The omnistat:A flexible continuous-culture system for prolonged experimental evolution

1. Microbial evolution experiments provide a powerful tool to unravel the molecular basis of adaptive evolution but their outcomes can be difficult to interpret, unless the selective forces that are applied during the experiment are carefully controlled. In this respect, experimental evolution in continuous cultures provides advantages over commonly used sequential batch-culture protocols because continuous cultures allow for more accurate control over the induced selective environment. However, commercial continuous-culture systems are large and expensive, while available DIY continuous-culture systems are not versatile enough to allow for multiple sensors and rigorous stirring. 2. We present a modular continuous-culture system that adopts the commonly used GL45 glass laboratory bottle as a bioreactor vessel. Our design offers three advantages: first, it is equipped with a large head plate, fitting two sensors and seven input/output ports, enabling the customization of the system for many running modes (chemostat, auxostat, etc.). Second, the bioreactor is small (25-250 ml), which makes it feasible to run many replicates in parallel. Third, bioreactor modules can be coupled by uni- or bi-directional flows to induce spatiotemporal variation in selection. These features result in a particularly flexible culturing platform that facilitates the investigation of a broad range of evolutionary and ecological questions. 3. To illustrate the versatility of our culturing system, we outline two evolution experiments that impose a temporally or spatially variable regime of selection. The first experiment illustrates how controlled temporal variation in resource availability can be utilized to select for anticipatory switching. The second experiment illustrates a spatially structured morbidostat setup that is designed to probe epistatic interactions between adaptive mutations. Furthermore, we demonstrate how sensor data can be used to stabilize selection pressures or track evolutionary adaptation. 4. Evolution experiments in which populations are exposed to controlled spatiotemporal variation, are essential to gain insight into the process of adaptation and the mechanisms that constrain evolution. Continuous-culture systems, like the one presented here, offer control over key environmental parameters and establish a well-defined regime of selection. As such, they create the opportunity to expose evolutionary constraints in the form of phenotypic trade-offs, contributing to a mechanistic understanding of adaptive evolution

Channeling anabolic side-products towards the production of non-essential metabolites: stable malate production in Synechocystis sp. PCC6803

[Image: see text] Powered by (sun)light to oxidize water, cyanobacteria can directly convert atmospheric CO(2) into valuable carbon-based compounds and meanwhile release O(2) to the atmosphere. As such, cyanobacteria are promising candidates to be developed as microbial cell factories for the production of chemicals. Nevertheless, similar to other microbial cell factories, engineered cyanobacteria may suffer from production instability. The alignment of product formation with microbial fitness is a valid strategy to tackle this issue. We have described previously the “FRUITS” algorithm for the identification of metabolites suitable to be coupled to growth (i.e., side products in anabolic reactions) in the model cyanobacterium Synechocystis. sp PCC6803. However, the list of candidate metabolites identified using this algorithm can be somewhat limiting, due to the inherent structure of metabolic networks. Here, we aim at broadening the spectrum of candidate compounds beyond the ones predicted by FRUITS, through the conversion of a growth-coupled metabolite to downstream metabolites via thermodynamically favored conversions. We showcase the feasibility of this approach for malate production using fumarate as the growth-coupled substrate in Synechocystis mutants. A final titer of ∼1.2 mM was achieved for malate during photoautotrophic batch cultivations. Under prolonged continuous cultivation, the most efficient malate-producing strain can maintain its productivity for at least 45 generations, sharply contrasting with other producing Synechocystis strains engineered with classical approaches. Our study also opens a new possibility for extending the stable production concept to derivatives of growth-coupled metabolites, increasing the list of suitable target compounds

Slr1670 from Synechocystis sp. PCC 6803 Is Required for the Re-assimilation of the Osmolyte Glucosylglycerol

