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"Walker, John"
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Structure of the dimeric ATP synthase from bovine mitochondria
The structure of the dimeric ATP synthase from bovine mitochondria determined in three rotational states by electron cryo-microscopy provides evidence that the proton uptake from the mitochondrial matrix via the proton inlet half channel proceeds via a Grotthus mechanism, and a similar mechanism may operate in the exit half channel. The structure has given information about the architecture and mechanical constitution and properties of the peripheral stalk, part of the membrane extrinsic region of the stator, and how the action of the peripheral stalk damps the side-to-side rocking motions that occur in the enzyme complex during the catalytic cycle. It also describes wedge structures in the membrane domains of each monomer, where the skeleton of each wedge is provided by three α-helices in the membrane domains of the b-subunit to which the supernumerary subunits e, f, and g and the membrane domain of subunit A6L are bound. Protein voids in the wedge are filled by three specifically bound cardiolipin molecules and two other phospholipids. The external surfaces of the wedges link the monomeric complexes together into the dimeric structures and provide a pivot to allow the monomer–monomer interfaces to change during catalysis and to accommodate other changes not related directly to catalysis in the monomer–monomer interface that occur in mitochondrial cristae. The structure of the bovine dimer also demonstrates that the structures of dimeric ATP synthases in a tetrameric porcine enzyme have been seriously misinterpreted in the membrane domains.
Journal Article
Break the rules! : the six counter-conventional mindsets of entrepreneurs that can help anyone change the world
\"The entrepreneurial path, whether for those who lead the journey or those who participate therein, is typically rocky at best, and the ones who make it through successful usually think about failure and challenge different from the rest of us. Drawing on two decades of research into what makes entrepreneurs entrepreneurs, and how they differ from the rest of us, Break the Rules! builds a compelling argument that successful entrepreneurs exhibit one or more of six counter-conventional mindsets that allow them to deal with, skate around, or overcome the daunting obstacles that stand in their way and take advantage of opportunities that present themselves along the entrepreneurial path. Surprisingly, however, these mindsets run counter to the conventional wisdom that's typically found in large and well-established companies. They fly in the face of what's taught in business schools about strategy, core competencies, target marketing, financing and more. The good news is this: each of the six break-the-rules mindsets can be learned, practiced, and ultimately built upon to assemble high-performing entrepreneurial teams and create thriving businesses that grow and prosper\"-- Provided by publisher.
Book
Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
Large sequences are integrated site specifically into the human genome without double-strand DNA cleavage.
Journal Article
Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression
Background
Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the
Tspan2
promoter.
Results
Quantitative RT-PCR showed loss of
Tspan2
mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the
Tspan2
CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the
Tspan2
CArG box displayed similar cell-specific loss of
Tspan2
mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of
Tspan2
in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders.
Conclusions
PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
Journal Article
In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels
Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting
PCSK9
, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.
Base editors are effective and safe for cholesterol reduction in non-human primates.
Journal Article
Advances in abscission signaling
A mechanistic overview of abscission signaling is presented to provide an easy entry point into the exciting field of research on how plants control shedding of organs.
Abstract
Abscission is a process in plants for shedding unwanted organs such as leaves, flowers, fruits, or floral organs. Shedding of leaves in the fall is the most visually obvious display of abscission in nature. The very shape plants take is forged by the processes of growth and abscission. Mankind manipulates abscission in modern agriculture to do things such as prevent pre-harvest fruit drop prior to mechanical harvesting in orchards. Abscission occurs specifically at abscission zones that are laid down as the organ that will one day abscise is developed. A sophisticated signaling network initiates abscission when it is time to shed the unwanted organ. In this article, we review recent advances in understanding the signaling mechanisms that activate abscission. Physiological advances and roles for hormones in abscission are also addressed. Finally, we discuss current avenues for basic abscission research and potentially lucrative future directions for its application to modern agriculture.
Journal Article