Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging

Cryo-electron tomography together with averaging of sub-tomograms containing identical particles can reveal the structure of proteins or protein complexes in their native environment. The resolution of this technique is limited by the contrast transfer function (CTF) of the microscope. The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF. Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination. We show that errors in defocus determination are well tolerated when correcting a series of tomograms collected at a range of defocus values. We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction. Using bacteriophage PRDI as a test sample, we demonstrate that this approach gives an improvement in the structure obtained by sub-tomogram averaging from cryo-electron tomograms.

Computational model of membrane fission catalyzed by ESCRT-III

ESCRT-III proteins catalyze membrane fission during multi vesicular body biogenesis, budding of some enveloped viruses and cell division. We suggest and analyze a novel mechanism of membrane fission by the mammalian ESCRT-III subunits CHMP2 and CHMP3. We propose that the CHMP2-CHMP3 complexes self-assemble into hemi-spherical dome-like structures within the necks of the initial membrane buds generated by CHMP4 filaments. The dome formation is accompanied by the membrane attachment to the dome surface, which drives narrowing of the membrane neck and accumulation of the elastic stresses leading, ultimately, to the neck fission. Based on the bending elastic model of lipid bilayers, we determine the degree of the membrane attachment to the dome enabling the neck fission and compute the required values of the protein-membrane binding energy. We estimate the feasible values of this energy and predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission. We support the computational model by electron tomography imaging of CHMP2-CHMP3 assemblies in vitro. We predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission.

Structure and assembly of immature HIV

The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled immature Gag lattice to approximate to 17-angstrom resolution. Gag is arranged in the immature virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein-protein interfaces involved in Gag assembly, and to identify the structural transformations associated with virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped viruses of variable sizes.

Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis

Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release-akin to its role in vesicle formation-and is not restricted to severing the thin membrane tether.

A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990–2010: A systematic analysis for the Global Burden of Disease Study 2010

BACKGROUND Quantification of the disease burden caused by different risks informs prevention by providing an account of health loss different to that provided by a disease-by-disease analysis. No complete revision of global disease burden caused by risk factors has been done since a comparative risk assessment in 2000, and no previous analysis has assessed changes in burden attributable to risk factors over time. METHODS We estimated deaths and disability-adjusted life years (DALYs; sum of years lived with disability [YLD] and years of life lost [YLL]) attributable to the independent effects of 67 risk factors and clusters of risk factors for 21 regions in 1990 and 2010. We estimated exposure distributions for each year, region, sex, and age group, and relative risks per unit of exposure by systematically reviewing and synthesising published and unpublished data. We used these estimates, together with estimates of cause-specific deaths and DALYs from the Global Burden of Disease Study 2010, to calculate the burden attributable to each risk factor exposure compared with the theoretical-minimum-risk exposure. We incorporated uncertainty in disease burden, relative risks, and exposures into our estimates of attributable burden. FINDINGS In 2010, the three leading risk factors for global disease burden were high blood pressure (7·0% [95% uncertainty interval 6·2-7·7] of global DALYs), tobacco smoking including second-hand smoke (6·3% [5·5-7·0]), and alcohol use (5·5% [5·0-5·9]). In 1990, the leading risks were childhood underweight (7·9% [6·8-9·4]), household air pollution from solid fuels (HAP; 7·0% [5·6-8·3]), and tobacco smoking including second-hand smoke (6·1% [5·4-6·8]). Dietary risk factors and physical inactivity collectively accounted for 10·0% (95% UI 9·2-10·8) of global DALYs in 2010, with the most prominent dietary risks being diets low in fruits and those high in sodium. Several risks that primarily affect childhood communicable diseases, including unimproved water and sanitation and childhood micronutrient deficiencies, fell in rank between 1990 and 2010, with unimproved water and sanitation accounting for 0·9% (0·4-1·6) of global DALYs in 2010. However, in most of sub-Saharan Africa childhood underweight, HAP, and non-exclusive and discontinued breastfeeding were the leading risks in 2010, while HAP was the leading risk in south Asia. The leading risk factor in Eastern Europe, most of Latin America, and southern sub-Saharan Africa in 2010 was alcohol use; in most of Asia, North Africa and Middle East, and central Europe it was high blood pressure. Despite declines, tobacco smoking including second-hand smoke remained the leading risk in high-income north America and western Europe. High body-mass index has increased globally and it is the leading risk in Australasia and southern Latin America, and also ranks high in other high-income regions, North Africa and Middle East, and Oceania. INTERPRETATION Worldwide, the contribution of different risk factors to disease burden has changed substantially, with a shift away from risks for communicable diseases in children towards those for non-communicable diseases in adults. These changes are related to the ageing population, decreased mortality among children younger than 5 years, changes in cause-of-death composition, and changes in risk factor exposures. New evidence has led to changes in the magnitude of key risks including unimproved water and sanitation, vitamin A and zinc deficiencies, and ambient particulate matter pollution. The extent to which the epidemiological shift has occurred and what the leading risks currently are varies greatly across regions. In much of sub-Saharan Africa, the leading risks are still those associated with poverty and those that affect children.

