Optimized fluorescent proteins for the rhizosphere-associated bacterium <i>Bacillus mycoides</i> with endophytic and biocontrol agent potential

Tracking of fluorescent protein (FP)-labelled rhizobacteria is a key prerequisite to gain insights into plant-bacteria interaction mechanisms. However, the performance of FPs mostly has to be optimized for the bacterial host and for the environment of intended application. We report on the construction of mutational libraries of the superfolder green fluorescent protein sfGFP and the red fluorescent protein mKate2 in the bacterium B. mycoides, which next to its potential as plant-biocontrol agent occasionally enters an endophytic lifestyle. By fluorescence-activated cell sorting and comparison of signal intensities at the colony and single-cell level, the variants sfGFP(SPS6) and mKate (KPS12) with significantly increased brightness were isolated. Their high applicability for plant-bacteria interaction studies was shown by confocal laser scanning microscopy tracking of FP-tagged B. mycoides strains after inoculation to Chinese cabbage plants in a hydroponic system. During the process of colonization, strain EC18 rapidly attached to plant roots and formed a multicellular matrix, especially at the branching regions of the root hair, which probably constitute entrance sites to establish an endophytic lifestyle. The universal applicability of the novels FPs was proven by expression from a weak promoter, dual-labelling of B. mycoides, and by excellent expression and detectability in additional soil- and rhizosphere-associated Bacillus species

Draft Genome Sequence of Bacillus mycoides M2E15, a Strain Isolated from the Endosphere of Potato

We present the draft genome sequence of Bacillus mycoides M2E15, a bacterium isolated from potato endosphere. Analysis of the 6.08-Mbp draft genome sequence identified 6,386 protein-encoding sequences, including potential plant growth promoting genes. Specifically, genes for proteins involved in phosphate utilization, iron acquisition, and bacteriocin production were identified

Automated, Point-of-Care mobile flow cytometry: Bringing the laboratory to the sample

Background: Innate effector cells are very responsive to infectious and inflammatory cues found in damaged and inflamed tissues. Their activation is a potential target to assess the state of the immune system. Unfortunately, these cells are very susceptible for ex-vivo activation, hampering accurate interpretation of flow cytometry data. Whether a brief window exists before ex-vivo activation starts to occur is currently unknown. Aims: 1) This study extensively investigated ex-vivo activation of innate effector cells over time. 2) We tested the feasibility of applying a mobile, automated, flow cytometry laboratory for out-of-hospital Point-of-Care analyses to minimize ex-vivo activation bias. Methods: 1) Ex-vivo neutrophil, eosinophil and monocyte activation in a blood collection tube over time and the reactivity to a formyl-peptide was investigated in a healthy cohort. 2) To facilitate fast, out-of-hospital analysis, application of the mobile flow cytometry was tested by placing an automated flow cytometer into a van. The stability of the setup was assessed by repetitively measuring laser alignment and fluorescence verification beads. Findings: 1) Immediately after venipuncture activation marker expression on neutrophils, eosinophils and monocyte subsets started to change in a time-dependent manner. 2) The mobile flow cytometry laboratory travelled over 3000 km, performing measurements at 19 locations with a median single-person-set-up time of 14 min. The laser alignment and fluorescence were stable during all experiments. Conclusions: Accurate flow data of innate immune cells are only obtained when ex-vivo activation is kept to minimum. The use of a mobile, fast, automated, flow cytometry laboratory for out-of-hospital Point-of-Care analyses provides new investigational and diagnostic possibilities outside major hospital flow cytometry laboratories

A positive neighborhood walkability is associated with a higher magnitude of leisure walking in adults upon COVID-19 restrictions: a longitudinal cohort study

Background: Previous cross-sectional and longitudinal observational studies revealed positive relationships between contextual built environment components and walking behavior. Due to severe restrictions during COVID-19 pandemic lockdowns, physical activity was primarily performed within the immediate living area. Using this unique opportunity, we evaluated whether built environment components were associated with the magnitude of change in walking activity in adults during COVID-19 restrictions. Methods: Data on self-reported demographic characteristics and walking behaviour were extracted from the prospective longitudinal Lifelines Cohort Study in the Netherlands of participants ≥ 18 years. For our analyses, we made use of the data acquired between 2014–2017 (n = 100,285). A fifth of the participants completed the questionnaires during COVID-19 restrictive policies in July 2021 (n = 20,806). Seven spatial components were calculated for a 500m and 1650m Euclidean buffer per postal code area in GIS: population density, retail and service destination density, land use mix, street connectivity, green space density, sidewalk density, and public transport stops. Additionally, the walkability index (WI) of these seven components was calculated. Using multivariable linear regression analyses, we analyzed the association between the WI (and separate components) and the change in leisure walking minutes/week. Included demographic variables were age, gender, BMI, education, net income, occupation status, household composition and the season in which the questionnaire was filled in. Results: The average leisure walking time strongly increased by 127 min/week upon COVID-19 restrictions. All seven spatial components of the WI were significantly associated with an increase in leisure walking time; a 10% higher score in the individual spatial component was associated with 5 to 8 more minutes of leisure walking/week. Green space density at the 500m Euclidean buffer and side-walk density at the 1650m Euclidean buffer were associated with the highest increase in leisure walking time/week. Subgroup analysis revealed that the built environment showed its strongest impact on leisure walking time in participants not engaging in leisure walking before the COVID-19 pandemic, compared to participants who already engaged in leisure walking before the COVID-19 pandemic. Conclusions: These results provide strong evidence that the built environment, corrected for individual-level characteristics, directly links to changes observed in leisure walking time during COVID-19 restrictions. Since this relation was strongest in those who did not engage in leisure walking before the COVID-19 pandemic, our results encourage new perspectives in health promotion and urban planning

