Distinct Intermediate States Research Articles

Unraveling the spatial organization and development of human thymocytes through integration of spatial transcriptomics and single-cell multi-omics profiling

The structural components of the thymus are essential for guiding T cell development, but a thorough spatial view is still absent. Here we develop the TSO-his tool, designed to integrate multimodal data from single-cell and spatial transcriptomics to decipher the intricate structure of human thymus. Specifically, we characterize dynamic changes in cell types and critical markers, identifying ELOVL4 as a mediator of CD4+ T cell positive selection in the cortex. Utilizing the mapping function of TSO-his, we reconstruct thymic spatial architecture at single-cell resolution and recapitulates classical cell types and their essential co-localization for T cell development; additionally, previously unknown co-localization relationships such as that of CD8αα with memory B cells and monocytes are identified. Incorporating VDJ sequencing data, we also delineate distinct intermediate thymocyte states during αβ T cell development. Overall, these insights enhance our understanding of thymic biology and may inform therapeutic interventions targeting T cell-mediated immune responses.

Efficient Dlx2-mediated astrocyte-to-neuron conversion and inhibition of neuroinflammation by NeuroD1.

In vivo astrocyte-to-neuron (AtN) conversion induced by overexpression of neural transcriptional factors has great potential for neural regeneration and repair. Here, we demonstrate that a single neural transcriptional factor, Dlx2, converts mouse striatal astrocytes into neurons in a dose-dependent manner. Lineage-tracing studies in Aldh1l1-CreERT2 mice confirm that Dlx2 can convert striatal astrocytes into DARPP32+ and Ctip2+ medium spiny neurons (MSNs). Time-course studies reveal a gradual conversion from astrocytes to neurons in 1 month, with a distinct intermediate state in between astrocytes and neurons. Interestingly, when Dlx2-infected astrocytes start to lose astrocytic markers, the other local astrocytes proliferate to maintain astrocytic levels in the converted areas. Unexpectedly, although Dlx2 efficiently reprograms astrocytes into neurons in the gray matter striatum, it also induces partial reprogramming of astrocytes in the white matter corpus callosum. Such partial reprogramming of white matter astrocytes is associated with neuroinflammation, which can be suppressed by the addition of NeuroD1. Our results highlight the importance of investigating AtN conversion in both the gray matter and white matter to thoroughly evaluate therapeutic potentials. This study also unveils the critical role of anti-inflammation by NeuroD1 during AtN conversion.

Charge Generation Dynamics in Organic Photovoltaic Blends under One-Sun-Equivalent Illumination Detected by Highly Sensitive Terahertz Spectroscopy.

Organic photovoltaic (OPV) devices attain high performance with nonfullerene acceptors by utilizing the synergistic dual channels of charge generation that originate from excitations in both the donor and acceptor materials. However, the specific intermediate states that facilitate both channels are subject to debate. To address this issue, we employ time-resolved terahertz spectroscopy with improved sensitivity (ΔE/E < 10-6), enabling direct probing of charge generation dynamics in a prototypical PM6:Y6 bulk heterojunction system under one-sun-equivalent excitation density. Charge generation arising from donor excitations is characterized with a rise time of ∼9 ps, while that from acceptor excitations shows a rise time of ∼18 ps. Temperature-dependent measurements further reveal notably distinct activation energies for these two charge generation pathways. Additionally, the two channels of charge generation can be substantially manipulated by altering the ratio of bulk to interfaces. These findings strongly suggest the presence of two distinct intermediate states: interfacial and intramoiety excitations. These states are crucial in mediating the transfer of electrons and holes, driving charge generation within OPV devices.

Marcus-like translocation kinetics of a knotted protein.

