Electrochemical nanoreactor provides a comprehensive view of isocitrate dehydrogenase cancer-drug kinetics

The ability to control enzyme cascades entrapped in a nanoporous electrode material (the “Electrochemical Leaf”, e-Leaf) has been exploited to gain detailed kinetic insight into the mechanism of an anti-cancer drug. Ivosidenib, used to treat acute myeloid leukemia, acts on a common cancer-linked variant of isocitrate dehydrogenase 1 (IDH1 R132H) inhibiting its “gain-of-function” activity—the undesired reduction of 2-oxoglutarate (2OG) to the oncometabolite 2-hydroxyglutarate (2HG). The e-Leaf quantifies the kinetics of IDH1 R132H inhibition across a wide and continuous range of conditions, efficiently revealing factors underlying the inhibitor residence time. Selective inhibition of IDH1 R132H by Ivosidenib and another inhibitor, Novartis 224, is readily resolved as a two-stage process whereby initial rapid non-inhibitory binding is followed by a slower step to give the inhibitory complex. These kinetic features are likely present in other allosteric inhibitors of IDH1/2. Such details, essential for understanding inhibition mechanisms, are not readily resolved in conventional steady-state kinetics or by techniques that rely only on measuring binding. Extending the new method and analytical framework presented here to other enzyme systems will be straightforward and should rapidly reveal insight that is difficult or often impossible to obtain using other methods

NADP(H)-dependent biocatalysis without adding NADP(H)

Significance Within cells, enzymes and cofactors catalyzing multistep processes (cascades) are often confined together—either in enclosures (e.g., organelles) or via physical association (metabolons). Nanoconfinement, offering potential general advantages for catalysis, can also be achieved by loading enzymes and their exchangeable cofactors into a porous, electrically conducting inorganic material, thereby enabling catalysis to be channeled, energized, and investigated electrochemically. Such nanoconfinement enables a cascade comprising electroactive ferredoxin NADP+ reductase and isocitrate dehydrogenase to be active for days, catalyzing exhaustive oxidation of bulk isocitrate by recycling trapped NADP(H) carried in on isocitrate dehydrogenase. Nanoconfinement massively increases the efficiency of cofactor-dependent cascade catalysis and has conceptual relevance for prebiotic evolution where complex organic molecules might have formed in gaps and cracks of minerals. Abstract Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP+/NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP+ reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo—the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 102 to 103-fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the “gain of function” reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers

Interactive biocatalysis achieved by driving enzyme cascades inside a porous conducting material

An emerging concept and platform, the electrochemical Leaf (e-Leaf), offers a radical change in the way tandem (multi-step) catalysis by enzyme cascades is studied and exploited. The various enzymes are loaded into an electronically conducting porous material composed of metallic oxide nanoparticles, where they achieve high concentration and crowding – in the latter respect the environment resembles that found in living cells. By exploiting efficient electron tunneling between the nanoparticles and one of the enzymes, the e-Leaf enables the user to interact directly with complex networks, rendering simultaneous the abilities to energise, control and observe catalysis. Because dispersion of intermediates is physically suppressed, the output of the cascade – the rate of flow of chemical steps and information – is delivered in real time as electrical current. Myriad enzymes of all major classes now become effectively electroactive in a technology that offers scalability between micro-(analytical, multiplex) and macro-(synthesis) levels. This Perspective describes how the e-Leaf was discovered, the steps in its development so far, and the outlook for future research and applications

A new record of Percursaria percursa (Ulvaceae, Ulvales) on the North Island, New Zealand

The filamentous green alga Percursaria percursa (Ulvaceae, Ulvales) was recorded for the first time on the North Island of New Zealand at mokoroa Estuary, Tauranga Harbour. This species is previously known within New Zealand from only two records, both from the South Island. In Tauranga Harbour, this species was restricted to anoxic estuarine sediments where mangrove forests had been mulched, and mulchate left in situ. Percursaria percursa was found intertwined with Ulva spp. and Rhizoclonium spp. Surveys of other North and South Island estuaries suggest that this alga, although occurring as part of nuisance green algal blooms in Tauranga Harbour, has only colonized human-impacted locations, and has not yet been observed in natural' estuarine ecosystems in New Zealand. As this species was found intertwined with other mat-forming filamentous green algae, it can easily be misidentified in the field, leading to both over- and under-reporting of species occurrence

Justice and conservation: The need to incorporate recognition

In light of the Aichi target to manage protected areas equitably by 2020, we ask how the conservation sector should define justice. We focus in particular on ‘recognition’, because it is the least well understood aspect of environmental justice, and yet highly relevant to conservation because of its concern with respect for local knowledge and cultures. In order to explore the meaning of recognition in the conservation context, we take four main steps. First, we identify four components of recognition to serve as our analytical framework: subjects of justice, the harms that constitute injustice, the mechanisms that produce injustices, and the responses to alleviate these. Secondly, we apply this framework to explore four traditions of thinking about recognition: Hegelian inter-subjectivity, critical theory, southern decolonial theory, and the capabilities approach. Thirdly, we provide three case studies of conservation conflicts highlighting how different theoretical perspectives are illustrated in the claims and practices of real world conservation struggles. Fourthly, we finish the paper by drawing out some key differences between traditions of thinking, but also important areas of convergence. The convergences provide a basis for concluding that conservation should look beyond a distributive model of justice to incorporate concerns for social recognition, including careful attention to ways to pursue equality of status for local conservation stakeholders. This will require reflection on working practices and looking at forms of intercultural engagement that, for example, respect alternative ways of relating to nature and biodiversity

