Other Biochemistry, Biophysics, and Structural Biology Commons^™

Analysis Of The Atypical Temperature Dependence And Conformational Changes During Turnover Of A Lactobacillus Chlorogenic Acid Esterase, Nathaniel Carl, Yianni Tsigaris, Dan Ji, Nora K. Anpree, Kellie K. Omori, Cedric P. Owens

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Many bacterial chlorogenic acid esterases (ChlEs) exhibit atypical temperature behavior, featuring activities that barely change with temperature and activity maxima that fall below the thermal denaturation point. This work focuses on a ChlE from Lactobacillus helveticus (Lh–ChlE), which has a flat temperature dependence. First, it was determined that conformational changes during Lh–ChlE turnover are not rate-limiting and that the overall rate depends on the chemical step at all temperatures. Next, Lh–ChlE’s temperature dependence was investigated using a conformational equilibrium model that assumes the existence of a temperature-dependent equilibrium between an active and an inactive conformation and an activation heat capacity …

Out Of This World: An Exoplanetary Workshop, Monica Storss, Bart Kuipers

ELO (un)supervised 2026

The workshop investigates how scientific constraints can productively defamiliarize human experience and create new frameworks for creativity and expression. Participants will produce original work while considering broader questions about environment, perception, and cultural formation in speculative contexts, and how the arts, sciences, and technology inform each other in relational multiplicities. This generative workshop explores how astronomical data from confirmed exoplanets can generate new poetic forms and linguistic constraints. Participants will examine how exoplanetary conditions can shape language, metaphor, and narrative structure. We'll develop from writing exercises that respond to non-terrestrial physical parameters: alternative light spectra, gravitational variations, and atmospheric compositions. …

Investigating Quinclorac Binding To Human Serum Albumin Using Spectroscopy And Molecular Docking, Le Nguyen, L. Hicks, L. Jodoin, S. Carby, D. S. Sanford, D. Benavides, A. Mishra, R. Yadav

Journal of the Arkansas Academy of Science

Human serum albumin (HSA) is the most abundant plasma protein and serves as a major transporter and reservoir for a wide range of endogenous ligands, including fatty acids, heme, bilirubin, prostaglandins, and metal ions, as well as numerous exogenous compounds, such as pharmaceuticals. HSA is a 66 kDa monomeric, multidomain protein containing two principal ligand-binding sites, Sudlow sites I and II. Sudlow site I contains Trp214, the sole tryptophan residue in HSA, whose intrinsic fluorescence provides a sensitive probe for studying ligand–protein interactions.

In this study, the interaction between HSA and the persistent organochlorine herbicide quinclorac (QNC) was investigated using …

Mapping Nucleosome Stability: Linking Oncogenic Structural Alterations To Long-Range Allosteric Communication, Augustine C. Onyema

Dissertations, Theses, and Capstone Projects

The nucleosome core particle (NCP) is essential for chromatin structure and function, serving as the fundamental unit of eukaryotic chromatin. Oncogenic mutations in core histones disrupt chromatin dynamics, altering DNA repair and transcription processes. How DNA communicates with histones, and how this information propagates through the histone, has been suggested to be a cooperative process. Here, I investigate the molecular consequences of two mutations, H2BE76K and H4R92T, using 36 µs of all-atom molecular dynamics simulations on Anton2. I also characterize the kinetic correlation within histone/DNA domains via conditional activity (CONDACT) using the dihedral angle as our degrees of …

Somatic Prosthetics For Planetary Resonance, Disha Dharesh Kumar R

Masters Theses

This thesis investigates how industrial design can transcend extractive technological paradigms in favor of relational interfaces that foster planetary attunement. Framing the climate crisis as a 'crisis of imagination', the research challenges the Western bifurcation of nature and culture by drawing on Indic cosmologies- which recognize stones, plants, and ecosystems as conscious at different levels and participants in a shared cosmic field. By synthesizing research in Biosemiotics, Quantum Information Pansycishm, and Neuroscience, the project redefines intelligence as a distributed, more-than-human phenomenon.

