Stable Intermediate State Research Articles

PlmCas12e Utilizes Glu662 to Prevent Cleavage Site Occupation by Positively Charged Residues Before Target Strand Cleavage.

CRISPR-Cas12e is a recently identified gene-editing tool mainly known because its relatively small size benefits cell delivery. Drastically different from Cas9, it creates a blunt-end double-strand breakage of the DNA via two cleavage sites; Cas12e produces a sticky-end double-strand breakage of the DNA through only one cleavage site in its RuvC domain, meaning two consecutive cleavage events first on the non-target strand (ntsDNA) and then the target strand (tsDNA). Though crucial for Cas12e's cleavage efficiency, the mechanism by which Cas12e loads tsDNA for the second cleavage remains elusive. Through molecular dynamics simulations and our recently matured traveling-salesman-based automated path-searching (TAPS) algorithm, we identified a series of positively charged residues (Arg856TSL, Arg768RuvC, Lys898TSL, Arg904TSL, Arg764RuvC) that guide the tsDNA backbone toward the cleavage site of wild-type PlmCas12e. Further simulations of the R856L and R904L mutants supported such observations. More interestingly, we found the key role of Glu662RuvC in coordinating Arg764RuvC, preventing its occupation of the cleavage site, and facilitating tsDNA cleavage. Additional simulations confirmed that mutating Glu662RuvC to valine disabled such coordination and created a stable intermediate state with Arg764RuvC occupying the cleavage site before tsDNA loading. These insights, revealing an elaborate mechanism of cleavage facilitation, offer essential guiding principles for future rational engineering of Cas12e into more efficient gene-editing tools.

Structure Function Studies of Photosystem II Using X-Ray Free Electron Lasers.

The structure and mechanism of the water-oxidation chemistry that occurs in photosystem II have been subjects of great interest. The advent of X-ray free electron lasers allowed the determination of structures of the stable intermediate states and of steps in the transitions between these intermediate states, bringing a new perspective to this field. The room-temperature structures collected as the photosynthetic water oxidation reaction proceeds in real time have provided important novel insights into the structural changes and the mechanism of the water oxidation reaction. The time-resolved measurements have also given us a view of how this reaction-which involves multielectron, multiproton processes-is facilitated by the interaction of the ligands and the protein residues in the oxygen-evolving complex. These structures have also provided a picture of the dynamics occurring in the channels within photosystem II that are involved in the transport of the substrate water to the catalytic center and protons to the bulk.

Unraveling the interplay between the leucine zipper and forkhead domains of FOXP2: Implications for DNA binding, stability and dynamics.

FOXP2 is a transcription factor associated with speech and language. Like other FOX transcription factors, it has a DNA binding region called the forkhead domain (FHD). This domain can exist as a monomer or a domain swapped dimer. In addition to the FHD, the leucine zipper region (LZ) of FOXP2 is also believed to be associated with both DNA binding and oligomerization. To better understand the relationship between DNA binding and oligomerization of FOXP2, we investigated its structure, stability and dynamics, focusing specifically on the FHD and the LZ. We did this by using two constructs: one containing the isolated FHD and one containing both the LZ and the FHD (LZ-END). We demonstrate in this work, that while the FHD maintains a monomeric form that is capable of binding DNA, the LZ-END undergoes a dynamic transition between oligomeric states in the presence of DNA. Our findings suggest that FOXP2's LZ domain influences DNA binding affinity through a change in oligomeric state. We show through hydrogen exchange mass spectroscopy that certain parts of the FHD and interlinking region become less dynamic when in the presence of DNA, confirming DNA binding and oligomerization in these regions. Moreover, the detection of a stable equilibrium intermediate state during LZ-END unfolding supports the idea of cooperation between these two domains. Overall, our study sheds light on the interplay between two FOXP2 domains, providing insight into the protein's ability to respond dynamically to DNA, and enriching our understanding of FOXP2's role in gene regulation.

