petiteFinder: an automated computer vision tool to compute Petite colony frequencies in baker's yeast.

BACKGROUND: Mitochondrial respiration is central to cellular and organismal health in eukaryotes. In baker's yeast, however, respiration is dispensable under fermentation conditions. Because yeast are tolerant of this mitochondrial dysfunction, yeast are widely used by biologists as a model organism to ask a variety of questions about the integrity of mitochondrial respiration. Fortunately, baker's yeast also display a visually identifiable Petite colony phenotype that indicates when cells are incapable of respiration. Petite colonies are smaller than their Grande (wild-type) counterparts, and their frequency can be used to infer the integrity of mitochondrial respiration in populations of cells. Unfortunately, the computation of Petite colony frequencies currently relies on laborious manual colony counting methods which limit both experimental throughput and reproducibility. RESULTS: To address these problems, we introduce a deep learning enabled tool, petiteFinder, that increases the throughput of the Petite frequency assay. This automated computer vision tool detects Grande and Petite colonies and computes Petite colony frequencies from scanned images of Petri dishes. It achieves accuracy comparable to human annotation but at up to 100 times the speed and outperforms semi-supervised Grande/Petite colony classification approaches. Combined with the detailed experimental protocols we provide, we believe this study can serve as a foundation to standardize this assay. Finally, we comment on how Petite colony detection as a computer vision problem highlights ongoing difficulties with small object detection in existing object detection architectures. CONCLUSION: Colony detection with petiteFinder results in high accuracy Petite and Grande detection in images in a completely automated fashion. It addresses issues in scalability and reproducibility of the Petite colony assay which currently relies on manual colony counting. By constructing this tool and providing details of experimental conditions, we hope this study will enable larger-scale experiments that rely on Petite colony frequencies to infer mitochondrial function in yeast.

Integrated Antigenic and Nucleic Acid Detection in Single Virions and Extracellular Vesicles with Viral Content.

Virion-mediated outbreaks are imminent and despite rapid responses, continue to cause adverse symptoms and death. Therefore, tunable, sensitive, high-throughput assays are needed to help diagnose future virion-mediated outbreaks. Herein, it is developed a tunable in situ assay to selectively enrich virions and extracellular vesicles (EVs) and simultaneously detect antigens and nucleic acids at a single-particle resolution. The Biochip Antigen and RNA Assay (BARA) enhanced sensitivities compared to quantitative reverse-transcription polymerase chain reaction (qRT-PCR), enabling the detection of virions in asymptomatic patients, genetic mutations in single virions, and enabling the continued long-term expression of viral RNA in the EV-enriched subpopulation in the plasma of patients with post-acute sequelae of the coronavirus disease of 2019 (COVID-19). BARA revealed highly accurate diagnoses of COVID-19 by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA in saliva and nasopharyngeal swab samples. Altogether, the single-particle detection of antigens and viral RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks.

The Metabolic Profile of Young, Watered Chickpea Plants Can Be Used as a Biomarker to Predict Seed Number under Terminal Drought.

Chickpea is the second-most-cultivated legume globally, with India and Australia being the two largest producers. In both of these locations, the crop is sown on residual summer soil moisture and left to grow on progressively depleting water content, finally maturing under terminal drought conditions. The metabolic profile of plants is commonly, correlatively associated with performance or stress responses, e.g., the accumulation of osmoprotective metabolites during cold stress. In animals and humans, metabolites are also prognostically used to predict the likelihood of an event (usually a disease) before it occurs, e.g., blood cholesterol and heart disease. We sought to discover metabolic biomarkers in chickpea that could be used to predict grain yield traits under terminal drought, from the leaf tissue of young, watered, healthy plants. The metabolic profile (GC-MS and enzyme assays) of field-grown chickpea leaves was analysed over two growing seasons, and then predictive modelling was applied to associate the most strongly correlated metabolites with the final seed number plant-1. Pinitol (negatively), sucrose (negatively) and GABA (positively) were significantly correlated with seed number in both years of study. The feature selection algorithm of the model selected a larger range of metabolites including carbohydrates, sugar alcohols and GABA. The correlation between the predicted seed number and actual seed number was R2 adj = 0.62, demonstrating that the metabolic profile could be used to predict a complex trait with a high degree of accuracy. A previously unknown association between D-pinitol and hundred-kernel weight was also discovered and may provide a single metabolic marker with which to predict large seeded chickpea varieties from new crosses. The use of metabolic biomarkers could be used by breeders to identify superior-performing genotypes before maturity is reached.