The research materializes as a speculative design artifact: a device that functions as a somatic prosthetic for planetary resonance. …

Novel Biomarker Discovery In Multiple Sclerosis, Manisha Gangasani

Honors Theses

Multiple Sclerosis (MS) is a chronic neuroinflammatory disorder affecting the brain, spinal cord, and optic nerve. Patients experience cognitive, motor, autonomic, and emotional symptoms that often overlap with other neurological conditions, making early diagnosis challenging. Current diagnostic criteria require evidence of lesions with dissemination in time and space. Racial disparities in MS outcomes are well documented, with Black individuals often exhibiting greater disease severity, higher relapse rates, and increased disability compared to White individuals. These differences likely reflect a combination of genetic, biological, environmental, and healthcare access factors, underscoring the need to improve early diagnosis and reduce inequities in care. …

Evaluation Of The Posterior Segment Of The Dromedary Camel’S Eye Using Spectral-Domain Optical Coherence Tomography (Sd-Oct), Amine Berghiche, Djallal Eddine Rahmoun, Nassiba Reghaissia, Allaoua Chaouki Bentazir, Mohamed Kamal Merai Abdel Maksoud

The Thai Journal of Veterinary Medicine

The dromedary camel possesses unique ocular adaptations enabling survival in harsh desert environments. This study aimed to characterize the posterior segment of the dromedary camel’s eye using spectral-domain optical coherence tomography (SD-OCT) and fundus photography. Fourteen healthy adult camels were sedated and imaged. High-resolution OCT images enabled clear visualization and measurement of the retina, choroid, and sclera. The retina exhibited well-defined layers, including the retinal pigment epithelium, photoreceptor layer, plexiform layers, and nerve fiber layer. Mean thicknesses of the retina, choroid, and sclera were 267 ± 2.54 μm, 299 ± 3.15 μm, and 155 ± 2.74 μm in the right …

Albumin-Binding Domains In Therapeutic Protein Engineering: A Structural And Computational Perspective On Rational Design, Matthew Argyle, Dallin M. Chipman, Anna Claire Woolley, Bradley C. Bundy, Dennis Della Corte

Faculty Publications

Therapeutic proteins face a critical pharmacokinetic challenge: rapid clearance from circulation limits their clinical efficacy. Albumin-binding domains (ABDs) offer an elegant solution by enabling therapeutic proteins to “hitchhike” on serum albumin’s favorable 19-day half-life through FcRn-mediated recycling. Clinical validation through approved therapeutics like ozoralizumab demonstrates the success of this approach, with preclinical studies showing fusion to an ABD extended half-life to 18 days. This review provides an analysis of ABD-fusion protein design, integrating structural biology, computational prediction, and rational engineering principles. We catalog the major classes of albumin-binding modalities, including bacterial three-helix bundle domains, engineered peptides, antibody-derived binders, and alternative …

Computational And Functional Characterization Of Terebrid Venom Peptides, Favour T. Achimba

Dissertations, Theses, and Capstone Projects

Venom peptides represent a natural repertoire of bioactive compounds that are potent modulators of ion channels. Teretoxins, venom peptides from terebrid snails, remain underexplored despite their structural and functional diversity. This thesis is the first to comprehensively explore the structure and function of teretoxins using a comparative computational sequence and structure model followed by experimental assays. The majority of the functional focus has been on identifying the ion channel molecular targets of uncharacterized teretoxins. Teretoxins, like other venom peptides, are hypothesized to manipulate the cellular function of a diversity of ion channels. Dysregulation of ion channels leads to several …

Benzothiazole Sulfones As A Tool For Peptide Modification And Cleavage, Phoebe R. Rubiio, Gavin A. Clausen, Zachary D. Ruighaver, Abigail F. Dalton, John P. Blobe, Amanda L. Moore, Christopher R. Shugrue

Chemistry Faculty Publications

We report the development of a new benzothiazole sulfone (BTS) tool for peptide modification and cleavage. Benzothiazole sulfones are electrophiles that rapidly react with thiols in nucleophilic aromatic substitution (SNAr) reactions. Alkyne-containing BTS compounds were incorporated onto azidolysine-containing peptides through copper-catalyzed azide-alkyne cycloadditions (CuAACs). BTS-containing peptides were found to react rapidly with two equivalents of thiol nucleophiles at room temperature in 30 min or less. BTS-containing peptides were conjugated to a variety of different compounds, including small molecules, fluorophores, and peptides. Alternatively, a BTS-based cleavable linker could also be synthesized with small molecule cargo, such as biotin, and …