Abstract 423: Cell state-specific genomics to define plasticity along the epithelial-mesenchymal transitioncontinuum

Abstract The epithelial-to-mesenchymal transition (EMT) is a biological process that allows epithelial cells to convert into a mesenchymal phenotype, acquiring increased mobility and the capacity to migrate. EMT has historically been represented as binary states; however, a growing consensus supports an EMT continuum involving multiple intermediate stable states. While we lack a comprehensive understanding of the EMT continuum, several reports indicate that the late stable states lead to optimal stem cell traits for cancer. In addition, it has been suggested that EMT heterogeneity within tumor cell populations is associated with poor patient prognosis. Hence, there is a clear need to define the genetic drivers of EM plasticity in heterogeneous cell populations along the EMT continuum. To fulfill this need, we will collect and analyze single cell RNA sequencing (scRNA-seq) data from EMT-cell-state specific genetic CRISPR screens on EMT-heterogeneous cell populations. Here, we apply a series of cloning techniques including polymerase chain reaction and Gibson ligation to engineer an adeno-associated virus (AAV) plasmid containing the avian TVA receptor gene under control of the N-cadherin promoter. Since N-cadherin is predominantly expressed in mesenchymal cells, this AAV enables us to generate cell lines that will exclusively express the TVA receptor when in a mesenchymal state. Consequently, we will be able to deliver mesenchymal-specific CRISPR gene edits via an avian retrovirus-derived RCAS system. Citation Format: Destinee K. Williams, Nicholas Hou. Cell state-specific genomics to define plasticity along the epithelial-mesenchymal transitioncontinuum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 423.

Buckling mediated by mobile localized elastic excitations.

Experiments reveal that structural transitions in thin sheets are mediated by the passage of transient and stable mobile localized elastic excitations. These "crumples" or "d-cones" nucleate, propagate, interact, annihilate, and escape. Much of the dynamics occurs on millisecond time scales. Nucleation sites correspond to regions where generators of the ideal unstretched surface converge. Additional stable intermediate states illustrate two forms of quasistatic inter-crumple interaction through ridges or valleys. These interactions create pairs from which extended patterns may be constructed in larger specimens. The onset of localized transient deformation with increasing sheet size is correlated with a characteristic stable crumple size, whose measured scaling with thickness is consistent with prior theory and experiment for localized elastic features in thin sheets. We offer a new theoretical justification of this scaling.

Continuously Tunable Optical Modulation Using Vanadium Dioxide Huygens Metasurfaces.

Efficient and dynamic light manipulation at small scale is highly desirable for many photonics applications. Active optical metasurfaces represent a useful way of achieving this due to their creative design potential, compact footprint, and low power consumption, paving the way toward the realization of chip-scale photonic devices with tunable optical functionality on demand. Here, we demonstrate a dynamically tunable, dual-function metasurface based on dielectric resonances in vanadium dioxide that is capable of independent active amplitude and phase control without the use of mechanical parts. Significant developments in the nanofabrication of vanadium dioxide have been shown to enable this metasurface. Gradual thermal control of the metasurface yields a computationally predicted continuously tuned amplitude modulation of 19 dB with negligible phase modulation and a continuously tuned phase modulation of 228° with negligible amplitude modulation, both at near-infrared wavelengths. Experimentally, a maximum continuously tuned amplitude modulation of 9.6 dB and phase modulation of 120° are shown, along with demonstration of stable intermediate states and repeated modulation without degradation. Reprogrammable optical functionality can thus be achieved in precisely engineered nanoantenna arrays for adaptive modulation of amplitude and phase of light for applications such as tunable holograms, lenses, and beam deflectors.

