ELM: super-resolution analysis of wide-field images of fluorescent shell structures.

It is often necessary to precisely quantify the size of specimens in biological studies. When measuring feature size in fluorescence microscopy, significant biases can arise due to blurring of its edges if the feature is smaller than the diffraction limit of resolution. This problem is avoided if an equation describing the feature's entire image is fitted to its image data. In this paper we present open-source software, ELM, which uses this approach to measure the size of spheroidal or cylindrical fluorescent shells with a precision of around 10 nm. This has been used to measure coat protein locations in bacterial spores and cell wall diameter in vegetative bacilli, and may also be valuable in microbiological studies of algae, fungi and viruses. ELM is available for download at https://github.com/quantitativeimaging/ELM

Ellipsoid Localisation Microscopy

Multilayered protein coats are crucial to the dormancy, robustness, and germination of bacterial spores. In Bacillus subtilis spores, the coat contains over 70 distinct proteins. Identifying which proteins reside in each layer may provide insight into their distinct functions. We present image analysis methods that determine the order and geometry of concentric protein layers by fitting a model description for a spheroidal fluorescent shell image to optical micrographs of spores incorporating fluorescent fusion proteins. The radius of a spherical protein shell can be determined with <10 nm error by fitting an equation to widefield fluorescence micrographs. Ellipsoidal shell axes can be fitted with comparable precision. The layer orders inferred for B. subtilis and B. megaterium are consistent with measurements in the literature. The aspect ratio of elongated spores and the tendency of some proteins to localize near their poles can be quantified, enabling measurement of structural anisotropy.We gratefully acknowledge support from MedImmune through the Beacon collaboration, the EPSRC Centre for Doctoral Training in Sensor Technologies and Applications (EP/L015889/1), and thank Clemens Kaminski, Romain Laine and Jose Casas-Finet for inspiring discussions.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.bpj.2015.09.02

Proteins Encoded by the gerP Operon Are Localized to the Inner Coat in Bacillus cereus Spores and Are Dependent on GerPA and SafA for Assembly.

The germination of Bacillus spores is triggered by certain amino acids and sugar molecules which permeate the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here, we use the ellipsoid localization microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex-lytic enzymes within the inner coat. We also reveal that the GerPA protein alone can localize in the absence of all other GerP proteins and that it has an essential role for the localization of all other GerP proteins within the spore. Its essential role is also demonstrated to be dependent on SafA, but not CotE, for localization, which is consistent with an inner coat location. GerP-null spores are shown also to have reduced permeability to fluorescently labeled dextran molecules compared to wild-type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability.IMPORTANCE The bacterial spore coat comprises a multilayered proteinaceous structure that influences the distribution, survival, and germination properties of spores in the environment. The results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ellipsoid localization microscopy (ELM) image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, health care, and environmental sectors.1. Engineering and Physical Sciences Research Council Centre for Doctoral Training in Sensor Technologies and Applications (EP/L015889/1) 2. Cambridge Nehru Scholarship 3. Raymond and Beverly Sackler Foundation 4. MedImmun

A Model for Simulating Life Histories of the Elderly: Model Design and Implementation Plans

This paper provides a strategy for the development of a model of life-cycle change in functional status, economic well-being, and family composition, with particular attention to persons aged 65 and older. The overall goal is to use the model as the basis for individual-level projections of the later life cycle, that is, microsimulation. Specifically, the scope of the project includes: 1. Specification and estimation of equations for the dynamics of functional status, nursing home occupancy, income and death among those aged 65+, using data from the 1982, 1984, and 1989 National Long-Term Care Survey (NLTCS) linked to Medicare data for 1982-1993, based on extensions of the Grade of Membership (GoM) framework; 2. Developing equations for year-to-year income streams, determined jointly with changes of marital status, for all ages represented in the cohorts to be simulated; 3. Estimating parameters governing the dynamics of family composition (existence and characteristics of spouse, parent[s] and child[ren]); 4. Integrating the results of the above modeling efforts in a microsimulation computer program with the capacity to dynamically simulate life histories, focussing on the elderly population; 5. Validating the model by comparing its results to actual data where possible, analyzing uncertainty attached to the output from the microsimulation model, and conducting sensitivity analyses using alternative assumptions regarding trends in model parameters; and 6. Using microsimulation, producing disaggregated projections of the elderly population and its characteristics, for example cohort profiles of active life expectancy, or comparisons over time in the health, family structure and economic well-being of the oldest-old

Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies.

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.Infinitus China Ltd

Shallow-water hydrothermal venting linked to the Palaeocene–Eocene Thermal Maximum

The Palaeocene–Eocene Thermal Maximum (PETM) was a global warming event of 5–6 °C around 56 million years ago caused by input of carbon into the ocean and atmosphere. Hydrothermal venting of greenhouse gases produced in contact aureoles surrounding magmatic intrusions in the North Atlantic Igneous Province have been proposed to play a key role in the PETM carbon-cycle perturbation, but the precise timing, magnitude and climatic impact of such venting remains uncertain. Here we present seismic data and the results of a five-borehole transect sampling the crater of a hydrothermal vent complex in the Northeast Atlantic. Stable carbon isotope stratigraphy and dinoflagellate cyst biostratigraphy reveal a negative carbon isotope excursion coincident with the appearance of the index taxon Apectodinium augustum in the vent crater, firmly tying the infill to the PETM. The shape of the crater and stratified sediments suggests large-scale explosive gas release during the initial phase of vent formation followed by rapid, but largely undisturbed, diatomite-rich infill. Moreover, we show that these vents erupted in very shallow water across the North Atlantic Igneous Province, such that volatile emissions would have entered the atmosphere almost directly without oxidation to CO2 and at the onset of the PETM

Electroweak Baryogenesis Using Baryon Number Carrying Scalars

We describe a new mechanism for the generation of the baryon asymmetry of the universe during a first order electroweak phase transition. The mechanism requires the existence of two (or more) baryon number carrying scalar fields with masses and CP violating mixing which vary with the Higgs field expectation value. This mechanism can be implemented using squarks in supersymmetric theories or using leptoquarks. Our central observation is that reflection of these scalars from a bubble wall can yield a significant net baryon number flux into the symmetric phase, balanced by a flux of opposite sign into the broken phase. For generic parameter choices, scalars with incident energies in a specific, but not narrow, range yield order one reflection asymmetries (between the probability of reflection of the scalars and of their antiparticles). The interesting energies are those for which there are two propagating scalars in the symmetric phase but only one in the broken phase. Electroweak sphaleron processes drive the baryon number in the symmetric phase toward zero, but do not act in the broken phase. Our estimate of the resulting baryon asymmetry is consistent with cosmological observations for a range of mass parameters and CP violating phases in a supersymmetric implementation, as long as the bubble walls are not too fast and not too thick.Comment: 41 pages, 7 postscript figures, in revtex. Version to appear in Nuclear Physics B. Several small changes; results improved by recent work of Hu, Moore, and Muelle