Design principles for site-selective hydroxylation by a Rieske oxygenase

Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C–H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions. We present high resolution crystal structures of SxtT and GxtA with the native β-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site. Ultimately, this structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C–H hydroxylation event. Substitution of these residues produces a SxtT variant that is fully adapted to exhibit the non-native site-selectivity and substrate scope of GxtA. Importantly, we also found that these selectivity regions are conserved in other structurally characterized Rieske oxygenases, providing a framework for predictively repurposing and manipulating Rieske oxygenases as biocatalysts.

Transition Therapy: Tackling the Ecology of Tumor Phenotypic Plasticity

Phenotypic switching in cancer cells has been found to be present across tumor types. Recent studies on Glioblastoma report a remarkably common architecture of four well-defined phenotypes coexisting within high levels of intra-tumor genetic heterogeneity. Similar dynamics have been shown to occur in breast cancer and melanoma and are likely to be found across cancer types. Given the adaptive potential of phenotypic switching (PHS) strategies, understanding how it drives tumor evolution and therapy resistance is a major priority. Here we present a mathematical framework uncovering the ecological dynamics behind PHS. The model is able to reproduce experimental results, and mathematical conditions for cancer progression reveal PHS-specific features of tumors with direct consequences on therapy resistance. In particular, our model reveals a threshold for the resistant-to-sensitive phenotype transition rate, below which any cytotoxic or switch-inhibition therapy is likely to fail. The model is able to capture therapeutic success thresholds for cancers where nonlinear growth dynamics or larger PHS architectures are in place, such as glioblastoma or melanoma. By doing so, the model presents a novel set of conditions for the success of combination therapies able to target replication and phenotypic transitions at once. Following our results, we discuss transition therapy as a novel scheme to target not only combined cytotoxicity but also the rates of phenotypic switching.

Emergence of order in condensates composed of multi-valent, multi-domain proteins

Many RNA-binding proteins (RBPs) that assemble into membraneless organelles, have a common architecture including disordered prion-like domain (PLD) and folded RNA-binding domain (RBD). An enrichment of PLD within the condensed phase gives rise to formation, on longer time scales, amyloid-like fibrils (aging). In this study, we employ coarse-grained Langevin dynamics simulations to explore the physical basis for the structural diversity in condensed phases of multi-domain RBPs. We discovered a highly cooperative first order transition between disordered (liquid-like) structures and an ordered (solid-like) phase whereby chains of PLD organize in fibrils with high nematic orientational order. Cooperativity of this liquid-solid transition makes fibril formation highly malleable to mutations or post-translational modifications. An interplay between homo-domain (PLD-PLD) and hetero-domain (PLD-RBD) interactions results in variety of structures with distinct spatial architectures. Our results provide a mechanistic understanding of how multi-domain RBPs could form assemblies with distinct structural and, potentially, material properties.

One Phase Well OPW : Unlock Shallow Reservoir Efficient and Economically by Eliminating Surface Casing

Tunu is a mature giant gas and condensate field locate in Mahakam Delta, East Kalimantan, Indonesia. The field has been in development for almost 30 years and currently has been considered as a mature field where to put a state of an economic well has become more challenging nowadays. The deeper zone of Tunu has no longer been considered as profitable to be produced and the current focus is more on the widespread shallow gas pocket located in the much shallower zone of Tunu. One phase well is architecture without 9-5/8" surface casing. OPW is one-section drilling using a diverter mode from surface to TD without using BOP. Historical for OPW is began from 2018, where drilling reservoir section using diverter mode in two-phase. In 2018 also succeeded in performing perforated surface casing. Due successfully in drilling operation using diverter and perforated surface casing, in 2019 drilling trials for OPW were carried out. Until now, the OPW architecture has become one of the common architecture used in drilling operations as an optimization effort. Until December 2020 PHM has completed 15+ OPW wells. A general comparison of OPW and SLA well is at the cost of constructing a well of approximately 200,000 - 300,000 US$. The disadvantages of OPW wells are more expensive in the mud and cement section when using a 9-1/2" hole, but in terms of the duration, OPW drilling time is more efficient up to 2-3 days. If viewed from the integrity of the OPW wells, from 15 OPW wells that have been completed, only 2 of them have SCP.