Influence of Post‐Treatment Temperature on the Stability and Swelling Behavior of Casein Microparticles

AbstractThis study investigates the influence of exposure time and post‐treatment temperature on the stability and swelling of casein microparticles prepared by depletion flocculation and film drying. Stability experiments with sodium dodecyl sulfate at 20 °C show that particles become more stable with higher post‐treatment temperature and longer exposure times. The two‐step decrease in decay rates in each case indicates a stable intermediate state structurally different from untreated and more strongly treated microparticles. The swelling curves of microparticles also show differences in three temperature ranges at 120 min exposure times each, which is analyzed with dynamic swelling models. At temperatures T ≤ 50 °C, the casein microparticles swell and decompose in a typical two‐step process. For post‐treatments in the temperature range between T = 60 and 70 °C, the swelling curves change their shape toward limited swelling to an equilibrium swelling value. The swelling curves of microparticles treated with temperatures at 80 and 90 °C show a typical overshooting, which can be analyzed with an adapted parallel swelling model and attributed to an elastic network of newly formed bonds.

Multilevel resistive switching with negative differential resistance in Al/NiO/ZnFe2O4/ITO ReRAM device

The Resistive Random Access Memory devices have emerged as an energy-efficient alternative to Von Neumann computers by enabling in-memory computing. Here we demonstrate bipolar resistive switching in thin films of Nickel oxide (NiO) and Zinc Ferrite (ZFO) using a simple Al/NiO/ZFO/ITO configuration, making them a possible candidate for the next generation memory devices. The fabricated device demonstrated excellent resistive switching behavior with high endurance for up to 1000 cycles, good retention for more than 104 s, and a good resistance ratio of HRS to LRS ∼102. Ohmic conduction was observed in the LRS, while in the HRS, along with ohmic conduction, space charge limited current (SCLC) and Schottky mechanisms were observed. Besides the LRS and HRS, a number of stable intermediate resistance states can also be obtained during the RESET process using different stop voltages, which makes the current device a multilevel resistive switching device.

Interdomain dynamics in human Replication Protein A regulates kinetics and thermodynamics of its binding to ssDNA.

Human Replication Protein A (hRPA) is a multidomain protein that interacts with ssDNA intermediates to provide the latter much-needed stability during DNA metabolism and maintain genomic integrity. Although the ssDNA organization with hRPA was studied recently through experimental means, characterizing the underlying mechanism at the atomic level remains challenging because of the dynamic domain architecture of hRPA and poorly understood heterogeneity of ssDNA-protein interactions. Here, we used a computational framework, precisely tailored to capture protein-ssDNA interactions, and investigated the binding of hRPA with a 60 nt ssDNA. Two distinct binding mechanisms are realized based on the hRPA domain flexibility. For a rigid domain architecture of hRPA, ssDNA binds sequentially with hRPA domains, resulting in slow association kinetics. The binding pathway involves the formation of stable and distinct intermediate states. On contrary, for a flexible domain architecture of hRPA, ssDNA binds synergistically to the A and B domains followed by the rest of hRPA. The domain dynamics in hRPA alleviates the free energy cost of domain orientation necessary for specific binding with ssDNA, leading to fast association kinetics along a downhill binding free energy landscape. An ensemble of free energetically degenerate intermediate states is encountered that makes it arduous to characterize them structurally. An excellent match between our results with the available experimental observations provides new insights into the rich dynamics of hRPA binding to ssDNA and in general paves the way to investigate intricate details of ssDNA-protein interactions, crucial for cellular functioning.

Meta-stable initial condition for improving the switching probability in azobenzene derivatives on surface

The trans isomer of azobenzene (AB) and its derivatives is the most abundant isomer at thermodynamic equilibrium and is known to switch between its trans and cis isomers when triggered by light, electrons/holes and electrical field in adlayers on surfaces. However, the equilibrium initial condition limits the operation of molecular switches on surface due to large activation barrier for switching (trans to cis), hence the switching probability is very low. We show in this article, condensation of meta-stable (non-equilibrium) cis state of azobenzene derivatives on graphite. When electrons/holes induced switching is performed on these molecular switches from the meta-stable initial condition, the switching probability is enhanced by several folds. The molecules are switching between two associated cis states, instead of trans–cis switching. The switching between the cis states has relatively low energy barrier as revealed by density functional theory (DFT) calculations is at the origin of the enhanced switching probability. We also observe that reversible trans–cis and cis–cis’ switching on graphite is mediated through a stable charged intermediate state, leading to a low threshold voltage for switching compared to neutral molecules.

