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Supplemental Digital Content is available in the text., Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

Size is a fundamental cellular trait that is important in determining phytoplankton physiological and ecological processes. Fossil coccospheres, the external calcite structure produced by the excretion of interlocking plates by the phytoplankton coccolithophores, can provide a rare window into cell size in the past. Coccospheres are delicate however and are therefore poorly preserved in sediment. We demonstrate a novel technique combining imaging flow cytometry and cross-polarised light (ISX+PL) to rapidly and reliably visually isolate and quantify the morphological characteristics of coccospheres from marine sediment by exploiting their unique optical and morphological properties. Imaging flow cytometry combines the morphological information provided by microscopy with high sample numbers associated with flow cytometry. High throughput imaging overcomes the constraints of labour-intensive manual microscopy and allows statistically robust analysis of morphological features and coccosphere concentration despite low coccosphere concentrations in sediments. Applying this technique to the fine-fraction of sediments, hundreds of coccospheres can be visually isolated quickly with minimal sample preparation. This approach has the potential to enable rapid processing of down-core sediment records and/or high spatial coverage from surface sediments and may prove valuable in investigating the interplay between climate change and coccolithophore physiological/ecological response.

We report a method for embedding cell-free protein synthesis reactions in macro-scale hydrogel materials without a free liquid phase. This paper focuses on methods of preparation for a variety of hydrogels and an investigation of the impact that the hydrogel material has on cell-free protein synthesis.

The uptake of microplastics into marine species has been widely documented across trophic levels. Feeding mode is suggested as playing an important role in determining different contamination loads across species, but this theory is poorly supported with empirical evidence. Here we use the two distinct feeding modes of the benthic polychaete, Hediste diversicolor (The Harbour Ragworm) (O.F. Müller, 1776), to test the hypothesis that filter feeding will lead to a greater uptake of microplastic particles than deposit feeding. Worms were exposed to both polyamide microfragments and microfibres in either water (as filter feeders) or sediment (as deposit feeders) for 1 week. No effect of exposure time was found between 1 day and 1 week (p0.19) but feeding mode was found to significantly affect the number of microfibres recovered from each worm (p0.001). When exposed to microfibers, filter feeding worms took up ≈15,000 % more fibres than deposit feeding worms (p0.001), whereas when feeding on microfragments there was no difference between feeding modes. Our data demonstrate that both feeding mode and particle characteristics significantly influence the uptake of microplastics by H. diversicolor. Using imaging flow cytometry, filter feeders were found to take up a broader size range of particles, with significantly more smaller and larger particles than deposit feeders (p0.05), commensurate with the range of plastics isolated from the guts of ragworms recovered from the environment. These results demonstrate that biological traits are useful in understanding the uptake of plastics into marine worms and warrant further exploration as a tool for understanding the bioaccessibility of plastics to marine organisms.

Decarbonisation of the transport sector is essential to mitigate anthropogenic climate change. Microbial metabolisms are already integral to the production of renewable, sustainable fuels and, building on that foundation, are being re-engineered to generate the advanced biofuels that will maintain mobility of people and goods during the energy transition. This review surveys the range of natural and engineered microbial systems for advanced biofuels production and summarises some of the techno-economic challenges associated with their implementation at industrial scales.

Bacteria modify their morphology in response to various factors including growth stage, nutrient availability, predation, motility and long-term survival strategies. Morphological changes may also be associated with specific physiological phenotypes such as the formation of dormant or persister cells in a “viable but non-culturable” (VBNC) state which frequently display different shapes and size compared to their active counterparts. Such dormancy phenotypes can display various degrees of tolerance to antibiotics and therefore a detailed understanding of these phenotypes is crucial for combatting chronic infections and associated diseases. Cell shape and size are therefore more than simple phenotypic characteristics; they are important physiological properties for understanding bacterial life-strategies and pathologies. However, quantitative studies on the changes to cell morphologies during bacterial growth, persister cell formation and the VBNC state are few and severely constrained by current limitations in the most used investigative techniques of flow cytometry (FC) and light or electron microscopy. In this study, we applied high-throughput Imaging Flow Cytometry (IFC) to characterise and quantify, at single-cell level and over time, the phenotypic heterogeneity and morphological changes in cultured populations of four bacterial species, Bacillus subtilis, Lactiplantibacillus plantarum, Pediococcus acidilactici and Escherichia coli. Morphologies in relation to growth stage and stress responses, cell integrity and metabolic activity were analysed. Additionally, we were able to identify and morphologically classify dormant cell phenotypes such as VBNC cells and monitor the resuscitation of persister cells in Escherichia coli following antibiotic treatment. We therefore demonstrate that IFC, with its high-throughput data collection and image capture capabilities, provides a platform by which a detailed understanding of changes in bacterial phenotypes and their physiological implications may be accurately monitored and quantified, leading to a better understanding of the role of phenotypic heterogeneity in the dynamic microbiome.