Contingency and selection in mitochondrial genome dynamics.

High frequencies of mutant mitochondrial DNA (mtDNA) in human cells lead to cellular defects that are associated with aging and disease. Yet much remains to be understood about the dynamics of the generation of mutant mtDNAs and their relative replicative fitness that informs their fate within cells and tissues. To address this, we utilize long-read single-molecule sequencing to track mutational trajectories of mtDNA in the model organism Saccharomyces cerevisiae. This model has numerous advantages over mammalian systems due to its much larger mtDNA and ease of artificially competing mutant and wild-type mtDNA copies in cells. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation in yeast. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that we show are applicable to more complex organisms like humans.

An Automated Tabletop Continuous Culturing System with Multicolor Fluorescence Monitoring for Microbial Gene Expression and Long-Term Population Dynamics.

Real-time monitoring of gene expression dynamics and population levels in a multispecies microbial community could enable the study of the role of changing gene expression patterns on eco-evolutionary outcomes. Here we report the design and validation of a unique experimental platform with an in situ fluorescence measurement system that has high dynamic range and temporal resolution and is capable of monitoring multiple fluorophores for long-term gene expression and population dynamics experiments. We demonstrate the capability of our system to capture gene expression dynamics in response to external perturbations in two synthetic genetic systems: a simple inducible genetic circuit and a bistable toggle switch. Finally, in exploring the population dynamics of a two species microbial community, we show that our system can capture the switch between competitive exclusion and long-term coexistence in response to different nutrient conditions.

Long-term mortality after primary percutaneous coronary intervention for high-risk myocardial infarction.

BACKGROUND: Primary percutaneous coronary intervention (PPCI) has evolved, including the introduction of stents and platelet glycoprotein IIb/IIIa receptor inhibitors (GPI). The effects of these changes and other variables on long-term survival for a single-centre service were studied. METHODS: A prospective database of clinical and angiographic variables were kept for patients treated with PPCI in Waikato Hospital from 1996 to 2006 (n=527). This was analysed with long-term mortality data. Survival was recorded using Kaplan-Meier curves. Multivariate analysis of factors at presentation, including ethnicity was performed. RESULTS: 5, 8 & 10-year survival rates were 76.5% (n=274), 72.7% (n=125) & 71.0% (n=19) respectively. Increased stent (42.8% vs. 84.1%, p<0.001) and GPI (39.6% vs. 73.3%, p<0.001) use was seen between early and late stages of the study. Stent use was associated with greater 5-year survival (80.5% vs. 70.8%, p=0.02), but GPI use was not. Multivariate analysis showed stent use independently predicted reduced mortality. Age, Maori ethnicity, renal failure and cardiogenic shock predicted higher mortality. CONCLUSIONS: Survival after PPCI remains high long-term. Stent and GPI use significantly increased. Stent, but not GPI, use was associated with improved survival. Maori ethnicity was under-represented in the study and is associated with worse long-term outcomes after myocardial infarction (MI).

Harvest Time Optimization for Combustion Quality of Different Miscanthus Genotypes across Europe.

