Comprehensive framework for human health risk assessment of nanopesticides

Nanopesticides are not only in an advanced state of research and development but have started to appear on the market. Industry and regulatory agencies need a consolidated and comprehensive framework and guidance for human health risk assessments. In this perspective we develop such a comprehensive framework by exploring two case studies from relevant product types: an active ingredient delivered with a nanocarrier system, and a nanoparticle as an active ingredient. For a nanocarrier system, three entities are tracked during the assessment: the nanocarrier–active ingredient complex, the empty nanocarrier remaining after the complete release of the active ingredient, and the released active ingredient. For the nanoparticle of pure active ingredient, only two entities are relevant: the nanoparticle and the released ions. We suggest important adaptations of the existing pesticide framework to determine the relevant nanopesticide entities and their concentrations for toxicity testing. Depending on the nature of the nanopesticides, additional data requirements, such as those pertaining to durability in biological media and potential for crossing biological barriers, have also been identified. Overall, our framework suggests a tiered approach for human health risk assessment, which is applicable for a range of nanopesticide products to support regulators and industry in making informed decisions on nanopesticide submissions. Brief summaries of suitable methods including references to existing standards (if available) have been included together with an analysis of current knowledge gaps. Our study is an important step towards a harmonized approach accepted by regulatory agencies for assessing nanopesticides

Viscous hydrophilic injection matrices for serial crystallography

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates – gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used for in meso membrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydro­gels as viscous injection matrices is described, namely sodium carb­oxy­methyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements

A cross-curricular physical activity intervention to combat cardiovascular disease risk factors in 11-14 year olds: 'Activity Knowledge Circuit'

Background: Cardiovascular disease is the leading cause of mortality worldwide. Risk factors associated with cardiovascular disease have been shown to track from childhood through to adulthood. Previous school-based physical activity interventions have demonstrated modest improvements to cardiovascular disease risk factors by implementing extra-curricular activities or improving current physical education curriculum. Few have attempted to increase physical activity in class-room taught curriculum subjects. This study will outline a school-based cross-curricular physical activity intervention to combat cardiovascular disease risk factors in 11-14 year old children. Method/Design: A South Wales Valley school of low socio-economic status has been selected to take part. Participants from year eight (12-13 years) are to be assigned to an intervention group, with maturation-matched participants from years seven (11-12 years) and nine (13-14 years) assigned to a control group. A cross-curricular physical activity intervention will be implemented to increase activity by two hours a week for 18 weeks. Participants will briskly walk 3200 m twice weekly during curriculum lessons (60 minutes duration). With the exception of physical education, all curriculum subjects will participate, with each subject delivering four intervention lessons. The intervention will be performed outdoors and on school premises. An indoor course of equal distance will be used during adverse weather conditions. Cardiovascular disease risk factors will be measured pre- and post-intervention for intervention and control groups. These will take place during physical education lessons and will include measures of stature, mass, waist, hip, and neck circumferences, together with skinfold measure's taken at four sites. Blood pressure will be measured, and fitness status assessed via the 20 m multi-stage fitness test. Questionnaires will be used to determine activity behaviour (physical activity questionnaire for adolescence), diet (seven day food diary) and maturation status. Fasting blood variables will include total cholesterol, lowdensity lipoprotein cholesterol, high density lipoprotein cholesterol, triglycerides, insulin, glucose, high-sensitivity C-reactive protein, interleukin-6, adiponectin, and fibrinogen. Motivational variables and psychological well-being will be assessed by questionnaire. Discussion: Our study may prove to be a cost effective strategy to increase school time physical activity to combat cardiovascular disease risk factors in children.</p

Dynamic catcher for stabilization of high-viscosity extrusion jets

A `catcher' based on a revolving cylindrical collector is described. The simple and inexpensive device reduces free-jet instabilities inherent to high-viscosity extrusion injection, facilitating delivery of microcrystals for serial diffraction X-ray crystallography

Influence of pump laser fluence on ultrafast myoglobin structural dynamics

International audienceHigh-intensity femtosecond pulses from an X-ray free-electron laser enable pump–probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1,2 . However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore 3–17 . As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern 18,19 whether this experimental approach 20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions 18,19 . Here we describe ultrafast pump–probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe–CO bond distance (predicted by recent quantum wavepacket dynamics 21 ) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump–probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump–probe experiments 20 such that mechanistically relevant insight emerges

Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL data de novo is complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms

Towards phasing using high X-ray intensity

X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential ‘bleaching’ of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed

Towards phasing using high X-ray intensity

X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential ‘bleaching’ of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed

Effects of self-seeding and crystal post-selection on the quality of Monte Carlo-integrated SFX data

Serial femtosecond crystallography (SFX) is an emerging method for data collection at free-electron lasers (FELs) in which single diffraction snapshots are taken from a large number of crystals. The partial intensities collected in this way are then combined in a scheme called Monte Carlo integration, which provides the full diffraction intensities. However, apart from having to perform this merging, the Monte Carlo integration must also average out all variations in crystal quality, crystal size, X-ray beam properties and other factors, necessitating data collection from thousands of crystals. Because the pulses provided by FELs running in the typical self-amplified spontaneous emission (SASE) mode of operation have very irregular, spiky spectra that vary strongly from pulse to pulse, it has been suggested that this is an important source of variation contributing to inaccuracies in the intensities, and that, by using monochromatic pulses produced through a process called self-seeding, fewer images might be needed for Monte Carlo integration to converge, resulting in more accurate data. This paper reports the results of two experiments performed at the Linac Coherent Light Source in which data collected in both SASE and self-seeded mode were compared. Importantly, no improvement attributable to the use of self-seeding was detected. In addition, other possible sources of variation that affect SFX data quality were investigated, such as crystal-to-crystal variations reflected in the unit-cell parameters; however, these factors were found to have no influence on data quality either. Possibly, there is another source of variation as yet undetected that affects SFX data quality much more than any of the factors investigated here