*Indicates a student co-author.
Chaudhary, Krishna N., Kyra I. Brosnahan, Lucas J. Gibson-Elias, Jose L. Moreno Jr., Briana L. Hickey, Richard J. Hooley, and Bethany G. Caulkins. “Investigation of the effects on proton relaxation times upon encapsulation in a water-soluble synthetic receptor.” Physical Chemistry Chemical Physics, issue 13, 2024
Abstract: Sequestration of small molecule guests in the cavity of a water-soluble deep cavitand host has a variety of effects on their NMR properties. The effects of encapsulation on the longitudinal (T1) and transverse (T2) relaxation times of the protons in variably sized guest molecules are analyzed here, using inversion recovery and spin-echo experiments. Sequestration of neutral organic species from the bulk solvent reduces the overall proton relaxation times, but the magnitude of this effect on different protons in the same molecule has a variety of contributors, from the motion of the guest when bound, to the position of the protons in the cavity and the magnetic anisotropy induced by the aromatic walls of the host. These subtle effects can have large consequences on the environment experienced by the bound guest, and this sheds light on the nature of small molecules in enclosed environments.
Zhang, Xinmi, Anabhra Singh, Kassandra Soriano Martinez, and Patrick M Ferree. “Direct Parental (DIPA) CRISPR in the jewel wasp, Nasonia vitripennis.” G3 Genes|Genomes|Genetics, vol. 14, issue 7, July 2024.
Abstract: While clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology has demonstrated remarkable promise as a gene-editing tool, its application in certain insects, such as the jewel wasp, Nasonia vitripennis, has been hindered by a lack of a tractable method for reagent delivery. Direct Parental (DIPA-) CRISPR recently emerged as a facile way to induce gene lesions because it involves adult injection with commercially available Cas9–sgRNA with no helper reagent. However, DIPA-CRISPR has so far been tested in only a few insects. Here, we have assessed the amenability of DIPA-CRISPR in N. vitripennis by targeting two eye pigmentation genes, cinnabar and vermilion, which function in the ommochrome pathway. Successful generation of lesions in both genes demonstrated the functionality of DIPA-CRISPR in N. vitripennis and its potential application to other genes, thereby expanding the range of insects suitable for this method. We varied two parameters, Cas9–sgRNA concentration and injection volume, to determine optimal injection conditions. We found that the larger injection volume coupled with either higher or lower reagent concentration was needed for consistent mutation production. However, DIPA-CRISPR yields an overall low mutation rate in N. vitripennis when compared to other tested insects, a characteristic that may be attributed to a proportionally low vitellogenic import efficiency in the jewel wasp. We discuss different factors that may be considered in determining when DIPA-CRISPR may be preferable over other reagent delivery methods.
Ferree, Patrick M., Jelena Blagojević, Andreas Houben, Cesar Martins, Vladimir A Trifonov, and Mladen Vujošević. “What is a B chromosome? Early definitions revisited.” G3 Genes|Genomes|Genetics, vol. 14, issue 6, June 2024.
Abstract: Since the discovery of B chromosomes, multiple different definitions of these selfish genetic elements have been put forth. We reconsidered early definitions in light of recently published studies. While there are many characteristics that vary among different B chromosomes, such as their evolutionary origins, size, segregation behaviors, gene content, and function, there is one defining trait of all B chromosomes: they are nonessential for the organism. The points raised here may be useful for framing future B chromosome studies and help guide the categorization of new chromosomal elements that are uncovered in genomic studies.
Zhang, Xinmi, and Patrick M. Ferree. “PSRs: Selfish chromosomes that manipulate reproductive development.” Seminars in Cell and Developmental Biology, vols. 159-160, June-July 2024, pp. 66-73.
Abstract: B chromosomes are intriguing “selfish” genetic elements, many of which exhibit higher-than-Mendelian transmission. This perspective highlights a group of B chromosomes known as Paternal Sex Ratio chromosomes (PSRs), which are found in several insects with haplo-diploid reproduction. PSRs harshly alter the organism’s reproduction to facilitate their own inheritance. A manifestation of this effect is the conversion of female destined individuals into males. Key to this conversion is the mysterious ability of PSRs to cause elimination of the sperm-inherited half of the genome during zygote formation. Here we discuss how PSRs were discovered, what is known about how they alter paternal chromatin dynamics to cause sex conversion, and how PSR-induced genome elimination is different from other forms of programmed genome elimination in different insects. PSRs also stand out because their DNA sequence compositions differ in remarkable ways from their insect’s essential chromosomes, a characteristic suggestive of interspecies origins. Broadly, we also highlight poorly understood aspects of PSR dynamics that need to be investigated.