Delayed harvest can improve the quality of miscanthus biomass for combustion and enhance the long-term sustainability of the crop, despite accompanying yield losses. The aim of this study is to identify the optimal harvesting time, which can deliver improved biomass quality for combustion of novel miscanthus genotypes at various sites across Europe, without high yield losses and without compromising their environmental performance. The relevant field trials were established as part of the European project OPTIMISC with 15 genotypes at six sites across Europe. For this study, the five highest yielding genotypes from each germplasm group and three sites with contrasting climatic conditions (Stuttgart, Germany; Adana, Turkey; and Moscow, Russia) were selected for assessment. The biomass samples were collected between August and March (depending on site) and subjected to mineral and ash content analysis. At Stuttgart, the delay in harvesting time led to a significant variation in combustion quality characteristics, such as N content (0.64-0.21%), ash content (5.15-2.60%), and ash sintering index (1.30-0.20). At Adana, the delay in harvesting time decreased the N content from 0.62 to 0.23%, ash content from 10.63 to 3.84%, and sintering index from 0.54 to 0.07. At Moscow, the impact of delay in harvesting was not significant, except for N, Mg, and ash sintering index. Overall, a delay in harvesting time improved the combustion quality characteristics of each genotype, but at the expense of yield. Yield losses of up to 49% in Stuttgart and Adana and 21% for Moscow were recorded, with variations between genotypes and sites. The harvesting time also affected nutrient offtake, which in turn influences the long-term environmental performance of the crop. The highest N, P, and K offtakes were recorded at Stuttgart for each harvesting time except for final harvest (March), where Moscow had the highest N offtake. This study describes the three criteria (biomass quality, yield losses, nutrient offtake) for determining the ideal harvesting time, which gives the best compromise between dry matter yields and biomass quality characteristics without negatively affecting the environmental performance of the crop.

Site-Specific Management of Miscanthus Genotypes for Combustion and Anaerobic Digestion: A Comparison of Energy Yields.

In Europe, the perennial C4 grass miscanthus is currently mainly cultivated for energy generation via combustion. In recent years, anaerobic digestion has been identified as a promising alternative utilization pathway. Anaerobic digestion produces a higher-value intermediate (biogas), which can be upgraded to biomethane, stored in the existing natural gas infrastructure and further utilized as a transport fuel or in combined heat and power plants. However, the upgrading of the solid biomass into gaseous fuel leads to conversion-related energy losses, the level of which depends on the cultivation parameters genotype, location, and harvest date. Thus, site-specific crop management needs to be adapted to the intended utilization pathway. The objectives of this paper are to quantify (i) the impact of genotype, location and harvest date on energy yields of anaerobic digestion and combustion and (ii) the conversion losses of upgrading solid biomass into biogas. For this purpose, five miscanthus genotypes (OPM 3, 6, 9, 11, 14), three cultivation locations (Adana, Moscow, Stuttgart), and up to six harvest dates (August-March) were assessed. Anaerobic digestion yielded, on average, 35% less energy than combustion. Genotype, location, and harvest date all had significant impacts on the energy yield. For both, this is determined by dry matter yield and ash content and additionally by substrate-specific methane yield for anaerobic digestion and moisture content for combustion. Averaged over all locations and genotypes, an early harvest in August led to 25% and a late harvest to 45% conversion losses. However, each utilization option has its own optimal harvest date, determined by biomass yield, biomass quality, and cutting tolerance. By applying an autumn green harvest for anaerobic digestion and a delayed harvest for combustion, the conversion-related energy loss was reduced to an average of 18%. This clearly shows that the delayed harvest required to maintain biomass quality for combustion is accompanied by high energy losses through yield reduction over winter. The pre-winter harvest applied in the biogas utilization pathway avoids these yield losses and largely compensates for the conversion-related energy losses of anaerobic digestion.

Could Miscanthus replace maize as the preferred substrate for anaerobic digestion in the United Kingdom? Future breeding strategies.

Fodder maize is the most commonly used crop for biogas production owing to its high yields, high concentrations of starch and good digestibility. However, environmental concerns and possible future conflict with land for food production may limit its long-term use. The bioenergy grass, Miscanthus, is a high-yielding perennial that can grow on marginal land and, with 'greener' environmental credentials, may offer an alternative. To compete with maize, the concentration of non-structural carbohydrates (NSC) and digestibility may need to be improved. Non-structural carbohydrates were quantified in 38 diverse genotypes of Miscanthus in green-cut biomass in July and October. The aim was to determine whether NSC abundance could be a target for breeding programmes or whether genotypes already exist that could rival maize for use in anaerobic digestion systems. The saccharification potential and measures of N P and K were also studied. The highest concentrations of NSC were in July, reaching a maximum of 20% DW. However, the maximum yield was in October with 300-400 g NSC plant-1 owing to higher biomass. The digestibility of the cell wall was higher in July than in October, but the increase in biomass meant yields of digestible sugars were still higher in October. Nutrient concentrations were at least twofold higher in July compared to November, and the abundance of potassium showed the greatest degree of variation between genotypes. The projected maximum yield of NSC was 1.3 t ha-1 with significant variation to target for breeding. Starch accumulated in the highest concentrations and continued to increase into autumn in some genotypes. Therefore, starch, rather than sugars, would be a better target for breeding improvement. If harvest date was brought forward to autumn, nutrient losses in non-flowering genotypes would be comparable to an early spring harvest.

