Local Structural Changes Research Articles

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Analysis of short-term and local scale variations in fish community structure in Dachen Island waters

Understanding the changes in community structure of fishery resources in island reef waters is crucial for effective fishery resource management, as these waters constitute a critical link in maintaining the sustainable development of offshore fishery resources. The study analyzed the structure of the fish community in the waters surrounding Dachen Island by using data collected during four voyage surveys conducted between September 2020 and April 2021. The study utilized co-occurrence network analysis, multiple regression trees, and functional diversity analysis to investigate the short-term and local scale changes in fish community structure. The results showed that the fish community in the waters adjacent to Dachen Island can be categorized into two groups: annual and stage-use species. The stage-use species include Scoliodon laticaudus, Trichiurus lepturus, Tetraodontidae, and others, which are used during the summer and autumn stages of bait migration. Additionally, Dasyatidae and others, which used during the winter and spring stages of reproductive migration. Furthermore, the study found that the habitats dominated by nearshore rocky habitats and distant deep habitats formed the local scale variation in community structure in island waters. The integrated analysis of species α-diversity and functional diversity provided a basis for understanding the mechanisms of biodiversity action. Our study aims to provide a theoretical framework for the development of fishery resource management strategies based on the life cycle of fish, and to serve as a reference for the prioritization of key protection areas for fishery resources in island and reef environments. Overall, the study’s findings can promote the understanding of community structure dynamics in island waters. Additionally, these findings can offer valuable insights into the restoration of habitats and the implementation of marine ranching activities in Dachen Island. Moreover, the outcomes can serve as a theoretical foundation for the development of appropriate management and conservation strategies for fisheries resources.

Phylogenetic and Evolutionary Analysis of Plant Small RNA 2'-O-Methyltransferase (HEN1) Protein Family.

HUA ENHANCER 1 (HEN1) is a pivotal mediator in protecting sRNAs from 3'-end uridylation and 3' to 5' exonuclease-mediated degradation in plants. Here, we investigated the pattern of the HEN1 protein family evolutionary history and possible relationships in the plant lineages using protein sequence analyses and conserved motifs composition, functional domain identification, architecture, and phylogenetic tree reconstruction and evolutionary history inference. According to our results, HEN1 protein sequences bear several highly conserved motifs in plant species retained during the evolution from their ancestor. However, several motifs are present only in Gymnosperms and Angiosperms. A similar trend showed for their domain architecture. At the same time, phylogenetic analysis revealed the grouping of the HEN1 proteins in the three main super clads. In addition, the Neighbor-net network analysis result provides some nodes have multiple parents indicating a few conflicting signals in the data, which is not the consequence of sampling error, the effect of the selected model, or the estimation method. By reconciling the protein and species tree, we considered the gene duplications in several given species and found 170 duplication events in the evolution of HEN1 in the plant lineages. According to our analysis, the main HEN1 superclass mostly showed orthologous sequences that illustrate the vertically transmitting of HEN1 to the main lines. However, in both orthologous and paralogs, we predicted insignificant structural deviations. Our analysis implies that small local structural changes that occur continuously during the folds can moderate the changes created in the sequence. According to our results, we proposed a hypothetical model and evolutionary trajectory for the HEN1 protein family in the plant kingdom.

Structural transitions in jammed asymmetric bidisperse granular packings

We study the local structural changes along the jamming transitions in asymmetric bidisperse granular packings. The local structure of the packing is assessed by the contact orientational order, tilde{Q}_{ell }, that quantifies the contribution of each contact configuration (Large–Large, Small–Small, Large–Small, Small–Large) in the jammed structure. The partial values of tilde{Q}_{ell } are calculated with respect to known ordered lattices that are fixed by the size ratio, delta , of the particles. We find that the packing undergoes a structural transition at phi _J, manifested by a sudden jump in the partial tilde{Q}_{ell }. Each contact configuration contributes to the jammed structure in a different way, changing with delta and concentration of small particles, X_{textrm{S}}. The results show not only that the packing undergoes a structural change upon jamming, but also that bidisperse packings exhibit local HCP and FCC structures also found in monodisperse packings. This suggests that the jammed structure of bidisperse systems is inherently endowed with local structural order. These results are relevant in understanding how the arrangement of particles determines the strength of bidisperse granular packings.Graphic abstract

Twisting and Protonation of Retinal Chromophore Regulate Channel Gating of Channelrhodopsin C1C2.

