Unambiguous Assignment Research Articles

Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

Facilitated structure verification of the biopharmaceutical peptide exenatide by 2D heteronuclear NMR maps

Exenatide is a peptide based anti-diabetic prescription medication. Until now, the literature has lacked a comprehensive atom-specific molecular characterization for this complex large peptide by NMR spectroscopy that can be effortlessly and rapidly utilized for biopharmaceutical structural veracity. Peptide structure verification by NMR is challenging and cumbersome when reliant on traditional proton-based methodology (through-bond and through-space proton connectivity) alone due to increasing complexity, low signal dispersion, and overlap. These challenges are overcome by using 2D heteronuclear (1H-13C and 1H-15N) maps that not only allow unambiguous signal assignment, but also condense the structural verification information within simplified peptide amide and carbon fingerprint maps. Here we report such simplified amide and carbon fingerprint maps for exenatide; made possible by the first ever comprehensive heteronuclear (1H,13C, and 15N) atom specific assignment of exenatide. These heteronuclear assignments were obtained without any isotopic enrichments i.e. at natural abundance, and hence are easily deployable as routine procedures. Furthermore, we compare the 2D heteronuclear maps of exenatide to a chemically identical peptide differing only in the isomerism of the Cα position of the first amino acid, [dHis1]-exenatide, to demonstrate the uniqueness of these maps. We show that despite deliberate changes in pH, temperature, and concentrations, the differences between the amide maps of exenatide and [dHis1]-exenatide are retained. The work presented here not only provides a facilitated structure verification of exenatide but also a framework for heteronuclear NMR data acquisition and signal assignment of large peptides, at natural abundance, in creating their respective unique 2D fingerprint maps.

Magnetoentropic mapping and computational modeling of cycloids and skyrmions in the lacunar spinels GaV4S8 and GaV4Se8

We report the feasibility of using magnetoentropic mapping for the rapid identification of magnetic cycloid and skyrmion phases in uniaxial systems, based on the GaV4S8 and GaV4Se8 model skyrmion hosts with easy-axis and easy-plane anisotropies respectively. We show that these measurements can be interpreted with the help of a simple numerical model for the spin Hamiltonian to yield unambiguous assignments for both single phase regions and phase boundaries. In the two lacunar spinel chemistries, we obtain excellent agreement between the measured magnetoentropic features and a minimal spin Hamiltonian built on Heisenberg exchange, single-ion anisotropy, and anisotropic Dzyaloshinskii-Moriya interactions. In particular, we identify characteristic high-entropy behavior in the cycloid phase that serves as a precursor to the formation of skyrmions at elevated temperatures and is a readily-measurable signature of this phase transition. Our results demonstrate that rapid magnetoentropic mapping guided by numerical modeling is an effective means of understanding the complex magnetic phase diagrams innate to skyrmion hosts. One notable exception is the observation of an anomalous, low-temperature high-entropy state in the easy-plane system GaV$_4$Se$_8$, which is not captured in the numerical model. Possible origins of this state are discussed.

Synthesis, Physicochemical Characteristics and Plausible Mechanism of Action of an Immunosuppressive Isoxazolo[5,4-e]-1,2,4-Triazepine Derivative (RM33).

Previous studies demonstrated strong anti-inflammatory properties of isoxazolo[5,4-e]-1,2,4-triazepine (RM33) in vivo. The aim of this investigation was to describe synthesis, determine physicochemical characteristics, evaluate biological activities in murine and human in vitro models, as well as to propose mechanism of action of the compound. The compound was devoid of cell toxicity up to 100 μg/mL against a reference A549 cell line. Likewise, RM33 did not induce apoptosis in these cells. The compound stimulated concanavalin A (ConA)-induced splenocyte proliferation but did not change the secondary humoral immune response in vitro to sheep erythrocytes. Nevertheless, a low suppressive effect was registered on lipopolysaccharide (LPS)-induced splenocyte proliferation and a stronger one on tumor necrosis factor alpha (TNFα) production by rat peritoneal cells. The analysis of signaling pathways elicited by RM33 in nonstimulated resident cells and cell lines revealed changes associated with cell activation. Most importantly, we demonstrated that RM33 enhanced production of cyclooxygenase 2 in LPS-stimulated splenocytes. Based on the previous and herein presented results, we conclude that RM33 is an efficient, nontoxic immune suppressor with prevailing anti-inflammatory action. Additionally, structural studies were carried out with the use of appropriate spectral techniques in order to unequivocally confirm the structure of the RM33 molecule. Unambiguous assignment of NMR chemical shifts of carbon atoms of RM33 was conducted thanks to full detailed analysis of 1H, 13C NMR spectra and their two-dimensional (2D) variants. Comparison between theoretically predicted chemical shifts and experimental ones was also carried out. Additionally, N-deuterated isotopologue of RM33 was synthesized to eliminate potentially disturbing frequencies (such as NH, NH2 deformation vibrations) in the carbonyl region of the IR (infrared) spectrum to confirm the presence of the carbonyl group.