It has been known that some proteins naturally form knots, wherein the amino acid chain is tied up in the protein in its functional form. However, much less is known about the transport kinetics of such knotted proteins as these are forced to traverse ultrasmall pores using an electric field. Previous computational studies indicate that the translocation of knotted protein can be blocked. Recent nanopore experiments demonstrated that localized electrical fields in the vicinity of a nanopore could drive the translocation coupled to the partial or complete unfolding of unknotted proteins without requiring chemical denaturants, enzymes, or a physical pulling force. Here we combine single-molecule experiments and all-atom MD simulations to study the electrical unfolding behavior of two knotted proteins, YibK and YbeA, for pore diameters (dpore) ranging from 1.5-7 nm. We found that YibK is ∼15 kBT more stable than YbeA, and that both proteins unfold through several distinct intermediate states and consist of two distinct denatured configurations. For pores larger than 3 nm diameters, the more stable YibK translocates faster than YbeA. In contrast, for pores below 3 nm in diameter, we found no evidence of translocation for YibK, and further, YbeA can translocate even for pore diameters below 2 nm. These observations can be explained by the conformation-translocation coupling mechanism which suggests that access to an intermediate and unfolded state is critical for the translocation (for dpore< dprotein). This can be achieved by controlling the external electric field without the need for an enzyme or a large mechanical pulling force. In the experiments with YbeA using pores having a diameter range of 3-4 nm, we observed that the translocation rate first increases by increasing voltage and then starts decreasing above a threshold voltage, suggesting Marcus-like translocation kinetics behavior.

Investigation of G-protein specificity and biased agonism at the beta-2 adrenergic receptor (β2AR).

The beta-2-adrenergic receptor (β2AR) is a prototypical G-protein coupled receptor (GPCR). In cardiomyocytes, β2AR couples to the G-protein subtypes Gs and Gi to regulate cardiac function. We used this system to investigate the molecular determinants of biased agonism and G-protein specificity. We developed a Gi-biased agonist, LM189, that we used to obtain the Cryo-EM structure of the β2AR-Gi complex. Spectroscopic investigations by electron paramagnetic resonance spectroscopy (EPR) and single-molecule FRET (smFRET) show that distinct intermediate states, differentially stabilized by balanced and biased ligands, contribute to G-protein specificity. Understanding the basis of specificity and bias is important to design safer drugs that target GPCRs with reduced side effects.

Conformational selection in the Cre CBD domain: Insights into intasome assembly.

During site-specific recombination tyrosine recombinases promote scission, insertion, and recombination of specific DNA sequences. The Cre/loxP system from P1 bacteriophage promotes viral DNA circularization after infection and equitable plasmid assortment during cell division. It acts through the formation of a tetramer (intasome) in which Cre monomers are bound in a head-to-tail fashion to a half loxP site, alternating between active and inactive conformations. Despite knowledge of the recombination mechanism and strand exchange order, information regarding target selection and intasome assembly is still missing. We used molecular dynamics simulations (MD) to assess the conformational flexibility of the CBD domain of Cre recombinase. The starting seeds for MD were structures with PDB ID 1q3u (an intasome) and 7rhy (a Cre monomer bound to a loxP half-site); these structures vary in the position of helix A. We simulated four replicas for each system for 5 microseconds in an explicit solvent model (TIP3P) with the software GROMACS using CHARMM36m at 300K and 1 atm. Van der Waals interactions were calculated using a 1.2 nm cutoff and PME for electrostatics, with a 4 fs timestep. Our results show that helix A can switch from an intasome-like to a monomer-like conformation, passing through distinct intermediate and unfolded states. These results suggest that the position of helix A is selected concomitantly with establishing monomer-monomer interactions during intasome assembly. Also, the flexibility of helix A could help to interpret NMR data in which the signals for this helix are missing.We acknowledge the computer resources provided by the LNS del Sureste de México through grant 202101023N, Laboratorio de Supercómputo y Visualización en Paralelo, and LANCAD through grants 99-2021 and 49-2022, and CONACyT (graduate school scholarship 858905, grant INF-2014-02-231504, grant A1-S-11842).

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.

Transcriptional networks of transient cell states during human prefrontal cortex development.

The human brain is divided into various anatomical regions that control and coordinate unique functions. The prefrontal cortex (PFC) is a large brain region that comprises a range of neuronal and non-neuronal cell types, sharing extensive interconnections with subcortical areas, and plays a critical role in cognition and memory. A timely appearance of distinct cell types through embryonic development is crucial for an anatomically perfect and functional brain. Direct tracing of cell fate development in the human brain is not possible, but single-cell transcriptome sequencing (scRNA-seq) datasets provide the opportunity to dissect cellular heterogeneity and its molecular regulators. Here, using scRNA-seq data of human PFC from fetal stages, we elucidate distinct transient cell states during PFC development and their underlying gene regulatory circuitry. We further identified that distinct intermediate cell states consist of specific gene regulatory modules essential to reach terminal fate using discrete developmental paths. Moreover, using in silico gene knock-out and over-expression analysis, we validated crucial gene regulatory components during the lineage specification of oligodendrocyte progenitor cells. Our study illustrates unique intermediate states and specific gene interaction networks that warrant further investigation for their functional contribution to typical brain development and discusses how this knowledge can be harvested for therapeutic intervention in challenging neurodevelopmental disorders.