The increase in population and the demand to study for various purposes has immensely contributed to classroom population in India. The varied interest among learners, their social background, attitude, and abilities, are to be looked by teacher. Teacher cannot blindfold to these differences. Heterogeneous classrooms are inevitable in India, which stands for Unity in Diversity. The aim to address the mixed atmosphere where each student feels welcomed and accepted with his/her capabilities under one roof becomes a challenge to a teacher. Hence answering this phenomenon is essential. This paper is divided into three parts. The first part discusses, what is a large class and heterogeneous class in Indian conditions in relation to other developing and developed countries. In the second part, discusses about the elements of heterogeneity. The third part presents the methodology used for this study such as informal interviews, discussions among 44 teachers from five colleges who teach to a large heterogeneous class. The collected data was analysed, how teachers manage those problems in the large classes given to them. As a result, the researcher also has given solution to manage those problems with the Eclectic Approach.

This research was performed in an Arts and science college of Indian ESL learners to explore the impact of mind mapping of vocabulary based retention strategy use. First year general English students were chosen. An experimental method was conducted; forty students have been taken as the subject. They were subjected to therapy in which the use of mind mapping approach instructed vocabulary selection through miming. The findings of the post-test acquired through the SPSS software showed at the end of instructions find that students can retain the words what they learned before. The findings of a delayed post-test after sometime disclosed that the long-term impact of the mind mapping approach was considerably effective in retaining the vocabulary by ESL learners in teaching this activity. One significant pedagogical implication of this research is that a more focused mind mapping approach of vocabulary can be very efficient at the general English classroom of ESL learners.

Botryococcus braunii is a colonial microalga that appears early in the fossil record and is a sensitive proxy of environmental and hydroclimatic conditions. Palaeozoic Botryococcus fossils which contribute up to 90% of oil shales and approximately 1% of crude oil, co-localise with diagnostic geolipids from the degradation of source-signature hydrocarbons. However more recent Holocene sediments demonstrate no such association. Consequently, Botryococcus are identified in younger sediments by morphology alone, where potential misclassifications could lead to inaccurate paleoenvironmental reconstructions. Here we show that a combination of flow cytometry and ancient DNA (aDNA) sequencing can unambiguously identify Botryococcus microfossils in Holocene sediments with hitherto unparalleled accuracy and rapidity. The application of aDNA sequencing to microfossils offers a far-reaching opportunity for understanding environmental change in the recent geological record. When allied with other high-resolution palaeoenvironmental information such as aDNA sequencing of humans and megafauna, aDNA from microfossils may allow a deeper and more precise understanding of past environments, ecologies and migrations.

Developing media to sustain cell growth and production is an essential and ongoing activity in bioprocess development. Modifications to media can often address host or product-specific challenges, such as low productivity or poor product quality. For other applications, systematic design of new media can facilitate the adoption of new industrially relevant alternative hosts. Despite manifold existing methods, common approaches for optimization often remain time and labor intensive. We present here a novel approach to conventional media blending that leverages stable, simple, concentrated stock solutions to enable rapid improvement of measurable phenotypes of interest. We applied this modular methodology to generate high-performing media for two phenotypes of interest: biomass accumulation and heterologous protein production, using high-throughput, milliliter-scale batch fermentations of Pichia pastoris as a model system. In addition to these examples, we also created a flexible open-source package for modular blending automation on a low-cost liquid handling system to facilitate wide use of this method. Our modular blending method enables rapid, flexible media development, requiring minimal labor investment and prior knowledge of the host organism, and should enable developing improved media for other hosts and phenotypes of interest.