Channelrhodopsins (ChRs) are light-gated ion channels and central optogenetic tools that can control neuronal activity with high temporal resolution at the single-cell level. Although their application in optogenetics has rapidly progressed, it is unsolved how their channels open and close. ChRs transport ions through a series of interlocking elementary processes that occur over a broad time scale of subpicoseconds to seconds. During these processes, the retinal chromophore functions as a channel regulatory domain and transfers the optical input as local structural changes to the channel operating domain, the helices, leading to channel gating. Thus, the core question on channel gating dynamics is how the retinal chromophore structure changes throughout the photocycle and what rate-limits the kinetics. Here, we investigated the structural changes in the retinal chromophore of canonical ChR, C1C2, in all photointermediates using time-resolved resonance Raman spectroscopy. Moreover, to reveal the rate-limiting factors of the photocycle and channel gating, we measured the kinetic isotope effect of all photoreaction processes using laser flash photolysis and laser patch clamp, respectively. Spectroscopic and electrophysiological results provided the following understanding of the channel gating: the retinal chromophore highly twists upon the retinal Schiff base (RSB) deprotonation, causing the surrounding helices to move and open the channel. The ion-conducting pathway includes the RSB, where inflowing water mediates the proton to the deprotonated RSB. The twisting of the retinal chromophore relaxes upon the RSB reprotonation, which closes the channel. The RSB reprotonation rate-limits the channel closing.

Ultrahigh‐Capacity Rocksalt Cathodes Enabled by Cycling‐Activated Structural Changes

AbstractMn‐redox‐based oxides and oxyfluorides are considered the most promising earth‐abundant high‐energy cathode materials for next‐generation lithium‐ion batteries. While high capacities are obtained in high‐Mn content cathodes such as Li‐ and Mn‐rich layered and spinel‐type materials, local structure changes and structural distortions ( often lead to voltage fade, capacity decay, and impedance rise, resulting in unacceptable electrochemical performance upon cycling. In the present study, structural transformations that exploit the high capacity of Mn‐rich oxyfluorides while enabling stable cycling, in stark contrast to commonly observed structural changes that result in rapid performance degradation, are reported. It is shown that upon cycling of a cation‐disordered rocksalt (DRX) cathode (Li1.1Mn0.8Ti0.1O1.9F0.1, an ultrahigh capacity of ≈320 mAh g−1 (energy density of ≈900 Wh kg−1) can be obtained through dynamic structural rearrangements upon cycling , along with a unique voltage profile evolution and capacity rise. At high voltage, the presence of Mn4+ and Li+ vacancies promotes local cation ordering, leading to the formation of domains of a “δ phase” within the disordered framework. On deep discharge, Mn4+ reduction, along with Li+ insertion transform the structure to a partially ordered DRX phase with a β′‐LiFeO2‐type arrangement. At the nanoscale, domains of the in situ formed phases are randomly oriented, allowing highly reversible structural changes and stable electrochemical cycling. These new insights not only help explain the superior electrochemical performance of high‐Mn DRXbut also provide guidance for the future development of Mn‐based, high‐energy density oxide, and oxyfluoride cathode materials.

In situ catalytic cells for x-ray absorption spectroscopy measurement.