Extending Miscanthus Cultivation with Novel Germplasm at Six Contrasting Sites.

Miscanthus is a genus of perennial rhizomatous grasses with C4 photosynthesis which is indigenous in a wide geographic range of Asian climates. The sterile clone, Miscanthus × giganteus (M. × giganteus), is a naturally occurring interspecific hybrid that has been used commercially in Europe for biomass production for over a decade. Although, M. × giganteus has many outstanding performance characteristics including high yields and low nutrient offtakes, commercial expansion is limited by cloning rates, slow establishment to a mature yield, frost, and drought resistance. In this paper, we evaluate the performance of 13 novel germplasm types alongside M. × giganteus and horticultural "Goliath" in trials in six sites (in Germany, Russia, The Netherlands, Turkey, UK, and Ukraine). Mean annual yields across all the sites and genotypes increased from 2.3 ± 0.2 t dry matter ha-1 following the first year of growth, to 7.3 ± 0.3, 9.5 ± 0.3, and 10.5 ± 0.2 t dry matter ha-1 following the second, third, and fourth years, respectively. The highest average annual yields across locations and four growth seasons were observed for M. × giganteus (9.9 ± 0.7 t dry matter ha-1) and interspecies hybrid OPM-6 (9.4 ± 0.6 t dry matter ha-1). The best of the new hybrid genotypes yielded similarly to M. × giganteus at most of the locations. Significant effects of the year of growth, location, species, genotype, and interplay between these factors have been observed demonstrating strong genotype × environment interactions. The highest yields were recorded in Ukraine. Time needed for the crop establishment varied depending on climate: in colder climates such as Russia the crop has not achieved its peak yield by the fourth year, whereas in the hot climate of Turkey and under irrigation the yields were already high in the first growing season. We have identified several alternatives to M. × giganteus which have provided stable yields across wide climatic ranges, mostly interspecies hybrids, and also Miscanthus genotypes providing high biomass yields at specific geographic locations. Seed-propagated interspecific and intraspecific hybrids, with high stable yields and cheaper reliable scalable establishment remain a key strategic objective for breeders.

Environmental Influences on the Growing Season Duration and Ripening of Diverse Miscanthus Germplasm Grown in Six Countries.

The development of models to predict yield potential and quality of a Miscanthus crop must consider climatic limitations and the duration of growing season. As a biomass crop, yield and quality are impacted by the timing of plant developmental transitions such as flowering and senescence. Growth models are available for the commercially grown clone Miscanthus x giganteus (Mxg), but breeding programs have been working to expand the germplasm available, including development of interspecies hybrids. The aim of this study was to assess the performance of diverse germplasm beyond the range of environments considered suitable for a Miscanthus crop to be grown. To achieve this, six field sites were planted as part of the EU OPTIMISC project in 2012 in a longitudinal gradient from West to East: Wales-Aberystwyth, Netherlands-Wageningen, Stuttgart-Germany, Ukraine-Potash, Turkey-Adana, and Russia-Moscow. Each field trial contained three replicated plots of the same 15 Miscanthus germplasm types. Through the 2014 growing season, phenotypic traits were measured to determine the timing of developmental stages key to ripening; the tradeoff between growth (yield) and quality (biomass ash and moisture content). The hottest site (Adana) showed an accelerated growing season, with emergence, flowering and senescence occurring before the other sites. However, the highest yields were produced at Potash, where emergence was delayed by frost and the growing season was shortest. Flowering triggers varied with species and only in Mxg was strongly linked to accumulated thermal time. Our results show that a prolonged growing season is not essential to achieve high yields if climatic conditions are favorable and in regions where the growing season is bordered by frost, delaying harvest can improve quality of the harvested biomass.

Leaves of the Arabidopsis maltose exporter1 mutant exhibit a metabolic profile with features of cold acclimation in the warm.

