Indigenous-led precision public health: a new starting point

Precision public healthcare has been applied to bring about positive change, narrowing the gap in healthcare inequity for Aboriginal peoples. Three such examples include the Mappa, Lyfe Languages, and Pilbra Faces projects, which were all developed through engagement and codesign with Indigenous Australians and each meet a distinct critical need. The Mappa project offers patients and healthcare providers with the necessary geographical information to navigate and maximally utilize available healthcare services. Lyfe Languages is a community driven translational tool that empowers indigenous languages in healthcare. The Pilbara Faces project aims to create a database of clinical measurements enabling better disease diagnosis and monitoring. These three projects have been integrated into a multi-faceted precision public health program, the Healthy Pilbara Project Initiative, acting synergistically to improve the lives of Aboriginal peoples living in Western Australia

Colloquium: Mechanical formalisms for tissue dynamics

The understanding of morphogenesis in living organisms has been renewed by tremendous progressin experimental techniques that provide access to cell-scale, quantitative information both on theshapes of cells within tissues and on the genes being expressed. This information suggests that ourunderstanding of the respective contributions of gene expression and mechanics, and of their crucialentanglement, will soon leap forward. Biomechanics increasingly benefits from models, which assistthe design and interpretation of experiments, point out the main ingredients and assumptions, andultimately lead to predictions. The newly accessible local information thus calls for a reflectionon how to select suitable classes of mechanical models. We review both mechanical ingredientssuggested by the current knowledge of tissue behaviour, and modelling methods that can helpgenerate a rheological diagram or a constitutive equation. We distinguish cell scale ("intra-cell")and tissue scale ("inter-cell") contributions. We recall the mathematical framework developpedfor continuum materials and explain how to transform a constitutive equation into a set of partialdifferential equations amenable to numerical resolution. We show that when plastic behaviour isrelevant, the dissipation function formalism appears appropriate to generate constitutive equations;its variational nature facilitates numerical implementation, and we discuss adaptations needed in thecase of large deformations. The present article gathers theoretical methods that can readily enhancethe significance of the data to be extracted from recent or future high throughput biomechanicalexperiments.Comment: 33 pages, 20 figures. This version (26 Sept. 2015) contains a few corrections to the published version, all in Appendix D.2 devoted to large deformation

Structural investigation of RNA-guided nuclease proteins

RNA-guided nuclease proteins exist in all three domains of life and perform a diverse array of essential cellular functions. In eukaryotes, RNA-guided Argonaute (Ago) proteins have garnered significant interest due to their role as the core protein in RNA interference (RNAi). Although the function of prokaryotic Argonautes (pAgos) remains elusive, recent studies suggest that pAgos may function as a novel form of host-defense through cleavage of foreign genetic elements. Prokaryotes have also evolved a diverse set of CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems as a form of adaptive immunity. Similar to Ago proteins, CRISPR-Cas systems utilize noncoding RNA-directed nuclease proteins for recognition and cleavage of invasive nucleic acid. Structural investigation of Argonaute and CRISPR-Cas nuclease proteins provides insight into their targeting mechanisms, assists in comparing these two immune systems, and aids in repurposing these enzymes for technological applications.We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-hydroxylated guide RNAs rather than the 5′-phosphorylated guides used by all known Argonautes. A 2.0-Å resolution crystal structure of an MpAgo–RNA complex reveals a guide strand binding site comprising residues that block 5′ phosphate interactions. A 3.2-Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA target advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9 of type II CRISPR-Cas systems, providing an efficient off-switch for Cas9-based applications. However, the mechanisms and specificities of these Acrs remain unknown. Here we show that AcrIIC1, the most diverse of the known Acr proteins, blocks DNA cutting by multiple divergent Cas9 homologs. A 1.5-Å resolution crystal structure of an AcrIIC1-Cas9 HNH domain complex revealed that AcrIIC1 binding occludes the HNH active site and blocks Cas9 conformational changes required for enzyme activity, halting cleavage on both strands of the target DNA. This mechanism functions in cell-based assays to block the genome editing activity of distinct Cas9 proteins and explains how a single protein has evolved broad inhibitory activity of CRISPR-Cas9

DNA recognition by an RNA-guided bacterial Argonaute.

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5'-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5' region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins

Conformational changes between MpAgo binary and ternary complexes.

<p>(A) A transparent cartoon representation of MpAgo bound to guide RNA only (PDB ID: 5I4A) with vector arrows, generated using PyMol, indicating conformational changes of MpAgo upon target binding. Black arrows represent the vector direction of the Linker L2, PAZ domain, and N domain away from the C-lobe. (B) The guide RNA (blue) from the MpAgo binary complex is overlaid with the guide RNA (orange) from the MpAgo ternary complex after alignment of the PIWI domains from the two structures. The black arrows show direction of conformational changes of the guide RNA upon target DNA (transparent red) binding.</p

DNA recognition by an RNA-guided bacterial Argonaute

<div><p>Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium <i>Marinitoga piezophila</i> (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.</p></div