Efficient One-Pot Microwave-Assisted Synthesis, Crystallographic, and Spectroscopic Characterization of Novel Antitumor and Antimicrobial (3E)-5-Hydroxy-1-Isopropyl-3-[(5-Methyl-2-Thienyl)Methylene]-5-Phenylpyrrolidin-2-One

A microwave-assisted, chemoselective synthesis of novel antitumor and antimicrobial (3E)-5-hydroxy-1-isopropyl-3-[(5-methyl-2-thienyl)methylene]-5-phenylpyrrolidin-2-one has been achieved via the solvent-free one-pot reaction of (3E)-3-[(5-methyl-2-thienyl)methylene]-5-phenylfuran-2(3H)-one with isopropylamine. The product is obtained in significant purity and yield under ecofriendly reaction conditions. The microwave technique surpasses conventional thermal heating approaches by accelerating the reaction in a clean, ecofriendly manner that avoids the use of organic or toxic solvents. The structural formula of the product is confirmed by crystallographic and spectroscopic characterization. X-ray single crystal diffraction reveals that the compound crystallizes in an orthorhombic centrosymmetric crystal form, with unambiguous assignment of the E-configuration for the C3–Cthienyl bond. The synthesized molecules have two centers of chirality in the hydroxypyrrolidin-2-one ring: 1) the carbon atom attached to nitrogen, the hydroxyl group, and the phenyl ring; 2) the nitrogen atom attached to the carbonyl carbon R3 group, the chiral carbon in the ring, and the covalent bond bearing the lone pair of electrons. The molecular geometry is also optimized using density functional theory calculations, and the results obtained are in good agreement with the experimental data. Evaluation of the biological and medicinal activity of the compound affords similar results to the reference data in antitumor treatment of human colon and breast cells, which can be attributed to the presence of the hydroxyl group, the heterocyclic motifs, and sulfur. Calculation of the molecular electrostatic potential locates the most electrophilic site near the hydroxyl group attached to the heterocyclic ring, which is consistent with the bioactivity results. The frontier molecular orbitals are also determined, finding that the energy difference between highest occupied molecular orbital and lowest unoccupied molecular orbital is −0.15228 eV. A mechanism is proposed in which an intramolecular nucleophilic attack occurs on the carbonyl carbon by the lone pair of electrons on the nitrogen atom, leading to ring closure with proton transfer to oxygen and final formation of the hydroxyl group.

Total Synthesis and Structural Reassignment of Laingolide A.

The asymmetric total synthesis of four diastereomers of laingolide A was achieved, which led to the unambiguous assignment of the stereochemistry of the natural product. The salient features of the convergent, fully stereocontrolled approach were a copper-catalysed stereospecific Kumada-type coupling, a Julia-Kocienski olefination and an RCM/alkene migration sequence to access the desired macrocyclic enamide.

A phasianid bird from the Pleistocene of Tainan: the very first avian fossil from Taiwan

Taiwan accommodates more than 600 avian species, including about 30 endemic ones. As yet, however, no fossil birds have been scientifically documented from Taiwan, so that the evolutionary origins of this diversified avifauna remain elusive. Here we report on the very first fossil bird from Taiwan. This Pleistocene specimen, a distal end of the left tarsometatarsus, shows diagnostic features of the galliform Phasianidae, including an asymmetric plantar articular facet trochlea metatarsi III. Our discovery of a Pleistocene phasianid from Taiwan opens a new perspective on studies of the evolution of the avifauna in Taiwan because the fossil shows that careful search for fossils in suitable localities has the potential of recovering avian remains. In general, East Asia has an extremely poor avian fossil record, especially if terrestrial birds are concerned, which impedes well-founded evolutionary scenarios concerning the arrival of certain groups in the area. The Phasianidae exhibit a high degree of endemism in Taiwan, and the new fossil presents the first physical evidence for the presence of phasianids on the island, some 400,000–800,000 years ago. The specimen belongs to a species the size of the three larger phasianids occurring in Taiwan today (Syrmaticus mikado, Lophura swinhoii, and Phasianus colchicus). Still, an unambiguous assignment to either of these species is not possible due to the incomplete nature of the left tarsometatarsus. Because the former two species are endemic to Taiwan, the fossil has the potential to yield the first data on their existence in the geological past of Taiwan if future finds allow identification on species-level.