The political decisions and policy leading to the Royal Australian Air Force having no fighters or interceptors for the coming war against Japan

The thesis provides an understanding of the ignored need for a modern air defence system for the Australian air force to meet the growing threat from Japan in the 1930s and early 1940s. The quality of advice provided to, and accepted by, Australian politicians was misleading and eliminated the need for fighters and interceptors despite glaring evidence to the contrary. Based on primary source material, including official documents, Allied and Axis pilot memoirs, popular aviation literature and newspaper and magazine articles and interviews, the thesis highlights the inability of Australian politicians to face the reality of the international situation.

Global, regional, and national disability-adjusted life years (DALYs) for 306 diseases and injuries and healthy life expectancy (HALE) for 188 countries, 1990–2013: Quantifying the epidemiological transition

Background The Global Burden of Disease Study 2013 (GBD 2013) aims to bring together all available epidemiological data using a coherent measurement framework, standardised estimation methods, and transparent data sources to enable comparisons of health loss over time and across causes, age–sex groups, and countries. The GBD can be used to generate summary measures such as disability-adjusted life-years (DALYs) and healthy life expectancy (HALE) that make possible comparative assessments of broad epidemiological patterns across countries and time. These summary measures can also be used to quantify the component of variation in epidemiology that is related to sociodemographic development. Methods We used the published GBD 2013 data for age-specific mortality, years of life lost due to premature mortality (YLLs), and years lived with disability (YLDs) to calculate DALYs and HALE for 1990, 1995, 2000, 2005, 2010, and 2013 for 188 countries. We calculated HALE using the Sullivan method; 95% uncertainty intervals (UIs) represent uncertainty in age-specific death rates and YLDs per person for each country, age, sex, and year. We estimated DALYs for 306 causes for each country as the sum of YLLs and YLDs; 95% UIs represent uncertainty in YLL and YLD rates. We quantified patterns of the epidemiological transition with a composite indicator of sociodemographic status, which we constructed from income per person, average years of schooling after age 15 years, and the total fertility rate and mean age of the population. We applied hierarchical regression to DALY rates by cause across countries to decompose variance related to the sociodemographic status variable, country, and time. Findings Worldwide, from 1990 to 2013, life expectancy at birth rose by 6·2 years (95% UI 5·6–6·6), from 65·3 years (65·0–65·6) in 1990 to 71·5 years (71·0–71·9) in 2013, HALE at birth rose by 5·4 years (4·9–5·8), from 56·9 years (54·5–59·1) to 62·3 years (59·7–64·8), total DALYs fell by 3·6% (0·3–7·4), and age-standardised DALY rates per 100 000 people fell by 26·7% (24·6–29·1). For communicable, maternal, neonatal, and nutritional disorders, global DALY numbers, crude rates, and age-standardised rates have all declined between 1990 and 2013, whereas for non–communicable diseases, global DALYs have been increasing, DALY rates have remained nearly constant, and age-standardised DALY rates declined during the same period. From 2005 to 2013, the number of DALYs increased for most specific non-communicable diseases, including cardiovascular diseases and neoplasms, in addition to dengue, food-borne trematodes, and leishmaniasis; DALYs decreased for nearly all other causes. By 2013, the five leading causes of DALYs were ischaemic heart disease, lower respiratory infections, cerebrovascular disease, low back and neck pain, and road injuries. Sociodemographic status explained more than 50% of the variance between countries and over time for diarrhoea, lower respiratory infections, and other common infectious diseases; maternal disorders; neonatal disorders; nutritional deficiencies; other communicable, maternal, neonatal, and nutritional diseases; musculoskeletal disorders; and other non-communicable diseases. However, sociodemographic status explained less than 10% of the variance in DALY rates for cardiovascular diseases; chronic respiratory diseases; cirrhosis; diabetes, urogenital, blood, and endocrine diseases; unintentional injuries; and self-harm and interpersonal violence. Predictably, increased sociodemographic status was associated with a shift in burden from YLLs to YLDs, driven by declines in YLLs and increases in YLDs from musculoskeletal disorders, neurological disorders, and mental and substance use disorders. In most country-specific estimates, the increase in life expectancy was greater than that in HALE. Leading causes of DALYs are highly variable across countries. Interpretation Global health is improving. Population growth and ageing have driven up numbers of DALYs, but crude rates have remained relatively constant, showing that progress in health does not mean fewer demands on health systems. The notion of an epidemiological transition—in which increasing sociodemographic status brings structured change in disease burden—is useful, but there is tremendous variation in burden of disease that is not associated with sociodemographic status. This further underscores the need for country-specific assessments of DALYs and HALE to appropriately inform health policy decisions and attendant actions.