Leyden, Melissa R., Brent Gowen, Rodrigo Gonzalez-Romero, Jose Maria Eirin-Lopez, Bo-Hyun Kim, Fumio Hayashi, Jay McCartney, Patrick C. Zhang, Miyoko Kubo-Irie, Jeffrey Shabanowitz, Donald F. Hunt, Patrick Ferree, Harold Kasinsky, and Juan Ausió. “Protamines and the sperm nuclear basic proteins Pandora’s Box of insects.” Biochemistry and Cell Biology, vol. 102, no. 3, June 2024, pp. 238-251.
Abstract: Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis,and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms.
Leconte, Aaron M. and Clair M. Colee*. Directed Evolution: A Primer. American Chemical Society, 2024.
Abstract: While the field of directed evolution is vast and varied, it comes from a simple principle: to apply the principles of biological evolution in the laboratory to generate useful molecules. This widely used technique has yielded blockbuster drugs, essential biological tools, and many more useful chemicals. Considering its impact, it's unsurprising that the 2018 Chemistry Nobel Prize was awarded for the use of directed evolution of proteins. Directed Evolution is an excellent starting point for any scientist interested in directed evolution. This primer aims to provide readers with the foundational knowledge and vocabulary that enables them to tackle the literature and begin designing their projects.
Colee*, Clair M., Nicole M. Oberlag*, Marcell Simon*, Owen S. Chapman*, Lyndsey C. Flanagan*, Edison S. Reid-McLaughlin*, Jordan A. Gewing-Mullins*, Synaida Maiche*, Devi F. Patel*, Andre R. O. Cavalcanti, and Aaron M. Leconte*. “Discovery of red-shifting mutations in firefly luciferase using a high-throughput biochemical approach.” Biochemistry, vol. 63, issue 6, pp. 733-742.
Abstract: Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.
External Grant: Leconte, Aaron, Principal Investigator. “Towards XNA PCR: Evaluating the Potential of Fusion Domains to Improve XNA Polymerases.” Research Corporation for Science Advancement, SEED Award, 2024-2026, $60,000.
Abstract: Xenonucleic acids ("XNA") are unnatural nucleic acids that often possess improved biotechnological properties. However, most valuable XNAs are often not readily synthesized by DNA polymerases, and so we, and many others, have worked to engineer DNA polymerases to enzymatically synthesize XNA. While significant progress has been made in the past decade to synthesize longer XNAs, amplification of XNA by PCR is still impractical, requiring high enzyme concentrations and overnight reactions only to synthesize short amplicons (<100 base pairs). Here, we propose a novel method for improving XNA polymerases, which we believe may significantly improve the ability of XNA polymerases to perform XNA PCR. We hope to use the requested funding to hire a research assistant who will be able to gather key preliminary data on this project and to assist in writing a proposal to NSF RUI to continue this work beyond the SEED award.
Dong, Emily, and Sarah Marzen. “Cognitive biases can move opinion dynamics from consensus to signatures of transient chaos.” American Journal of Physics, vol. 92, issue 10, October 2024, pp. 801-808.
Abstract: Interest in how democracies form consensus has increased recently, with statistical physics and economics approaches both suggesting that there is convergence to a fixed point in belief networks, but with fluctuations in opinions when there are “stubborn” voters. We modify a model of opinion dynamics in which agents are fully Bayesian to account for two cognitive biases: confirmation bias and in-group bias. Confirmation bias occurs when the received information is considered to be more likely to align with the receiver's beliefs. In-group bias occurs when the receiver further considers the information to be more likely when the receiver's beliefs and the sender's beliefs are aligned. We find that when there are no cognitive biases, a network of agents always converges to complete consensus. With confirmation bias alone, polarization can occur. With both biases present, consensus and polarization are possible, but when agents attempt to counteract confirmation bias, there can be signatures of transient chaos and ongoing opinion fluctuations. Based on this simple model, we conjecture that complex opinion fluctuations might be a generic feature of opinion dynamics when agents are Bayesian with biases.
Marzen, Sarah. “Time Delays Improve Performance of Certain Neural Networks.” Physics Magazine, July 22, 2024.
Abstract: Both the predictive power and the memory storage capability of an artificial neural network called a reservoir computer increase when time delays are added into how the network processes signals, according to a new model.
