. We also studied changes in the microbial community structure in response to glucose enrichments and analyzed the transcription of Prochlorocccus genes involved in carbon metabolism and photosynthesis. Our results showed a diel variation for glucose assimilation in Prochlorococcus, with maximum assimilation at midday and minimum at midnight (~2-fold change), which was different from that of the total microbial community. This suggests that the timing in glucose assimilation in Prochlorococcus is coupled to photosynthetic light reactions producing energy, it being more convenient for Prochlorococcus to show maximum glucose uptake precisely when the rest of microbial populations have their minimum glucose uptake. Many transcriptional responses to glucose enrichment occurred after 12- and 24-h periods, but community composition did not change. High-light Prochlorococcus strains were the most impacted by glucose addition, with transcript-level increases observed for genes in pathways for glucose metabolism, such as the pentose phosphate pathway, the Entner-Doudoroff pathway, glycolysis, respiration, and glucose transport. While Prochlorococcus C assimilation from glucose represented less than 0.1% of the bacterium's photosynthetic C fixation, increased assimilation during the day and glcH gene upregulation upon glucose enrichment indicate an important role of mixotrophic C assimilation by natural populations of Prochlorococcus. IMPORTANCE Several studies have demonstrated that Prochlorococcus, the most abundant photosynthetic organism on Earth, can assimilate organic molecules, such as amino acids, amino sugars, ATP, phosphonates, and dimethylsulfoniopropionate. This autotroph can also assimilate small amounts of glucose, supporting the hypothesis that Prochlorococcus is mixotrophic. Our results show, for the first time, a diel variability in glucose assimilation by natural populations of Prochlorococcus with maximum assimilation during midday. Based on our previous results, this indicates that Prochlorococcus could maximize glucose uptake by using ATP made during the light reactions of photosynthesis. Furthermore, Prochlorococcus showed a different timing of glucose assimilation from the total population, which may offer considerable fitness advantages over competitors "temporal niches." Finally, we observed transcriptional changes in some of the genes involved in carbon metabolism, suggesting that Prochlorococcus can use both pathways previously proposed in cyanobacteria to metabolize glucose.
Assuntos
Organofosfonatos , Prochlorococcus , Prochlorococcus/genética , Prochlorococcus/metabolismo , Glucose/metabolismo , Água do Mar , Carbono/metabolismo , Aminoácidos/metabolismo , Trifosfato de Adenosina/metabolismo , Amino Açúcares/metabolismoRESUMO
Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles - small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.
Assuntos
Vesículas Extracelulares , Prochlorococcus , Synechococcus , Synechocystis , EcossistemaRESUMO
BACKGROUND: The growing proportion of elderly intensive care patients constitutes a public health challenge. The benefit of critical care in these patients remains unclear. We compared outcomes in elderly versus very elderly subjects receiving mechanical ventilation. METHODS: In total, 5,557 mechanically ventilated subjects were included in our post hoc retrospective analysis, a subgroup of the VENTILA study. We divided the cohort into 2 subgroups on the basis of age: very elderly subjects (age ≥ 80 y; n = 1,430), and elderly subjects (age 65-79 y; n = 4,127). A propensity score on being very elderly was calculated. Evaluation of associations with 28-d mortality was done with logistic regression analysis. RESULTS: Very elderly subjects were clinically sicker as expressed by higher SAPS II scores (53 ± 18 vs 50 ± 18, P < .001), and their rates of plateau pressure < 30 cm H2O were higher, whereas other parameters did not differ. The 28-d mortality was higher in very elderly subjects (42% vs 34%, P < .001) and remained unchanged after propensity score adjustment (adjusted odds ratio 1.31 [95% CI 1.16-1.49], P < .001). CONCLUSIONS: Age was an independent and unchangeable risk factor for death in mechanically ventilated subjects. However, survival rates of very elderly subjects were > 50%. Denial of critical care based solely on age is not justified. (ClinicalTrials.gov registration NCT02731898.).
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Estado Terminal , Respiração Artificial , Idoso , Humanos , Unidades de Terapia Intensiva , Estudos Retrospectivos , Fatores de Risco , Escore Fisiológico Agudo SimplificadoRESUMO
The expression of five genes involved in nitrogen assimilation in cyanobacteria, namely glnA, glsF, icd, ntcA, and glnB, encoding three key enzymes from that pathway (glutamine synthetase, glutamate synthase, isocitrate dehydrogenase) and two regulatory proteins (NtcA and PII), was studied in this work. Their changes under different conditions were analyzed by quantitative real-time RT-PCR. Nutrient limitation induced clear modifications on the expression of most studied genes: lack of nitrogen provoked an initial increase, followed by a marked decrease; in the cases of phosphorus and iron starvation, a general, stronger expression decrease was observed, particularly striking in the case of iron. Darkness and addition of the photosynthethic inhibitors DCMU and DBMIB also had a strong effect on gene expression. Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, provoked a sharp increase in icd expression. These results, together with previous studies, suggest that 2-oxoglutarate could be the molecule utilized by Prochlorococcus to sense the C/N balance. Besides, our results confirm the different regulation of nitrogen assimilation in Prochlorococcus with regard to other cyanobacteria.
Assuntos
Carbono/metabolismo , Genes Bacterianos , Nitrogênio/metabolismo , Prochlorococcus/genética , Transporte de Elétrons/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/fisiologia , Ferro/metabolismo , Deficiências de Ferro , Luz , Redes e Vias Metabólicas/genética , Fósforo/deficiência , Fósforo/metabolismo , Fotossíntese/genética , Prochlorococcus/metabolismo , Percepção de Quorum/genética , Especificidade da Espécie , Inanição/genética , Inanição/metabolismoRESUMO
This study was designed to test the role of general and selective task instructions when processing documents, which vary as regards trustworthiness and position toward a conflicting topic. With selective task instructions, we refer to concrete guidelines as how to read the texts and how to select appropriate documents and contents, in contrast to general task instructions. Sixty-one secondary school students were presented with four different conflicting documents in an electronic learning environment and were told to write an essay based on the information from the texts. Only half of the students were told to only use information from two out of the four texts to write their essay (i.e., selective condition). As predicted, students told to focus on specific documents and not use all of them for the assigned task (i.e., selective condition) better discriminated the quality of documents and type of information for the task.
RESUMO
A methodology for the automatic production of quantum mechanical/molecular mechanical (QM/MM) models of retinal-binding rhodopsin proteins and subsequent prediction of their spectroscopic properties has been proposed recently by some of the authors. The technology employed for the evaluation of the excitation energies is called Automatic Rhodopsin Modeling (ARM), and it involves the use of the complete active space self-consistent field (CASSCF) method followed by a multiconfiguration second-order perturbation theory (in particular, CASPT2) calculation of external correlation energies. Although it was shown that ARM is capable of successfully reproducing and predicting spectroscopic property trends in chromophore-embedding protein sets, practical applications of such technology are limited by the high computational costs of the multiconfiguration perturbation theory calculations. In the present work we benchmark the more affordable multiconfiguration pair-density functional theory (MC-PDFT) method whose accuracy has been recently validated for retinal chromophores in the gas phase, indicating that MC-PDFT could potentially be used to analyze large (e.g., few hundreds) sets of rhodopsin proteins. Here, we test this theory for a set of rhodopsin QM/MM models whose experimental absorption maxima (λ a max) have been measured. The results indicate that MC-PDFT may be employed to calculate λ a max values for this important class of photoresponsive proteins.