A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations.

Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.

Closing the gap to effective gene drive in Aedes aegypti by exploiting germline regulatory elements.

CRISPR/Cas9-based homing gene drives have emerged as a potential new approach to mosquito control. While attempts have been made to develop such systems in Aedes aegypti, none have been able to match the high drive efficiency observed in Anopheles species. Here we generate Ae. aegypti transgenic lines expressing Cas9 using germline-specific regulatory elements and assess their ability to bias inheritance of an sgRNA-expressing element (kmosgRNAs). Four shu-Cas9 and one sds3-Cas9 isolines can significantly bias the inheritance of kmosgRNAs, with sds3G1-Cas9 causing the highest average inheritance of ~86% and ~94% from males and females carrying both elements outcrossed to wild-type, respectively. Our mathematical model demonstrates that sds3G1-Cas9 could enable the spread of the kmosgRNAs element to either reach a higher (by ~15 percentage point) maximum carrier frequency or to achieve similar maximum carrier frequency faster (by 12 generations) when compared to two other established split drive systems.

A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias.

CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (wGDe). Here, through an analysis using this linkage we show that in males inheritance bias of wGDe did not occur by homing, rather through increased propagation of the donor drive element. We test the same wGDe drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.

Daisy-chain gene drives: The role of low cut-rate, resistance mutations, and maternal deposition.

The introgression of genetic traits through gene drive may serve as a powerful and widely applicable method of biological control. However, for many applications, a self-perpetuating gene drive that can spread beyond the specific target population may be undesirable and preclude use. Daisy-chain gene drives have been proposed as a means of tuning the invasiveness of a gene drive, allowing it to spread efficiently into the target population, but be self-limiting beyond that. Daisy-chain gene drives are made up of multiple independent drive elements, where each element, except one, biases the inheritance of another, forming a chain. Under ideal inheritance biasing conditions, the released drive elements remain linked in the same configuration, generating copies of most of their elements except for the last remaining link in the chain. Through mathematical modelling of populations connected by migration, we have evaluated the effect of resistance alleles, different fitness costs, reduction in the cut-rate, and maternal deposition on two alternative daisy-chain gene drive designs. We find that the self-limiting nature of daisy-chain gene drives makes their spread highly dependent on the efficiency and fidelity of the inheritance biasing mechanism. In particular, reductions in the cut-rate and the formation of non-lethal resistance alleles can cause drive elements to lose their linked configuration. This severely reduces the invasiveness of the drives and allows for phantom cutting, where an upstream drive element cuts a downstream target locus despite the corresponding drive element being absent, creating and biasing the inheritance of additional resistance alleles. This phantom cutting can be mitigated by an alternative indirect daisy-chain design. We further find that while dominant fitness costs and maternal deposition reduce daisy-chain invasiveness, if overcome with an increased release frequency, they can reduce the spread of the drive into a neighbouring population.

The Challenges in Developing Efficient and Robust Synthetic Homing Endonuclease Gene Drives.

Making discrete and precise genetic changes to wild populations has been proposed as a means of addressing some of the world's most pressing ecological and public health challenges caused by insect pests. Technologies that would allow this, such as synthetic gene drives, have been under development for many decades. Recently, a new generation of programmable nucleases has dramatically accelerated technological development. CRISPR-Cas9 has improved the efficiency of genetic engineering and has been used as the principal effector nuclease in different gene drive inheritance biasing mechanisms. Of these nuclease-based gene drives, homing endonuclease gene drives have been the subject of the bulk of research efforts (particularly in insects), with many different iterations having been developed upon similar core designs. We chart the history of homing gene drive development, highlighting the emergence of challenges such as unintended repair outcomes, "leaky" expression, and parental deposition. We conclude by discussing the progress made in developing strategies to increase the efficiency of homing endonuclease gene drives and mitigate or prevent unintended outcomes.

Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source.

The increasing prevalence of insecticide resistance and the ongoing global burden of vector-borne diseases have encouraged new efforts in mosquito control. For Aedes aegypti, the most important arboviral vector, integration rates achieved in Cas9-based knock-ins so far have been rather low, highlighting the need to understand gene conversion patterns and other factors that influence homology-directed repair (HDR) events in this species. In this study, we report the effects of sequence mismatches or donor template forms on integration rates. We found that modest sequence differences between construct homology arms [DNA sequence in the donor template which resembles the region flanking the target cut] and genomic target comprising 1.2% nucleotide dissimilarity (heterology) significantly reduced integration rates. While most integrations (59-88%) from plasmid templates were the result of canonical [on target, perfect repair] HDR events, no canonical events were identified from other donor types (i.e. ssDNA, biotinylated ds/ssDNA). Sequencing of the transgene flanking region in 69 individuals with canonical integrations revealed 60% of conversion tracts to be unidirectional and extend up to 220 bp proximal to the break, though in three individuals bidirectional conversion of up to 725 bp was observed.

