Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.     

Rap1 Can Bypass the FAK-Src-Paxillin Cascade to Induce Cell Spreading and Focal Adhesion Formation

We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.     

Health-Related Quality of Life,Self-Efficacy and Enjoyment Keep the Socially Vulnerable Physically Active in Community-Based Physical Activity Programs: A Sequential Cohort Study

Physical inactivity is most commonly found in socially vulnerable groups. Dutch policies target these groups through community-based health-enhancing physical activity (CBHEPA) programs. As robust evidence on the effectiveness of this approach is limited, this study investigated whether CBHEPA programs contribute to an increase in and the maintenance of physical activity in socially vulnerable groups. In four successive cohorts, starting at a six-month interval, 268 participants from 19 groups were monitored for twelve months in seven CBHEPA programs. Data collection was based on repeated questionnaires. Socio-economic indicators, program participation and coping ability were measured at baseline. Physical activity, health-related quality of life and on-going program participation were measured three times. Self-efficacy and enjoyment were measured at baseline and at twelve months. Statistical analyses were based on a quasi-RCT design (independent t-tests), a comparison of participants and dropouts (Mann-Whitney test), and multilevel modelling to assess change in individual physical activity, including group level characteristics. Participants of CBHEPA programs are socially vulnerable in terms of low education (48.6%), low income (52.4%), non-Dutch origin (64.6%) and health-related quality of life outcomes. Physical activity levels were not below the Dutch average. No increase in physical activity levels over time was observed. The multilevel models showed significant positive associations between health-related quality of life, self-efficacy and enjoyment, and leisure-time physical activity over time. Short CBHEPA programs (10–13 weeks) with multiple trainers and gender-homogeneous groups were associated with lower physical activity levels over time. At twelve months, dropouts'' leisure-time physical activity levels were significantly lower compared to continuing participants, as were health-related quality of life, self-efficacy and enjoyment outcomes. BMI and care consumption scored significantly higher among dropouts. In conclusion, Dutch CBHEPA programs reach socially vulnerable, but not necessarily inactive, groups in terms of socio-economic and health-related quality of life outcomes. Our findings suggest that CBHEPA programs particularly contribute to physical activity maintenance in socially vulnerable groups, rather than to an increase in physical activity behaviour over time.     

Food availability affects the strength of mutualistic host–microbiota interactions in Daphnia magna

The symbiotic gut microbial community is generally known to have a strong impact on the fitness of its host. Nevertheless, it is less clear how the impact of symbiotic interactions on the hosts'' fitness varies according to environmental circumstances such as changes in the diet. This study aims to get a better understanding of host–microbiota interactions under different levels of food availability. We conducted experiments with the invertebrate, experimental model organism Daphnia magna and compared growth, survival and reproduction of conventionalized symbiotic Daphnia with germ-free individuals given varying quantities of food. Our experiments revealed that the relative importance of the microbiota for the hosts'' fitness varied according to dietary conditions. The presence of the microbiota had strong positive effects on Daphnia when food was sufficient or abundant, but had weaker effects under food limitation. Our results indicate that the microbiota can be a potentially important factor in determining host responses to changes in dietary conditions. Characterization of the host-associated microbiota further showed that Aeromonas sp. was the most prevalent taxon in the digestive tract of Daphnia.     

Evaluation of injury thresholds for predicting severe head injuries in vulnerable road users resulting from ground impact via detailed accident reconstructions

Biomechanics and Modeling in Mechanobiology - The aim of this study was to evaluate the effectiveness of various head injury criteria and associated risk functions in prediction of vulnerable road...     

Precise Control of Phase Separation Enables 12% Efficiency in All Small Molecule Solar Cells

Compared to conjugated polymers, small‐molecule organic semiconductors present negligible batch‐to‐batch variations, but presently provide comparatively low power conversion efficiencies (PCEs) in small‐molecular organic solar cells (SM‐OSCs), mainly due to suboptimal nanomorphology. Achieving precise control of the nanomorphology remains challenging. Here, two new small‐molecular donors H13 and H14 , created by fluorine and chlorine substitution of the original donor molecule H11 , are presented that exhibit a similar or higher degree of crystallinity/aggregation and improved open‐circuit voltage with IDIC‐4F as acceptor. Due to kinetic and thermodynamic reasons, H13 ‐based blend films possess relatively unfavorable molecular packing and morphology. In contrast, annealed H14 ‐based blends exhibit favorable characteristics, i.e., the highest degree of aggregation with the smallest paracrystalline π–π distortions and a nanomorphology with relatively pure domains, all of which enable generating and collecting charges more efficiently. As a result, blends with H13 give a similar PCE (10.3%) as those made with H11 (10.4%), while annealed H14 ‐based SM‐OSCs have a significantly higher PCE (12.1%). Presently this represents the highest efficiency for SM‐OSCs using IDIC‐4F as acceptor. The results demonstrate that precise control of phase separation can be achieved by fine‐tuning the molecular structure and film formation conditions, improving PCE and providing guidance for morphology design.     

Plant community composition steers grassland vegetation via soil legacy effects

Soil legacy effects are commonly highlighted as drivers of plant community dynamics and species co‐existence. However, experimental evidence for soil legacy effects of conditioning plant communities on responding plant communities under natural conditions is lacking. We conditioned 192 grassland plots using six different plant communities with different ratios of grasses and forbs and for different durations. Soil microbial legacies were evident for soil fungi, but not for soil bacteria, while soil abiotic parameters did not significantly change in response to conditioning. The soil legacies affected the composition of the succeeding vegetation. Plant communities with different ratios of grasses and forbs left soil legacies that negatively affected succeeding plants of the same functional type. We conclude that fungal‐mediated soil legacy effects play a significant role in vegetation assembly of natural plant communities.