In catalysis, determining the relationship between the dynamic electronic and atomic structure of the catalysts and the catalytic performance under actual reaction conditions is essential to gain a deeper understanding of the reaction mechanism since the structure evolution induced by the absorption of reactants and intermediates affects the reaction activity. Hard x-ray spectroscopy methods are considered powerful and indispensable tools for the accurate identification of local structural changes, for which the development of suitable in situ reaction cells is required. However, the rational design and development of spectroscopic cells is challenging because a balance between real rigorous reaction conditions and a good signal-to-noise ratio must be reached. Here, we summarize the in situ cells currently used in the monitoring of thermocatalysis, photocatalysis, and electrocatalysis processes, focusing especially on the cells utilized in the BL14W1-x-ray absorption fine structure beamline at the Shanghai Synchrotron Radiation Facility, and highlight recent endeavors on the acquisition of improved spectra under real reaction conditions. This review provides a full overview of the design of in situ cells, aiming to guide the further development of portable and promising cells. Finally, perspectives and crucial factors regarding in situ cells under industrial operating conditions are proposed.

Correlation between Local Structure and Reaction Mechanism in Disordered Rock-Salt-Type Lithium–Manganese Oxides

First-principles (FP) calculations of disordered rock-salt-type lithium–manganese oxides were performed by assuming a quasi-random structure model with the composition of Li16Mn16O32. The effects of randomness in the crystal structure and the changes in local atomic configurations during the Li extraction process on electronic structures, phase stability, and electrochemical properties were investigated by the FP calculations. The calculations indicate that the randomness of local atomic configurations, which originates from the disordered structure, makes it possible for Mn and O sites to take on a variety of electronic states. Analyses of densities of states, magnetic moments of Mn atoms, and Bader charges indicate the possibility that not only Mn but also oxygen atoms contribute to the redox reaction. The migration of Li and Mn is found to appear from octahedral to tetrahedral sites at a high state of charge. The FP calculations suggest that the change in local atomic configurations and electronic structures causes the transition in voltage profiles from sloping to 3 V plateau shape as the cycle progresses, which is characteristic of spinel-related positive electrode materials.

Quantifying changes in local basal ganglia structural connectivity in the 6-hydroxydopamine model of Parkinson's Disease using correlational tractography.

In recent years, tractography based on diffusion magnetic resonance imaging (dMRI) has become a popular tool for studying microstructural changes resulting from brain diseases like Parkinson's Disease (PD). Quantitative anisotropy (QA) is a parameter that is used in deterministic fiber tracking as a measure of connection between brain regions. It remains unclear, however, if microstructural changes caused by lesioning the median forebrain bundle (MFB) to create a Parkinsonian rat model can be resolved using tractography based on ex-vivo diffusion MRI. This study aims to fill this gap and enable future mechanistic research on structural changes of the whole brain network rodent models of PD. Specifically, it evaluated the ability of correlational tractography to detect structural changes in the MFB of 6-hydroxydopamine (6-OHDA) lesioned rats. The findings reveal that correlational tractography can detect structural changes in lesioned MFB and differentiate between the 6-OHDA and control groups. Imaging results are supported by behavioral and histological evidence demonstrating that 6-OHDA lesioned rats were indeed Parkinsonian. The results suggest that QA and correlational tractography is appropriate to examine local structural changes in rodent models of neurodegenerative disease. More broadly, we expect that similar techniques may provide insight on how disease alters structure throughout the brain, and as a tool to optimize therapeutic interventions.

Na3 Zr2 Si2 PO12 Solid Electrolyte Membrane for High-Performance Seawater Battery.

Seawater batteries (SWBs) have gained tremendous interest in the electrochemical energy storage research field because of their low cost, natural abundance, and potential use for long-duration energy storage. Advancing a SWB to demonstration projects is plagued by the poor electrochemical performance stemming from the poor interfaces of the solid electrolyte (SE), as well as the structural and chemical instabilities and sluggish ionic transport properties. In this study, the anode compartment of a surrogate SWB is constructed with a Na | SE | hard carbon configuration, and tailored dopants are introduced into the Nasicon-type Na3 Zr2 Si2 PO12 (NZSP) SE membrane. After doping with TiO2 , a much more densely packed pellet with uniformly distributed porous structure is obtained. Changes in surface chemistry and local structure in the bulk are observed, which are believed to contribute to the improved ionic conductivity and higher critical current density of the TiO2 -doped NZSP. Stable cycling performance with reversible capacities based on different Na storage mechanisms are also demonstrated.