When subjected to mild salt stress, the cyanobacterium Synechocystis sp. PCC 6803 produces small amounts of glycerol through an as of yet unidentified pathway. Here, we show that this glycerol is a degradation product of the main osmolyte of this organism, glucosylglycerol (GG). Inactivation of ggpS, encoding the first step of GG-synthesis, abolished de novo synthesis of glycerol, while the ability to hydrolyze exogenously supplied glucoslylglycerol was unimpaired. Inactivation of glpK, encoding glycerol kinase, had no effect on glycerol synthesis. Inactivation of slr1670, encoding a GHL5-type putative glycoside hydrolase, abolished de novo synthesis of glycerol, as well as hydrolysis of GG, and led to increased intracellular concentrations of this osmolyte. Slr1670 therefore presumably displays GG hydrolase activity. A gene homologous to the one encoded by slr1670 occurs in a wide range of cyanobacteria, proteobacteria, and archaea. In cyanobacteria, it co-occurs with genes involved in GG-synthesis

WhatsApp-assisted health education intervention: a systematic

The acknowledgment of social media as a strategy in health education is essential, providing benefits to health promotion and the prevention and treatment of comorbidities. This study assessed the contribution of WhatsApp use on the prognosis of medical conditions and its impact on enhancing the quality of life. The review protocol used PubMed and Web of Science as databases, followed PRISMA-E 2012 guidelines, was registered in PROSPERO (Nº2021232688), and its risk of bias was analyzed Cochrane Collaboration\u27s tool for assessing the risk of bias. Thirteen impact studies (n = 1653 participants) were selected and showed evidence on metabolic parameters such as reducing blood pressure (7.6%); body mass index-BMI (7.6%); glycated haemoglobin-HbA1c (15.4%); resting heart rate (7.6%); increase in static balance increase on balance (7.6%) and reduced body weight (7.6%). Furthermore, specific findings on each intervention were demonstrated, such as diminished rates of relapse in substance users (7.6%); increased populational knowledge on chronic diseases (23%) associated with a decrease in the potential complications (7.6%), and a rise on treatment adhesion (7.6%); impact on the follow-up of pregnant patients (7.6%) and progress on oral hygiene (15.4%). Therefore, it was concluded that this intervention strategy demonstrated significant magnitude in reducing the progression and complication of the assessed disorders

Exploiting Day- and Night-Time Metabolism of Synechocystis sp. PCC 6803 for Fitness-Coupled Fumarate Production around the Clock

Cyanobacterial cell factories are widely researched for the sustainable production of compounds directly from CO2. Their application, however, has been limited for two reasons. First, traditional approaches have been shown to lead to unstable cell factories that lose their production capability when scaled to industrial levels. Second, the alternative approaches developed so far are mostly limited to growing conditions, which are not always the case in industry, where nongrowth periods tend to occur (e.g., darkness). We tackled both by generalizing the concept of growth-coupled production to fitness coupling. The feasibility of this new approach is demonstrated for the production of fumarate by constructing the first stable dual-strategy cell factory. We exploited circadian metabolism using both systems and synthetic biology tools, resulting in the obligatorily coupling of fumarate to either biomass or energy production. Resorting to laboratory evolution experiments, we show that this engineering approach is more stable than conventional methods

Alignment of microbial fitness with engineered product formation: obligatory coupling between acetate production and photoautotrophic growth

Background: Microbial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines. However, the sustained productivity of genetically engineered strains is often a challenge, as spontaneous non-producing mutants tend to grow faster and take over the population. Novel strategies to prevent this issue of strain instability are urgently needed. Results: In this study, we propose a novel strategy applicable to all microbial production systems for which a genome-scale metabolic model is available that aligns the production of native metabolites to the formation of biomass. Based on well-established constraint-based analysis techniques such as OptKnock and FVA, we developed an in silico pipeline—FRUITS—that specifically ‘Finds Reactions Usable in Tapping Side-products’. It analyses a metabolic network to identify compounds produced in anabolism that are suitable to be coupled to growth by deletion of their re-utilization pathway(s), and computes their respective biomass and product formation rates. When applied to Synechocystis sp. PCC6803, a model cyanobacterium explored for sustainable bioproduction, a total of nine target metabolites were identified. We tested our approach for one of these compounds, acetate, which is used in a wide range of industrial applications. The model-guided engineered strain shows an obligatory coupling between acetate production and photoautotrophic growth as predicted. Furthermore, the stability of acetate productivity in this strain was confirmed by performing prolonged turbidostat cultivations. Conclusions: This work demonstrates a novel approach to stabilize the production of target compounds in cyanobacteria that culminated in the first report of a photoautotrophic growth-coupled cell factory. The method developed is generic and can easily be extended to any other modeled microbial production system