Marzen, Sarah, Paul M. Riechers, and James P. Crutchfield. “Complexity-calibrated Benchmarks for Machine Learning Reveal When Next-Generation Reservoir Computer Predictions Succeed and Mislead.” Scientific Reports, March 28, 2023.
Abstract: Recurrent neural networks are used to forecast time series in finance, climate, language, and from many other domains. Reservoir computers are a particularly easily trainable form of recurrent neural network. Recently, a “next-generation” reservoir computer was introduced in which the memory trace involves only a finite number of previous symbols. We explore the inherent limitations of finite-past memory traces in this intriguing proposal. A lower bound from Fano's inequality shows that, on highly non-Markovian processes generated by large probabilistic state machines, next-generation reservoir computers with reasonably long memory traces have an error probability that is at least ~ 60% higher than the minimal attainable error probability in predicting the next observation. More generally, it appears that popular recurrent neural networks fall far short of optimally predicting such complex processes. These results highlight the need for a new generation of optimized recurrent neural network architectures. Alongside this finding, we present concentration- of-measure results for randomly-generated but complex processes. One conclusion is that large probabilistic state machines—specifically, large Ïμ-machines—are key to generating challenging and structurally-unbiased stimuli for ground-truthing recurrent neural network architectures.
Seifert, Gabriella, Ava Sealander, Sarah Marzen, and Michael Levin. “From reinforcement learning to agency: Frameworks for understanding basal cognition.” BioSystems, vol. 235, January 2024.
Abstract: Organisms play, explore, and mimic those around them. Is there a purpose to this behavior? Are organisms just behaving, or are they trying to achieve goals? We believe this is a false dichotomy. To that end, to understand organisms, we attempt to unify two approaches for understanding complex agents, whether evolved or engineered. We argue that formalisms describing multiscale competencies and goal-directedness in biology (e.g., TAME), and reinforcement learning (RL), can be combined in a symbiotic framework. While RL has been largely focused on higher-level organisms and robots of high complexity, TAME is naturally capable of describing lower-level organisms and minimal agents as well. We propose several novel questions that come from using RL/TAME to understand biology as well as ones that come from using biology to formulate new theory in AI. We hope that the research programs proposed in this piece shape future efforts to understand biological organisms and also future efforts to build artificial agents.
Marzen, Sarah. “The Best of Both Worlds: Joint Engineering of Living and Artificial Neural Networks for Context-Dependent Learning and Prediction.” Office of Naval Research, Multidisciplinary University Research Initiatives (MURI) Program, 2024.
Abstract: Our multidisciplinary university research team, composed of experts in biophysics, physics, chemistry, neuromorphic engineering, and neuroscience, is set to revolutionize computing by developing Hybrid Adaptive Networks (HANs) that integrate the strengths of Living Neural Networks (LNNs) and Artificial Neural Networks (ANNs). These HANs are designed to combine the best of both worlds: the adaptive, context-sensitive, and energy-efficient learning capabilities inherent in LNNs, with the speed, precision, and reproducibility of ANNs. The unique approach aims to create novel computing architectures that go beyond the limitations of traditional AI&ML systems, enabling advanced computing through synergy of biological and artificial systems.
Lundberg, Joyce, and Donald A. McFarlane. “Time-transgressive microbial diversity in a tropical bat guano accumulation, Deer Cave, Mulu, Borneo.” International Journal of Speleology, vol. 53, issue 3, 2024.
Van Rentergem, Guy, Joyce Lundberg, Donald A. McFarlane, and Warren Roberts. “A field-tested protocol for the measurement and mapping of bat guano deposition rate in caves.” Acta Chiropterologica, vol. 26, no. 1, June 2024, pp. 133-141.
Abstract: Measurement of bat guano deposition rate in caves can be an important research tool for estimation of colony size, for monitoring the record of long-term bat population trends, and for allied studies of guano invertebrate ecology, environmental contaminants, and paleoecology. However, previously published methodologies have lacked consistency. In the context of our recent studies of insectivorous bat guano deposition rates in Deer Cave, Sarawak, Borneo, we review some past studies and offer suggestions for best practises, along with proposed experimental design considerations for future studies (e. g., design of guano catchers, optimum deployment of catchers in relation to specific site characteristics, data reporting standards, and examples of mapping techniques). Consistent techniques will facilitate inter-site comparisons, and determination of intra-site changes over time. As a case study, and the first publication of detailed mapping of spatial variability in guano deposition rates, we present spatially explicit guano deposition rates for Deer Cave, ranging up to 88 g (dry weight)/m2 /day in the main cave, and up to 540 g (dry weight)/m2/day in the northern extension.