Comparing The Differences Of Hsp90Α And Hsp90Β In Native Protein Complex Incorporation, Daryna Serediuk

Honors Theses and Capstones

Epichaperomes are long-lasting, stable assemblies of chaperones, co-chaperones, and associated factors that facilitate cell survival in maladaptive cellular states. Their inhibition has emerged as an attractive therapeutic strategy for the treatment of cancer and neurodegenerative diseases (Rodina et al., 2016). Epichaperomes are hallmarks of robust proliferation in cancer cell populations across many cancer types and are abundantly present in several stem cell varieties, including induced pluripotent stem cells, human embryonic kidney cells, and cancer stem cells (Kishinevsky et al., 2018).

The core protein of the eukaryotic heat shock protein machinery, Heat Shock Protein 90 (Hsp90), is a central chaperone that …

Design And Advancement Of Analytical Frameworks For In Vivo Single-Photon Calcium Imaging To Characterize Temporal- And Treatment-Dependent Neural Adaptations To Opioid Exposure And Withdrawal In The Medial Prefrontal Cortex, Alexia R. Alsum Dr.

Theses and Dissertations--Pharmacy

Despite the availability of several approved medications for opioid use disorder (OUD), it remains a significant public health concern characterized by persistent neurobiological adaptations that drive relapse and impede successful recovery. The medial prefrontal cortex (mPFC), a region critical for executive control and decision-making, undergoes pronounced functional changes during chronic opioid exposure and withdrawal, yet the cellular mechanisms underlying these adaptations remain poorly understood. This dissertation aims to investigate longitudinal alterations in mPFC neural activity across opioid exposure and withdrawal using in vivo single-photon calcium imaging and newly developed computational frameworks.

Following standard preprocessing to extract and normalize calcium traces, …

From Sequence To Allostery: How Amino Acid Differences Rewire Coupled Conformational Heterogeneity In The Ptp Enzyme Family, Shivani Sharma

Dissertations, Theses, and Capstone Projects

Protein Tyrosine Phosphatases (PTPs) are critical regulators of cellular signaling, and their activity is often modulated through allosteric mechanisms. Allostery is defined as a chemical or conformational change in one site of the protein due to a binding event at another site. Although several studies have investigated aspects of allostery in PTPs, the mechanistic foundations underlying allosteric regulation remain poorly understood. Despite a highly conserved catalytic domain across PTPs, regions with low or no conservation suggest potential "rewiring" of allosteric networks. This raises key questions about the universality versus individuality of allosteric mechanisms across the PTP family.

In this work, …

Advanced Interactions Of Directed Energy With Retinal Pigment Epithelial Cells And Their Photochemical And Photothermal Responses, Jin B. Ha

Theses & Dissertations

Excess optical radiation can injure the retina through either rapid heat deposition (photothermal damage) or cumulative photo‑oxidative stress (photochemical damage), yet current laser‑safety limits treat these mechanisms as independent. To test that assumption, this dissertation integrated systematic wavelength, duration and temperature‑controlled laser exposures with live/dead fluorescence imaging in a melanin‑loaded hTERT‑RPE1 monolayer model. First, concurrent 447 nm (blue) and 2 µm (infra‑red) beams delivered for 200 s demonstrated a strong thermal–photochemical synergy: each beam alone was sub‑threshold (≤25 % lethality) but together produced 100 % RPE death without exceeding 50 °C, proving that modest heating markedly lowers the photon dose …

A Cancer Education Needs Assessment: Informing Middle-Aged Female Patients About The Relationships Between Obesity And Women’S Health Concerns In The Reproductive System, Breast, And Endometrial Health, Batul Mirza

MUSC Theses and Dissertations

Obesity significantly impacts women’s health, particularly among middle-aged women, by increasing the risk of hormone-sensitive cancers such as breast, endometrial, and reproductive system cancers. This study examines the educational needs of this demographic group regarding obesity-related cancer risks and explores effective intervention strategies. Obesity-induced mechanisms – hormonal imbalances, chronic inflammation, and insulin resistance – drive cancer susceptibility, emphasizing the need for targeted health education. The study employs a qualitative design, which includes interviews with subject matter experts (SMEs) and surveys of middle-aged women. The goal is to assess awareness, perceived barriers, and preferred learning methods. Findings suggest that with many …