Transparent, Multivalued Transistors Enabled By Area‐Selective Optical Doping on Ga‐Doped IZTO Thin Films

AbstractMultivalued transistors are demonstrated on wafer‐scale, sputtered gallium‐doped indium‐zinc‐tin‐oxide (Ga‐doped IZTO) thin films by area‐selective optical doping. Cation elemental doping and ultraviolet (UV) exposure are representative techniques to control the carrier concentrations of metal oxide semiconductors. In this work, both doping techniques are exploited to design a pseudo‐heterojunction on a monolithic Ga‐doped IZTO thin film to generate multivalued electrical characteristics. The resulting semiconductor channel possessed two distinct work functions (i.e., smaller work function on the UV‐exposed area with respect to the masked Ga‐doped IZTO area), which resulted in sequential activation with two turn‐on voltages under a gate voltage sweep. Consequently, a stable intermediate state is generated for the ternary operation of the devices. This work further demonstrates the fabrication of transparent ternary device arrays by utilizing transparent elements including indium tin oxide (ITO), Ga‐doped IZTO, SiO2, and glass as electrodes, semiconducting channels, dielectric layers, and substrates, respectively.

Rotating asymmetric solitons in competing nonlinear media

We predict a novel family of off-center localized nonlinear modes in a rotating optical system. The whispering-gallery-like solitons bifurcate out from the symmetric fundamental solitons through a symmetry breaking. They can appear as localized spots, nearly flat-top beams and crescent-like patterns extending over the entire range of polar angles. At critical rotation frequency, asymmetric solitons transform into vortex solitons, whose topological charge gradually increases with the growth of the propagation constant. Asymmetric solitons rotate around the origin persistently during propagation and preserve their shape over arbitrary distance in almost the whole existence domain. Thus, we put forward the first example of stable asymmetric intermediate states in optical systems that bridge the symmetric fundamental solitons and the symmetric vortex solitons with different topological charges, through the variance of the propagation constant and rotation frequency.

Stability of intermediate states in the surface lithiation process of silicene

We have studied using density functional theory the Li-silicene system, mainly in the saturated region (0.11>X(Li)<1.0) which is critical to understand the solid electrolyte interface layer (SEI) formation. The electronic and vibrational properties of the intermediate states of lithiated silicene were calculated. Two models were used for the Li atoms on the silicene surface: Li on top of Si (perpendicular) and Li on top of the hexagon center (centered). The centered configurations have lower Eb values up to X(Li) = 0.33. However, the vibrational properties showed instabilities for lithium in the hexagon center. An intermediate state at X(Li) = 0.38 was found to be dynamically stable. A metallic to semiconductor transition is observed at X(Li) = 0.33, and X(Li) = 0.46. Few ordered vacancies or vacancies in the dilute region exhibit vibrational stability and gap opening. Elastic stiffness tensor for different concentrations of Li in the silicene-Li system were calculated, a critical concentration of X(Li)=0.333 was determined for the change of the mechanical properties behavior. silicel has a slightly higher 2D Young‘s moduli than silicene. However, vacancies in silicel produce a substantial decrement in mechanical properties.

The Denaturant- and Mutation-Induced Disassembly of Pseudomonas aeruginosa Hexameric Hfq Y55W Mutant.

Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qβ host factor (Hfq) from mesophilic Pseudomonas aeruginosa (Pae). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states. With an extremely low pH of 1.4, a low ionic strength, and decreasing protein concentration, the accumulated trimers and dimers turn into monomers. Also, we report on the structural features of monomeric Hfq resulting from a triple mutation (D9A/V43R/Y55W) within the inter-subunit surface of the protein. This globular and rigidly packed monomer displays a high thermostability and an oligomer-like content of the secondary structure, although its urea resistance is much lower.