Identification of neutrophil phenotype categories in geriatric hip fracture patients aids in personalized medicine

OBJECTIVES: The number of geriatric hip fracture patients is high and expected to rise in the coming years, and many are frail and at risk for adverse outcomes. Early identification of high-risk patients is crucial to balance treatment and optimize outcome, but remains challenging. Previous research in patients with multitrauma suggested that neutrophil phenotype analysis could aid in early identification of high-risk patients. This pilot study investigated the feasibility and clinical value of neutrophil phenotype analysis in geriatric patients with a hip fracture. METHODS: A prospective study was conducted in a regional teaching hospital in the Netherlands. At the emergency department, blood samples were collected from geriatric patients with a hip fracture and analyzed using automated flow cytometry. Flow cytometry data were processed using an automated clustering algorithm. Neutrophil activation data were compared with a healthy control cohort. Neutrophil phenotype categories were assessed based on two-dimensional visual assessment of CD16/CD62L expression. RESULTS: Blood samples from 45 geriatric patients with a hip fracture were included. Neutrophils showed an increased activation profile and decreased responsiveness to formyl peptides when compared to healthy controls. The neutrophil phenotype of all patients was categorized. The incidence of severe adverse outcome was significantly different between the different categories ( P = 0.0331). Moreover, patients with neutrophil phenotype category 0 developed no severe adverse outcomes. CONCLUSIONS: Using point-of-care fully automated flow cytometry to analyze the neutrophil compartment in geriatric hip fracture patients is feasible and holds clinical value in determining patients at risk for adverse outcome. This study is a first step toward immuno-based precision medicine for identifying geriatric hip fracture patients that are deemed fit for surgery

The distributed ASCI supercomputer project

The Distributed ASCI Supercomputer (DAS) is a homogeneous wide-area distributed system consisting of four cluster computers at different locations. DAS has been used for research on communication software, parallel languages and programming systems, schedulers, parallel applications, and distributed applications. The paper gives a preview of the most interesting research results obtained so far in the DAS project

What makes us drink? : Alcohol consumption in the rat in connection with reward and cognition

Alcohol use disorder (AUD; often referred to as alcohol addiction or alcoholism) is a chronic relapsing brain disorder characterized by loss of control over alcohol intake. Alcohol is among the most widely used substances in the world, and even though only a minority of the people who regularly consume alcohol develop AUD, this still amounts to approximately 76 million people worldwide. Importantly, it is still unclear why some individuals are more at risk for AUD than others. The development of AUD is thought to involve a complex interaction between personality characteristics, the environment of an individual and long-lasting alcohol-induced changes in brain function. The overarching aim of this thesis was to determine which factors contribute to the individual vulnerability for AUD, using studies in rats. One of the main findings is that in populations of rats, we could identify animals that voluntarily drink low or high quantities of alcohol. We observed that a high voluntary alcohol consumption was related to a higher motivation to obtain alcohol and a loss of control over alcohol seeking, a key characteristic of AUD. Moreover, cognitive assessments showed that high alcohol consumption was related to reward-directed behaviour in decision making tasks and an enhanced focus on reward cues. In addition, we observed that a disrupted social development increases alcohol consumption during adulthood. With regard to the brain mechanisms involved, we investigated the role of the mesolimbic dopamine system, which is thought to play an important role in addictive behaviour. We found that functional activity of the mesolimbic dopamine system is of great importance for the motivation to obtain alcohol, but not for the individual level of alcohol consumption. Interestingly, the mesolimbic dopamine system was more sensitive to rewards and reward cues in rats that have been exposed to alcohol during adolescence. In conclusion, the results in this thesis contribute to the understanding of the behavioural, cognitive, social and brain mechanisms that underlie the risk for AUD. Therefore, these studies may help the development of improved treatment strategies to restore control over alcohol use

What makes us drink?: Alcohol consumption in the rat in connection with reward and cognition

Alcohol use disorder (AUD; often referred to as alcohol addiction or alcoholism) is a chronic relapsing brain disorder characterized by loss of control over alcohol intake. Alcohol is among the most widely used substances in the world, and even though only a minority of the people who regularly consume alcohol develop AUD, this still amounts to approximately 76 million people worldwide. Importantly, it is still unclear why some individuals are more at risk for AUD than others. The development of AUD is thought to involve a complex interaction between personality characteristics, the environment of an individual and long-lasting alcohol-induced changes in brain function. The overarching aim of this thesis was to determine which factors contribute to the individual vulnerability for AUD, using studies in rats. One of the main findings is that in populations of rats, we could identify animals that voluntarily drink low or high quantities of alcohol. We observed that a high voluntary alcohol consumption was related to a higher motivation to obtain alcohol and a loss of control over alcohol seeking, a key characteristic of AUD. Moreover, cognitive assessments showed that high alcohol consumption was related to reward-directed behaviour in decision making tasks and an enhanced focus on reward cues. In addition, we observed that a disrupted social development increases alcohol consumption during adulthood. With regard to the brain mechanisms involved, we investigated the role of the mesolimbic dopamine system, which is thought to play an important role in addictive behaviour. We found that functional activity of the mesolimbic dopamine system is of great importance for the motivation to obtain alcohol, but not for the individual level of alcohol consumption. Interestingly, the mesolimbic dopamine system was more sensitive to rewards and reward cues in rats that have been exposed to alcohol during adolescence. In conclusion, the results in this thesis contribute to the understanding of the behavioural, cognitive, social and brain mechanisms that underlie the risk for AUD. Therefore, these studies may help the development of improved treatment strategies to restore control over alcohol use