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013

Background The Global Burden of Disease, Injuries, and Risk Factor study 2013 (GBD 2013) is the first of a series of annual updates of the GBD. Risk factor quantification, particularly of modifiable risk factors, can help to identify emerging threats to population health and opportunities for prevention. The GBD 2013 provides a timely opportunity to update the comparative risk assessment with new data for exposure, relative risks, and evidence on the appropriate counterfactual risk distribution. Methods Attributable deaths, years of life lost, years lived with disability, and disability-adjusted life-years (DALYs) have been estimated for 79 risks or clusters of risks using the GBD 2010 methods. Risk–outcome pairs meeting explicit evidence criteria were assessed for 188 countries for the period 1990–2013 by age and sex using three inputs: risk exposure, relative risks, and the theoretical minimum risk exposure level (TMREL). Risks are organised into a hierarchy with blocks of behavioural, environmental and occupational, and metabolic risks at the first level of the hierarchy. The next level in the hierarchy includes nine clusters of related risks and two individual risks, with more detail provided at levels 3 and 4 of the hierarchy. Compared with GBD 2010, six new risk factors have been added: handwashing practices, occupational exposure to trichloroethylene, childhood wasting, childhood stunting, unsafe sex, and low glomerular filtration rate. For most risks, data for exposure were synthesised with a Bayesian meta-regression method, DisMod-MR 2.0, or spatial-temporal Gaussian process regression. Relative risks were based on meta-regressions of published cohort and intervention studies. Attributable burden for clusters of risks and all risks combined took into account evidence on the mediation of some risks such as high body-mass index (BMI) through other risks such as high systolic blood pressure and high cholesterol. Findings All risks combined account for 57·2% (95% uncertainty interval [UI] 55·8–58·5) of deaths and 41·6% (40·1–43·0) of DALYs. Risks quantified account for 87·9% (86·5–89·3) of cardiovascular disease DALYs, ranging to a low of 0% for neonatal disorders and neglected tropical diseases and malaria. In terms of global DALYs in 2013, six risks or clusters of risks each caused more than 5% of DALYs: dietary risks accounting for 11·3 million deaths and 241·4 million DALYs, high systolic blood pressure for 10·4 million deaths and 208·1 million DALYs, child and maternal malnutrition for 1·7 million deaths and 176·9 million DALYs, tobacco smoke for 6·1 million deaths and 143·5 million DALYs, air pollution for 5·5 million deaths and 141·5 million DALYs, and high BMI for 4·4 million deaths and 134·0 million DALYs. Risk factor patterns vary across regions and countries and with time. In sub-Saharan Africa, the leading risk factors are child and maternal malnutrition, unsafe sex, and unsafe water, sanitation, and handwashing. In women, in nearly all countries in the Americas, north Africa, and the Middle East, and in many other high-income countries, high BMI is the leading risk factor, with high systolic blood pressure as the leading risk in most of Central and Eastern Europe and south and east Asia. For men, high systolic blood pressure or tobacco use are the leading risks in nearly all high-income countries, in north Africa and the Middle East, Europe, and Asia. For men and women, unsafe sex is the leading risk in a corridor from Kenya to South Africa. Interpretation Behavioural, environmental and occupational, and metabolic risks can explain half of global mortality and more than one-third of global DALYs providing many opportunities for prevention. Of the larger risks, the attributable burden of high BMI has increased in the past 23 years. In view of the prominence of behavioural risk factors, behavioural and social science research on interventions for these risks should be strengthened. Many prevention and primary care policy options are available now to act on key risks.