BCL6-dependent TCF-1+ progenitor cells maintain effector and helper CD4 T cell responses to persistent antigen

Abstract Shortly after priming, the fate of activated CD4 T cells is segregated into BCL6+ follicular helper T (Tfh) and BCL6− effector (Teff) cells. However, it remains unknown how these subsets are sustained in the presence of chronic antigen stimulation. Using a combination of single cell- and population-based approaches, we show that in chronic viral infection, activated CD4 T cells differentiate into BCL6-dependent TCF-1+ progenitor cells with superior capacity to expand and give rise to both Teff and Tfh. They share properties with progenitor-exhausted CD8 T cells and are required for the continued generation of Teff cells as antigen persists. In response to tumors, an analogous CD4 T cell population develops in draining lymph nodes. Our study reveals the heterogeneity and plasticity of CD4 T cells upon encountering persistent antigen and highlights their population dynamics through a stable bipotent intermediate state.

Abstract P4-07-19: Quantifying epithelial-mesenchymal tumor heterogeneity for prediction of patient prognosis based on EMT state

Abstract Background: Triple Negative Breast Cancer (TNBC) is an aggressive and heterogeneous subtype characterized by ER/PR/HER2 negative status. Much of the disease potential and aggressive nature of this subtype derives from inter- and intra-tumoral heterogeneity, which makes developing targeted therapies challenging. A key contributor to both heterogeneity in TNBC and later stage chemo-resistance and metastasis is the Epithelial-to-Mesenchymal transition (EMT). This developmental program is frequently exploited in the context of cancer to increase migratory abilities, invasiveness, metastatic potential, and resistance to chemotherapy. Indeed, EMT has been demonstrated and linked to poor prognosis and decreased survival in many solid cancer types. Cells have been found to reside in multiple stable intermediate states along the EMT spectrum, which confer increased aggressive, metastatic, and chemoresistance attributes to a heterogeneous tumor through increased stem-like characteristics. Identifying and targeting this disease-potentiating population in patient tumors is a major hurdle in overcoming metastatic disease. Knowledge gap: Despite major advances in our understanding, the contributions of EMT research to improvements in diagnostic pathology or cancer therapy have been minimal. One reason for this gap stems from our inability to accurately detect and quantify epithelial-mesenchymal heterogeneity in primary tumor specimens. Secondly, the significance of recently identified intermediate or partial EMT states to predicting tumor prognosis and therapy response are unclear. Approach &amp; Results: To study the role of various states within the EMT spectrum and their regulatory networks, the heterogeneous breast cancer cell line, SUM149PT, was used to derive six single cell clones encompassing the spectrum of EMT states, from epithelial to mesenchymal. Interrogation of this model system in vivo has revealed increased tumor growth and metastatic potential in the intermediate EMT states when compared to the extreme epithelial and mesenchymal states. To further elucidate EMT states in vivo, we employ a 6-marker multi-round immunofluorescence-based staining approach to identify cells that reside in various states along the EMT spectrum. We subsequently used an entropy-based approach and nearest-neighbor analysis on these tumors with the purpose of scoring heterogeneity and overall EMT state. Notably, this analysis segregated stromal infiltrates and their contributions to aggregate EMT scoring, which has been a major hurdle in using EMT as a scoring metric in patient samples. Overall, SUM149 clone-derived tumors held true to the relative EMT states of the starting cell populations; intermediate-derived tumors displayed high heterogeneity while epithelial and mesenchymal clone-derived tumors had lower levels of heterogeneity, despite retaining different EMT scores. Decoupling of heterogeneity and EMT state in this way provides two metrics to assess potential metastatic ability of a tumor. This staining method and analysis has been successfully applied in a preliminary set of patient tumors, showing promise for these two factors, E-M Heterogeneity and EMT score, as a tumor prognostic indicator to inform therapeutic decision-making. Conclusions: EMT tumor states and EMT-derived intra-tumoral heterogeneity play an important role in tumor metastasis and disease progression. Here, we have validated a multiplexed staining approach to quantify these metrics within a tumor, while segregating out stromal infiltrating cells. In the future, this staining and quantification shows promise as a means of predicting patient prognosis and informing potential treatment options based on targeting EMT states Citation Format: Meredith S Brown, Behnaz Abdollahi, Nevena Ognjenovic, Kristen E Muller, Saeed Hassanpour, Diwakar R Pattabiraman. Quantifying epithelial-mesenchymal tumor heterogeneity for prediction of patient prognosis based on EMT state [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-19.