Low-Coordinate Iron Chalcogenolates and Their Complexes with Diethyl Ether and Ammonia.

Treatment of Fe{N(SiMe3)2}2 with 2 equiv of the appropriate phenol or thiol affords the dimers {Fe(OC6H2-2,6-But2-4-Me)2}2 (1) and {Fe(OC6H3-2,6-But2)2}2 (2) or the monomeric Fe{SC6H3-2,6-(C6H3-2,6-Pri2)2}2 (3) in moderate to excellent yields. Recrystallization of 1 and 2 from diethyl ether gives the corresponding three-coordinate ether complexes Fe(OC6H3-2,6-But2-4-Me)2(OEt2) (4) and Fe(OC6H3-2,6-But2)2(OEt2) (5). In contrast, no diethyl ether complex is formed by the dithiolate 3. The 1H NMR spectra of 4 and 5 show equilibria between the ether complexes and the base-free dimers. A comparison of these spectra with those of the dimeric 1 and 2 allows an unambiguous assignment of the paramagnetically shifted signals. Treatment of 1 with excess ammonia gives the tetrahedral diammine Fe(OC6H2-2,6-But2-4-Me)2(NH3)2 (6). Ammonia is strongly coordinated in 6, with no apparent loss of ammine ligand either in solution or upon heating under low pressure. In contrast, significantly weaker ammonia coordination is observed when dithiolate 3 is treated with excess ammonia, which gives the diammine Fe{SC6H3-2,6-(2,6-Pri2-C6H3)2}2(NH3)2 (7). Complex 7 readily loses ammonia either in solution or under reduced pressure to give the monoammine complex Fe{SC6H3-2,6-(2,6-Pri2-C6H3)2}2(NH3) (8). The weak binding of ammonia by iron thiolate 7 reflects the likely behavior of the proposed iron-sulfur active site in nitrogenases, where release of ammonia is required to close the catalytic cycle.

Increasing Top-Down Mass Spectrometry Sequence Coverage by an Order of Magnitude through Optimized Internal Fragment Generation and Assignment.

A major limitation of intact protein fragmentation is the lack of sequence coverage within proteins' interiors. We show that collisionally activated dissociation (CAD) produces extensive internal fragmentation within proteins' interiors that fill the existing gaps in sequence coverage, including disulfide loop regions that cannot be characterized using terminal fragments. A barrier to the adoption of internal fragments is the lack of methods for their generation and assignment. To provide these, we explore the effects of protein size, mass accuracy, internal fragment size, CAD activation energy, and data preprocessing upon the production and identification of internal fragments. We also identify and mitigate the major source of ambiguity in internal fragment identification, which we term "frameshift ambiguity." Such ambiguity results from sequences containing any "middle" portion surrounded by the same composition on both termini, which upon fragmentation can produce two internal fragments of identical mass, yet out of frame by one or more amino acids (e.g., TRAIT producing TRAI or RAIT). We show that such instances permit the a priori assignment of the middle sequence portion. This insight and our optimized methods permit the unambiguous assignment of greater than 97% of internal fragments using only the accurate mass. We show that any remaining ambiguity in internal fragment assignment can be removed by consideration of fragmentation propensities or by (pseudo)-MS3. Applying these methods resulted in a 10-fold and 43-fold expanded number of identified ions, and a concomitant 7- and 16-fold increase in fragmentation sites, respectively, for native and reduced forms of a disease-associated SOD1 variant.

Hiphop: Improved paternity assignment among close relatives using a simple exclusion method for biallelic markers.