Weissella paramesenteroides has potential as an industrial biocatalyst due to its ability to produce lactic acid. A novel strain of W. paramesenteroides was isolated from ensiled sorghum. The genome was sequenced using a hybrid assembly of Oxford Nanopore and Illumina data to produce a 2-Mbp genome and 22-kbp plasmid sequence.

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10−3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

The Southern Ocean occupies 14% of the Earth’s surface and plays a fundamental role in the global carbon cycle and climate. It provides a direct connection to the deep ocean carbon reservoir through biogeochemical processes that include surface primary productivity, remineralization at depth and the upwelling of carbon-rich water masses. However, the role of these different processes in modulating past and future air–sea carbon flux remains poorly understood. A key period in this regard is the Antarctic Cold Reversal (ACR, 14.6–12.7 kyr bp), when mid- to high-latitude Southern Hemisphere cooling coincided with a sustained plateau in the global deglacial increase in atmospheric CO2. Here we reconstruct high-latitude Southern Ocean surface productivity from marine-derived aerosols captured in a highly resolved horizontal ice core. Our multiproxy reconstruction reveals a sustained signal of enhanced marine productivity across the ACR. Transient climate modelling indicates this period coincided with maximum seasonal variability in sea-ice extent, implying that sea-ice biological feedbacks enhanced CO2 sequestration and created a substantial regional marine carbon sink, which contributed to the plateau in CO2 during the ACR. Our results highlight the role Antarctic sea ice plays in controlling global CO2, and demonstrate the need to incorporate such feedbacks into climate–carbon models.

Fossil coccospheres provide a wealth of information on cellular traits that can be compared directly to the living coccosphere such as cell size. Cell size is critical to ecosystem dynamics and particle sinking which has implications on the carbon cycle. However, cell size reconstruction is hindered by the poor preservation of coccospheres as coccospheres often disintegrate into individual coccoliths. Although palaeoecological information can be attained from individual coccoliths, assumptions must be made when correlating cell size to coccolith size. We demonstrate a novel technique using imaging flow cytometry to rapidly and reliably sort coccospheres from marine sediment by exploiting their unique optical and morphological properties. Imaging flow cytometry combines the functional insight of morphological information provided by microscopy with high sample numbers that are associated with flow cytometry. High throughput imaging overcomes the constraints of laborious manual microscopy enabling the analysis of sediments containing low concentrations of coccospheres that would simply not be feasible to manually hunt for coccospheres. By applying this technique to the fine fraction of sediments, hundreds of coccospheres can be isolated without the need for additional sample processing. Morphological information of individual coccospheres is obtained and graphical and statistical information can be extracted. This approach lends itself perfectly to rapid processing of down-core sediment samples or high spatial coverage from core-top samples and may prove valuable in investigating the interplay between a changing climate and coccolithophore response.

Current efforts to examine and quantify so-called ‘biomarkers’ present in polar ice samples offer exciting potential as biological and biochemical proxies for past climate and ocean dynamics. Here we present a new rapid and easily replicable method to provide measurements of the microscopic particulate content of ice samples from polar environments. Using an Amnis® Imagestream® Imaging Flow Cytometer, melted snow and ice samples from Patriot Hills in the Ellsworth Mountains, Antarctica were analysed for their particulate (biological and non-biological) content. Selective use of a nucleic acid stain pre-treatment allows for a straightforward gating strategy that resolves both autofluorescent and non-autofluorescent biological material in sample replicates. In the Patriot Hills samples this method clearly identifies marine picoplankton, along with non-biological particulates such as tephra and minerogenic material. Crucially, the 60x Brightfield images provided by the Imagestream offer a significant additional capability above standard flow cytometry systems; each object identified by the machine can be visually differentiated (automatically or manually) from particulates with similar fluorescence properties. Back-trajectory analysis with the NOAA Hybrid Single-Particle Lagrangian Integrated Trajectory (HySPLIT) model indicates that these ice-bound marine organisms originate from the Weddell and Amundsen-Bellingshausen Seas. This technique, when paired with established chemical and biochemical methods, shows considerable potential in providing valuable information about the nature and origin of aerosols and biomarker signals trapped in past ice layers.

