A new family of CRISPR-type V nucleases with C-rich PAM recognition.

Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a T-rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich (5'-CCY-3') PAM. The organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR-associated nucleases may be harnessed as genome editing reagents.

Implications of variability on medical device chemical equivalence assessment.

Chemical equivalence testing can be used to assess the biocompatibility implications of a materials or manufacturing change for a medical device. This testing can provide a relatively facile means to evaluate whether the change may result in additional or different toxicological concerns. However, one of the major challenges in the interpretation of chemical equivalence data is the lack established criteria for determining if two sets of extractables data are effectively equivalent. To address this gap, we propose a two-part approach based upon a relatively simple statistical model. First, the probability of a false positive conclusion, wherein there is an incorrectly perceived increase for a given analyte in the comparator relative to the baseline device, can be reduced to a prescribed level by establishing an appropriate acceptance criterion for the ratio of the observed means. Second, the probability of a false negative conclusion, where an actual increase in a given analyte cannot be discerned from the test results, can be minimized by specifying a limiting value of applicability based on the margin of safety (MoS) of the analyte. This approach provides a quantitative, statistically motivated method to interpret chemical equivalence data, despite the relatively high intrinsic variability and small number of replicates typically associated with a chemical characterization evaluation.

Modeling extraction of medical device polymers for biocompatibility evaluation.

Extraction testing is critical for biocompatibility evaluation of medical devices, whether to generate samples for biological testing or form the basis for toxicological risk assessment. However, it is not always clear how to compare extraction testing between different extraction conditions and sample geometries. We employ a physics-based model to elucidate the theoretical impact of extraction conditions, sample geometry and material properties on extraction efficiency (M/M0) and extract concentration (C/C0) for single-step and iterative/exhaustive extraction test methods. The model is specified by three parameters: thermodynamic contributions (Ψ), kinetic contributions (τ), and number of extraction iterations (N). We find that over the range of typical parameters for single-step extractions, M/M0 only approaches one (complete exhaustion) for relatively large values of Ψ (≥10) and τ(≥1). Further, the model suggests that test article geometry and solvent volume can have a dramatic and sometimes opposing effect on M/M0 and C/C0. Our results imply that iterative extractions can be approximated as a single-step extraction with scaled parameters Ψ' = ΨN and τ' = τN. The model provides a framework to reduce the biocompatibility evaluation test burden by optimizing test article and extraction condition selection and guiding development of new test protocols.

Real-world effectiveness of benralizumab: Results from the ZEPHYR 1 Study.

BACKGROUND: Real-world evidence characterizing the clinical outcomes and economic impact on patients with severe eosinophilic asthma treated with benralizumab is limited. OBJECTIVE: To characterize patients with severe asthma treated with benralizumab and assess its clinical and economic impact in the United States. METHODS: A pre-post benralizumab comparison was performed using a large US insurance claims database between November 2016 and November 2019. The primary cohort included patients with asthma aged 12 years or more with 2 or more records of benralizumab. Secondary cohorts included persistent users (6 or more records of benralizumab), patients switching to benralizumab from mepolizumab or omalizumab, and stratified by Medicaid vs non-Medicaid. Exacerbations, concomitant medications, and exacerbation-related health care resource utilization (HCRU) and costs were compared in the 12-month periods pre- and post-benralizumab initiation (index). RESULTS: Of the 204 patients in the primary cohort, mean age at index was 45.3 years and 68.6% were of female sex. The patients experienced a significant 55% reduction in rates of exacerbations post-benralizumab initiation (3.25 pre-index vs 1.47 post-index per person-year; P < .001), and 41% of the patients had no exacerbations post-benralizumab initiation. The proportion of oral corticosteroid-dependent patients decreased from 82% to 50% (P < .001). Reductions in HCRU were 42%, 46%, and 57% for asthma exacerbation-related inpatient hospitalizations, emergency department, and outpatient visits, respectively (all P < .001). Exacerbation-related costs decreased by $6439 ($13,559 vs $7120; P < .001). Similar results for all outcomes were observed for the persistent cohort, switch cohorts, and Medicaid vs non-Medicaid cohorts. CONCLUSION: Patients with severe asthma treated with benralizumab experienced clinical and economic benefits in the real world, as demonstrated by the reduction in exacerbations and HCRU.

PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage.

In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422-603 amino acids) CRISPR-Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5' T- or C-rich PAM sequence triggers dsDNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in Escherichia coli and find that some but not all can. Altogether, our findings show that miniature Cas12f nucleases can protect against invading dsDNA like much larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.

Computational investigation of enhanced properties in functionalized carbon nanotube doped polyvinyl alcohol gel electrolyte systems.

Recently, functionalized carbon nanotubes (fCNTs) were shown to increase the mechanical strength, thermal stability, and ionic conductivity in polyvinyl alcohol (PVA) based gel electrolytes (GE) for Zn ion batteries. However, questions remain about the origin of the property enhancement and the interactions between components of GEs. In this work, we employ density functional theory calculations to analyze the interactions between fCNT, PVA, and Zn ions. CNTs with increasing numbers of carboxyl (-COOH) functional groups and PVA chains with varying lengths were studied. We found that increasing the number of -COOH on the CNTs enhanced the adsorption energies (Eads) of PVA, and Eads also increased as the number of monomers increased. We then modelled the coordination of a Zn ion in fCNT-PVA complexes. Our results suggest that strong fCNT-PVA interactions contribute to the enhanced mechanical strength, while the enhanced ionic conductivity is partly owing to weak Zn adsorption.

A practical way to enhance the synthesis of N8- from an N3- precursor, studied by both computational and experimental methods.

Polymeric nitrogen (PN) belongs to a general family of materials containing all-nitrogen molecules or clusters. Although it is rare and challenging to synthesize PN members, they are attracting increasing scientific attention due to their high energy storage capacity and possible use as a green catalyst. A few theoretical calculations predicted the possible PN phases from N2 gas, but they all require extremely high pressures and temperatures to synthesize. In this work, a practical way to synthesize N8 polymeric nitrogen from an N3- precursor is elucidated using density functional theory calculations. The detailed mechanism, , is determined. The calculated energy barriers indicate that the first step is the rate-limiting step. This result guides us to rationally synthesize N8 under UV (254 nm) irradiation, chosen based on the calculated absorption spectrum for the azide anion. As expected, UV irradiation enhances N8 yields by nearly four times. This provides an interesting route to the scalable synthesis of high energy density N8 compounds.

Mapping the genomic landscape of CRISPR-Cas9 cleavage.

RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

Seeding fiber amplifiers with piecewise parabolic phase modulation for high SBS thresholds and compact spectra.

We propose using piecewise parabolic phase modulation of the seed laser for suppressing stimulated Brillouin scattering (SBS) in a fiber amplifier. Simulations are run with a 9 m passive fiber. Compared with random phase modulation and 0-π pseudo-random phase modulation, the piecewise parabolic phase waveform yields a higher SBS threshold per unit bandwidth. If the bandwidth is defined as the range of frequencies containing 85% of the total power, the threshold for parabolic phase modulation is 1.4 times higher than the threshold for the five- or seven-bit pseudo-random modulation format. If the bandwidth is defined more tightly, e.g., the range of frequencies containing 95% of the total power, the threshold for parabolic phase modulation is three times higher. For both cases, achieving a bandwidth of 1.5 GHz requires a maximum phase shift of ~30 radians. All of the waveforms are compared on the basis of the bandwidth required of the phase moduator. The coherence functions are calculated in order to compare their suitability for coherent combining.

Concurrent modifications in the three homeologs of Ms45 gene with CRISPR-Cas9 lead to rapid generation of male sterile bread wheat (Triticum aestivum L.).