EXAFS investigations on the pressure induced local structural changes of GeSe2 glass under different hydrostatic conditions

Pressure-induced transformations in glassy GeSe2 have been studied using the x-ray absorption spectroscopy. Experiments have been carried out at the scanning-energy beamline BM23 (European Synchrotron Radiation Facility) providing a micrometric x-ray focal spot up to pressures of about 45 GPa in a diamond anvil cell. Both Se and Ge K-edge experiments were performed under different hydrostatic conditions identifying the metallization onsets by accurate determinations of the edge shifts. The semiconductor-metal transition was observed to be completed around 20 GPa when neon was used as a pressure transmitting medium (PTM), while this transition was slightly shifted to lower pressures when no PTM was used. Accurate double-edge extended x-ray absorption fine structure (EXAFS) refinements were carried out using advanced data-analysis methods. EXAFS data-analysis confirmed the trend shown by the edge shifts for this disordered material, showing that the transition from tetrahedral to octahedral coordination for Ge sites is not fully achieved at 45 GPa. Results of present high pressure EXAFS experiments have shown the absence of significant neon incorporation into the glass within the pressure range up to 45 GPa.

Non-Noble FeCrOx Bimetallic Nanoparticles for Efficient NH3 Decomposition

Ammonia has the advantages of being easy to liquefy, easy to store, and having a high hydrogen content of 17.3 wt%, which can be produced without COx through an ammonia decomposition using an appropriate catalyst. In this paper, a series of FeCr bimetallic oxide nanocatalysts with a uniform morphology and regulated composition were synthesized by the urea two-step hydrolysis method, which exhibited the high-performance decomposition of ammonia. The effects of different FeCr metal ratios on the catalyst particle size, morphology, and crystal phase were investigated. The Fe0.75Cr0.25 sample exhibited the highest catalytic activity, with an ammonia conversion of nearly 100% at 650 °C. The dual metal catalysts clearly outperformed the single metal samples in terms of their catalytic performance. Besides XRD, XPS, and SEM being used as the means of the conventional characterization, the local structural changes of the FeCr metal oxide catalysts in the catalytic ammonia decomposition were investigated by XAFS. It was determined that the Fe metal and FeNx of the bcc structure were the active species of the ammonia-decomposing catalyst. The addition of Cr successfully prevented the Fe from sintering at high temperatures, which is more favorable for the formation of stable metal nitrides, promoting the continuous decomposition of ammonia and improving the decomposition activity of the ammonia. This work reveals the internal relationship between the phase and structural changes and their catalytic activity, identifies the active catalytic phase, thus guiding the design and synthesis of catalysts for ammonia decomposition, and excavates the application value of transition-metal-based nanocomposites in industrial catalysis.

Molecular Conformation and Intermolecular Interactions of Linear and Cyclic Amylose Derivatives in Solution

AbstractFour kinds of rigid and semiflexible ring polymers are synthesized from flexible cyclic amylose. Their conformational properties in dilute solution are determined from dimensional and hydrodynamic properties in terms of the wormlike ring model. The helix rise per residue h of some ring polymers is larger or smaller than that for the corresponding linear chains, indicating that the local helical structure of the ring polymer is not the same as that for the linear chain. Intermolecular interactions detected by the second virial coefficient and chiral recognition ability of some ring polymers are different from the linear chain, suggesting the local structural change causes the intermolecular interactions. The study also finds that rigid ring polymers can form liquid crystal phase at high concentration. X‐ray diffraction results suggest that the ring chains form pseudo rodlike conformation in the liquid crystal phase, showing that intermolecular excluded volume effects significantly influence the conformation of the polymer chains.

