Recent advances in mycobacterial membrane protein large 3 inhibitor drug design for mycobacterial infections.

INTRODUCTION: Tuberculosis and nontuberculous mycobacterial infections are notoriously difficult to treat, requiring long-courses of intensive multi-drug therapies associated with adverse side effects. To identify better therapeutics, whole cell screens have identified novel pharmacophores, a surprisingly high number of which target an essential lipid transporter known as MmpL3. AREAS COVERED: This paper summarizes what is known about MmpL3, its mechanism of lipid transport and therapeutic potential, and provides an overview of the different classes of MmpL3 inhibitors currently under development. It further describes the assays available to study MmpL3 inhibition by these compounds. EXPERT OPINION: MmpL3 has emerged as a target of high therapeutic value. Accordingly, several classes of MmpL3 inhibitors are currently under development with one drug candidate (SQ109) having undergone a Phase 2b clinical study. The hydrophobic character of most MmpL3 series identified to date seems to drive antimycobacterial potency resulting in poor bioavailability, which is a significant impediment to their development. There is also a need for more high-throughput and informative assays to elucidate the precise mechanism of action of MmpL3 inhibitors and drive the rational optimization of analogues.

CRISPR/Cas9-mediated tetra-allelic mutation of the 'Green Revolution' SEMIDWARF-1 (SD-1) gene confers lodging resistance in tef (Eragrostis tef).

Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF-1 (SD-1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD-1 edited tef lines was achieved in two tef cultivars by Agrobacterium-mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next-generation sequencing had at least two or more alleles of SD-1 mutated. Of these, 83% had tetra-allelic frameshift mutations in the SD-1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T1 and T2 generations revealed that the sd-1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd-1 plants. Measurements of lodging, in greenhouse-grown plants, showed that sd-1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9-mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.

Rapid, One-Step Synthesis of α-Ketoacetals via Electrophilic Etherification.

Herein, we report a rapid, one-step synthesis of α-ketoacetals via electrophilic etherification of α-alkoxy enolates and monoperoxyacetals. Methyl, primary, and secondary α-ketoacetals were obtained in 44-63% yields from tetrahydropyranyl substrates; using methyl tetrahydropyranyl, alkyl tetrahydropyranyl, or methyl tetrahydrofuranyl peroxyacetals, however, methyl and primary products were isolated in 66-90% yields. The present method is applied to C-O bond formation at tertiary carbons, via alkyl and methyl peroxyacetals, with yields of 25-65%. Intermolecular "alkoxyl" transfer, from peroxyacetal to α-alkoxy enolate, relies heavily on decreased steric bulk surrounding the peroxide bond and site of etherification; additionally, we found the α-OCH3 group to be critical in ensuring product formation. α-Ketoacetals demonstrated excellent reactivity, as selective, nucleophilic attack at the unprotected carbonyl furnished α-hydroxy acetals in 80-100% yields; subsequent hydrolysis of the foregoing compounds provided α-hydroxy aldehydes in yields of 58-90%.

Advances in Agrobacterium transformation and vector design result in high-frequency targeted gene insertion in maize.

CRISPR-Cas is a powerful DNA double-strand break technology with wide-ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knockouts in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here, we report an efficient, selectable marker-free site-specific gene insertion in maize using Agrobacterium infection. Advancements in maize transformation and new vector design enabled increase of targeted insertion frequencies by two orders of magnitude in comparison to conventional Agrobacterium-mediated delivery. Importantly, these advancements allowed not only a significant improvement of the frequency, but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium-mediated transformation.

Electrophilic Etherification of α-Heteroaryl Carbanions with Monoperoxyacetals as a Route to Ketene O,O- and N,O-Acetals.

Alkyl ketene acetals are useful reactants in a variety of synthetic processes, and yet, there are limited routes to their formation as isolable products. We now report the successful synthesis and isolation of heteroaryl ketene acetals through intermolecular transfer of alkoxyl (δ+OR) from electrophilic peroxides to lithiated benzofurans, indoles, and pyridines. Primary and secondary peroxyacetals enable selective transfer of the nonanomeric alkoxy group in moderate to high yield; substrates bearing an electron-donating substituent show enhanced reactivity toward electrophilic oxygen. Heteroaryl ketene acetals are remarkably stable throughout traditional purification techniques; the superior stability of ketene N,O-acetals compared to ketene O,O-acetals is presumably due to increased aromaticity of the indole and pyridine structures. The presented method overcomes typical problems associated with alkyl ketene acetal synthesis as reported products withstood workup and flash column chromatography procedures.

CRISPR-Cas9-mediated 75.5-Mb inversion in maize.

CRISPR-Cas is a powerful double-strand-break technology with wide-ranging applications from gene discovery to commercial product development. Thus far, this tool has been almost exclusively used for gene knockouts and deletions, with a few examples of gene edits and targeted gene insertions. Here, we demonstrate the application of CRISPR-Cas9 technology to mediate targeted 75.5-Mb pericentric inversion in chromosome 2 in one of the elite maize inbred lines from Corteva Agriscience. This inversion unlocks a large chromosomal region containing substantial genetic variance for recombination, thus providing opportunities for the development of new maize varieties with improved phenotypes.