KEY MESSAGE: Hexaploid bread wheat is not readily amenable to traditional mutagenesis approaches. In this study, we show efficient utilization of CRISPR-Cas system and Next Generation Sequencing for mutant analysis in wheat. Identification and manipulation of male fertility genes in hexaploid bread wheat is important for understanding the molecular basis of pollen development and to obtain novel sources of nuclear genetic male sterility (NGMS). The maize Male sterile 45 (Ms45) gene encodes a strictosidine synthase-like enzyme and has been shown to be required for male fertility. To investigate the role of Ms45 gene in wheat, mutations in the A, B and D homeologs were produced using CRISPR-Cas9. A variety of mutations in the three homeologs were recovered, including a plant from two different genotypes each with mutations in all three homeologs. Genetic analysis of the mutations demonstrated that all three wheat Ms45 homeologs contribute to male fertility and that triple homozygous mutants are required to abort pollen development and achieve male sterility. Further, it was demonstrated that a wild-type copy of Ms45 gene from rice was able to restore fertility to these wheat mutant plants. Taken together, these observations provide insights into the conservation of MS45 function in a polyploid species. Ms45 based NGMS can be potentially utilized for a Seed Production Technology (SPT)-like hybrid seed production system in wheat.

Ba4B8TeO19: A UV Nonlinear Optical Material.

We report a new noncentrosymmetric barium tellurium borate, Ba4B8TeO19 that has potential ultraviolet (UV) nonlinear optical (NLO) applications. Ba4B8TeO19 was synthesized by a flux method and crystallizes in the noncentrosymmetric space group Cc. The material exhibits a framework structure of [B8O17]∞ double layers connected to distorted TeO6 octahedra. Second harmonic generation (SHG) measurements at 1064 and 532 nm on polycrystalline Ba4B8TeO19 indicate that the title compound is phase-matchable (type I) with a moderate SHG response (1 × KH2PO4 at 1064 nm and 0.2 × ß-BaB2O4 at 532 nm). In addition, a short absorption edge (210 nm) was measured. Using density functional theory calculations, we show that the SHG response originates from contributions from O 2p and Te 5s states at the valence and conduction band edges. Finally, by computing the linear optical properties, we find that this compound displays a moderate birefringence of 0.055 at 1064 nm and 0.059 at 532 nm, necessary conditions for phase-matching in UV NLO materials.

VALUE OF FRACTAL ANALYSIS OF OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY IN VARIOUS STAGES OF DIABETIC RETINOPATHY.

PURPOSE: To use fractal dimensional analysis to investigate retinal vascular disease patterns in patients with diabetic retinopathy using spectral domain optical coherence tomography angiography. METHODS: A retrospective study was conducted which included 49 eyes from 26 control subjects and 58 eyes from 35 patients known to have diabetic retinopathy. Of the 58 eyes with known retinopathy, 31 were categorized as nonproliferative diabetic retinopathy (13 mild, 9 moderate, and 9 severe) and 27 were categorized as proliferative diabetic retinopathy. Optical coherence tomography angiography images were acquired using the RTVue XR Avanti (Optovue, Inc). Automated segmentation was obtained through both the superficial and deep capillary plexuses for each eye. Grayscale optical coherence tomography angiography images were standardized and binarized using ImageJ (National Institutes of Health). Fractal box-counting analyses were conducted using Fractalyse (ThéMA). Fractal dimensions (FDs) and correlation coefficient of the superficial and deep capillary plexuses were compared between control eyes and those in various stages of diabetic retinopathy. RESULTS: The superficial and deep capillary plexuses from diabetic and control eyes were analyzed. The average FD for diabetic eyes was significantly lower than in control eyes in the superficial plexus (P = 2.4 × 10) and in the deep capillary plexus (P = 1.87 × 10 ) with a more statistically significant difference noted in the deep capillary plexus. When analyzing diabetic patients without edema noted on optical coherence tomography, the FD was significantly reduced in the superficial (P = 0.001) and deep (P = 1.49 × 10) plexuses. When analyzing diabetic patients with edema noted on optical coherence tomography, the FD was significantly reduced in the superficial (P = 2.0 × 10) and deep (P = 1.85 × 10) plexuses. CONCLUSION: The optical coherence tomography angiography FD is significantly lower in both superficial and deep capillary plexuses in eyes with all stages studied of diabetic retinopathy. The results were more often significant for the deep capillary plexus. The use of fractal analysis provides an objective criterion to assess microvascular disease burden in diabetic retinopathy.