Assignment of parentage with molecular markers is most difficult when the true parents have close relatives in the adult population. Here, we present an efficient solution to that problem by extending simple exclusion approaches to parentage analysis with single nucleotide polymorphic markers (SNPs). We augmented the previously published homozygote opposite test (hot), which counts mismatches due to the offspring and candidate parent having different homozygous genotypes, with an additional test. In this case, parents homozygous for the same SNP are incompatible with heterozygous offspring (i.e., "Homozygous Identical Parents, Heterozygous Offspring are Precluded": hiphop). We tested this approach in a cooperatively breeding bird, the superb fairy-wren, Malurus cyaneus, where rates of extra-pair paternity are exceptionally high, and where paternity assignment is challenging because breeding males typically have first-order adult relatives in their neighbourhood. Combining the tests and conditioning on the maternal genotype with a set of 1376 autosomal SNPs always allowed us to distinguish a single most likely sire from his relatives, and also to identify cases where the true sire must have been unsampled. In contrast, if just the hot test was used, we failed to identify a single most-likely sire in 2.5% of cases. Resampling enabled us to create guidelines for the number of SNPs required when first-order relatives coexist in the mating pool. Our method, implemented in the R package hiphop, therefore provides unambiguous parentage assignments even in systems with complex social organisation. We also identified a suite of Z- and W-linked SNPs that always identified sex correctly.

Online Monitoring of Isomeric Reaction Intermediates.

Ephemeral intermediates often hold the key to a more detailed understanding of chemical reaction pathways. Online methods to unambiguously identify the structure of such molecular entities, in particular in the presence of multiple isomers, are scarce. This paper presents a methodology that allows real-time monitoring of isomeric solution-phase reaction intermediates of continuous-flow reactions by coupling a microfluidic chip-reactor to a cryogenic ion trap triple mass spectrometer. The technique combines the excellent reaction control associated with microfluidic chips with the unique specificity and sensitivity of infrared photodissociation (IRPD) spectroscopy, which allows for an unambiguous structural assignment of gaseous ions based on their IR fingerprint. It represents a valuable extension to the instrumentation for online-analysis of reactive intermediates and proves particularly valuable whenever the sensitivity of NMR is not sufficient. After a brief description of the experimental approach, illustrative examples are provided to highlight the application of the chip-IRPD setup for mechanistic studies, particularly for stereoselective processes. The article concludes with an outlook on future challenges and perspectives.

R/G Value-A Numeric Index of Individual Periodontal Health and Oral Microbiome Dynamics.

The dysbiosis of oral microbiome (OM) precedes the clinical signs of periodontal disease. Its simple measure thus could indicate individuals at risk of periodontitis development; however, such a tool is still missing. Up to now, numerous microbial taxa were associated with periodontal health or periodontitis. The outputs of most studies could, nevertheless, be slightly biased from following two reasons: First, the healthy group is often characterized only by the absence of the disease, but the individuals could already suffer from dysbiosis without any visible signs. Second, the healthy/diseased OM characteristics are frequently determined based on average data obtained for whole groups of periodontally healthy persons versus patients. Especially in smaller sets of tested individuals the typical individual variability can thus complicate the unambiguous assignment of oral taxa to respective state of health. In this work the taxonomic composition of OM was evaluated for 20 periodontally healthy individuals and 15 patients with chronic periodontitis. The narrowed selection set of the most diseased patients (confirmed by clinical parameters) and the most distant group of healthy individuals with the lowest probability of dysbiosis was determined by clustering analysis and used for identification of marker taxa. Based on their representation in each individual oral cavity we proposed the numeric index of periodontal health called R/G value. Its diagnostic potential was further confirmed using independent set of 20 periodontally healthy individuals and 20 patients with periodontitis with 95 percent of samples assigned correctly. We also assessed the individual temporal OM dynamics in periodontal health and we compared it to periodontitis. We revealed that the taxonomic composition of the system changes dynamically but generally it ranges within values typical for periodontal health or transient state, but far from values typical for periodontitis. R/G value tool, formulated from individually evaluated data, allowed us to arrange individual OMs into a continuous series, instead of two distinct groups, thus mimicking the gradual transformation of a virtual person from periodontal health to disease. The application of R/G value index thus represents a very promising diagnostic tool for early prediction of persons at risk of developing periodontal disease.