Intermediate stable states in substance use

Many people across the world use potentially addictive legal and illegal substances, but evidence suggests that not all use leads to heavy use and dependence, as some substances are used moderately for long periods of time. Here, we empirically examine, the stability of and transitions between three substance use states: zero-use, moderate use, and heavy use. We investigate two large datasets from the US and the Netherlands on yearly usage and change of alcohol, nicotine, and cannabis. Results, which we make available through an extensive interactive tool, suggests that there are stable moderate use states, even after meeting criteria for a positive diagnosis of substance abuse or dependency, for both alcohol and cannabis use. Moderate use of tobacco, however, was rare. We discuss implications of recognizing three states rather than two states as a modeling target, in which the moderate use state can both act as an intervention target or as a gateway between zero use and heavy use.

Stability of Intermediate States in Surface Lithiation Process of Silicene

Stability of Intermediate States in Surface Lithiation Process of Silicene

Structural and dynamical determinants of a β-sheet-enriched intermediate involved in amyloid fibrillar assembly of human prion protein

The conformational conversion of the cellular prion protein (PrPC) into a misfolded, aggregated and infectious scrapie isoform is associated with prion disease pathology and neurodegeneration. Despite the significant number of experimental and theoretical studies the molecular mechanism regulating this structural transition is still poorly understood. Here, via Nuclear Magnetic Resonance (NMR) methodologies we investigate at the atomic level the mechanism of the human HuPrP(90–231) thermal unfolding and characterize the conformational equilibrium between its native structure and a β-enriched intermediate state, named β-PrPI. By comparing the folding mechanisms of metal-free and Cu2+-bound HuPrP(23–231) and HuPrP(90–231) we show that the coupling between the N- and C-terminal domains, through transient electrostatic interactions, is the key molecular process in tuning long-range correlated μs–ms dynamics that in turn modulate the folding process. Moreover, via thioflavin T (ThT)-fluorescence fibrillization assays we show that β-PrPI is involved in the initial stages of PrP fibrillation, overall providing a clear molecular description of the initial phases of prion misfolding. Finally, we show by using Real-Time Quaking-Induced Conversion (RT-QuIC) that the β-PrPI acts as a seed for the formation of amyloid aggregates with a seeding activity comparable to that of human infectious prions.

A Conductive Dinuclear Cuprous Complex Mimicking the Active Edge Site of the Copper(100)/(111) Plane for Selective Electroreduction of CO2 to C2H4 at Industrial Current Density.

Inorganic solids are a kind of important catalysts, and their activities usually come from sparse active sites, which are structurally different from inactive bulk. Therefore, the rational optimization of activity depends on studying these active sites. Copper is a widely used catalyst and is expected to be a promising catalyst for the electroreduction of CO2 to C2H4. Here, we report a conductive dinuclear cuprous complex with a short Cu···Cu contact for the electroreduction of CO2 to C2H4. By using 1H-[1,10]phenanthrolin-2-one and Cu(I) ions, a dinuclear cuprous complex [Cu2(ophen)2] (Cuophen) with a remarkable conductivity (3.9 × 10-4 S m-1) and a short intramolecular Cu···Cu contact (2.62 Å) was obtained. Such a short Cu···Cu contact is close to the distance of 2.54 Å between 2 adjacent Cu atoms in the edge of the copper(100)/(111) plane. Detailed examination of Cuophen revealed a high activity for the electroreduction of CO2 to C2H4 with a Faradaic efficiency of 55(1)% and a current density of 580 mA cm-2, and no obvious degradation was observed over 50 h of continuous operation. Comparing the properties and mechanisms of Cuophen and 2 other copper complexes with different Cu···Cu distances, we found that the shorter Cu···Cu distance is conducive not only for a *CO species to bridge 2 copper ions into a more stable intermediate transition state but also for C-C coupling.