Mixed-Metal Carbonate Fluorides as Deep-Ultraviolet Nonlinear Optical Materials.

Noncentrosymmetric mixed-metal carbonate fluorides are promising materials for deep-ultraviolet (DUV) nonlinear optical (NLO) applications. We report on the synthesis, characterization, structure-property relationships, and electronic structure calculations on two new DUV NLO materials: KMgCO3F and Cs9Mg6(CO3)8F5. Both materials are noncentrosymmetric (NCS). KMgCO3F crystallizes in the achiral and nonpolar NCS space group P6̅2m, whereas Cs9Mg6(CO3)8F5 is found in the polar space group Pmn21. The compounds have three-dimensional structures built up from corner-shared magnesium oxyfluoride and magnesium oxide octahedra. KMgCO3F (Cs9Mg6(CO3)8F5) exhibits second-order harmonic generation (SHG) at both 1064 and 532 nm incident radiation with efficiencies of 120 (20) × α-SiO2 and 0.33 (0.10) × ß-BaB2O4, respectively. In addition, short absorption edges of <200 and 208 nm for KMgCO3F and Cs9Mg6(CO3)8F5, respectively, are observed. We compute the electron localization function and density of states of these two compounds using first-principles density functional theory, and show that the different NLO responses arise from differences in the denticity and alignment of the anionic carbonate units. Finally, an examination of the known SHG active AMCO3F (A = alkali metal, M = alkaline earth metal, Zn, Cd, or Pb) materials indicates that, on average, smaller A cations and larger M cations result in increased SHG efficiencies.

Polar Oxides without Inversion Symmetry through Vacancy and Chemical Order.

One synthetic modality for materials discovery proceeds by forming mixtures of two or more compounds. In transition metal oxides (TMOs), chemical substitution often obeys Vegard's principle, and the resulting structure and properties of the derived phase follow from its components. A change in the assembly of the components into a digital nanostructure, however, can stabilize new polymorphs and properties not observed in the constituents. Here we formulate and demonstrate a crystal-chemistry design approach for realizing digital TMOs without inversion symmetry by combining two centrosymmetric compounds, utilizing periodic anion-vacancy order to generate multiple polyhedra that together with cation order produce a polar structure. We next apply this strategy to two brownmillerite-structured TMOs known to display centrosymmetric crystal structures in their bulk, Ca2Fe2O5 and Sr2Fe2O5. We then realize epitaxial (SrFeO2.5)1/(CaFeO2.5)1 thin film superlattices possessing both anion-vacancy order and Sr and Ca chemical order at the subnanometer scale, confirmed through synchrotron-based diffraction and aberration corrected electron microscopy. Through a detailed symmetry analysis and density functional theory calculations, we show that A-site cation ordering lifts inversion symmetry in the superlattice and produces a polar compound. Our results demonstrate how control of anion and cation order at the nanoscale can be utilized to produce acentric structures markedly different than their constituents and open a path toward novel structure-based property design.

Targeted mutagenesis of a conserved anther-expressed P450 gene confers male sterility in monocots.

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.

Interplay of Cation Ordering and Ferroelectricity in Perovskite Tin Iodides: Designing a Polar Halide Perovskite for Photovoltaic Applications.

