An mRNA-based platform for the delivery of pathogen-specific IgA into mucosal secretions.

Colonization of the gut and airways by pathogenic bacteria can lead to local tissue destruction and life-threatening systemic infections, especially in immunologically compromised individuals. Here, we describe an mRNA-based platform enabling delivery of pathogen-specific immunoglobulin A (IgA) monoclonal antibodies into mucosal secretions. The platform consists of synthetic mRNA encoding IgA heavy, light, and joining (J) chains, packaged in lipid nanoparticles (LNPs) that express glycosylated, dimeric IgA with functional activity in vitro and in vivo. Importantly, mRNA-derived IgA had a significantly greater serum half-life and a more native glycosylation profile in mice than did a recombinantly produced IgA. Expression of an mRNA encoded Salmonella-specific IgA in mice resulted in intestinal localization and limited Peyer's patch invasion. The same mRNA-LNP technology was used to express a Pseudomonas-specific IgA that protected from a lung challenge. Leveraging the mRNA antibody technology as a means to intercept bacterial pathogens at mucosal surfaces opens up avenues for prophylactic and therapeutic interventions.

Therapeutic enzyme engineering using a generative neural network.

Enhancing the potency of mRNA therapeutics is an important objective for treating rare diseases, since it may enable lower and less-frequent dosing. Enzyme engineering can increase potency of mRNA therapeutics by improving the expression, half-life, and catalytic efficiency of the mRNA-encoded enzymes. However, sequence space is incomprehensibly vast, and methods to map sequence to function (computationally or experimentally) are inaccurate or time-/labor-intensive. Here, we present a novel, broadly applicable engineering method that combines deep latent variable modelling of sequence co-evolution with automated protein library design and construction to rapidly identify metabolic enzyme variants that are both more thermally stable and more catalytically active. We apply this approach to improve the potency of ornithine transcarbamylase (OTC), a urea cycle enzyme for which loss of catalytic activity causes a rare but serious metabolic disease.

One pot facile transformation of CO2 to an unusual 3-D nano-scaffold morphology of carbon.

An electrosynthesis is presented to transform CO2 into an unusual nano and micron dimensioned morphology of carbon, termed Carbon Nano-Scaffold (CNS) with wide a range of high surface area graphene potential usages including batteries, supercapacitors, compression devices, electromagnetic wave shielding and sensors. Current CNS value is over $323 per milligram. The morphology consists of a series of asymmetric 20 to 100 nm thick flat multilayer graphene platelets 2 to 20 µm long orthogonally oriented in a 3D neoplasticism-like geometry, and appears distinct from the honeycomb, foam, or balsa wood cell structures previously attributed to carbon scaffolds. The CNS synthesis splits CO2 by electrolysis in molten carbonate and has a carbon negative footprint. It is observed that transition metal nucleated, high yield growth of carbon nanotubes (CNTs) is inhibited in electrolytes containing over 50 wt% of sodium or 30 wt% of potassium carbonate, or at electrolysis temperatures less than 700 °C. Here, it is found that a lower temperature of synthesis, lower concentrations of lithium carbonate, and higher current density promotes CNS growth while suppressing CNT growth. Electrolyte conditions of 50 wt% sodium carbonate relative to lithium carbonate at an electrolysis temperature of 670 °C produced over 80% of the CNS desired product at 85% faradaic efficiency with a Muntz brass cathode and an Inconel anode.

Calcium metaborate induced thin walled carbon nanotube syntheses from CO2 by molten carbonate electrolysis.

An electrosynthesis is presented to transform the greenhouse gas CO2 into an unusually thin walled, smaller diameter morphology of Carbon Nanotubes (CNTs). The transformation occurs at high yield and coulombic efficiency of the 4-electron CO2 reduction in a molten carbonate electrolyte. The electrosynthesis is driven by an unexpected synergy between calcium and metaborate. In a pure molten lithium carbonate electrolyte, thicker walled CNTs (100-160 nm diameter) are synthesized during a 4 h CO2 electrolysis at 0.1 A cm-2. At this low current density, CO2 without pre-concentration is directly absorbed by the air (direct air capture) to renew and sustain the carbonate electrolyte. The addition of 2 wt% Li2O to the electrolyte produces thinner, highly uniform (50-80 nm diameter) walled CNTs, consisting of ~ 75 concentric, cylindrical graphene walls. The product is produced at high yield (the cathode product consists of > 98% CNTs). It had previously been demonstrated that the addition of 5-10 wt% lithium metaborate to the lithium carbonate electrolyte boron dopes the CNTs increasing their electrical conductivity tenfold, and that the addition of calcium carbonate to a molten lithium carbonate supports the electrosynthesis of thinner walled CNTs, but at low yield (only ~ 15% of the product are CNTs). Here it is shown that the same electrolysis conditions, but with the addition of 7.7 wt% calcium metaborate to lithium carbonate, produces unusually thin walled CNTs uniform (22-42 nm diameter) CNTs consisting of ~ 25 concentric, cylindrical graphene walls at a high yield of > 90% CNTs.

Recent Advances in Solar Thermal Electrochemical Process (STEP) for Carbon Neutral Products and High Value Nanocarbons.

Climate change represents one of the most important environmental issues of our time. Due to high levels of anthropogenic CO2 emissions, atmospheric CO2 has for the first time ever exceeded 415 ppm and has increased from 315 ppm in 1950. An annual increase in atmospheric CO2 of ∼2 ppm is equal to a net increase of ∼15.6 billion tons of CO2. The combustion of fossil fuels for electricity and transportation is still the main reason accounting for the CO2 accumulation. On the top of that, fossil fuels are widely used in our modern industry for the productions of indispensable social staples. For instance, the millennia old thermal reduction of iron ore by charcoal or baked coal (3C + 2Fe2O3 → 4Fe + 3CO2) continues as the main method for the production of iron. The artificial fertilizer ammonia boosts the global population and is mainly produced from the Haber-Bosch process, in which hydrogen is generated via steam reforming of methane (CH4 + 2H2O → 4H2 + CO2). Sequestration and diminution of CO2 require the development of a portfolio of technologies on (1) efficient and long-term harvesting of renewable energy, that is, solar, not only for electricity but also directly as the energy force in vital chemical processes, wherever possible, (2) carbon-neutral processes to replace current industrial processes that emit vast amounts of CO2, such as iron and ammonia production, and (3) new, low-cost technologies for CO2 capture and conversion with particular interests in the exploration of CO2 as the feedstock for fuels or other valuable chemicals and materials. To this end, we conducted some studies on the sustainable synthesis of ammonia and iron with net-zero CO2 emissions and large-scale CO2 capture and conversion into fuels and high value nanocarbon products via electrolysis in molten salt(s) with the introduction of the Solar Thermal Electrochemical Process (STEP). In STEP, solar UV-visible energy is focused on a photovoltaic device that generates the electricity to drive the electrolysis, while concurrently the solar thermal energy is focused on a second system to generate heat for the electrolysis cell. The utilization of the full spectrum of sunlight in STEP results in a higher solar energy efficiency than other solar conversion processes. STEP has been applied to conduct (1) CO2-free ammonia synthesis from nitrogen and water with the aid of nano-Fe2O3 in a molten hydroxide electrolyte, (2) CO2-free production of iron via electrochemical reduction of iron ore in molten carbonate, (3) CO2 capture and conversion into nanostructured carbon products as well as fuels in molten or mixed molten electrolytes, and (4) organic electrosynthesis of benzoic acid from benzene without overoxidizing into CO2. In this Account, we highlight some recent achievements in these topics and propose that using STEP is a highly efficient strategy for saving energy and, consequently, the environment. STEP is an ideal tool that can theoretically be applied to all endothermic reactions.

Dependence on the Pyrimidine Biosynthetic Enzyme DHODH Is a Synthetic Lethal Vulnerability in Mutant KRAS-Driven Cancers.

Activating KRAS mutations are major oncogenic drivers in multiple tumor types. Synthetic lethal screens have previously been used to identify targets critical for the survival of KRAS mutant cells, but their application to drug discovery has proven challenging, possibly due in part to a failure of monolayer cultures to model tumor biology. Here, we report the results of a high-throughput synthetic lethal screen for small molecules that selectively inhibit the growth of KRAS mutant cell lines in soft agar. Chemoproteomic profiling identifies the target of the most KRAS-selective chemical series as dihydroorotate dehydrogenase (DHODH). DHODH inhibition is shown to perturb multiple metabolic pathways. In vivo preclinical studies demonstrate strong antitumor activity upon DHODH inhibition in a pancreatic tumor xenograft model.

A Robust Multiplex Mass Spectrometric Assay for Screening Small-Molecule Inhibitors of CD73 with Diverse Inhibition Modalities.

CD73/Ecto-5'-nucleotidase is a membrane-tethered ecto-enzyme that works in tandem with CD39 to convert extracellular adenosine triphosphate (ATP) into adenosine. CD73 is highly expressed on various types of cancer cells and on infiltrating suppressive immune cells, leading to an elevated concentration of adenosine in the tumor microenvironment, which elicits a strong immunosuppressive effect. In preclinical studies, targeting CD73 with anti-CD73 antibody results in favorable antitumor effects. Despite initial studies using antibodies, inhibition of CD73 catalytic activity using small-molecule inhibitors may be more effective in lowering extracellular adenosine due to better tumor penetration and distribution. To screen small-molecule libraries, we explored multiple approaches, including colorimetric and fluorescent biochemical assays, and due to some limitations with these assays, we developed a mass spectrometry (MS)-based assay. Only the MS-based assay offers the sensitivity and dynamic range required for screening small-molecule libraries at a substrate concentration close to the Km value of substrate and for evaluating the mode of binding of screening hits. To achieve a throughput suitable for high-throughput screening (HTS), we developed a RapidFire-tandem mass spectrometry (RF-MS/MS)-based multiplex assay. This assay allowed a large diverse compound library to be screened at a speed of 1536 reactions per 40-50 min.

Tracking airborne CO2 mitigation and low cost transformation into valuable carbon nanotubes.

Primary evidence of the direct uptake of atmospheric CO2 and direct transformation into carbon nanotubes, CNTs, is demonstrated through isotopic labeling, and provides a new high yield route to mitigate this greenhouse gas. CO2 is converted directly to CNTs and does not require pre-concentration of the airbone CO2. This C2CNT (CO2 to carbon nanotube) synthesis transforms CO2-gas dissolved in a 750 °C molten Li2CO3, by electrolysis, into O2-gas at a nickel electrode, and at a steel cathode into CNTs or carbon or nanofibers, CNFs. CNTs are synthesized at a 100-fold price reduction compared to conventional chemical vapour deposition, CVD, synthesis. The low cost conversion to a stable, value-added commodity incentivizes CO2 removal to mitigate climate change. The synthesis allows morphology control at the liquid/solid interface that is not available through conventional CVD synthesis at the gas/solid interface. Natural abundance (12)CO2 forms hollow CNTs, while equivalent synthetic conditions with heavier (13)CO2 favours closed core CNFs, as characterized by Raman, SEM and TEM. Production ease is demonstrated by the first synthesis of a pure (13)C multiwalled carbon nanofiber.

Carbon Nanotubes Produced from Ambient Carbon Dioxide for Environmentally Sustainable Lithium-Ion and Sodium-Ion Battery Anodes.

The cost and practicality of greenhouse gas removal processes, which are critical for environmental sustainability, pivot on high-value secondary applications derived from carbon capture and conversion techniques. Using the solar thermal electrochemical process (STEP), ambient CO2 captured in molten lithiated carbonates leads to the production of carbon nanofibers (CNFs) and carbon nanotubes (CNTs) at high yield through electrolysis using inexpensive steel electrodes. These low-cost CO2-derived CNTs and CNFs are demonstrated as high performance energy storage materials in both lithium-ion and sodium-ion batteries. Owing to synthetic control of sp(3) content in the synthesized nanostructures, optimized storage capacities are measured over 370 mAh g(-1) (lithium) and 130 mAh g(-1) (sodium) with no capacity fade under durability tests up to 200 and 600 cycles, respectively. This work demonstrates that ambient CO2, considered as an environmental pollutant, can be attributed economic value in grid-scale and portable energy storage systems with STEP scale-up practicality in the context of combined cycle natural gas electric power generation.

One-Pot Synthesis of Carbon Nanofibers from CO2.

Carbon nanofibers, CNFs, due to their superior strength, conductivity, flexibility, and durability have great potential as a material resource but still have limited use due to the cost intensive complexities of their synthesis. Herein, we report the high-yield and scalable electrolytic conversion of atmospheric CO2 dissolved in molten carbonates into CNFs. It is demonstrated that the conversion of CO2 → CCNF + O2 can be driven by efficient solar, as well as conventional, energy at inexpensive steel or nickel electrodes. The structure is tuned by controlling the electrolysis conditions, such as the addition of trace transition metals to act as CNF nucleation sites, the addition of zinc as an initiator and the control of current density. A less expensive source of CNFs will facilitate its adoption as a societal resource, and using carbon dioxide as a reactant to generate a value added product such as CNFs provides impetus to consume this greenhouse gas to mitigate climate change.

Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

Sungas Instead of Syngas: Efficient Coproduction of CO and H2 with a Single Beam of Sunlight.

The electrolytic coproduction of CO and H2 is achieved from air, water, and a single beam of sunlight rather than from fossil fuels. H2 and CO cosynthesis is driven by a single concentrator photovoltaic to simultaneously drive molten hydroxide and molten carbonate electrolyses. The carbon neutral process captures carbon without the need for the preconcentration of atmospheric carbon dioxide.

Poly(ethylene glycol) as a scaffold for high-affinity open-channel blockers of the mouse nicotinic acetylcholine receptor.

High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1-5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1-5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore.

Advances in understanding the mechanism and improved stability of the synthesis of ammonia from air and water in hydroxide suspensions of nanoscale Fe2O3.

We report a mechanism of electrochemical ammonia (NH3) production via an iron intermediate in which H2 and NH3 are cogenerated by different electron-transfer pathways. Solar thermal can contribute to the energy to drive this synthesis, resulting in a STEP, solar thermal electrochemical process, for NH3. Enhancements are presented to this carbon dioxide (CO2)-free synthesis, which uses suspensions of nano-Fe2O3 in high-temperature hydroxide electrolytes at nickel and Monel electrodes. In a 200 °C molten eutectic Na(0.5)K(0.5)OH electrolyte, the 3 Faraday efficiency per mole of synthesized NH3, η(NH3), increases with decreasing current density, and at j(electrolysis) = 200, 25, 2, and 0.7 mA cm(-2), η(NH3) = 1%, 7%, 37%, and 71%, respectively. At 200 mA cm(-2), over 90% of applied current drives H2, rather than NH3, formation. Lower temperature supports greater electrolyte hydration. At 105 °C in the hydrated Na(0.5)K(0.5)OH electrolyte, η(NH3) increases and then is observed to be highly stable at η(NH3) = 24(+2)%.

RETRACTED: Ammonia synthesis. Ammonia synthesis by N2 and steam electrolysis in molten hydroxide suspensions of nanoscale Fe2O3.

The Haber-Bosch process to produce ammonia for fertilizer currently relies on carbon-intensive steam reforming of methane as a hydrogen source. We present an electrochemical pathway in which ammonia is produced by electrolysis of air and steam in a molten hydroxide suspension of nano-Fe2O3. At 200°C in an electrolyte with a molar ratio of 0.5 NaOH/0.5 KOH, ammonia is produced at 1.2 volts (V) under 2 milliamperes per centimeter squared (mA cm(-2)) of applied current at coulombic efficiency of 35% (35% of the applied current results in the six-electron conversion of N2 and water to ammonia, and excess H2 is cogenerated with the ammonia). At 250°C and 25 bar of steam pressure, the electrolysis voltage necessary for 2 mA cm(-2) current density decreased to 1.0 V.

Fabrication of VB2/air cells for electrochemical testing.

A technique to investigate the properties and performance of new multi-electron metal/air battery systems is proposed and presented. A method for synthesizing nanoscopic VB2 is presented as well as step-by-step procedure for applying a zirconium oxide coating to the VB2 particles for stabilization upon discharge. The process for disassembling existing zinc/air cells is shown, in addition construction of the new working electrode to replace the conventional zinc/air cell anode with a the nanoscopic VB2 anode. Finally, discharge of the completed VB2/air battery is reported. We show that using the zinc/air cell as a test bed is useful to provide a consistent configuration to study the performance of the high-energy high capacity nanoscopic VB2 anode.

STEP wastewater treatment: a solar thermal electrochemical process for pollutant oxidation.

A solar thermal electrochemical production (STEP) pathway was established to utilize solar energy to drive useful chemical processes. In this paper, we use experimental chemistry for efficient STEP wastewater treatment, and suggest a theory based on the decreasing stability of organic pollutants (hydrocarbon oxidation potentials) with increasing temperature. Exemplified by the solar thermal electrochemical oxidation of phenol, the fundamental model and experimental system components of this process outline a general method for the oxidation of environmentally stable organic pollutants into carbon dioxide, which is easily removed. Using thermodynamic calculations we show a sharply decreasing phenol oxidation potential with increasing temperature. The experimental results demonstrate that this increased temperature can be supplied by solar thermal heating. In combination this drives electrochemical phenol removal with enhanced oxidation efficiency through (i) a thermodynamically driven decrease in the energy needed to fuel the process and (ii) improved kinetics to sustain high rates of phenol oxidation at low electrochemical overpotential. The STEP wastewater treatment process is synergistic in that it is performed with higher efficiency than either electrochemical or photovoltaic conversion process acting alone. STEP is a green, efficient, safe, and sustainable process for organic wastewater treatment driven solely by solar energy.

ATP-sensitive potassium channels exhibit variance in the number of open channels below the limit predicted for identical and independent gating.

In small cells containing small numbers of ion channels, noise due to stochastic channel opening and closing can introduce a substantial level of variability into the cell's membrane potential. Negatively cooperative interactions that couple a channel's gating conformational change to the conformation of its neighbor(s) provide a potential mechanism for mitigating this variability, but such interactions have not previously been directly observed. Here we show that heterologously expressed ATP-sensitive potassium channels generate noise (i.e., variance in the number of open channels) below the level possible for identical and independent channels. Kinetic analysis with single-molecule resolution supports the interpretation that interchannel negative cooperativity (specifically, the presence of an open channel making a closed channel less likely to open) contributes to the decrease in noise. Functional coupling between channels may be important in modulating stochastic fluctuations in cellular signaling pathways.

STEP cement: Solar Thermal Electrochemical Production of CaO without CO2 emission.

New molten salt chemistry allows solar thermal energy to drive calcium oxide production without any carbon dioxide emission. This is accomplished in a one pot synthesis, and at lower projected cost than the existing cement industry process, which after power production, is the largest contributor to anthropogenic greenhouse gas emissions.