BACKGROUND: Arabidopsis plants accumulate maltose from starch breakdown during cold acclimation. The Arabidopsis mutant, maltose excess1-1, accumulates large amounts of maltose in the plastid even in the warm, due to a deficient plastid envelope maltose transporter. We therefore investigated whether the elevated maltose level in mex1-1 in the warm could result in changes in metabolism and physiology typical of WT plants grown in the cold. PRINCIPAL FINDINGS: Grown at 21 °C, mex1-1 plants were much smaller, with fewer leaves, and elevated carbohydrates and amino acids compared to WT. However, after transfer to 4 °C the total soluble sugar pool and amino acid concentration was in equal abundance in both genotypes, although the most abundant sugar in mex1-1 was still maltose whereas sucrose was in greatest abundance in WT. The chlorophyll a/b ratio in WT was much lower in the cold than in the warm, but in mex1-1 it was low in both warm and cold. After prolonged growth at 4 °C, the shoot biomass, rosette diameter and number of leaves at bolting were similar in mex1-1 and WT. CONCLUSIONS: The mex1-1 mutation in warm-grown plants confers aspects of cold acclimation, including elevated levels of sugars and amino acids and low chlorophyll a/b ratio. This may in turn compromise growth of mex1-1 in the warm relative to WT. We suggest that elevated maltose in the plastid could be responsible for key aspects of cold acclimation.

Door to balloon times: streamlining admission for primary percutaneous coronary intervention.

AIMS: Primary angioplasty is superior to thrombolysis in ST elevation myocardial infarction (STEMI). This advantage is dependent on how quickly angioplasty can be performed. Several strategies have been suggested to cut door to balloon (D2B) times. We aimed to audit and reorganise the admission process to accelerate D2B times, in Waikato Hospital, New Zealand. METHODS: The admission process for STEMI was audited. Three changes were made. One step in the catheterisation lab activation system (referral to cardiology registrar on call) was removed. Single call pager activation of the catheterisation lab team was adopted. Feedback of timing performance data by email and printout was established. Timing data were collected for 6 months before and after these changes. RESULTS: After the admission process was changed 88.5% of patients had a D2B time <90 min vs 63.6% before. Median D2B times were reduced from 74.5 min to 59 min (p=0.09). Median time from admission to arrival at cath lab was reduced from 50 min to 35 min (p=0.019). CONCLUSIONS: Relatively minor changes in admission process, without new resources, can lead to reductions in door to balloon times. This was achieved in a hospital with a selective PPCI policy and modest annual volume of PPCI.

Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans.

A role for the cAMP-dependent pathway in regulation of the cell wall in the model yeast Saccharomyces cerevisiae has recently been demonstrated. In this study we report the results of a phenotypic analysis of a Candida albicans mutant, characterized by a constitutive activation of the cAMP pathway due to deletion of PDE2, the gene encoding the high cAMP-affinity phosphodiesterase. Unlike wild-type strains, this mutant has an increased sensitivity to cell wall and membrane perturbing agents such as SDS and CFW, and antifungals such as amphotericin B and flucytosine. Moreover, the mutant is characterized by an altered sensitivity and a significantly reduced tolerance to fluconazole. The mutant's membrane has around 30% higher ergosterol content and the cell wall glucan was 22% lower than in the wild-type. These cell wall and membrane changes are manifested by a considerable reduction in the thickness of the cell wall, which in the mutant is on average 60-65 nm, compared to 80-85 nm in the wild-type strains as revealed by electron microscopy. These results suggest that constitutive activation of the cAMP pathway affects cell wall and membrane structure, and biosynthesis, not only in the model yeast S. cerevisiae but also in the human fungal pathogen C. albicans.

Isoamyl alcohol-induced morphological change in Saccharomyces cerevisiae involves increases in mitochondria and cell wall chitin content.

Isoamyl alcohol reduced growth and induced filament formation in Saccharomyces cerevisiae. Isoamyl alcohol-induced filamentation was accompanied by an almost threefold greater increase in the specific activity of succinate dehydrogenase than in untreated cells, which suggested that isoamyl alcohol treatment caused the cells to produce more mitochondria than in normal yeast form proliferation. This was supported by measuring the dry weight of purified, isolated mitochondria. Filaments have an increased chitin content which is distributed over the majority of their surface, and is not confined to bud scars and the chitin ring between mother and daughter cells as in yeast-form cells.