Science is key to developing sustainable products and solutions. But scientists also need to work more with governments, industry and society to help implement those solutions.

Multifactorial approaches can quickly and efficiently model complex, interacting natural or engineered biological systems in a way that traditional one-factor-at-a-time experimentation can fail to do. We applied a Design of Experiments (DOE) approach to model ethanol biosynthesis in yeast, which is well-understood and genetically tractable, yet complex. Six alcohol dehydrogenase (ADH) isozymes catalyze ethanol synthesis, differing in their transcriptional and post-translational regulation, subcellular localization, and enzyme kinetics. We generated a combinatorial library of all ADH gene deletions and measured the impact of gene deletion(s) and environmental context on ethanol production of a subset of this library. The data were used to build a statistical model that described known behaviors of ADH isozymes and identified novel interactions. Importantly, the model described features of ADH metabolic behavior without explicit a priori knowledge. The method is therefore highly suited to understanding and optimizing metabolic pathways in less well-understood systems.

Smart materials are able to alter one or more of their properties in response to defined stimuli. Our ability to design and create such materials, however, does not match the diversity and specificity of responses seen within the biological domain. We propose that relocation of molecular phenomena from living cells into hydrogels can be used to confer smart functionality to materials. We establish that cell-free protein synthesis can be conducted in agarose hydrogels, that gene expression occurs throughout the material and that co-expression of genes is possible. We demonstrate that gene expression can be controlled transcriptionally (using in gel gene interactions) and translationally in response to small molecule and nucleic acid triggers. We use this system to design and build a genetic device that can alter the structural property of its chassis material in response to exogenous stimuli. Importantly, we establish that a wide range of hydrogels are appropriate chassis for cell-free synthetic biology, meaning a designer may alter both the genetic and hydrogel components according to the requirements of a given application. We probe the relationship between the physical structure of the gel and in gel protein synthesis and reveal that the material itself may act as a macromolecular crowder enhancing protein synthesis. Given the extensive range of genetically encoded information processing networks in the living kingdom and the structural and chemical diversity of hydrogels, this work establishes a model by which cell-free synthetic biology can be used to create autonomic and adaptive materials.Significance statementSmart materials have the ability to change one or more of their properties (e.g. structure, shape or function) in response to specific triggers. They have applications ranging from light-sensitive sunglasses and drug delivery systems to shape-memory alloys and self-healing coatings. The ability to programme such materials, however, is basic compared to the ability of a living organism to observe, understand and respond to its environment. Here we demonstrate the relocation of biological information processing systems from cells to materials. We achieved this by operating small, programmable genetic devices outside the confines of a living cell and inside hydrogel matrices. These results establish a method for developing materials functionally enhanced with molecular machinery from biological systems.

ObjectiveExtracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signalling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterisation of endogenously produced EVs is still in its infancy. Due to their genetic tractability and opportunities for live imaging, zebrafish represent an ideal but under-used model to investigate endogenous EVs. The overall aim of this study was to establish a transgenic zebrafish model to allow the in vivo identification, tracking and extraction of endogenous EVs produced by different cell types.Approach and ResultsUsing a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labelled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Importantly, we demonstrate the utility of this model by showing that cardiomyocytes, endothelial cells and macrophages exchange EVs in the adult heart and that ischaemic injury models dynamically alter EV production.ConclusionsWe have developed a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labelling approach allows cell type-specific tracing of EV origin without bias towards the expression of individual protein markers and will allow detailed future examination of their function.