Owing to its ideal semiconducting band gap and good carrier-transport properties, the fully inorganic perovskite CsSnI3 has been proposed as a visible-light absorber for photovoltaic (PV) applications. However, compared to the organic-inorganic lead halide perovskite CH3NH3PbI3, CsSnI3 solar cells display very low energy conversion efficiency. In this work, we propose a potential route to improve the PV properties of CsSnI3. Using first-principles calculations, we examine the crystal structures and electronic properties of CsSnI3, including its structural polymorphs. Next, we purposefully order Cs and Rb cations on the A site to create the double perovskite (CsRb)Sn2I6. We find that a stable ferroelectric polarization arises from the nontrivial coupling between polar displacements and octahedral rotations of the SnI6 network. These ferroelectric double perovskites are predicted to have energy band gaps and carrier effective masses similar to those of CsSnI3. More importantly, unlike nonpolar CsSnI3, the electric polarization present in ferroelectric (CsRb)Sn2I6 can effectively separate the photoexcited carriers, leading to novel ferroelectric PV materials with potentially enhanced energy conversion efficiency.

Controlling the H to T' structural phase transition via chalcogen substitution in MoTe2 monolayers.

New materials exhibiting reversible structural transitions are desired for a variety of applications, yet they are difficult to identify and stabilize. Monolayer MoTe2 has emerged as a good candidate as it displays a small energy difference between a semiconducting H and semimetallic T' phase; however, switching between the two phases is difficult. Here, we propose using chalcogen alloying to overcome this challenge. Using first principles density functional theory calculations, we investigate 7 MoTe2-xXx alloys (X = N, P, Sb, F, Br, I, and Se) at three concentrations. We find that the energy difference between the H and T' phases is dependent on the chemistry, size, and concentration of the dopant atom, providing significant control over the stability of the two phases. From the thermodynamic stability of these compounds, we show that several can be stabilized under the appropriate experimental conditions. We also find that P-alloying enhances the chemical reactivity of the basal plane towards a variety of adsorbates. Finally, we show that mechanical strain makes it is easier to stabilize and dynamically switch between the two states than in unalloyed MoTe2. Our results suggest that Te substitution in monolayer MoTe2 is a way to induce and control a reversible structural phase transition in this two-dimensional material system.

Phenolic composition of pomegranate peel extracts using an liquid chromatography-mass spectrometry approach with silica hydride columns.

The peels of different pomegranate cultivars (Molla Nepes, Parfianka, Purple Heart, Wonderful and Vkunsyi) were compared in terms of phenolic composition and total phenolics. Analyses were performed on two silica hydride based stationary phases: phenyl and undecanoic acid columns. Quantitation was accomplished by developing a liquid chromatography with mass spectrometry approach for separating different phenolic analytes, initially in the form of reference standards and then with pomegranate extracts. The high-performance liquid chromatography columns used in the separations had the ability to retain a wide polarity range of phenolic analytes, as well as offering beneficial secondary selectivity mechanisms for resolving the isobaric compounds, catechin and epicatechin. The Vkunsyi peel extract had the highest concentration of phenolics (as determined by liquid chromatography with mass spectrometry) and was the only cultivar to contain the important compound punicalagin. The liquid chromatography with mass spectrometry data were compared to the standard total phenolics content as determined by using the Folin-Ciocalteu assay.

Electronic, Crystal Chemistry, and Nonlinear Optical Property Relationships in the Dugganite A3B3CD2O14 Family.

A family of six nonlinear optical (NLO) materials, A3B3CD2O14 (A = Sr, Ba, or Pb; B = Mg or Zn; C = Te or W; and D = P or V), has been synthesized and characterized. In addition to the synthesis and crystal structures, comprehensive characterization of these compounds includes second harmonic generation (SHG) measurements, theoretical calculations, infrared and diffuse reflectance spectroscopies, and thermogravimetric measurements. We find that all of the reported materials are SHG-active at 1064 nm, with responses ranging from 2.8 to 13.5 × KDP, and exhibit absorption edges in the mid- to deep-ultraviolet regime. By systematically replacing the A, B, C, and D cations, we are able to tune these properties and investigate the role of different NLO-active structural units in producing the SHG responses. Specifically, our electronic structure calculations reveal that the presence of Pb(2+) on the A-site and Te(6+) on the C-site is critical for generating a large SHG response. The synthesis and structure-property relationships described in this family of materials will enable the design and discovery of new NLO materials.