Expanding the CRISPR Toolbox in Culicine Mosquitoes: In Vitro Validation of Pol III Promoters.

CRISPR-Cas9-based "gene drive" technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date-and an expanded molecular toolbox with which to build them-will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives.

Complex systems, part I: why 42 is rarely, if ever, the ultimate answer.

We describe the different categories of systems and systems thinking, and illustrate why almost all clinical interactions constitute simultaneously complex and complicated systems, so-called "wicked systems". We also discuss why they are not amenable to quantitative analysis. With the use of comparisons and illustrations we show some of the problems that reductionist metrics create, and support concerns regarding quantitative fallacy. The systematic correlation of data in medicine was one of the earliest achievements of the discipline. Recently, however, the overwhelming bias towards an evidence base, which relies almost entirely on randomised controlled trials, has created a reductionist view that often excludes important aspects of medicine and, in particular, surgery. We must now move away from thinking that is controlled by the "tyranny of metrics" to embrace complex-systems thinking, and work across disciplines. We outline the arguments for this and give clinical examples from oral and maxillofacial surgery.

Making and working of a new electronic resource for patients, carers and professionals: maxfacts.uk.

Many maxillofacial patients have serious short, medium, or long-term problems, as well as having to make informed decisions about often life-changing interventions. Validated comprehensive information, at the right time and the right level for a diverse group of users (patients, carers, and professionals), is vital if patients are to make a serious contribution to their treatment. We describe the development of an online resource for this purpose. Maxfacts.uk aims to cover every aspect of oral and maxillofacial surgery and care, from neck dissection and ballistic wounds to physiotherapy and texture-modified foods. The principles of design, and the multilayered structure, interface, and functions of such a multiuser website are outlined, including accessibility and engagement. The maxfacts model and design could easily be adapted for other users with similar, complex, long-term needs.

Ternary graph as a questionnaire: a new approach to assessment of quality of life?

This study was prompted by awareness of the importance of research into quality of life (QoL) for patients with diseases of the head and neck, the important part questionnaires currently play in this field, and awareness of the "questionnaire fatigue" experienced by many patients. Our multidisciplinary research group raised coincidental awareness of the widespread use of ternary graphs in the sciences, social sciences, and humanities as a graphical tool for quantitative, semiquantitative, or purely graphical characteristics of ternary mixtures. We explored how the basic properties of ternary graphs could be translated into an interactive electronic tool as an alternative to conventional questionnaires. We have described how this was done, and offered open access to an interactive ternary-graph based (self) assessment tool, specifically designed for the needs of patients with conditions of the head and neck. Finally, have we made open-source code available for those who may wish to adapt or develop the tool for further applications.

Stability over 36 months of a new liquid 10% polyclonal immunoglobulin product (IgPro10, Privigen) stabilized with L-proline.

BACKGROUND AND OBJECTIVES: IgPro10 (Privigen) is a new liquid intravenous immunoglobulin (IVIG) product that is formulated with 250 mM L-proline at pH 4.8. A 3-year study was performed to assess its stability. MATERIALS AND METHODS: Physicochemical, biological and immunological parameters of Privigen were assessed during storage under controlled conditions over up to 36 months at 25 degrees C. RESULTS: Privigen was shown to be stable after storage for 3 years at room temperature (25 degrees C). Of all parameters tested, only a few showed changes during storage. The appearance of the solution complied with the specifications given by the European Pharmacopeia over the full study period, with a single exception. The IgG fraction in Privigen displayed high purity (> or = 98%), which did not change during storage over 36 months. No relevant amounts of aggregated IgG molecules were formed in Privigen samples and the ratio between monomers and dimers shifted slightly towards the equilibrium over time. Testing of reference antibody contents and the Fc function demonstrated that the biological activity and effector functions of Privigen were preserved over the full study period of 36 months. CONCLUSION: Thirty-six months room temperature stability of Privigen was achieved at pH 4.8 with an innovative formulation containing the physiological stabilizer L-proline.

Estimation of carbon-carbon bond lengths and medium-range internuclear distances by solid-state nuclear magnetic resonance.

We describe magic-angle-spinning NMR methods for the accurate determination of internuclear dipole-dipole couplings between homonuclear spins-(1/2) in the solid state. The new sequences use symmetry principles to treat the effect of magic-angle sample-rotation and resonant radio frequency fields. The pulse-sequence symmetries generate selection rules which reduce the interference of undesirable interactions and improve the robustness of the pulse sequences with respect to chemical shift anisotropies. We show that the pulse sequences may be used to estimate distances between 13C spins in organic solids, including bond lengths in systems with large chemical shift anisotropies, such as conjugated systems. For bond-length measurements, the precision of the method is +/-2 pm with a systematic overestimate of the internuclear distance by 3 +/- 1 pm. The method is expected to be a useful tool for investigating structural changes in macromolecules.