Foldit Education Mode, Brett Schulz

Electronic Theses and Dissertations

Foldit education mode is a puzzle campaign within the protein folding video game Foldit. Education mode, initially designed as a classroom supplement for undergraduate biochemistry courses, has been expanded to provide a more comprehensive learning tool to players without a background in biochemistry. The edited and added levels teach players about protein biochemistry through playing puzzles designed to convey information and gameplay mechanics. The new Foldit education mode campaign features 49 levels grouped into 11 categories. The campaign has 6 new levels designed specifically for education mode and 4 levels adapted from the traditional Foldit campaign for use within education …

Structural And Functional Lessons Learned About Regulation Of Enterotoxigenic E. Coli Virulence Regulatory Factor, Rns, Jessica Dianne Tolbert

Dartmouth College Ph.D Dissertations

Antibiotic resistance has exacerbated the global health crisis of diarrheal diseases caused by enteric bacterial pathogens. Investigating pathogens, such as Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae (cholera), helps to uncover mechanisms for the inhibition of virulence gene expression. A notable member of the AraC transcription factor family in ETEC, Rns, regulates virulence factors including pili and fimbriae, which are essential for adherence and colonization of the intestinal epithelial layer. This microbe–host interaction initiates the release of toxins, leading to the water and ion loss in clinically diagnosed as diarrhea.

In the canonical N-terminal domain of Rns, mutagenesis of the …

Effect Of Glycation On The Binding Interactions Of Serum Albumin And Silver Nanoparticles, Megan Kirk

Celebrating Scholarship and Creativity Day (2018-)

Nanoparticles are particles ranging from 1-100 nanometers in size. Most nanoparticles, upon contact with biological matrices, are immediately coated with proteins, forming the protein corona, which can alter the structure and function of proteins. Despite the tremendous advancement of nanotechnology over the last decade, the interactions of nanoparticles with proteins are not well characterized. This project investigates the effect of glycation on the binding interactions of HSA, the most abundant protein in human blood, with silver nanoparticles using various spectroscopic techniques. This is important because in diabetic patients, HSA is exposed to high concentrations of glucose over extended periods of …

A Qualitative Nmr And Histological Analysis Of Adipocere Formation In Different Aqueous Environments Using Sus Scrofa Models, Mia M. Sorrentino

Student Theses

Adipocere is a wax-like crust that has the potential to form over corpses in moist, humid, and anaerobic environments, preserving them in a process similar to that of mummification. The substance can be identified within 3-6 months postmortem and is characterized by the ratio of saturated fatty acids to unsaturated fatty acids. Palmitic acid and stearic acid are the most abundant in comparison to myristic and oleic acids. The goal of this study was to investigate factors affecting adipocere formation in different aqueous environments. Pork belly samples were submerged in water samples collected from different places around New York City …

Metabolic Transitions In Embryos Of Artemia Franciscana: Regulation And Consequences For Oxidative Stress, Daniel Anthony Arabie

LSU Doctoral Dissertations

Embryos of Artemia franciscana survive harsh conditions in diapause and anoxia-induced quiescence for years by undergoing deep metabolic transitions. I investigated the relationship between phosphorylation of pyruvate dehydrogenase (PDH) and carbon flux to the tricarboxylic acid (TCA) cycle during metabolic depression. The difference in catalytic activity of the PDH complex (PDC) in maximally phosphorylated and dephosphorylated samples was 16.5-fold. Western blot analysis confirmed the changes in phosphorylation of PDH. Analysis of PDH expression in diapause and post-diapause embryos via phospho-PDH showed no significant differences. Investigation of post-diapause embryos under anoxia showed that phosphorylation of PDH decreased and activated the enzyme. …

First-In-Class Peptide Molecules Targeting The Mien1 Cancer Signaling Pathway, For Which No Inhibitors Have Been Identified To Date., Amit Tripathi, Jamboor K. Vishwanatha

Research Symposium

Background: Migration and Invasion Enhancer 1 (MIEN1) is a critical oncogene that drives cancer cell migration, invasion, and epithelial-to-mesenchymal transition (EMT) in various cancers. MIEN1 overexpression is linked to aggressive tumor progression and poor prognosis. Despite its established role in cancer biology, targeted therapeutic options remain unexplored. This study aimed to identify and characterize first-in-class therapeutic molecules targeting the MIEN1 signaling pathway to inhibit cancer progression.