Well-characterized promoter collections for synthetic biology applications are not always available in industrially relevant hosts. We developed a broadly applicable method for promoter identification in atypical microbial hosts that requires no a priori understanding of cis-regulatory element structure. This novel approach combines bioinformatic filtering with rapid empirical characterization to expand the promoter toolkit and uses machine learning to improve the understanding of the relationship between DNA sequence and function. Here, we apply the method in Geobacillus thermoglucosidasius, a thermophilic organism with high potential as a synthetic biology chassis for industrial applications. Bioinformatic screening of G. kaustophilus, G. stearothermophilus, G. thermodenitrificans, and G. thermoglucosidasius resulted in the identification of 636 100 bp putative promoters, encompassing the genome-wide design space and lacking known transcription factor binding sites. Eighty of these sequences were characterized in vivo, and activities covered a 2-log range of predictable expression levels. Seven sequences were shown to function consistently regardless of the downstream coding sequence. Partition modeling identified sequence positions upstream of the canonical -35 and -10 consensus motifs that were predicted to strongly influence regulatory activity in Geobacillus, and artificial neural network and partial least squares regression models were derived to assess if there were a simple, forward, quantitative method for in silico prediction of promoter function. However, the models were insufficiently general to predict pre hoc promoter activity in vivo, most probably as a result of the relatively small size of the training data set compared to the size of the modeled design space.

Infections after placement of cardiac implantable electronic devices (CIEDs) are associated with substantial morbidity and mortality. There is limited evidence on prophylactic strategies, other than the use of preoperative antibiotics, to prevent such infections.We conducted a randomized, controlled clinical trial to assess the safety and efficacy of an absorbable, antibiotic-eluting envelope in reducing the incidence of infection associated with CIED implantations. Patients who were undergoing a CIED pocket revision, generator replacement, or system upgrade or an initial implantation of a cardiac resynchronization therapy defibrillator were randomly assigned, in a 1:1 ratio, to receive the envelope or not. Standard-of-care strategies to prevent infection were used in all patients. The primary end point was infection resulting in system extraction or revision, long-term antibiotic therapy with infection recurrence, or death, within 12 months after the CIED implantation procedure. The secondary end point for safety was procedure-related or system-related complications within 12 months.A total of 6983 patients underwent randomization: 3495 to the envelope group and 3488 to the control group. The primary end point occurred in 25 patients in the envelope group and 42 patients in the control group (12-month Kaplan-Meier estimated event rate, 0.7% and 1.2%, respectively; hazard ratio, 0.60; 95% confidence interval [CI], 0.36 to 0.98; P = 0.04). The safety end point occurred in 201 patients in the envelope group and 236 patients in the control group (12-month Kaplan-Meier estimated event rate, 6.0% and 6.9%, respectively; hazard ratio, 0.87; 95% CI, 0.72 to 1.06; P0.001 for noninferiority). The mean (±SD) duration of follow-up was 20.7±8.5 months. Major CIED-related infections through the entire follow-up period occurred in 32 patients in the envelope group and 51 patients in the control group (hazard ratio, 0.63; 95% CI, 0.40 to 0.98).Adjunctive use of an antibacterial envelope resulted in a significantly lower incidence of major CIED infections than standard-of-care infection-prevention strategies alone, without a higher incidence of complications. (Funded by Medtronic; WRAP-IT ClinicalTrials.gov number, NCT02277990.).

The judicious choice of promoter to drive gene expression remains one of the most important considerations for synthetic biology applications. Constitutive promoter sequences isolated from nature are often used in laboratory settings or small-scale commercial production streams, but unconventional microbial chassis for new synthetic biology applications require well-characterized, robust and orthogonal promoters. This review provides an overview of the opportunities and challenges for synthetic promoter discovery and design, including molecular methodologies, such as saturation mutagenesis of flanking regions and mutagenesis by error-prone PCR, as well as the less familiar use of computational and statistical analyses for de novo promoter design.

Prevalence was reduced, and early evidence indicates herd immunity., In 2008, a national human papillomavirus (HPV) immunization program using a bivalent vaccine against HPV types 16 and 18 was implemented in Scotland along with a national surveillance program designed to determine the longitudinal effects of vaccination on HPV infection at the population level. Each year during 2009–2013, the surveillance program conducted HPV testing on a proportion of liquid-based cytology samples from women undergoing their first cervical screening test for precancerous cervical disease. By linking vaccination, cervical screening, and HPV testing data, over the study period we found a decline in HPV types 16 and 18, significant decreases in HPV types 31, 33, and 45 (suggesting cross-protection), and a nonsignificant increase in HPV 51. In addition, among nonvaccinated women, HPV types 16 and 18 infections were significantly lower in 2013 than in 2009. Our results preliminarily indicate herd immunity and sustained effectiveness of the bivalent vaccine on virologic outcomes at the population level.