Crystal engineering in two dimensions: An approach to molecular nanopatterning

We describe a surprising cooperative adsorption process observed by scanning tunneling microscopy (STM) at the liquid���solid interface. The process involves the association of a threefold hydrogen-bonding unit, trimesic acid (TMA), with straight-chain aliphatic alcohols of varying length (from C7 to C30), which coadsorb on highly oriented pyrolytic graphite (HOPG) to form linear patterns. In certain cases, the known TMA “flower pattern” can coexist temporarily with the linear TMA−alcohol patterns, but it eventually disappears. Time-lapsed STM imaging shows that the evolution of the flower pattern is a classical ripening phenomenon. The periodicity of the linear TMA−alcohol patterns can be modulated by choosing alcohols with appropriate chain lengths, and the precise structure of the patterns depends on the parity of the carbon count in the alkyl chain. Interactions that lead to this odd−even effect are analyzed in detail. The molecular components of the patterns are achiral, yet their association by hydrogen bonding leads to the formation of enantiomeric domains on the surface. The interrelation of these domains and the observation of superperiodic structures (moiré patterns) are rationalized by considering interactions with the underlying graphite surface and within the two-dimensional crystal of the adsorbed molecules. Comparison of the observed two-dimensional structures with the three-dimensional crystal structures of TMA−alcohol complexes determined by X-ray crystallography helps reveal the mechanism of molecular association in these two-component systems.

Catalytic transformation of aliphatic alcohols to corresponding esters in O2 under neutral conditions using visible-light irradiation

Selective oxidation of aliphatic alcohols under mild and base-free conditions is a challenging process for organic synthesis. Herein, we report a one-pot process for the direct oxidative esterification of aliphatic alcohols that is significantly enhanced by visible-light irradiation at ambient temperatures. The new methodology uses heterogenerous photocatalysts of gold���palladium alloy nanoparticles on a phosphate-modified hydrotalcite support and molecular oxygen as a benign oxidant. The alloy photocatalysts can absorb incident light, and the light-excited metal electrons on the surface of metal nanoparticles can activate the adsorbed reactant molecules. Tuning the light intensity and wavelength of the irradiation can remarkably change the reaction activity. Shorter wavelength light (<550 nm) drives the reaction more efficiently than light of longer wavelength (e.g., 620 nm), especially at low temperatures. The phosphate-exchanged hydrotalcite support provides sufficient basicity (and buffer) for the catalytic reactions; thus, the addition of base is not required. The photocatalysts are efficient and readily recyclable. The findings reveal the first example of using “green” oxidants and light energy to drive direct oxidative esterification of aliphatic alcohols under base-free, mild conditions.

Pheromone-mediated mass trapping and population diversion as strategies for suppressing Carpophilus spp. (Coleoptera: Nitidulidae) in Australian stone fruit orchards

1 Five experiments were conducted during 1995-99 in stone fruit orchards on the Central Coast and in inland New South Wales, Australia, on the use of synthetic aggregation pheromones and a coattractant to suppress populations of the ripening fruit pests Carpophilus spp. (Coleoptera: Nitidulidae). 2 Perimeter-based suppression traps baited with pheromone and coattractant placed at 3m intervals around small fruit blocks, caught large numbers of Carpophilus spp. Very small populations of Carpophilus spp. occurred within blocks, and fruit damage was minimal. 3 Carpophilus spp. populations in stone fruit blocks 15-370m from suppression traps were also small and non-damaging, indicating a large zone of pheromone attractivity. 4 Pheromone/coattractant-baited suppression traps appeared to divert Carpophilus spp. from nearby (130 m) ripening stone fruit. Ten metal drums containing decomposing fruit, baited with pheromone and treated with insecticide, attracted Carpophilus spp. and appeared to reduce populations and damage to ripening fruit at distances of 200-500 m. Populations and damage were significantly greater within 200m of the drums and may have been caused by ineffective poisoning or poor quality/overcrowding of fruit resources in the drums. 5 Suppression of Carpophilus spp. populations using synthetic aggregation pheromones and a coattractant appears to be a realistic management option in stone fruit orchards. Pheromone-mediated diversion of beetle populations from ripening fruit may be more practical than perimeter trapping, but more research is needed on the effective range of Carpophilus pheromones and the relative merits of trapping compared to attraction to insecticide-treated areas.