Double-quantum-filtered rotational-resonance MAS NMR in the presence of large chemical shielding anisotropies.

Double-quantum filtration under rotational resonance MAS NMR conditions where the chemical shielding anisotropies involved exceed the differences in isotropic chemical shielding is considered by means of numerical simulations and (13)C MAS NMR experiments. The responses of two different pulse sequences, suitable for double-quantum filtration specifically under rotational resonance conditions, to large chemical shielding anisotropies are compared. In the presence of large chemical shielding anisotropies a very recently introduced pulse sequence (T. Karlsson, M. Edén, H. Luthman, and M. H. Levitt, J. Magn. Reson. 145, 95-107, 2000) suffers losses in double-quantum-filtration efficiencies. The double-quantum-filtration efficiency of another pulse sequence (N. C. Nielsen, F. Creuzet, R. G. Griffin, and M. H. Levitt, J. Chem. Phys. 96, 5668-5677, 1992) is less afflicted by the presence of large chemical shielding anisotropies. Both sequences deliver double-quantum-filtered lineshapes that sensitively reflect chemical shielding tensor orientations. It is further shown that double-quantum-filtered rotational-resonance lineshapes of spin systems composed of more than two spins offer a suitable experimental approach for determining chemical shielding tensor orientations for cases where conventional rotational-resonance experiments are not applicable due to the presence of additional background resonances.

Irrelevant response effects improve serial learning in serial reaction time tasks.

Tones were introduced into a serial reaction time (SRT) task to serve as redundant response effects. Experiment 1 showed that the tones improved serial learning with a 10-element stimulus sequence, but only if the tone effects were mapped onto the responses contingently. Experiment 2 demonstrated that switching to noncontingent response-effect mapping increased SRT only when participants had previously adapted to contingent response-effect mapping. In Experiment 3, the beneficial influence of contingent tone effects on serial learning occurred only when there was sufficient time between the response effects and the next imperative stimuli. The results are discussed in terms of the ideomotor principle. It is claimed that an internal representation of the to-be-produced tone effects develops and gains control over the execution of the response sequence.

Double-quantum filtration under rotational-resonance conditions: numerical simulations and experimental results

The dependence of the performance of a recently introduced pulse sequence to achieve double-quantum excitation under the n = 1 rotational-resonance condition (T. Karlsson, M. Eden, H. Luthman, and M. H. Levitt, 2000, J. Magn. Reson. 145, 95-107) on different spin-system properties is investigated by means of numerical simulations and (13)C MAS NMR experiments. For spin systems where chemical shielding anisotropies amount to only an insignificant fraction of the isotropic chemical shielding difference, high efficiencies are found for large and small dipolar coupling interactions. In the presence of significant chemical shielding anisotropies the overall efficiencies decrease and become strongly dependent on the duration of the excitation period. It is demonstrated that those spin-system parameters which are sensitively encoded in the lineshapes of a conventional n = 1 rotational-resonance spectrum are similarly sensitively encoded in the corresponding rotational-resonance double-quantum-filtered lineshapes and may be quantitatively recovered by iterative lineshape-fitting approaches. In certain favorable circumstances, the in-built selectivity of the rotational-resonance double-quantum-filtration approach permits successful application of the experiment on spin systems with more than two spins. Copyright 2000 Academic Press.

Magnitudes and orientations of interaction tensors determined from rotational resonance MAS NMR lineshapes of a four-(13)C-spin system

Possibilities and limitations of iterative lineshape fitting approaches for the complete determination of magnitudes and orientations of NMR interaction tensors in a four-(13)C-spin system from MAS NMR experiments are investigated. The availability of fast and numerically accurate computational methods is an important prerequisite. The model compound chosen for this investigation is the monoammonium salt of maleic acid. Various selectively and fully (13)C-labeled versions of this compound permit a stepwise reduction of the number of unknown parameters, necessary to fully describe the four-(13)C-spin system in the uniformly (13)C-labeled maleate moiety. This stepwise procedure allows one to monitor reliability and accuracy of multiparameter fits of the four-(13)C-spin system itself, as well as to characterize limitations and requirements for such fitting procedures. Satisfactory (1)H-decoupling performance is an essential experimental requirement; TPPM decoupling yields n = 1, 2 rotational resonance (13)C MAS NMR lineshapes suitable for analysis by iterative lineshape fitting methods. It is demonstrated that assumptions about "typical" chemical shielding tensor orientations, even if not deviating much from the real orientations, lead to severe errors in internuclear distance determinations. Copyright 1999 Academic Press.

Dynamic hydrogen disorder in yttrium-dihydride phases as seen by variable-temperature 89Y CP MAS NMR.