OTIMIZAÇÃO GEOMÉTRICA DE ESCOAMENTOS COM CONVECÇÃO FORÇADA SOBRE UM ARRANJO TRIANGULAR DE CORPOS ROMBUDOS QUADRADOS

O presente estudo consiste na avaliação numérica de um arranjo triangular de corpos rombudos submetidos a um escoamento transiente, bidimensional, incompressível, laminar e com convecção forçada. A avaliação geométrica é realizada através do método Design Construtal. O problema possui uma restrição dada pela soma da área dos corpos rombudos e dois graus de liberdade: ST/D (razão da distância entre o centro dos corposrombudos posteriores e a dimensão de suas arestas), SL/D (razão da distância longitudinal entre os centros dos corpos rombudos, frontal e posteriores, e a dimensão de suas arestas). Para todas as simulações são considerados números de Reynolds e Prandtl constantes, ReD=100 e Pr = 0.71. As equações de conservação de massa, quantidade de movimento e energia são resolvidas através do Método dos Volumes Finitos (FVM). O principal objetivo é avaliaro efeito dos graus de liberdade sobre o coeficiente de arrasto (CD) e o número de Nusselt (NuD) entre os corpos rombudos e o escoamento circundante, assim como, os valores ótimos de ST/D para seis razões de ST/D = 1.5, 2.0, 2.5, 3.0, 3.5 e 4.0 e seis passos longitudinais SL/D= 1.5, 2.0, 2.5, 3.0, 3.5 e 4.0. Os resultados mostraram que a alteração da razão ST/D possuem influência sobre os coeficientes de arrasto e número de Nusselt do arranjo triangular de corpos rombudos, bem como, sobre as geometrias que conduzem aos melhoresdesempenhos fluido dinâmico e térmico do escoamento.Palavras-chave: Design Construtal, convecção forçada, arranjo triangular, escoamento externo, corpos rombudos

OTIMIZAÇÃO GEOMÉTRICA DE ESCOAMENTOS COM CONVECÇÃO FORÇADA SOBRE UM ARRANJO DE QUATRO CILINDROS

O presente estudo consiste na avaliação numérica de um arranjo formado por quatro cilindros submetidos a um escoamento transiente, bidimensional,incompressível, laminar e com convecção forçada. A avaliação geométrica é realizada através do método Design Construtal. O problema possui uma restrição dada pela soma da área dos cilindros e três graus de liberdade: ST1/D (razão entre o passo transversal dos cilindros frontais e o diâmetro dos cilindros), ST2/D (razão entre o passo transversal dos cilindros posteriores e o diâmetro dos cilindros) e SL/D (razão entre o passo longitudinal dos cilindros frontais e posteriores e o diâmetro dos cilindros). Para todas as simulações são considerados números de Reynolds e Prandtl constantes, ReD = 100 e Pr = 0.71. As equações de conservação de massa, quantidade de movimento e energia são resolvidas através do Método dos Volumes Finitos (FVM). O principal objetivo é avaliar o efeito dos graus de liberdade sobre o coeficiente dearrasto (C D) e o número de Nusselt (NuD) entre os cilindros e o escoamentocircundante, assim como, os valores ótimos de ST2/D para três razões de ST1/D = 1.5, 3.0 e 4.0 , sendo estes resultados obtidos para a razão SL/D = 1.5 e 4.0. Os resultados mostraram que a alteração da razão ST1/D e ST2/D possui grande influência sobre os coeficientes de arrasto e número de Nusselt do arranjo formado pelos quatro cilindros.Keywords: Design Construtal, convecção forçada, arranjo quatro cilindros, escoamento externo, estudo numérico