\u03b1v\u03b23 Integrin induces partial EMT independent of TGF-\u03b2 signaling

Epithelial–mesenchymal transition (EMT) plays a pivotal role for tumor progression. Recent studies have revealed the existence of distinct intermediate states in EMT (partial EMT); however, the mechanisms underlying partial EMT are not fully understood. Here, we demonstrate that αvβ3 integrin induces partial EMT, which is characterized by acquiring mesenchymal phenotypes while retaining epithelial markers. We found αvβ3 integrin to be associated with poor survival in patients with lung adenocarcinoma. Moreover, αvβ3 integrin-induced partial EMT promoted migration, invasion, tumorigenesis, stemness, and metastasis of lung cancer cells in a TGF-β-independent fashion. Additionally, TGF-β1 promoted EMT progression synergistically with αvβ3 integrin, while a TGF-β signaling inhibitor showed no effect on αvβ3 integrin-induced partial EMT. Meanwhile, the microRNA-200 family abolished the αvβ3 integrin-induced partial EMT by suppressing αvβ3 integrin cell surface expression. These findings indicate that αvβ3 integrin is a key inducer of partial EMT, and highlight a new mechanism for cancer progression.

The Transport Cycle of a Sodium/Proton Antiporter

Sodium/proton antiporters, also known as sodium/proton exchangers, are essential secondary active transporters that are involved in pH homeostasis and regulation of sodium concentration and cell volume. They belong to the monovalent cation/proton antiporter families (CPA1 and CPA2) which all appear to share a common fold, spanning prokaryotic to mammalian family members. They obey general principles of secondary active transporters, namely they switch between at least two conformations in accordance with the alternating access model, the major states (outward facing (OF) and inward facing (OF)) are rooted in structurally breaking an inverted repeat symmetry, and they transduce energy from a driving ion gradient to vectorial substrate transport through conformational cycles. However, these broad principles constrain but do not immediately provide insights into the molecular mechanism of sodium/proton antiport. We have been using molecular dynamics simulations (long equilibrium MD, free energy calculations, enhanced sampling for rare events, constant pH simulations) in combination with experimental techniques such as X-ray crystallography and functional measurements to investigate key steps in the transport cycle of the prokaryotic CPA2 transporter TtNapA. A competitive binding mechanism between protons and sodium emerges from an analysis of the thermodynamics of the OF and IF states and naturally explains the experimentally observed activity peak in a narrow pH range. Direct computational sampling of the conformational transitions shows that NapA can undergo fast (sub-microsecond) conformational changes in either the proton or sodium-loaded states and reveals a distinct meta-stable intermediate state. Taken together, the information from all the major steps in the complete transport cycle can then form the basis for multi-scale kinetic models of the physiological function of the antiporter.

Single-cell transcriptomics identifies divergent developmental lineage trajectories during human pituitary development

The anterior pituitary gland plays a central role in regulating various physiological processes, including body growth, reproduction, metabolism and stress response. Here, we perform single-cell RNA-sequencing (scRNA-seq) of 4113 individual cells from human fetal pituitaries. We characterize divergent developmental trajectories with distinct transitional intermediate states in five hormone-producing cell lineages. Corticotropes exhibit an early intermediate state prior to full differentiation. Three cell types of the PIT-1 lineage (somatotropes, lactotropes and thyrotropes) segregate from a common progenitor coexpressing lineage-specific transcription factors of different sublineages. Gonadotropes experience two multistep developmental trajectories. Furthermore, we identify a fetal gonadotrope cell subtype expressing the primate-specific hormone chorionic gonadotropin. We also characterize the cellular heterogeneity of pituitary stem cells and identify a hybrid epithelial/mesenchymal state and an early-to-late state transition. Here, our results provide insights into the transcriptional landscape of human pituitary development, defining distinct cell substates and subtypes and illustrating transcription factor dynamics during cell fate commitment.