McFarlane, Donald, and Joyce Lundberg. “Rates of diagenesis of tropical insectivorous bat guano accumulations: implications for potential paleoenvironmental reconstruction.” International Journal of Speleology, vol. 53, issue 1, January 2024, pp. 39-49.
Ahmad Affandi, Nurfarhana Alyssa, Norasikin Ahmad Ludin, Mirratul Mukminah Junedi, Lim Chin Haw, and Kathleen Purvis-Roberts. “Analysing temporal factor in dynamic life cycle assessment of solar photovoltaic system.” Solar Energy, vol. 270, issue 1, March 2024.
Abstract: Static life cycle assessment is a mature and widely used tool for quantifying the environmental performance of a solar photovoltaic system. The system’s lifetime is estimated to last for 25 years but there are cases that the whole system could ultimately be replaced or dysfunctional. This study proposed a dynamic life cycle model integrating time-varying factors to assess the cause affecting photovoltaic system performance over its expected lifetime and compare the scenario with a static life cycle analysis. Results show that the unpredictable weather in Malaysia causes a consistent drop in the module’s degradation rate, which significantly affects the system’s lifetime. A fixed assumption over a system’s lifespan could lead to a 20 % difference in the overall impact assessment. However, it does not significantly affect the static outcome, with only 0.05 years increment to the system’s energy payback time and 1.3 years longer on its return on investment. Conclusively, dynamic data changes the result by 5 % for environmental and 8 % for financial impact assessment. This proves that the unknown value hidden from the basic assumption in the static model can be defined by implementing the temporal elements into the dynamic model.
Felcher, Nathan, Daisy Achiriloaie, Brian Lee, Ryan McGorty, and Janet Sheung. “Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks.” Journal of Visualized Experiments (JoVE), January 26, 2024.
Abstract: Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner’s guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.
Harmon, Isabela P., Emily A. McCabe, Madeleine R. Verguna, Julia Weinstein, Hannah L. Graves, Clare M. Boldt, Deijah D. Bradley, June Lee, Jessica M. Maurice, and Tessa K. Solomon-Lane. “Multiple behavioral mechanisms shape development in a highly social cichlid fish.” Physiology & Behavior, vol. 278, no. 1, May 2024.
Abstract: Early-life social experiences shape adult phenotype, yet the underlying behavioral mechanisms remain poorly understood. We manipulated early-life social experience in the highly social African cichlid fish Astatotilapia burtoni to investigate the effects on behavior and stress axis function in juveniles. Juveniles experienced different numbers of social partners in stable pairs (1 partner), stable groups (6 fish; 5 partners), and socialized pairs (a novel fish was exchanged every 5 days; 5 partners). Treatments also differed in group size (groups vs. pairs) and stability (stable vs. socialized). We then measured individual behavior and water-borne cortisol to identify effects of early-life experience. We found treatment differences in behavior across all assays: open field exploration, social cue investigation, dominant behavior, and subordinate behavior. Treatment did not affect cortisol. Principal components (PC) analysis revealed robust co-variation of behavior across contexts, including with cortisol, to form behavioral syndromes sensitive to early-life social experience. PC1 (25.1 %) differed by social partner number: juveniles with more partners (groups and socialized pairs) were more exploratory during the social cue investigation, spent less time in the territory, and were more interactive as dominants. PC5 (8.5 %) differed by stability: socialized pairs were more dominant, spent less time in and around the territory, were more socially investigative, and had lower cortisol than stable groups or pairs. Observations of the home tanks provided insights into the social experiences that may underlie these effects. These results contribute to our understanding of how early-life social experiences are accrued and exert strong, lasting effects on phenotype.
Smiley, Kristina O., Kathleen M. Munley, Krisha Aghi, Sara E. Lipshutz, Tessa M. Patton, Devaleena S. Pradhan, and Tessa K. Solomon-Lane. “Sex diversity in the 21st century: Concepts, frameworks, and approaches for the future of neuroendocrinology.” Hormones and Behavior, vol. 157, 2024.