Efficacy of multispecies pheromone lures for Carpophilus davidsoni Dobson and Carpophilus mutilatus Erichson (Coleoptera: Nitidulidae)

Orchard experiments were conducted in southern New South Wales to evaluate the efficacy of multispecies pheromone lures in trapping two economically important species of Carpophilus. Captures of Carpophilus davidsoni Dobson and Carpophilus mutilatus Erichson in traps baited with aggregation pheromones of both species or a three-way lure that also included the pheromone of Carpophilus hemipterus (L.) were not significantly different from captures in traps baited with conspecific pheromones. Carpophilus davidsoni and C. mutilatus were cross-attracted to traps baited with the pheromone of the other species, but numbers were significantly reduced compared with traps baited with conspecific or heterospecific pheromones. Multispecies lures will improve prospects for the commercial use of synthetic aggregation pheromones in Carpophilus beetle management in stone-fruit orchards.

Pheromone-trapping of Carpophilus spp. (Coleoptera: Nitidulidae) in stone fruit orchards near Gosford, New South Wales: Fauna, seasonality and effect of insecticides

Traps baited with synthetic aggregation pheromones of Carpophilus hemipterus (L.), Carpophilus mutilatus Erichson and Carpophilus davidsoni Dobson and fermenting bread dough were used to identify the fauna and monitor the seasonal abundance of Carpophilus spp. in insecticide treated peach and nectarine orchards in the Gosford area of coastal New South Wales. In four orchards 67 178 beetles were trapped during 1994–1995, with C. davidsoni (82%) and Carpophilus gaveni (Dobson) (12.2%) dominating catches. Five species (C. hemipterus, C. mutilatus, Carpophilus marginellus Motschulsky, Carpophilus humeralis (F.) and an unidentified species) each accounted for 0.2–3.2% of trapped beetles. Carpophilus davidsoni was most abundant during late September–early October but numbers declined rapidly during October, usually before insecticides were applied. Spring populations of Carpophilus spp. were very large in 1994–1995 (1843–2588 per trap per week). However, despite a preharvest population decline of approximately 95% and 2–11 applications of insecticide, 14–545 beetles per trap per week (above the arbitrary fruit damage threshold of 10 beetles per trap per week) were recorded during the harvest period and fruit damage occurred at three of the four orchards. Lower preharvest populations in 1995–1996 (< 600 per trap per week) and up to six applications of insecticide resulted in < 10 beetles per trap per week during most of the harvest period and minimal or no fruit damage. The implications of these results for the integrated management of Carpophilus spp. in coastal and inland areas of southeastern Australia are discussed.

An improved coattractant for pheromone trapping of Carpophilus spp. (Coleoptera: Nitidulidae)

Fermenting apple juice (FAJ) contained within polyacrylamide granules was an effective pheromone coattractant for Carpophilus davidsoni in trapping experiments conducted in stone fruit orchards in southern New South Wales. Fermenting apple juice-baited traps captured as many beetles as traps baited with the 'standard' coattractant fermenting bread dough (FBD), either alone or in combination with aggregation pheromone. Increasing the interval of FAJ replacement to 2 weeks instead of 1 week, as is necessary for FBD, did not reduce trapping efficiency. Replacement of FAJ every three weeks did not affect captures of C. davidsoni in one experiment but did reduce captures of Carpophilus mutilatus. In a second experiment, captures of C. davidsoni were also reduced. Fermenting apple juice contained within polyacrylamide granules replaced at fortnightly intervals is an effective, convenient and practical pheromone coattractant for Carpophilus spp.

Phenology of Carpophilus spp. (Coleoptera: Nitidulidae)in stone fruit orchards as determined by pheromone trapping: Implications for prediction of crop damage

Traps baited with synthetic aggregation pheromone and fermenting bread dough were used to monitor seasonal incidence and abundance of the ripening fruit pests, Carpophilus hemipterus (L.), C. mutilatus Erichson and C. davidsoni Dobson in stone fruit orchards in the Leeton district of southern New South Wales during five seasons (1991-96). Adult beetles were trapped from September-May, but abundance varied considerably between years with the amount of rainfall in December-January having a major influence on population size and damage potential during the canning peach harvest (late February-March). Below average rainfall in December-January was associated with mean trap catches of < 10 beetles/trap/week in low dose pheromone traps during the harvest period in 1991/92 and 1993/94 and no reported damage to ripening fruit. Rainfall in December-January 1992/93 was more than double the average and mean trap catches ranged from 8-27 beetles/week during the harvest period with substantial damage to the peach crop. December-January rainfall was also above average in 1994/95 and 1995/96 and means of 50-300 beetles/trap/week were recorded in high dose pheromone traps during harvest periods. Carpophilus spp. caused economic damage to peach crops in both seasons. These data indicate that it may be possible to predict the likelihood of Carpophilus beetle damage to ripening stone fruit in inland areas of southern Australia, by routine pheromone-based monitoring of beetle populations and summer temperatures and rainfall.