Probing an intermediate state by X-ray absorption near-edge structure in nickel-doped 2LiBH4–MgH2 reactive hydride composite at moderate temperature

Transition metal nickel (Ni) efficiently provokes an enhanced hydrogen desorption kinetics of the 2LiBH4–MgH2 reactive hydride composite (RHC) system by in-situ formed MgNi3B2. However, the detailed information about the complete evolution process of Ni in the 2LiBH4–MgH2 system is still insufficient and deserves further research. Herein, an interesting phenomenon that inconspicuous exothermic process coupled with unimpressive hydrogen-releasing behavior at a relatively moderate temperature range (150–300 °C) was found in the typical 2LiBH4–MgH2 RHC system when doped with nanosized Ni/C derived from the pyrolysis of MOFs-74-Ni. The X-ray absorption near-edge structure spectra of 2LiBH4–MgH2–15%Ni/C sample under various temperatures confirm that chemical valence of Ni presents a distinct intermediate state. After that, phase analysis indicates that Ni most likely reacts with LiBH4 to form a temporary Ni4B3, and a series of meticulous supplementary data were calculated to identify the authenticity of the proposed assumption. More precisely, the experimental calorific value (15.43 mJ) and hydrogen release capacity (0.47 wt%) are highly consistent with the theoretical calculation value (15.66–15.99 mJ and 0.48 wt%) of the proposed reaction. It is revealed that the Ni4B3 intermediate phase will further transform into catalytic active MgNi3B2 species, which provokes a significantly enhanced kinetic behavior and also high hydrogen storage capacity (above 9.0 wt%) of the 2LiBH4–MgH2 RHC system. Finally, the heterogeneous nucleation mechanism of the MgNi3B2 catalytic active substance is also collaboratively verified through careful scanning electron microscope and high-resolution transmission electron microscopy observations. This work ideally reveals the complete evolution process of Ni in the 2LiBH4–MgH2 RHC system by the detection of a nanosized intermediate state. More importantly, it provides ideas and inspirations for the other nanosized catalytic complex hydride systems.

Shape-Dependent Macromolecular Crowding on the Thermodynamics and Microsecond Conformational Dynamics of Protein Unfolding Revealed at the Single-Molecule Level

One of the main differences in the intercellular environment compared to the laboratory condition is the presence of macromolecular crowders of various compositions, sizes, and shapes. In this article, we have contemplated a systematic shape dependency of macromolecular crowders on the thermodynamics and microsecond conformational fluctuation dynamics of protein unfolding by taking human serum albumin (HSA) as the model protein and similar-sized crowders, namely, dextran-40, ficoll-70, and PEG-35 as macromolecular crowders of different shapes, to mimic the cell environment. We observed that dextran-40 and ficoll-70 counteract the thermal denaturation and PEG-35 assists it. A complete thermodynamic analysis suggests that the stabilization by dextran-40 and ficoll-70 occurs mainly through stabilizing entropic effect, which is somewhat counteracted by the destabilizing enthalpic effect, in line with what is expected from the traditional interpretation of excluded volume and soft interaction. Surprisingly, the destabilizing effect of PEG-35 is not through unfavorable interaction but through a destabilizing entropic effect, which is opposite to the excluded volume prediction. Our speculation is that the modulation of the associated water structure due to crowder-induced distortion plays a crucial role in modulating the entropic component. Moreover, while a two-state model can approximate the overall thermal denaturation of HSA in the absence and presence of various crowders, the thermal denaturation profile of domain III of HSA involves a distinct intermediate state. The active-site dynamics of HSA are altered significantly in the presence of all the three differently shaped crowders used in the study. Rod-shaped dextran-40 and spherical ficoll-70 hinder the microsecond conformational dynamics of domain III of HSA in all states except the intermediate state. Mesh-like PEG-35 in addition to hindering the microsecond conformational dynamics shifts the intermediate state from 40 to 30 °C. Overall, our results provide new insight into deciphering the mechanism of crowder-induced changes in protein. Through our interpretation, we not only explain the unfavorable entropic contribution but also provide a physical basis to explain the entropy-enthalpy compensation.