SNP panels for the estimation of dairy breed proportion and parentage assignment in African crossbred dairy cattle

BackgroundUnderstanding the relationship between genetic admixture and phenotypic performance is crucial for the optimization of crossbreeding programs. The use of small sets of informative ancestry markers can be a cost-effective option for the estimation of breed composition and for parentage assignment in situations where pedigree recording is difficult. The objectives of this study were to develop small single nucleotide polymorphism (SNP) panels that can accurately estimate the total dairy proportion and assign parentage in both West and East African crossbred dairy cows.MethodsMedium- and high-density SNP genotype data (Illumina BovineSNP50 and BovineHD Beadchip) for 4231 animals sampled from African crossbreds, African Bos taurus, European Bos taurus, Bos indicus, and African indigenous populations were used. For estimating breed composition, the absolute differences in allele frequency were calculated between pure ancestral breeds to identify SNPs with the highest discriminating power, and different combinations of SNPs weighted by ancestral origin were tested against estimates based on all available SNPs. For parentage assignment, informative SNPs were selected based on the highest minor allele frequency (MAF) in African crossbred populations assuming two Scenarios: (1) parents were selected among all the animals with known genotypes, and (2) parents were selected only among the animals known to be a parent of at least one progeny.ResultsFor the medium-density genotype data, SNPs selected for the largest differences in allele frequency between West African indigenous and European Bos taurus breeds performed best for most African crossbred populations and achieved a prediction accuracy (r2) for breed composition of 0.926 to 0.961 with 200 SNPs. For the high-density dataset, a panel with 70% of the SNPs selected on their largest difference in allele frequency between African and European Bos taurus performed best or very near best across all crossbred populations with r2 ranging from 0.978 to 0.984 with 200 SNPs. In all African crossbred populations, unambiguous parentage assignment was possible with ≥ 300 SNPs for the majority of the panels for Scenario 1 and ≥ 200 SNPs for Scenario 2.ConclusionsThe identified low-cost SNP assays could overcome incomplete or inaccurate pedigree records in African smallholder systems and allow effective breeding decisions to produce progeny of desired breed composition.

Microcosamine A, Microgrewiapine A and Microgrewiapine B: three homochiral alkaloids?

An asymmetric synthesis of microcosamine A, microgrewiapine A and microgrewiapine B ( Microcos paniculata alkaloids) is reported. Conjugate addition of lithium ( S )- N -benzyl- N -(α-methylbenzyl)amide to tert -butyl crotonate followed by enolate oxidation generates the C(2) and C(3) stereogenic centres of the targets, with subsequent diastereoselective intramolecular reductive amination being used to form the piperidine ring, simultaneously producing the C(6) stereogenic centre. 1 H NMR 3 J coupling constant and nOe analyses allow unambiguous assignment of relative configuration of the alkaloids. Comparison of specific rotation data for microcosamine A and microgrewiapine B is consistent with both possessing the absolute (2 S ,3 R ,6 S )-configuration. For microgrewiapine A, conflicting data regarding the absolute configuration are revealed: in the isolation study a Mosher’s analysis concludes a (2 S ,3 R ,6 S )-configuration, but comparison of specific rotation data suggests a (2 R ,3 S ,6 R )-configuration. Thus it is promulgated it is imprudent to suggest the absolute configuration of this natural product; its re-isolation is required for resolution of this problem.

Assignment of Absolute Configuration of Bromoallenes by Vacuum-Ultraviolet Circular Dichroism (VUVCD).

A new application of vacuum-ultraviolet circular dichroism (VUVCD), which enables the measurement of CD spectra in the vacuum-ultraviolet region (140–200 nm), for the assignment of the absolute configurations of bromoallenes is described. Bromoallene moieties are found in natural products obtained from many marine organisms. To date, the absolute configuration of bromoallenes has been assigned almost exclusively with Lowe’s rule, which is based on specific rotation. However, exceptions to Lowe’s rule have been reported arising from the presence of other substituents with large specific rotations. For the unambiguous assignment of the absolute configuration of the bromoallene moiety with its characteristic absorption wavelength at 180–190 nm due to the π–π* transition, VUVCD was applied to four pairs of bromoallene diastereomers prepared by modifying the synthetic scheme of omaezallene. The VUVCD spectra clearly showed positive or negative Cotton effects around 180–190 nm according to the configuration of the bromoallene employed, revealing the potential of VUVCD for determining absolute stereochemistry.