CRISPR-Cas9 Editing in Maize: Systematic Evaluation of Off-target Activity and Its Relevance in Crop Improvement.

CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits has evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in a complex plant genome. Our approach pairs computational prediction with genome-wide biochemical off-target detection followed by validation in maize plants. Our results reveal high frequency (up to 90%) on-target editing with no evidence of off-target cleavage activity when guide RNAs were bioinformatically predicted to be specific. Predictable off-target edits were observed but only with a promiscuous guide RNA intentionally designed to validate our approach. Off-target editing can be minimized by designing guide RNAs that are different from other genomic locations by at least three mismatches in combination with at least one mismatch occurring in the PAM proximal region. With well-designed guides, genetic variation from Cas9 off-target cleavage in plants is negligible, and much less than inherent variation.

Biomimetic composites by surface-initiated polymerization of cyclic lactones at anorganic bone: preparation and in vitro evaluation of osteoblast and osteoclast competence.

Biomimetic composites were constructed using anorganic bone to initiate the polymerization of cyclic lactones. The resulting anorganic bone/polylactone composites preserve the inorganic structure and the mechanical properties of the original bone. Thermal conditions used to prepare the anorganic bone were shown to control the surface functionalities, surface area, and crystallinity, all of which influence the rates of subsequent polymerizations. Thermal pretreatment of anorganic bone was examined as a function of time and temperature, ranging from 400°C to 800°C. Polymerization rates of different monomers were also compared. Additionally, in vitro evaluations of anorganic bone/poly-L-lactide and anorganic bone/polyglycolide composites for osteoblast and osteoclast competence suggest that these composites are good candidates for potential in vivo use, since both composites promoted osteoblast differentiation. The anorganic bone/poly-L-lactide composite also promoted osteoclast differentiation.

Artificial chromosome formation in maize (Zea mays L.).

We report on the construction of maize minichromosomes using shuttle vectors harboring native centromeric segments, origins of replication, selectable marker genes, and telomeric repeats. These vectors were introduced into scutellar cells of maize immature embryos by microprojectile bombardment. Several independent transformation events were identified containing minichromosomes in addition to the normal diploid complement of 20 maize chromosomes. Immunostaining indicated that the minichromosomes recruited centromeric protein C, which is a specific component of the centromere/kinetochore complex. Minichromosomes were estimated to be 15-30 Mb in size based on cytological measurements. Fluorescent in situ hybridization (FISH) showed that minichromosomes contain the centromeric, telomeric, and exogenous unique marker sequences interspersed with maize retrotransposons. Minichromosomes were detected for at least a year in actively dividing callus cultures, providing evidence for their stability through numerous cell cycles. Plants were regenerated and minichromosomes were detected in root tips, providing confirmation of their normal replication and transmission during mitosis and through organogenesis. Assembly of maize artificial chromosomes may provide a tool to study centromere function and a foundation for developing new high capacity vectors for plant functional genomics and breeding.

Asymmetric Synthesis of 1,2-dioxanes: Approaches to the Peroxyplakoric Acids.

The stereospecific intramolecular alkylation of a hydroperoxyacetal provides the basis for the first asymmetric synthesis of the dioxane propionate core of the peroxyplakorates. Chemoselective hydrometallation of an alkyne in the presence of a peroxide is used to introduce a synthon for the polyunsaturated side chains of the peroxyplakorates. The route suggests a general solution for the 1,2-dioxane unit in many peroxide natural products.

Fragmentation of carbonyl oxides by N-oxides: an improved approach to alkene ozonolysis.

[Structure: see text] Ozonolysis of alkenes in the presence of amine N-oxides results in the direct formation of aldehydes. This reaction, which appears to involve an unprecedented trapping and fragmentation of the short-lived carbonyl oxide intermediates, avoids the hazards associated with generation and isolation of ozonides or other peroxide products.

Light-response quantitative trait loci identified with composite interval and eXtreme array mapping in Arabidopsis thaliana.

Genetic analysis of natural variation in ecotypes of Arabidopsis thaliana can facilitate the discovery of new genes or of allelic variants of previously identified genes controlling physiological processes in plants. We mapped quantitative trait loci (QTL) for light response in recombinant inbred lines (RILs) derived from the Columbia and Kashmir accessions via two methods: composite interval mapping and eXtreme array mapping (XAM). After measuring seedling hypocotyl lengths in blue, red, far-red, and white light, and in darkness, eight QTL were identified by composite interval mapping and five localized near photoreceptor loci. Two QTL in blue light were associated with CRY1 and CRY2, two in red light were near PHYB and PHYC, and one in far-red light localized near PHYA. The RED2 and RED5 QTL were verified in segregating lines. XAM was tested for the identification of QTL in red light with pools of RILs selected for extreme phenotypes. Thousands of single feature polymorphisms detected by differential DNA hybridized to high-density oligo-nucleotide arrays were used to estimate allele frequency differences between the pools. The RED2 QTL was identified clearly; differences exceeded a threshold of significance determined by simulations. The sensitivities of XAM to population type and size and genetic models were also determined by simulation analysis.