Abstract: Sex is ubiquitous and variable throughout the animal kingdom. Historically, scientists have used reductionist methodologies that rely on a priori sex categorizations, in which two discrete sexes are inextricably linked with gamete type. However, this binarized operationalization does not adequately reflect the diversity of sex observed in nature. This is due, in part, to the fact that sex exists across many levels of biological analysis, including genetic, molecular, cellular, morphological, behavioral, and population levels. Furthermore, the biological mechanisms governing sex are embedded in complex networks that dynamically interact with other systems. To produce the most accurate and scientifically rigorous work examining sex in neuroendocrinology and to capture the full range of sex variability and diversity present in animal systems, we must critically assess the frameworks, experimental designs, and analytical methods used in our research. In this perspective piece, we first propose a new conceptual framework to guide the integrative study of sex. Then, we provide practical guidance on research approaches for studying sex-associated variables, including factors to consider in study design, selection of model organisms, experimental methodologies, and statistical analyses. We invite fellow scientists to conscientiously apply these modernized approaches to advance our biological understanding of sex and to encourage academically and socially responsible outcomes of our work. By expanding our conceptual frameworks and methodological approaches to the study of sex, we will gain insight into the unique ways that sex exists across levels of biological organization to produce the vast array of variability and diversity observed in nature.
Thomson, Diane M. “Using demographic modeling to develop post-fire restoration strategies for a native shrub in a sage scrub community.” Restoration Ecology, vol. 32, issue 8, November 2024.
Abstract: Mediterranean-climate shrublands are key biodiversity hotspots and carbon storage pools, but are increasingly threatened by climate change, non-native species, and altered fire regimes. Fires are important to historic shrubland disturbance cycles but can also promote non-native plants, which may limit post-fire native shrub recovery. Increasing drought with climate change could also reduce post-fire shrub regeneration. I developed a stochastic, individual-based demographic model (IBM) for the native shrub Artemisia californica, parameterized from an experimental removal of non-native annuals after a 2013 fire in southern California. The IBM simulated A. californica recovery for 7 years after fire under different rainfall conditions (drought or pre-drought) and non-native removal strategies (from no years to all 7 years). Drought lowered A. californica canopy volume 7 years after fire by 90% or more. Rainfall in the second year after fire, when most A. californica germination occurred, had particularly strong effects on final canopy cover. Non-native removal in all 7 years increased canopy volume by three times under drought conditions and 3.5 times under pre-drought conditions. Targeting non-native removal in the first 2 years proved nearly as effective, achieving from 88% (drought) to 95% (pre-drought) the benefits of removal in all 7 years. In sum, low rainfall may be the most important limitation on post-fire shrub recovery, but removal of non-natives in years of pulsed shrub recruitment can be an effective restoration strategy even under drought conditions. More generally, this study illustrates how demographic models can help optimize the targeting of scarce management and restoration resources.
Hamilton, Rachael E., Ellen A. Berkley, Iain K. Laufer, Madeline E. Ruos, Brandon Smith, Anthony N. Dahi, Timothy J. Gallagher, Vanessa Justo, Flannery Warner and Anna G. Wenzel. “Brønsted Acid-Catalyzed, Asymmetric Allenoate Claisen Reaction.” Journal of Organic Chemistry, vol. 89, issue 24, 2024, pp. 18654 – 18659.
Abstract: An auxiliary-based protocol is described for an asym-metric allenoate Claisen rearrangement. Silicated tosic acid (10 mol %) was used as an inexpensive, user-friendly catalyst. Stereochemicalanalysis revealed a preferential attack at the si face of prostereogenicolefin. The amine auxiliary was readily hydrolyzed and can be isolatedfrom the reaction mixture (85−87% recovery). The resultingunsaturated β-keto esters were isolated in ≤99% yield and 98% ee.Diastereoselective examples provided a 96:4 syn:anti ratio of theresulting vicinal stereocenters.
Lew, Maria J. H., Jacob A. Kelber, Mary J. Van Vleet, Adam R. Johnson, Elizabeth R. Jarvo, and Nancy S. B. Williams. “A σ-Poor, π-Rich Pyridonate Pincer Ligand Designed To Enhance C–H Oxidative Addition at Platinum Group Metals: Experimental and Computational Studies.” Organometallics, vol. 43, issue 15, pp. 1630-1639.
Abstract: A strong π-donor, weak σ-donor pyridonate pincer complex of platinum is reported, a rare but important electronic ligand structure for promoting C–H activation in late transition metals. Protonation of the complex 2,6-bis(diethylaminomethyl)-4-pyridonate platinum(II) methyl occurs initially at oxygen, with a second proton equivalent affording methane. Methyl triflate similarly methylates initially with oxygen and then platinum. A computational study indicates that this terdentate pyridonate pincer greatly enhances the ability of late transition metals to cleave the C–H bond of methane compared to a classic NCN pincer. With iridium bound to this π-donor pincer, the oxidative addition of methane is computationally predicted to be thermodynamically favorable.