Variable-temperature solid-state MAS NMR studies on some yttrium-dihydride phases YH2+x are reported and yield evidence that 89Y CP MAS NMR techniques are an experimentally feasible route to investigate order-disorder phenomena in such metal-hydride phases.

Order-disorder phenomena determined by high-resolution powder diffraction: the structures of tetrakis(trimethylsilyl)methane C

The compounds tetrakis(trimethylsilyl)methane C[Si(CH(3))(3)](4) (TC) and tetrakis(trimethylsilyl)silane Si[Si(CH(3))(3)](4) (TSi) have crystal structures with the molecules in a cubic closed-packed (c.c.p.) stacking. At room temperature both structures have space group Fm{\bar 3}m (Z = 4) with a = 13.5218 (1) Å, V = 2472.3 (1) Å(3) for TSi, and a = 12.8902 (2) Å, V = 2141.8 (1) Å(3) for TC. X-ray scattering data can be described by a molecule with approximately sixfold orientational disorder, ruling out a structure with free rotating molecules. Upon cooling, TSi exhibits a first-order phase transition at T(c) = 225 K, as is characterized by a jump of the lattice parameter of Deltaa = 0.182 Å and by an exothermal maximum in differential scanning calorimetry (DSC) with DeltaH = 11.7 kJ mol(-1) and DeltaS = 50.0 J mol(-1) K(-1). The structure of the low-temperature phase is refined against X-ray powder data measured at 200 K. It has space group P2(1)3 (Z = 4), a = 13.17158 (6) Å and V = 2285.15 (2) Å(3). The molecules are found to be ordered as a result of steric interactions between neighboring molecules, as is shown by analyzing distances between atoms and by calculations of the lattice energy in dependence on the orientations of the molecules. TC has a phase transition at T(c1) = 268 K, with Deltaa(1) = 0.065 Å, DeltaH(1) = 3.63 kJ mol(-1) and DeltaS(1) = 13.0 J mol(-1) K(-1). A second first-order phase transition occurs at T(c2) = 225 K, characterized by Deltaa(2) = 0.073 Å, DeltaH(2) = 6.9 kJ mol(-1) and DeltaS(2) = 30.0 J mol(-1) K(-1). The phase transition at higher temperature has not been reported previously. New NMR experiments show a small anomaly in the temperature dependence of the peak positions in NMR to occur at T(c2). Rietveld refinements were performed for the low-temperature phase measured at T = 150 K [space group P2(1)3, lattice parameter a = 12.609 (3) Å], and for the intermediate phase measured at T = 260 K [space group Pa{\bar 3}, lattice parameter a = 12.7876 (1) Å]. The low-temperature phase of TC is formed isostructural to the low-temperature phase of TSi. In the intermediate phase the molecules exhibit a twofold orientational disorder.

Iterative lineshape fitting of MAS NMR spectra: a tool to investigate homonuclear J coupling in isolated spin pairs.

Possibilities and limitations of iterative lineshape fitting procedures of MAS NMR spectra of isolated homonuclear spin pairs, aiming at determination of magnitudes and orientations of the various interaction tensors, are explored. Requirements regarding experimental MAS NMR spectra as well as simulation and fitting procedures are discussed. Our examples chosen are the isolated 31P spin pairs in solid Na4P2O7. 10H2O, (1), and Cd(NO3)2. 2PPh3, (2). In both cases the two 31P chemical shielding tensors in the molecular unit are related by C2 symmetry, and determination of the orientations of these two tensors in the molecular frame is possible. In addition, aspects of homonuclear J coupling will be addressed. For 1, both magnitude and sign of 2Jiso(31P, 31P) (Jiso = -19.5 +/- 2.5 Hz) are obtained; for 2, (Jiso = +139 +/- 3 Hz) anisotropy of J with an orientation of the J-coupling tensor collinear, or nearly collinear, with the dipolar coupling tensor can be excluded, while absence or presence of anisotropy of J with any other relative orientation of the J-coupling tensor cannot be determined.

29Si CP/MAS NMR of phosphorus-bearing organosilicon compounds.

While liquid-state 29Si NMR of phosphorus-bearing organosilicon compounds with more than one phosphorus per molecule can take advantage of the presence of J-coupling nJ(31P29Si) for purposes of structural assignment from J-coupling patterns, conventional 29Si CP/MAS spectra of such molecular solids do not reveal structural details in a straightforward manner. For such compounds it is necessary to obtain 29Si CP/MAS spectra under conditions of simultaneous 1H- and 31P-high power decoupling in order to derive reliable 29Si chemical shift information. 29Si CP/MAS NMR spectra, obtained with and without 31P high power decoupling during the acquisition time, of several organosilicon compounds containing SixPy (x = 1-10, y = 1-10) moieties are reported.