Plasmid targeting and destruction by the DdmDE bacterial defence system.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes.

Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.

Piezoelectric Ceramics of the (1 - x)Bi0.50Na0.50TiO3-xBa0.90Ca0.10TiO3 Lead-Free Solid Solution: Chemical Shift of the Morphotropic Phase Boundary, a Case Study for x = 0.06.

Research and development of lead-free piezoelectric materials are still the hottest topics in the field of piezoelectricity. One of the most promising lead-free family of compounds to replace lead zirconate-titanate for actuators is that of Bi0.50Na0.50TiO3 (BNT) based solid solutions. The pseudo-binary (1 - x)Bi0.50Na0.50TiO3-xBa1 - yCayTiO3 system has been proposed for high temperature capacitors and not yet fully explored as piezoelectric material. In this work, the solid solution with x = 0.06 and y = 0.10 was obtained by two different synthesis routes: solid state and Pechini, aiming at using reduced temperatures, both in synthesis (<800 °C) and sintering (<1150 °C), while maintaining appropriated piezoelectric performance. Crystal structure, ceramic grain size, and morphology depend on the synthesis route and were analyzed by X-ray diffraction, together with scanning and transmission electron microscopy. The effects of processing and ceramic microstructure on the structural, dielectric, ferroelectric, and piezoelectric properties were discussed in terms of a shift of the Morphotropic Phase Boundary, chemically induced by the synthesis route.

Towards Lead-Free Piezoceramics: Facing a Synthesis Challenge.

The search for electroceramic materials with enhanced ferro-pyro-piezoelectric properties and revealing the perovskite type structure has been the objective of a significant number of manuscripts reported in the literature. This has been usually carried out by proposing the synthesis and processing of new compounds and solid solution series. In this work, several methods to obtain ferro-pyro-piezoelectric families of materials featuring the well-known ABO3 perovskite structure (or related) such as BaTiO3, Ba1-xCaxTi1-yZryO3, (Bi0.5Na0.5)TiO3, (K0.5Na0.5)NbO3 and their solid solutions with different cations either in the A or B positions, are presented. For this kind of materials, the challenge for obtaining a single phase compound with a specific grain size and morphology and, most importantly, with the adequate stoichiometry, will also be discussed. The results reviewed herein will be discussed in terms of the tendency of working with softer conditions, i.e., lower temperature and shorter reaction times, also referred to as soft-chemistry.

Perfil lipídico por trimestres de gestación en una población de mujeres colombianas / Quarterly lipid profiles in a population of pregnant Colombian women

Introducción: el embarazo se acompaña de cambios en el metabolismo materno de las lipoproteínas para satisfacer las demandas nutricionales del feto. El objetivo de este trabajo fue determinar el comportamiento del perfil lipídico durante los diferentes trimestres en una población de gestantes colombianas. Metodología: el colesterol total, las lipoproteínas y los triglicéridos fueron estudiados en 422 mujeres durante los tres trimestres del embarazo. Colesterol, triglicéridos y lipoproteínas fueron medidos en suero por métodos enzimáticos. Resultados: los triglicéridos (128,2±85,8; 238,6±130,2; 168,7 mg/dL), el colesterol total (180,3±70,8; 235,8± 72,5; 269,9±107,5 mg/dL), las lipoproteínas de muy baja densidad (25,6±17,2; 47,5±25,8; 59,9±31,7 mg/dL) y las lipoproteínas de baja densidad (123,8±69,2; 158,3±77,6; 185±102,6 mg/dL), aumentaron trimestre a trimestre, mientras las lipoproteínas de alta densidad, descendieron (31,02±13,4; 30,04±12,8; 25,8±10,3 mg/dL). Las diferencias en las variables fueron estadísticamente significativas trimestre a trimestre (todas las p < 0,001). Conclusiones: estos resultados muestras cambios metabólicos en el perfil lipídico durante el embarazo, los datos pueden ser de interés médico para hacer seguimiento a aquellas gestantes con perfil lipídico alterado, diabetes gestacional, hipertensión u otras enfermedades relacionadas.