Understanding the Electronic Structure Basis for N2 Binding to FeMoco: A Systematic Quantum Mechanics/Molecular Mechanics Investigation.

The FeMo cofactor (FeMoco) of Mo nitrogenase is responsible for reducing dinitrogen to ammonia, but it requires the addition of 3-4 e-/H+ pairs before N2 even binds. A binding site at the Fe2/Fe3/Fe6/Fe7 face of the cofactor has long been suggested based on mutation studies, with Fe2 or Fe6 nowadays being primarily discussed as possibilities. However, the nature of N2 binding to the cofactor is enigmatic as the metal ions are coordinatively saturated in the resting state with no obvious binding site. Furthermore, the cofactor consists of high-spin Fe(II)/Fe(III) ions (antiferromagnetically coupled but also mixed-valence delocalized), which are not known to bind N2. This suggests that an Fe binding site with a different molecular and electronic structure than the resting state must be responsible for the experimentally known N2 binding in the E4 state of FeMoco. We have systematically studied N2 binding to Fe2 and Fe6 sites of FeMoco at the broken-symmetry QM/MM level as a function of the redox-, spin-, and protonation state of the cofactor. The local and global electronic structure changes to the cofactor taking place during redox events, protonation, Fe-S cleavage, hydride formation, and N2 coordination are systematically analyzed. Localized orbital and quasi-restricted orbital analysis via diamagnetic substitution is used to get insights into the local single Fe ion electronic structure in various states of FeMoco. A few factors emerge as essential to N2 binding in the calculations: spin-pairing of dxz and dyz orbitals of the N2-binding Fe ion, a coordination change at the N2-binding Fe ion aided by a hemilabile protonated sulfur, and finally hydride ligation. The results show that N2 binding to E0, E1, and E2 models is generally unfavorable, likely due to the high-energy cost of stabilizing the necessary spin-paired electronic structure of the N2-binding Fe ion in a ligand environment that clearly favors high-spin states. The results for models of E4, however, suggest a feasible model for why N2 binding occurs experimentally in the E4 state. E4 models with two bridging hydrides between Fe2 and Fe6 show much more favorable N2 binding than other models. When two hydrides coordinate to the same Fe ion, an increased ligand-field splitting due to octahedral coordination at either Fe2 or Fe6 is found. This altered ligand field makes it easier for the Fe ion to acquire a spin-paired electronic structure with doubly occupied dxz and dyz orbitals that backbond to a terminal N2 ligand. Within this model for N2 binding, both Fe2 and Fe6 emerge as possible binding site scenarios. Due to steric effects involving the His195 residue, affecting both the N2 ligand and the terminal SH- group, distinguishing between Fe2 and Fe6 is difficult; furthermore, the binding depends on the protonation state of His195.

Application of floating wetlands for the improvement of degraded urban waters: Findings from three multi-year pilot-scale installations

Floating Treatment Wetlands (FTWs) are an emerging ecological engineering technology being applied the restoration of eutrophic urban water bodies. Documented water-quality benefits of FTW include nutrient removal, transformation of pollutants, and reduction in bacterial contamination. However, translating findings from short-duration lab and mesocosm scale experiments, into sizing criteria that might be applied to field installations is not straightforward. This study presents the results of three well established (>3 years) pilot-scale (40–280 m2) FTW installations in Baltimore, Boston, and Chicago. We quantify annual phosphorus removal through harvesting of above-ground vegetation and find an average removal rate of 2 g-P m−2. In our own study and in a review of literature, we find limited evidence of enhanced sedimentation as a pathway for phosphorus removal. In addition to water-quality benefits, FTW planted with native species, provide valuable wetland habitat; and theoretically improve ecological function. We document efforts to quantify the local effect of FTW installations on benthic and sessile macroinvertebrates, zooplankton, bloom-forming cyanobacteria, and fish. Data from these three projects suggest that, even on a small scale, FTW produce localized changes in biotic structure that reflect improving environmental quality. This study provides a simple and defensible method for sizing FTW for nutrient removal in eutrophic waterbodies. We propose several key research pathways which would advance our understanding of the effects FTW have on the ecosystem they are deployed in.

