Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments

Structural Changes in Isometrically Contracting Insect Flight Muscle Trapped following a Mechanical Perturbation

The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5–6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ∼40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ∼98% of strong-binding acto-myosin attachments present after a length perturbation are confined to “target zones” of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model for contraction in IFM that may be applicable to contraction in other types of muscle

Evolution of pathogenicity and sexual reproduction in eight Candida genomes

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.publishe

Children’s and adolescents’ rising animal-source food intakes in 1990–2018 were impacted by age, region, parental education and urbanicity

Animal-source foods (ASF) provide nutrition for children and adolescents’ physical and cognitive development. Here, we use data from the Global Dietary Database and Bayesian hierarchical models to quantify global, regional and national ASF intakes between 1990 and 2018 by age group across 185 countries, representing 93% of the world’s child population. Mean ASF intake was 1.9 servings per day, representing 16% of children consuming at least three daily servings. Intake was similar between boys and girls, but higher among urban children with educated parents. Consumption varied by age from 0.6 at <1 year to 2.5 servings per day at 15–19 years. Between 1990 and 2018, mean ASF intake increased by 0.5 servings per week, with increases in all regions except sub-Saharan Africa. In 2018, total ASF consumption was highest in Russia, Brazil, Mexico and Turkey, and lowest in Uganda, India, Kenya and Bangladesh. These findings can inform policy to address malnutrition through targeted ASF consumption programmes.publishedVersio

Incident type 2 diabetes attributable to suboptimal diet in 184 countries

The global burden of diet-attributable type 2 diabetes (T2D) is not well established. This risk assessment model estimated T2D incidence among adults attributable to direct and body weight-mediated effects of 11 dietary factors in 184 countries in 1990 and 2018. In 2018, suboptimal intake of these dietary factors was estimated to be attributable to 14.1 million (95% uncertainty interval (UI), 13.8–14.4 million) incident T2D cases, representing 70.3% (68.8–71.8%) of new cases globally. Largest T2D burdens were attributable to insufficient whole-grain intake (26.1% (25.0–27.1%)), excess refined rice and wheat intake (24.6% (22.3–27.2%)) and excess processed meat intake (20.3% (18.3–23.5%)). Across regions, highest proportional burdens were in central and eastern Europe and central Asia (85.6% (83.4–87.7%)) and Latin America and the Caribbean (81.8% (80.1–83.4%)); and lowest proportional burdens were in South Asia (55.4% (52.1–60.7%)). Proportions of diet-attributable T2D were generally larger in men than in women and were inversely correlated with age. Diet-attributable T2D was generally larger among urban versus rural residents and higher versus lower educated individuals, except in high-income countries, central and eastern Europe and central Asia, where burdens were larger in rural residents and in lower educated individuals. Compared with 1990, global diet-attributable T2D increased by 2.6 absolute percentage points (8.6 million more cases) in 2018, with variation in these trends by world region and dietary factor. These findings inform nutritional priorities and clinical and public health planning to improve dietary quality and reduce T2D globally.publishedVersio

Kepler K2 Campaign 9: II. First space-based discovery of an exoplanet using microlensing

We report on the discovery of a bound exoplanetary microlensing event from a blind search of data gathered from Campaign 9 of the Kepler K2 mission (K2C9). K2-2016-BLG-0005Lb is a densely sampled, binary caustic-crossing microlensing event with caustic entry and exit points that are resolved in the K2C9 data, enabling the lens-source relative proper motion to be measured. We have fitted a binary microlens model to the K2 dataset, and to simultaneous observations from the Optical Gravitational Lensing Experiment (OGLE-IV), Canada-France-Hawaii Telescope (CFHT), Microlensing Observations in Astrophysics (MOA-2), the Korean Microlensing Telescope Network (KMTNet), and the United Kingdom InfraRed Telescope (UKIRT). Whilst the ground-based data only sparsely sample the binary caustic, they provide a clear detection of parallax that allows us to break completely the microlensing mass-position-velocity degeneracy and measure the planet's mass directly. We find a host mass of $0.58\pm0.03 ~{\rm M}_\odot$ and a planetary mass of $1.1 \pm 0.1 ~{\rm M_J}$. The system lies at a distance of $5.2 \pm 0.2~$kpc from Earth towards the Galactic bulge. The projected physical separation of the planet from its host is found to be $4.2 \pm 0.3~$au which, for circular orbits, corresponds to $a = 4.4^{+1.9}_{-0.4}~$au and period $P = 13^{+9}_{-2}~$yr, making K2-2016-BLG-0005Lb a close Jupiter analogue. Though previous exoplanet microlensing events have included space-based data, this event is the first bound microlensing exoplanet to be discovered from space-based data. Even through a space telescope not designed for microlensing studies, this result highlights the advantages for exoplanet microlensing discovery that come from continuous, high-cadence temporal sampling that is possible from space. (Abridged).Comment: 17 pages. Submitted to MNRA

<i>Kepler K2</i> Campaign 9: II. First space-based discovery of an exoplanet using microlensing

We present K2-2016-BLG-0005Lb, a densely sampled, planetary binary caustic-crossing microlensing event found from a blind search of data gathered from Campaign 9 of the Kepler K2 mission (K2C9). K2-2016-BLG-0005Lb is the first bound microlensing exoplanet discovered from space-based data. The event has caustic entry and exit points that are resolved in the K2C9 data, enabling the lens–source relative proper motion to be measured. We have fitted a binary microlens model to the Kepler data, and to simultaneous observations from multiple ground-based surveys. Whilst the ground-based data only sparsely sample the binary caustic, they provide a clear detection of parallax that allows us to break completely the microlensing mass–position–velocity degeneracy and measure the planet’s mass directly. We find a host mass of 0.58 ± 0.04M⊙ and a planetary mass of 1.1 ± 0.1MJ. The system lies at a distance of 5.2 ± 0.2 kpc from Earth towards the Galactic bulge, more than twice the distance of the previous most distant planet found by Kepler. The sky-projected separation of the planet from its host is found to be 4.2 ± 0.3 au which, for circular orbits, deprojects to a host separation $a = 4.4^{+1.9}_{-0.4}$ au and orbital period $P = 13^{+9}_{-2}$ yr. This makes K2-2016-BLG-0005MLb a close Jupiter analogue orbiting a low-mass host star. According to current planet formation models, this system is very close to the host mass threshold below which Jupiters are not expected to form. Upcoming space-based exoplanet microlensing surveys by NASA’s Nancy Grace Roman Space Telescope and, possibly, ESA’s Euclid mission, will provide demanding tests of current planet formation models