Methods: To develop novel bio-active peptides, we leveraged conserved immunoreceptor tyrosine-based activation motif (ITAM) and prenylation motifs within the MIEN1 protein sequence. Using rational peptide design, we identified and synthesized bioactive peptides, namely LA3IK …

Ivy Proteins As Guardians Of Bacterial Cell Wall Integrity: Regulation Of Lytic Transglycosylases Explored, Aamna Aijaz, Trinity E. Alamutu, Vy T. Dao, Nathaniel R. Davis, Michael D. Landers, Nykolas K. Mackevicius, Liliana Ortiz, Irina F. Padilla-Montoya, Kacy J. Smith, Maria L. Snyder, Anthony J. Stefanie, Blaise M. Williams, Thomas C. Leeper

The Kennesaw Journal of Undergraduate Research

Lytic transglycosylases (LTs) are bacterial enzymes involved in biosynthesis and maintenance of the peptidoglycan cell wall. LTs are reported to be regulated by two paralogs of Inhibitor of Vertebrate Lysozymes: Ivyp1 and Ivyp2. This study investigated the regulatory dynamics between the soluble LT (SltB1), and Ivy paralogs as putative regulators in Pseudomonas aeruginosa (PA). The secondary objective was to delineate the critical roles played by a glutamic acid at position 135 in SltB1 and a histidine at position 62 in Ivyp1 in governing their molecular interaction through site directed mutagenesis. Isolated SltB1 shows no lytic activity in our assay, in …

Fast 3d Printing Of Fine, Continuous, And Soft Fibers Via Embedded Solvent Exchange, Wonsik Eom, Mohammad Tanver Hossain, Vidush Parasramka, Jeongmin Kim, Ryan W. Y. Siu, Kate A. Sanders, Dakota Piorkowski, Andrew Lowe, Hyun Gi Koh, Michael F. L. De Volder, Douglas S. Fudge, Randy H. Ewoldt, Sameh H. Tawfick

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Nature uses fibrous structures for sensing and structural functions as observed in hairs, whiskers, stereocilia, spider silks, and hagfish slime thread skeins. Here, we demonstrate multi-nozzle printing of 3D hair arrays having freeform trajectories at a very high rate, with fiber diameters as fine as 1.5 µm, continuous lengths reaching tens of centimeters, and a wide range of materials with elastic moduli from 5 MPa to 3500 MPa. This is achieved via 3D printing by rapid solvent exchange in high yield stress micro granular gel, leading to radial solidification of the extruded polymer filament at a rate of 2.33 μm/s. …

Human Serum Albumin Interaction With Organochlorine Pesticides, D. S. Sanford, R. Yadav, A. Mishra

Journal of the Arkansas Academy of Science

Human serum albumin (HSA) is the most abundant blood plasma protein and plays a crucial role in drug transport and physiological homeostasis. HSA has a highly flexible structure consisting of three domains, each with multiple binding sites lined with hydrophobic and hydrophilic residues. The structural properties of HSA make it a robust reservoir and receptor for various endogenous and exogenous compounds, including therapeutic drugs. Organochlorine pesticides (OCPs) are a subclass of pesticides characterized by their chlorine content. Once widely used, these compounds remain persistent in the environment and biological systems despite regulatory restrictions in many developed countries. The strong ligand-binding …

Raman Spectroscopy For Rapid Quantification Of Macronutrients In Heirloom Beans, K. Chandler Williams

Graduate Theses, Dissertations, and Problem Reports (ETD)

Quantification of macronutrients in food requires a large investment in facilities and instrumentation as well as ongoing expenses of labor, reagents, and hazardous waste disposal. These costs pose a significant barrier to determining macronutrient quantities in the thousands of uncharacterized landrace and heirloom crops and discourage traditional crop breeding projects from ensuring that their selected crops maintain desirable macronutrient profiles in addition to target agronomic traits. Our objective was to test whether a hand-held Raman spectrometer—a technology which has found use in plant disease diagnostics but remains largely overlooked in nutrient quantification-- could provide a feasible means for quantifying the …