Distinct intermediate states in the isostructural R−3m phase of the topological insulator Bi2Se3 at high pressure

The electronic state of layered three-dimensional topological insulators at high pressure is a compelling but a puzzling hot topic. The open question is the structural origins for the pressure-induced novel physics of electronic topological transition (ETT), topological superconductivity, and Majorana fermions in the same isostructural $R\text{\ensuremath{-}}3m$ phase. Here, we report a combined investigation on the local structure and bulk electronic state of topological insulator $\mathrm{B}{\mathrm{i}}_{2}\mathrm{S}{\mathrm{e}}_{3}$ using x-ray diffraction, high quality x-ray absorption fine-structure spectroscopies both at the $\mathrm{Bi}\phantom{\rule{0.16em}{0ex}}{L}_{3}$ edge and at the Se K edge, and first-principles theoretical calculations. We have found three pressure-induced distinct intermediate states in the isostructural rhombohedral phase of $\mathrm{B}{\mathrm{i}}_{2}\mathrm{S}{\mathrm{e}}_{3}$. The bulk electronic structure of the $R\text{\ensuremath{-}}3m$ phase was calculated based on the experimental structure. The corresponding distinct electronic states provide the origins of ETT at \ensuremath{\sim}3 GPa and the metallization at 7--9.5 GPa in the $R\text{\ensuremath{-}}3m$ phase of $\mathrm{B}{\mathrm{i}}_{2}\mathrm{S}{\mathrm{e}}_{3}$. Our results demonstrate that the local structure plays a critical role in the electronic states of topological insulator $\mathrm{B}{\mathrm{i}}_{2}\mathrm{S}{\mathrm{e}}_{3}$.

A Novel Approach for Quantifying Cancer Cells Showing Hybrid Epithelial/Mesenchymal States in Large Series of Tissue Samples: Towards a New Prognostic Marker.

In cancer biology, epithelial-to-mesenchymal transition (EMT) is associated with tumorigenesis, stemness, invasion, metastasis, and resistance to therapy. Evidence of co-expression of epithelial and mesenchymal markers suggests that EMT should be a stepwise process with distinct intermediate states rather than a binary switch. In the present study, we propose a morphological approach that enables the detection and quantification of cancer cells with hybrid E/M states, i.e., which combine partially epithelial (E) and partially mesenchymal (M) states. This approach is based on a sequential immunohistochemistry technique performed on the same tissue section, the digitization of whole slides, and image processing. The aim is to extract quantitative indicators able to quantify the presence of hybrid E/M states in large series of human cancer samples and to analyze their relationship with cancer aggressiveness. As a proof of concept, we applied our methodology to a series of about a hundred urothelial carcinomas and demonstrated that the presence of cancer cells with hybrid E/M phenotypes at the time of diagnosis is strongly associated with a poor prognostic value, independently of standard clinicopathological features. Although validation on a larger case series and other cancer types is required, our data support the hybrid E/M score as a promising prognostic biomarker for carcinoma patients.

Phenotypic and microRNA characterization of the neglected CD24+ cell population in MCF-7 breast cancer 3-dimensional spheroid culture.

In vitro 3-dimensional (3D) spheroid culture has been widely used as model to enrich CD44CD24 cancer stem cells (CSC) with high aldehyde dehydrogenase 1 (ALDH1) activity. Although CD24 subpopulation was known to be present in 3D spheroids and may influence cancer drug therapies, its characteristics and CSC properties were not well defined. In this study, CD24 population from the Michigan Cancer Foundation-7 (MCF-7) spheroid was sorted and subjected to spheroid formation test, stem cell markers immunofluorescence, invasion and migration test, as well as microRNA expression profiling. Sorted MCF-7 CD24 cells from primary spheroids were able to reform its 3D spheroid shape after 7 days in nonadherent culture conditions. In contrast to the primary spheroids, the expression of SOX-2, CD44, CD49f, and Nanog was dim in MCF-7 CD24 cells. Remarkably, MCF-7 CD24 cells were found to show high expression of ALDH1 protein which may have resulted in these cells exhibiting higher resistance against doxorubicin and cisplatin when compared with that of the parental cells. Moreover, microRNA profiling has shown that the absence of CSC properties was consistent with the downregulation of major CSCs-related pathways including Hedgehog, wingless-related integration site (Wnt), and microtubule associated protein kinase (MAPK) signaling pathways. However, the upregulated pathways such as adherens junctions, focal adhesion, and tight junction suggest that CD24 cells were probably at an epithelial-like state of cell transition. In conclusion, neglected CD24 cells in MCF-7 spheroid did not exhibit typical breast CSCs properties. The presence of miRNAs and their analyzed pathways suggested that these cells could be a distinct intermediate cell state in breast CSCs.