The Atomic Drill Bit: Precision Controlled Atomic Fabrication of 2D Materials.

The ability to deterministically fabricate nanoscale architectures with atomic precision is the central goal of nanotechnology, whereby highly localized changes in the atomic structure can be exploited to control device properties at their fundamental physical limit. Here, an automated, feedback-controlled atomic fabrication method is reported and the formation of 1D-2D heterostructures in MoS2 is demonstrated through selective transformations along specific crystallographic orientations. The atomic-scale probe of an aberration-corrected scanning transmission electron microscope (STEM) is used, and the shape and symmetry of the scan pathway relative to the sample orientation are controlled. The focused and shaped electron beam is used to reliably create Mo6 S6 nanowire (MoS-NW) terminated metallic-semiconductor 1D-2D edge structures within a pristine MoS2 monolayer with atomic precision. From these results, it is found that a triangular beam path aligned along the zig-zag sulfur terminated (ZZS)direction forms stable MoS-NW edge structures with the highest degree of fidelity without resulting in disordering of the surrounding MoS2 monolayer. Density functional theory (DFT) calculations and ab initio molecular dynamic simulations (AIMD) are used to calculate the energetic barriers for the most stable atomic edge structures and atomic transformation pathways. These discoveries provide an automated method to improve understanding of atomic-scale transformations while opening a pathway toward more precise atomic-scale engineering of materials.

Pseudouridimycin-A Potent Nucleoside Inhibitor of the RNA Polymerase Beta Prime Subunit of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococcus, GAS), a Gram-positive bacterium, is a major cause of mild to severe life-threatening infections. Antibacterial resistance to penicillin and macrolides poses a major threat in the treatment of GAS and necessitates alternate drugs and newer antibiotics. In this direction, nucleotide-analog inhibitors (NIAs) have emerged as important antiviral, antibacterial, and antifungal agents. Pseudouridimycin (PUM), a nucleoside analogue inhibitor discovered from the soil bacterium Streptomyces sp., has proven to be effective against multidrug-resistant S. pyogenes. However, the mechanism of its activity remains elusive. In this study, subunits of the RNA polymerase of GAS have been identified as targets for PUM inhibition and the binding regions have been mapped to the N-terminal domain of the β' subunit, using computational methods. The antibacterial activity of PUM against macrolide-resistant GAS was evaluated. PUM showed effective inhibition at 0.1-1 μg/mL concentration, which was higher when compared to earlier reports. The molecular interaction between PUM and the RNA polymerase β'-N terminal subunit was investigated using isothermal titration calorimetry (ITC), circular dichorism (CD), and intrinsic fluorescence spectroscopy. The thermodynamic characterization by ITC showed an affinity constant of 6.175 × 105 M-1 denoting a moderate affinity. Fluorescence studies revealed that the interaction of protein-PUM was spontaneous in nature and follows a static quenching of tyrosine signals from the protein. The near- and far-UV CD spectral analysis concluded that PUM induced local tertiary structural changes in the protein, predominantly contributed by aromatic amino acids rather than notable changes in the secondary structure. Hence PUM could be a promising lead drug target for macrolide-resistant strains of S. pyogenes and enable eradication of pathogen in the host system.

The Dynamic Interactions of a Multitargeting Domain in Ameloblastin Protein with Amelogenin and Membrane.