Chilling- And Dark-Regulated Photoprotection In Miscanthus, An Economically Important C4 Grass, Jared Haupt, Katarzyna Glowacka

Department of Biochemistry: Faculty Publications

Tolerance of chilling dictates the geographical distribution, establishment, and productivity of C4 crops. Chilling reduces enzyme rate, limiting the sink for the absorbed light energy leading to the need for quick energy dissipation via non-photochemical quenching (NPQ). Here, we characterize NPQ upon chilling in three Miscanthus accessions representing diverse chilling tolerance in C4 grasses. High chilling tolerant accessions accumulate substantial amounts of zeaxanthin during chilling nights in both field and growth chamber settings. Chilling-induced zeaxanthin accumulation in the dark enhances rate of NPQ induction by 66% in the following morning. Based on our data, the emerging ways for the unique …

Analysis Of Natural Structures And Chemical Mapping Data Reveals Local Stability Compensation In Rna, Robert L. Cornwell-Arquitt, Riley Nigh, Michael T. Hathaway, Joseph D. Yesselman, David A. Hendrix

Department of Biochemistry: Faculty Publications

RNA molecules adopt complex structures that perform essential biological functions across all forms of life, making them promising candidates for therapeutic applications. However, our ability to design new RNA structures remains limited by an incomplete understanding of their folding principles. While global metrics such as the minimum free energy are widely used, they are at odds with naturally occurring structures and incompatible with established design rules. Here, we introduce local stability compensation (LSC), a principle that RNA folding is governed by the local balance between destabilizing loops and their stabilizing adjacent stems, challenging the focus on global energetic optimization. Analysis …

Polymeric Sumo-2/3 Chain Signals Are Antagonistically Regulated By The Sumo-Targeted Ubiquitin E3 Ligase Rnf4 And The Sumo-Specific Isopeptidase Senp6, Shaniah A. Irving M.A., Xiang-Dong Zhang Ph.D.

Biology Theses

Polymeric SUMO-2/3 chain modification is critical for chromosome segregation, DNA damage repair, genome stability, stress responses, and protein degradation. However, how these signals are regulated remains poorly understood. Both the SUMO-targeted ubiquitin E3 ligase (STUbL) RNF4 and the SUMO-specific isopeptidase SENP6 recognize poly-SUMO-2/3 chains through multiple SUMO-interacting motifs (SIMs). RNF4 contains four tandem SIMs within a short (~80 amino acids) region, whereas SENP6 possesses eight SIMs spanning a much larger (~500 amino acids) domain.

Our preliminary results show that overexpressing an RNF4 N-terminal fragment containing four tandem SIMs (wild type, WT), but not an inactive SIMs mutant (Mut), increases levels …

Student Comprehension Of Biochemistry In A Flipped Classroom Format, Ed Harris, Evan A. Schroder, Teryn J. Berks

Department of Biochemistry: Faculty Publications

Concept-heavy courses such as Biochemistry in life and physical science curricula are challenging for many college-aged students. It is easy for students to disengage in a lecture and not learn the subject matter while in class. To improve student learning and participation, we employed a flipped format for the first half of the course and compared learning outcomes and attitudes with the traditional lecture in the second half of the course. The experimental course was an upper-level biochemistry course taken primarily by juniors (49%), seniors (40%), and some graduate students (10%) at the college level. Our results indicate that lower-performing …

Predicting Mutation-Induced Allosteric Changes In Structures And Conformational Ensembles Of The Abl Kinase Using Alphafold2 Adaptations With Alanine Sequence Scanning, Nishank Raisinghani, Mohammed Alshahrani, Grace Gupta, Gennady M. Verkhivker

Mathematics, Physics, and Computer Science Faculty Articles and Research

Despite the success of AlphaFold2 approaches in predicting single protein structures, these methods showed intrinsic limitations in predicting multiple functional conformations of allosteric proteins and have been challenged to accurately capture the effects of single point mutations that induced significant structural changes. We examined several implementations of AlphaFold2 methods to predict conformational ensembles for state-switching mutants of the ABL kinase. The results revealed that a combination of randomized alanine sequence masking with shallow multiple sequence alignment subsampling can significantly expand the conformational diversity of the predicted structural ensembles and capture shifts in populations of the active and inactive ABL states. …