Pushing the Boundaries Through Successful Delivery of Highly Challenging ERD Well in Offshore East Java, Indonesia

With the far-reaching reservoir target coupled with other surface constraint including fix well slot coordinate and pre-determined conductor size, the longest well with 2.5 ERD Index in Offshore East Java was pioneered. The team has big task in hand to ensure all aspect of ERD well engineering and construction are being addressed properly within the fast-paced time frame given. One of the approaches strategized by the team is to split the high angle big hole size long interval of middle section into two casing string which was not the common architecture applied in the other offset wells. The objective was to ensure that the middle section of the well will not be compromised and avoid complication in the deeper section of the well. Worth to mention that the middle section consists combination of challenging lithology that deserve the right solution to avoid unwanted problem. There are highly kartsitified carbonate formation, shale and sand interbedded formation, and thick time dependent shale formation. To mitigate the challenges previously mentioned, intermediate section which is normally drilled and isolated with 17-1/2" hole × 13-3/8" casing in previous wells, now separated into two sections which require enlargement: 17-1/2" to 20" and 14-3/4" to 17-1/2". This paper focuses on 14-3/4" × 17-1/2" which is the most challenging underreaming operation in this well and the first of its kind in this field application. Adding to the fact that the inclination reach 75 degree in this section, SOBM and RSS BHA are deployed to mitigate the torque and drag issue. State of the art modelling tool is also used by team to define effectively match BHA and drilling parameter with minimal lateral vibration and stick slip for this section Apart from drilling stage, the enlarged hole size requires a condition to have uncommon casing size and specification, 16" intermediate semi flush liner connection and 13-3/8" full flush intermediate casing connection to ensure sufficient annular area and less restriction during running to bottom. The relentless effort to secure one the most critical ERD well construction phase has really paid off by allowing the next phase of operation to be executed as per plan thus assuring the overall well objective is met.

Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex

We sequenced maxicircles from T. cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T. cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T. cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T. cruzi is a complex of species composed by group 1 that contains clades A (TcI), B (TcIII)and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist in T. cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T. cruzi.

A mechatronic shape display based on auxetic materials

Shape displays enable people to touch simulated surfaces. A common architecture of such devices uses a mechatronic pin-matrix. Besides their complexity and high cost, these matrix displays suffer from sharp edges due to the discreet representation which reduces their ability to render a large continuous surface when sliding the hand. We propose using an engineered auxetic material actuated by a smaller number of motors. The material bends in multiple directions, feeling smooth and rigid to touch. A prototype implementation uses nine actuators on a 220 mm square section of material. It can display a range of surface curvatures under the palm of a user without aliased edges. In this work we use an auxetic skeleton to provide rigidity on a soft material and demonstrate the potential of this class of surface through user experiments.

Arquitectura común: aprendiendo de los habitantes y sus prácticas cotidianas

Over the last decade it has been possible to see growing ties between several architectural groups and urban communities located mainly in territories marked by decay, informality, and inequality. This process has generated a progressive recognition of the value that the daily practices of inhabitants and their communities have in the production of new ways of living, which poses new challenges for the development of the area. Starting from a description and analysis of a neighborhood improvement experience, self-managed by the inhabitants, this article addresses this challenge by proposing the formation of a common architecture, understood as a process of production of spatiality, supported by communalization dynamics that are open to new learnings that incorporate the everyday knowledge of the inhabitants and their communities.

Calcium Release Channels: Structure and Function of IP3 Receptors and Ryanodine Receptors

Ca2+-release channels are giant membrane proteins that control the release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. The two members, ryanodine receptors (RyRs) and inositol-1,4,5-trisphosphate Receptors (IP3Rs), are evolutionarily related and are both activated by cytosolic Ca2+. They share a common architecture, but RyRs have evolved additional modules in the cytosolic region. Their massive size allows for the regulation by tens of proteins and small molecules, which can affect the opening and closing of the channels. In addition to Ca2+, other major triggers include IP3 for the IP3Rs, and depolarization of the plasma membrane for a particular RyR subtype. Their size has made them popular targets for study via electron microscopic methods, with current structures culminating near 3Å. The available structures have provided many new mechanistic insights int the binding of auxiliary proteins and small molecules, how these can regulate channel opening, and the mechanisms of disease-associated mutations. They also help scrutinize previously proposed binding sites, as some of these are now incompatible with the structures. Many questions remain around the structural effects of post-translational modifications, additional binding partners, and the higher-order complexes these channels can make in situ. This review summarizes our current knowledge about the structures of Ca2+-release channels and how this informs on their function.