Synthesis and structural elucidation of a series of isoflavones-based on FPR antagonists

Synthesis and structural elucidation of a series of isoflavones-based on FPR antagonists

Rapid and unambiguous assignment of proton-deficient N-acyl amidine indole alkaloid by a combination of calculation methods

The unprecedently rearranged 6/5/8/6/5 pentacyclic monoterpenoid indole alkaloid voafriamidine from Voacanga africana contains an uncommon N-acyl amidine structural motif that forms a unique 2,4-diazatricycle-[6,5,11,8,03,7] tetradecane skeleton. Despite the lack of proper NMR correlations for the related quaternary carbon and nitrogen, the complex structure was finally elucidated by the combination of ACD/Structure Elucidator, density functional theory (DFT) calculations of the NMR spectra and electronic circular dichroism (ECD) analyses.

Laser-Induced Fluorescence Spectroscopy of Large Secondary Alkoxy Radicals: Part II. Rotational and Fine Structure.

Selected vibronic bands of the B̃ ← X̃ laser-induced fluorescence (LIF) spectra of jet-cooled 2-pentoxy and 2-hexoxy, including the origin and CO-stretch bands, have been measured with rotational resolution and analyzed using (1) an effective Hamiltonian that comprises a rotational part and a spin-rotation (SR) part (the "isolated-states model") and (2) a recently developed Hamiltonian in which the nearly degenerate à and X̃ states are treated together (the "coupled-states model") (see Liu, J., J. Chem. Phys. 2018, 148, 124112). The observed rotational and fine structures of the strongest vibronic bands have first been simulated using a genetic algorithm with the isolated-states model. The parameters for the simulation include rotational constants for both the X̃ and B̃ states, which can be calculated from the electronic structure theory, as well as the electronic SR constants of the X̃ state and the transition dipole moments (TDMs), both of which are predicted based on their transferability in an "orbital-fixed coordinate system" using iso-propoxy as the reference molecule. Quantum chemistry calculations suggest that the lowest two electronic (X̃ and Ã) states of secondary alkoxy radicals have small energy separations on the order of 100 cm-1 (see Part I of this series: J. Phys. Chem. A 2021, DOI: 10.1021/acs.jpca.0c10662). The electron configurations of these two nearly degenerate states have been determined by comparing the experimentally determined rotational constants and the TDMs to the ones predicted for the X̃ and à states. The experimental LIF spectra were also simulated with the coupled-states model, in which the effective spin-orbit (SO) constants (aζed) and the SO-free separation between the à and the X̃ states (ΔE0) have been determined. Molecular constants derived from fitting the rotational and fine structures of the experimental LIF spectra enabled unambiguous assignment of the observed vibronic bands to specific conformers of 2-pentoxy and 2-hexoxy as reported in Part I.

Elastic properties assessment in the multiferroic BiFeO3 by pump and probe method

We have performed elasticity measurements in the bulk multiferroic BiFeO3 (BFO) using acoustical pump and probe spectroscopy. The sound velocities of the (quasi)-longitudinal and two (quasi)-transverse acoustic waves along three independent directions of the (110) surface have been measured. Moreover, one surface wave and one longitudinal wave propagating perpendicular to the surface have been detected. Based on initial input values of the six independent Cij elastic constants determined by our density functional theory calculations and our eleven experimental velocities, the numerical resolution of the acoustic equations allows us to determine all the Cij elastic constants of BFO. The propagation direction dependence of volume and surface waves phase velocities allows the unambiguous assignment of the waves and, hence, the polarization of phonons.

Structural Insights into the Diamide Modulation of Ryanodine Receptor

Diamide insecticides, developed by pesticide giants DuPont, Bayer and Syngenta, are among the top-selling insecticides globally, with annual sales >USD$2 billion. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). However, due to heavy usage, many pests have developed strong resistance, posing a threat on food safety in many countries. Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2 Å local resolution map allows for the unambiguous assignment of the CHL binding site, which is located in the pseudo-voltage-sensor domain of RyR. The molecule induces a conformational change by affecting the S4-S5 linker, triggering channel opening. The binding site is further corroborated with data from cellular and drosophila mutagenesis studies, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammal RyRs. Importantly, our data reveals that several pests have developed resistance via two mechanisms, either by mutating key residues involved in binding or by introducing steric hindrance. Mutations in human RyRs are associated with inherited myopathies. Based on the activities of diamides against the mutant mammalian RyRs, we propose to develop therapeutic molecules targeting the allosteric diamide-binding site with the aim to treat human myopathies. Our findings provide a foundation for developing novel pesticides to overcome the resistance crisis, and to decrease toxicity for humans and beneficial insects. It also highlights an allosteric site that can be targeted for novel therapeutics in humans.