Design construtal aplicado a um canal com aletas submetido a convecção forçada

O propósito deste trabalho é apresentar um estudo numérico de um canal bidimensional com duas aletas retangulares submetido a um escoamento no regime laminar com transferência de calor por convecção forçada avaliando a geometria das aletas através do método Design Construtal. Os objetivos principais são maximizar a taxa de transferência de calore ao mesmo tempo minimizar a diferença de pressões entre a entrada e saída do canal para diferentes dimensões das duas aletas do canal, considerando os mesmos números de Reynolds (ReH= 100) e Prandtl (Pr = 0,71). O problema é submetido a três restrições dadas pelas áreas do canal, aletas e área máxima de ocupação de cada aleta.O sistema ainda possui três graus de liberdade dados pela razão entre altura e comprimento do canal, que é mantida fixa H/L = 0,0625, a razão entre altura e comprimento da aleta frontal (H3/L3) situada na superfície inferior do canal e a razão entre altura e comprimento da aleta posterior (H4/L4) localizada na superfície superior do canal. Os dois últimos graus de liberdade foram variados neste estudo. Para a abordagem numérica do problema, as equações de conservação de massa, quantidade de movimento e energia são resolvidas com o método dos volumes finitos (MVF). Os resultados mostraram que uma razão de H4/L4 igual a 3,0 é a que melhor atende ao multi-objetivo proposto.Também foi observado que razões de H4/L4 mais próximas de 0,25 tem melhor desempenho fluido dinâmico,sendo a razão entre o melhor e o pior caso fluidodinâmico igual a 28,79,enquanto que razões de H4/L4 próximas à 4.0 tem melhor desempenho térmico, com desempenho 60,62% superior comparado ao pior caso

Análise do impacto das intervenções que modificam o risco cardiovascular em pacientes com critérios de síndrome metabólica: uma revisão sistemática / Impact of interventions that modify cardiovascular risk in patients with metabolic syndrome criteria: a systematic review

Estudos epidemiológicos apontam relação direta entre os componentes da Síndrome Metabólica (SM) e o desenvolvimento de Doenças Cardiovasculares (DCV). O presente estudo visou avaliar as contribuições do arsenal terapêutico atual para SM com benefícios na modulação do perfil cardiovascular em longo prazo. O protocolo de revisão cumpriu as diretrizes do PRISMA-E 2012, com registro no PROSPERO (Nº CRD42020180215), os artigos foram coletados a partir de PUBMED e MEDLINE, priorizando ensaios clínicos randomizados publicados a partir do ano de 2010. O viés foi analisado pela ferramenta Cochrane Collaboration’s tool for assessing risk of bias. Os desfechos de interesse incluíram alterações no desfecho composto por mortalidade cardiovascular, Infarto Agudo do Miocárdio Não Fatal e Acidente Vascular Encefálico Não Fatal (MACE); mortalidade geral; arritmias; Insuficiência Cardíaca; Doenças Arteriais Coronarianas e alterações metabólicas que interferem em DCV. Em 32 ensaios clínicos randomizados elegíveis (215.936 participantes), com idade média de 60 anos e seguimento médio de 2,11 anos, verificou-se impacto entre as intervenções dos estudos com ao menos um desfecho de interesse.  As estratégias denotaram eficácia em minimizar sintomas, progressão e/ou agravamento da SM e reduziram o risco de MACE (50%); mortalidade geral e hospitalização por IC (31,25%); com efeitos metabólicos em perda de peso (53,1¨%), pressão arterial (34,4%), perfil lipídico (47%), resultado composto renal (6,25%), Circunferência Abdominal (29%), proteína C reativa (6,25%), hemoglobina glicada (25%); efeitos benéficos em biomarcadores cardiovasculares (19%), disfunção endotelial (15,6%) e tromboembolismo venoso (3%). Concluímos que as terapias recentes para SM apontaram magnitude significativa em DCV, com melhora no prognóstico e sobrevida dos pacientes.