The enamel matrix protein Ameloblastin (Ambn) has critical physiological functions, including regulation of mineral formation, cell differentiation, and cell-matrix adhesion. We investigated localized structural changes in Ambn during its interactions with its targets. We performed biophysical assays and used liposomes as a cell membrane model. The xAB2N and AB2 peptides were rationally designed to encompass regions of Ambn that contained self-assembly and helix-containing membrane-binding motifs. Electron paramagnetic resonance (EPR) on spin-labeled peptides showed localized structural gains in the presence of liposomes, amelogenin (Amel), and Ambn. Vesicle clearance and leakage assays indicated that peptide-membrane interactions were independent from peptide self-association. Tryptophan fluorescence and EPR showed competition between Ambn-Amel and Ambn-membrane interactions. We demonstrate localized structural changes in Ambn upon interaction with different targets via a multitargeting domain, spanning residues 57 to 90 of mouse Ambn. Structural changes of Ambn following its interaction with different targets have relevant implications for the multifunctionality of Ambn in enamel formation.

Engineering the lymph node environment promotes antigen-specific efficacy in type 1 diabetes and islet transplantation

Antigen-specific tolerance is a key goal of experimental immunotherapies for autoimmune disease and allograft rejection. This outcome could selectively inhibit detrimental inflammatory immune responses without compromising functional protective immunity. A major challenge facing antigen-specific immunotherapies is ineffective control over immune signal targeting and integration, limiting efficacy and causing systemic non-specific suppression. Here we use intra-lymph node injection of diffusion-limited degradable microparticles that encapsulate self-antigens with the immunomodulatory small molecule, rapamycin. We show this strategy potently inhibits disease during pre-clinical type 1 diabetes and allogenic islet transplantation. Antigen and rapamycin are required for maximal efficacy, and tolerance is accompanied by expansion of antigen-specific regulatory T cells in treated and untreated lymph nodes. The antigen-specific tolerance in type 1 diabetes is systemic but avoids non-specific immune suppression. Further, microparticle treatment results in the development of tolerogenic structural microdomains in lymph nodes. Finally, these local structural and functional changes in lymph nodes promote memory markers among antigen-specific regulatory T cells, and tolerance that is durable. This work supports intra-lymph node injection of tolerogenic microparticles as a powerful platform to promote antigen-dependent efficacy in type 1 diabetes and allogenic islet transplantation.

Experimental reconstruction of membrane transporter evolution.

Cellular homeostasis relies on solutes crossing cell membranes against their concentration gradients. Membrane transporters catalyze this energetically unfavorable movement using energy from the downhill movement of ions. Often, related transporters couple to either Na+ or H+ gradients; it can be difficult to glean the mechanistic reasons between such differences because the ability to use ions with such different physicochemical properties might require many changes in the transporter. Since evolutionary adaptations to environmental constraints such as salt concentrations and pH likely gave rise to the switch between sodium- and proton-motive forces, we hypothesized that phylogenetic reconstruction of ion-coupling may be a method to study such complex changes. We used phylogenetic analysis to reconstruct the evolution of prokaryotic glutamate transporters and deduce stepwise amino acid sequence changes resulting in the emergence of H+-coupled transporters from Na+-coupled ancestors. The analysis suggests that in addition to changing protonatable residues, introducing H+-dependence increases the hydrophobicity of previously polar regions proximal to the substrate and Na+ binding sites. To contextualize these changes, we used ancestral sequence reconstruction (ASR) to infer transporter sequences before and after the emergence of H+-coupling. We expressed and purified the inferred ancestral transporters, enabling their biochemical characterization and high-resolution cryo-EM structures. Surprisingly, the ancestral transporter before the emergence of H+-coupling shows Na+-independent substrate binding even though its Na+-binding pockets are structurally near-identical to Na+-coupled homologs. The emergence of H+-coupling preserves the protein architecture while subtly restructuring these critical regions. Our results suggest that changes in local structure and complex global allosteric properties underlie the diversification of the ion-motive forces used within the glutamate transporter family. The approach we developed might be broadly applicable to dissecting complex allosteric properties of membrane proteins.