Importance of the Active Site "Canopy" Residues in an O_2-Tolerant [NiFe]-Hydrogenase

The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a “canopy” above the bimetallic center, closest to the site at which exogenous agents CO and O_2 interact, substrate H_2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H_2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N–metal distance of 4.4 Å): its replacement with lysine lowers the H_2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H_2/D_2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H_2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H_2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O_2 tolerance by stabilizing the oxidized resting Ni^(III)–OH state (“Ni-B”). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe

An Estimation of the Entomological Inoculation Rate for Ifakara: A Semi-Urban Area in a Region of Intense Malaria Transmission in Tanzania.

An entomological study on vectors of malaria and their relative contribution to Plasmodium falciparum transmission in the semi-urban area of Ifakara, south-eastern Tanzania, was conducted. A total of 32 houses were randomly sampled from the area and light trap catches (LTC) performed in one room in each house every 2 weeks for 1 year. A total of 147 448 mosquitoes were caught from 789 LTC; 26 134 Anopheles gambiae s.l., 615 A. funestus, 718 other anophelines and 119 981 culicines. More than 60% of the total A. gambiae s.l. were found in five (0.6%) LTCs, with a maximum of 5889 caught in a single trap. Of 505 A. gambiae s.l. speciated by polymerase chain reaction, 91.5% were found to be A. arabiensis. Plasmodium falciparum sporozoite enzyme-linked immunosorbent assay tests were performed on 10 108 anopheles mosquitoes and 39 (0.38%) were positive. Entomological inoculation rate (EIR) estimates were generated using a standard method and an alternative method that allows the calculation of confidence intervals based on a negative binomial distribution of sporozoite positive mosquitoes. Overall EIR estimates were similar; 31 vs. 29 [95% confidence interval (CI): 19, 44] infectious bites per annum, respectively. The EIR ranged from 4 (95% CI: 1, 17) in the cool season to 108 (95% CI: 69, 170) in the wet season and from 54 (95% CI: 30, 97) in the east of the town to 15 (95% CI: 8, 30) in the town centre. These estimates show large variations over short distances in time and space. They are all markedly lower than those reported from nearby rural areas and for other parts of Tanzania

Commentary: Use of BACTRAC Proteomic Database-Uromodulin Protein Expression during Ischemic Stroke

Introduction: Uromodulin (UMOD) is a glycoprotein expressed by the epithelial cells of the thick ascending limb of Henle\u27s loop in the kidney. Research has shown that increased uromodulin expression may be associated with lower risk of cardiovascular disease in adults. Utilizing the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) (clinicaltrials.gov NCT03153683), a continuously enrolling tissue bank, we aimed to examine the associations between serum uromodulin, age, and high BMI (BMI\u3e 25) and its relationship to stroke in patients. Methods: Arterial blood distal and proximal to the thrombus was collected during a thrombectomy procedure using the BACTRAC protocol and sent to Olink (Boston, MA) to determine proteomic expression via proximity extension assay. Uromodulin expression was recorded and analyzed using two tailed T-tests and linear regressions. Results: The relationship between systemic and intracranial uromodulin, age, high BMI and hypertension were assessed. Systemic and intracranial uromodulin decreased with age (p\u3c 0.0001 and r²= 0.343, p= 0.0416 and r²= 0.102) respectively. Systemic uromodulin expression increased with BMI\u3e 25 (p= 0.014). Presence of hypertension decreased uromodulin’s expression systemically (p= 0.018) and intracranially (p= 0.007). Conclusions: Uromodulin was increased significantly in overweight patients, decreased significantly in older patients, and decreased in patients with hypertension. The increase in uromodulin in people with high BMI could be a protective reaction of the kidney to worsening conditions that make ischemic stroke more likely, with a goal of delaying dangerous outcomes. The decreased expression of uromodulin in older adults could be associated with the decline of general kidney function that accompanies aging. Hypertension can contribute to an AKI by decreasing perfusion to the kidney, therefore decreasing kidney function and uromodulin production. Further analyses are needed to understand the role of uromodulin following ischemic stroke

Indymedia and the long story of rebellion against neoliberal capitalism

Indymedia was born of the anti-globalisation movement of the late 1990s and quickly spread to become a social movement in its own right. This article reflects on how prescient the claims of the anti-globalisation movement have proven to be and how 20 years ago Indymedia and the anti-globalisation movement predicted there was trouble ahead and that neoliberalism was a central part of the problem. It notes how a history of struggle and protest emanating from the days of Indymedia has developed over time building a counter-politics that is becoming ever wiser about the multiple intersectional harms of capitalism and ever more sophisticated in its political response. The challenge is what comes next

Natural and synthetic 2-oxoglutarate derivatives are substrates for oncogenic variants of human isocitrate dehydrogenase 1 and 2

Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutarate (2OG) to (2R)-hydroxyglutarate. However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3- and C4-alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of WT IDH1/2. Absorbance-based, NMR, and electrochemical assays were employed to monitor WT IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of WT IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates than 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl–, 3-butyl–, and 3-benzyl–substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts