2020 Beckman Symposium

In the battle between magnetism and superconductivity, magnetism wins so often that only a handful of magnetic superconductors are known. In this context, here we report our characterization of YbxPt5P, - providing experimental evidence for the possible coexistence of both magnetism and superconductivity in this material. Comparison of samples with different Yb stoichiometry shows that the relative strengths of magnetism and superconductivity can be tuned. The likely coexistence and tunability of such strongly competing electronic states, along with the presence of strong spin orbit coupling due to the large amount of Pt present, makes YbxPt5P a remarkable material and opens the door to a new family of magnetic superconductors.

Carbon dioxide emissions, known to exacerbate climate change, have been increasing rapidly over the past century. One strategy to alleviate this issue is carbon capture and utilization (CCU), in which some of the abundant atmospheric CO₂ is used as a carbon source for the production of valuable compounds. An attractive target is sodium acrylate, the building block of superabsorbent sodium polyacrylate found in hygiene products and many other common goods. Currently, sodium acrylate is synthesized by sequential oxidations of propylene over heterogeneous catalysts at high temperatures. Researchers have instead sought a one-step process coupling CO₂ and ethylene, which is more sustainable and uses much less expensive starting materials. This method could consume a large quantity of atmospheric CO₂. However, coupling with ethylene is not spontaneous. Homogeneous nickel catalysts have shown great promise for enabling this reaction, but the reported systems suffer from low efficiency. The main obstacle is a very stable nickelalactone intermediate, which contains a rigid Ni–pre-acrylate ring that resists the release of free sodium acrylate from the catalyst. My project aims to develop nickel complexes which include an N-heterocyclic carbene (NHC) in a supporting bidentate ligand. The characteristic strong electron donation and steric imposition of NHCs are expected to destabilize nickelalactones and to promote ring-opening, thereby enabling efficient catalytic production of sodium acrylate. Initial results support the instability of a simple bis(NHC) nickelalactone, and experiments testing sodium acrylate production are underway. Computational investigation of NHC-containing ligands with varying electronic and steric effects also helps to elucidate their capability to support the target reaction. This family of ligands could be the key to efficient nickel catalysts coupling CO₂ and ethylene for sodium acrylate production, thus contributing to global CCU efforts.

Allenes, compounds containing two double bonds across three contiguous carbons, are versatile synthetic intermediates due to their substituent loading capacity and ability to generate one or more new stereogenic centers through axial-to-central chirality transfer. This structural motif is also present in over 150 natural products and a variety of bioactive molecules. Despite their prevalence and distinct reactivity, catalytic methods to access chiral 1,3-disubstituted allenes with high levels of selectivity from achiral starting materials remains a goal in chemical synthesis.

We have developed a mild and general strategy for the highly selective semi-reduction of prochiral 1,3-enynes. A diverse array of enantioenriched 1,3-disubstituted allenes are furnished in up to 98% yield and 99% enantioenrichment. This reaction is enabled by chiral copper hydride catalysts in the presence of water as a benign and abundant proton source. Additional applications of this method further illustrate the advantage of this simple protocol for the selective synthesis of mono-deuterated allenes and chiral 2,5-dihydropyrroles.

My lab is developing a bacterial strain that will catalyze the conversion of carbon dioxide to fuels and products using electrons from an electrode as the sole source of reducing power. This process would represent a new form of metabolism – electroautotrophy. Our platform for developing an electroautotrophic strain is Shewanella oneidensis, a metal-reducing bacterium that already possesses the capacity to exchange electrons with extracellular electrodes. The first key challenge in this project is to create an efficient pathway for electron flow from the electrode to intracellular electron carriers. This requires reversing the direction of electron flow through the native extracellular electron transfer pathway in S. oneidensis. We have demonstrated proof-of-concept for the inward electron transfer pathway by engineering S. oneidensis to catalyze the intracellular reduction of acetoin to 2,3-butanediol using an electrode as the electron source. We are currently working to improve flux through the inward electron transfer pathway by over-expressing the essential components. The second key challenge is to transplant a carbon fixation pathway into S. oneidensis, which is a heterotroph. We have expressed the missing components of the Calvin-Benson-Bassham cycle for carbon fixation in S. oneidensis and are currently working to functionalize the pathway through adaptive laboratory evolution. Moving forward, we will combine the modifications required for inward electron transfer and for carbon fixation to create the electroautotrophic strain.

Current cancer management follows a multipronged approach that include surgery, radiation, and chemotherapy. There is a pressing need for a platform technology to provide precision chemotherapy screening, drug delivery detection, and real-time therapy efficacy monitoring. We have developed single walled carbon nanotube (SWNT) sensors for the detection of chemotherapeutics, more recently for temozolomide (TMZ) and its byproduct 5-Amino-4-imidazolecarboxamide (AIC) with sensitivity at 5-500 μM and in vitro viability up to 7 days in TMZ treated glioblastoma cells. Sensors for irinotecan, cisplatin, and lomustine were developed with sensitivities at 50 μM. H2O2 sensors were used to measure the therapy efficacy of gemcitabine and irinotecan in pancreatic ductal adenocarcinoma in vitro and in vivo. Multiplexing these sensors could yield insights into delivery, diffusion, metabolism, and efficacy of chemotherapeutics in hours or days. Other strategies are being developed such as ratiometric approaches and wavelength-induced frequency filtering to improve performance and sensitivity. A fiber optic platform was developed as a minimally invasive form factor to integrate the sensor hydrogel, optical waveguide, and detection system. As a physician-scientist in NYC, my research rapidly shifted to the role of convalescent plasma (CP) in COVID-19. CP can be used to passively transfer antibodies from recently recovered patients to actively infected treatment-resistant patients. Initial studies revealed antibody neutralizing activity peaked at 31-35 days post symptom onset. Initial outcomes in severe and life-threatening COVID-19 hospitalized patients show CP as a potentially efficacious treatment for non-intubated patients. Retrospective study of 427 CP transfusion events yielded 12.9% of transfusion reaction rate with vast majority due to COVID-19 disease not CP. Increased risk was identified in patients with blood group B or cancer, and decreased risk for patients in their 80s.

Infrared spectroscopy and microscopy directly couples to molecular functional groups, providing chemical information without extrinsic labels. However, conventional infrared spectroscopy and microscopy have two main limitations: lack of spatial resolution due to Abbe’s diffraction limit, and restriction of applications in the aqueous conditions due to strong infrared attenuation. On the other hand, many meaningful biological processes and chemical transformation happen in the liquid phase. The understanding of them requires insights from the nanoscale. With the support from Beckman Foundation, we have developed the liquid-phase peak force infrared (LiPFIR) microscopy to deliver both label-free super spatial resolution at 10 nm and compatibility with liquid-phase measurement. We have demonstrated the method on a range of samples, from tracking the surface re-organization of polymers in organic solvent, hydrogen-deuterium isotope exchange and ethanol-induced denaturation of proteins, to imaging of cellular structures in aqueous phases. The LiPFIR microscopy bypasses the two limitations of infrared spectroscopy and microscopy. It will enable label-free spectroscopic investigations of chemical reactions and biomolecular transformations from the tens of nanometer scale.

Protein structural disorder helps avoid misfolding and aggregation, but in impeding protein folding might also impede function. The balance between these two selective pressures on protein biophysics might vary among species as a function of the effectiveness of weak selection.  We predicted the Intrinsic Structural Disorder (ISD) of Pfam domains across 118 fully-sequenced vertebrate species and estimated the effect of species identity on to control for differences in Pfam composition across species. We compared this to each species’ Codon Adaptation Index of Species (CAIS), a metric we developed to quantify for effectiveness of selection from synonymous codon usage, corrected for total genomic GC content and amino acid composition, to be comparable across species.
Simple correlations between ISD and CAIS indicate that well-adapted species tend to have high ISD (Spearman’s R = 0.67, p < 2e-16). To correct for phylogenetic confounding and resulting pseudoreplication, we transformed CAIS and ISD species effect data using Phylogenetic Independent Contrasts (PIC). Phylogenetically controlled linear models confirmed that better-adapted species have higher ISD (Spearman’s R = 0.43, p = 1.2e-7). This relationship is conserved across protein domains dated older than 2.1 Gya and protein domains dated younger than 1.5 Gya, though the relationship is stronger in recent domains than in ancient. This indicates selection for higher disorder, with the greater benefit to more recently emerged vertebrate protein domains.

Effective locomotion in nature happens by transitioning between multiple modes (e.g., walk, run, climb, slither). Despite this, far more mechanistic understanding of terrestrial locomotion has been on how to generate and stabilize around near-steady-state movement in a single mode. Unlike flight and swimming with aero- and hydrodynamics, a major challenge in understanding terrestrial locomotor transitions is the lack of methods to measure and model how animals actively make use of physical interaction with complex terrain. As a result, robots cannot robustly traverse complex terrain, an ability required for critical applications like search and rescue in earthquake rubble and extraterrestrial exploration over Martian rocks. Our BYI work focuses on using an energy landscape approach to elucidate principles of terrestrial locomotor transitions via integration of biomechanics, sensorimotor control, and terrain physics. Recently, we discovered that an energy landscape approach helps understand probabilistic locomotor transitions. In complex terrain, animals’ and robots’ locomotor modes are attracted to basins of a potential energy landscape. We have been developing new methods to enable experiments and modeling of how animals and robots actively cross potential energy barriers and hop between basins to make locomotor transitions. These include: (1) terrain platforms and tracking techniques to quantify animal locomotor transitions and terrain interaction, (2) robots with animal-like motion as physical models, and (3) force sensors to measure physical interaction and understand provide feedback control. I will highlight two recent discoveries enabled by these tools: to make locomotor transitions and traverse complex terrain, (1) legged insects actively adjust head, body, and appendages to lower potential energy barriers; (2) besides vision, limbless snakes must use mechanical sensing to adjust body form and control terrain contact to maintain propulsion and stability.

Over 100 distinct alkane monooxygenase (AlkB) enzymes containing a covalently bound, or fused, rubredoxin domain were identified and analyzed using bioinformatic techniques. One such protein was cloned as a full-length protein and truncated with its rubredoxin domain deleted. Its catalytic activity was evaluated, and its interaction with exogenous electron transfer partners was examined. These experiments shed light on the diversity of AlkB structure and reactivity, and the interaction of AlkBs with electron transfer partners.

Many experimental approaches rely on controlling gene expression in select subsets of cells within an individual animal. However, reproducibly targeting transgene expression to specific fractions of a genetically defined cell type is challenging. We developed Sparse Predictive Activity through Recombinase Competition (SPARC), a generalizable toolkit that can express any effector in precise proportions of post-mitotic cells in Drosophila. Using this approach, we demonstrate targeted expression of many effectors in several cell types and apply these tools to calcium imaging of individual neurons, optogenetic manipulation of sparse cell populations in vivo, and genomic profiling of mutant and control neuronal subtypes harvested from the same animals.

Auxin is a hormone crucial in nearly every aspect of plant growth and development. Advancing our understanding of auxin signaling in maize and other crop plants may provide needed tools for agricultural scientists to help feed a growing population in the face of global climate change. However, existing tools for detecting and measuring auxin level and location have significant shortcomings that can limit their utility in plants. We are working to build and test a novel auxin biosensor, ShadowAuxin, capable of accurate quantification and tracking of auxin in live plants. Our biosensor design relies on the dimerization of two fluorescent proteins (a donor and an acceptor) capable of Förster Resonance Energy Transfer (FRET) when in close proximity. Our biosensor utilizes a quenching fluorescent protein acceptor in the FRET pair; when this pair are held in close proximity by fused dimerization domains, the donor fluorescent signal should dim. Pilot experiments in yeast cells have shown that quenching FRET can be measured via fluorescence flow cytometry, and have revealed the importance of dimer-domain choice and carefully controlled protein expression level. A new pair of human-hormone inducible promoters has enabled us to precisely control the expression level of donor and acceptor proteins in order to maximize FRET quenching. This system allowed observation of quenching FRET without significant cellular toxicity; however we observed a maximum of only 12.9% decrease in fluorescent signal. This is likely due to off-target dimerization and imperfect intracellular stoichiometry of the fluorescent proteins. Therefore, current and future experiments focus on increasing FRET efficiency by adjusting promoter strength and testing new sets of heterodimerization domains. Ultimately, this quenching FRET system will be coupled to an auxin-sensing domain to generate a biosensor that relies on single-color fluorescence measurements and provides rapid response and wide dynamic range.

Extrachromosomal circular DNA (eccDNA) comprises products of genomic recombination that cause somatic changes in gene length and sequence. These circular molecules are derived from canonical linear chromosomal loci, expanding the diversity in coding and regulatory capacity within eukaryotic genomes and transcriptomes. Using a brand-new multidisciplinary approach to investigate eccDNA-mediated allelic diversity, I have identified various coding regions of eccDNA biogenesis, such as Titin and Mucin loci. In order to systematically investigate the biological implications and mechanisms of eccDNA formation, this project will focus on Titin eccDNAs as a prototype for eccDNA-mediated chromosomal rearrangements. Titin (TTN) is an extremely large protein that is responsible for the passive elasticity of muscle, functioning as a molecular spring. This single gene (TTN) is expressed in various isoforms, each with its own associated “spring constant.” It has been assumed that Titin protein diversity results from complex mechanisms dependent on alternative RNA splicing. However, there is thus far no comprehensive alternative-RNA-splicing framework that accounts for the full spectrum of TTN diversity. Preliminary data obtained in the past years of support suggest a novel mechanism of TTN diversity involving circular-DNA excision that generates recombinant TTN loci at the genomic level. Here, I report TTN-eccDNA formation as a potential mechanism for modulating overall myofibrillar passive tension. Results from investigating this novel mechanism for the modulation of cardiovascular stiffness will revolutionize basic, clinical, and therapeutic approaches to cardiovascular diseases. The AOB Award has been instrumental in advancing the field of eccDNA, in general, and in providing a missing key element in DCM biology, consideration of which may drive a reevaluation of current therapies.