Single-Cell Transcriptomics Uncovers Glial Progenitor Diversity and Cell Fate Determinants during Development and Gliomagenesis.

The identity and degree of heterogeneity of glial progenitors and their contributions to brain tumor malignancy remain elusive. By applying lineage-targeted single-cell transcriptomics, we uncover an unanticipated diversity of glial progenitor pools with unique molecular identities in developing brain. Our analysis identifies distinct transitional intermediate states and their divergent developmental trajectories in astroglial and oligodendroglial lineages. Moreover, intersectional analysis uncovers analogous intermediate progenitors during brain tumorigenesis, wherein oligodendrocyte-progenitor intermediates are abundant, hyper-proliferative, and progressively reprogrammed toward a stem-like state susceptible to further malignant transformation. Similar actively cycling intermediate progenitors are prominent components in human gliomas with distinct driver mutations. We further unveil lineage-driving networks underlying glial fate specification and identify Zfp36l1 as necessary for oligodendrocyte-astrocyte lineage transition and glioma growth. Together, our results resolve the dynamic repertoire of common and divergent glial progenitors during development and tumorigenesis and highlight Zfp36l1 as a molecular nexus for balancing glial cell-fate decision and controlling gliomagenesis.

Distinctive single-channel properties of α4β2-nicotinic acetylcholine receptor isoforms.

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2)2β2- and LS-(α4β2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2)2β2-nAChR exhibit a single conductance state, whereas LS-(α4β2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site’s role in mediating LS-(α4β2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings.

Identification of the tumour transition states occurring during EMT.

In cancer, the epithelial-to-mesenchymal transition (EMT) is associated with tumour stemness, metastasis and resistance to therapy. It has recently been proposed that, rather than being a binary process, EMT occurs through distinct intermediate states. However, there is no direct in vivo evidence for this idea. Here we screen a large panel of cell surface markers in skin and mammary primary tumours, and identify the existence of multiple tumour subpopulations associated with different EMT stages: from epithelial to completely mesenchymal states, passing through intermediate hybrid states. Although all EMT subpopulations presented similar tumour-propagating cell capacity, they displayed differences in cellular plasticity, invasiveness and metastatic potential. Their transcriptional and epigenetic landscapes identify the underlying gene regulatory networks, transcription factors and signalling pathways that control these different EMT transition states. Finally, these tumour subpopulations are localized in different niches that differentially regulate EMT transition states.

Salt Effects on the Thermodynamics of a Frameshifting RNA Pseudoknot under Tension

Because of the potential link between −1 programmed ribosomal frameshifting and response of a pseudoknot (PK) RNA to force, a number of single-molecule pulling experiments have been performed on PKs to decipher the mechanism of programmed ribosomal frameshifting. Motivated in part by these experiments, we performed simulations using a coarse-grained model of RNA to describe the response of a PK over a range of mechanical forces (fs) and monovalent salt concentrations (Cs). The coarse-grained simulations quantitatively reproduce the multistep thermal melting observed in experiments, thus validating our model. The free energy changes obtained in simulations are in excellent agreement with experiments. By varying f and C, we calculated the phase diagram that shows a sequence of structural transitions, populating distinct intermediate states. As f and C are changed, the stem–loop tertiary interactions rupture first, followed by unfolding of the 3′-end hairpin (I⇌F). Finally, the 5′-end hairpin unravels, producing an extended state (E⇌I). A theoretical analysis of the phase boundaries shows that the critical force for rupture scales as (logCm)α with α=1(0.5) for E⇌I (I⇌F) transition. This relation is used to obtain the preferential ion–RNA interaction coefficient, which can be quantitatively measured in single-molecule experiments, as done previously for DNA hairpins. A by-product of our work is the suggestion that the frameshift efficiency is likely determined by the stability of the 5′-end hairpin that the ribosome first encounters during translation.