Thermal conductivity of Fe-Si alloys and thermal stratification in Earth’s core

Light elements in Earth’s core play a key role in driving convection and influencing geodynamics, both of which are crucial to the geodynamo. However, the thermal transport properties of iron alloys at high-pressure and -temperature conditions remain uncertain. Here we investigate the transport properties of solid hexagonal close-packed and liquid Fe-Si alloys with 4.3 and 9.0 wt % Si at high pressure and temperature using laser-heated diamond anvil cell experiments and first-principles molecular dynamics and dynamical mean field theory calculations. In contrast to the case of Fe, Si impurity scattering gradually dominates the total scattering in Fe-Si alloys with increasing Si concentration, leading to temperature independence of the resistivity and less electron–electron contribution to the conductivity in Fe-9Si. Our results show a thermal conductivity of ∼100 to 110 W⋅m⁻¹⋅K⁻¹ for liquid Fe-9Si near the topmost outer core. If Earth’s core consists of a large amount of silicon (e.g., > 4.3 wt %) with such a high thermal conductivity, a subadiabatic heat flow across the core–mantle boundary is likely, leaving a 400- to 500-km-deep thermally stratified layer below the core–mantle boundary, and challenges proposed thermal convection in Fe-Si liquid outer core.

The geodynamo of Earth’s core is thought to be mainly driven by compositional (chemical) convection associated with the crystallization and light-element release of the inner core as well as thermal convection driven by a superadiabatic heat flow across the core–mantle boundary (CMB). The relative importance of these energy sources to the geodynamo, however, remains uncertain (1). The magnitudes of these energy sources can change throughout the evolution of the planet. The thermal gradient across the CMB can be constrained from both heat flow of the core and mantle, where a subadiabatic heat flow out of the core may hinder thermal convection and cause a thermally stratified layer at the top of the outer core (2). A global nonadiabatic structure at the top of the core has been inferred from seismic observations and geomagnetic fluctuations (3, 4), where the mechanisms for the origin rely on accurate determinations of the CMB heat flow and the core conductivity. Based on seismological observations and high-pressure and -temperature (P-T) mineral physics results, Earth’s outer and inner core are mainly composed of Fe (∼85 wt %) alloyed with Ni (∼5 wt %) and ∼8 to 10 wt % and 4–5 wt % of light elements, respectively, such as Si, O, S, C, and H (5–10). The effects of the candidate light elements on the electrical resistivity (ρ_e) and thermal conductivity (κ) of iron and their partitioning between the inner and outer core at relevant P-T conditions are thus of great importance for understanding the thermal state of the core as well as the generation and evolution of Earth’s magnetic field (2, 9, 11, 12). The thermal conductivity of the constituent core alloy controls the heat flow of the core, while the electrical resistivity of the constituent Fe alloy determines the ohmic dissipation rate of the magnetic field.Extensive studies on iron’s transport properties have been conducted via experiments and calculations (e.g., refs. 13–21), and recent studies report a thermal conductivity of ∼100 W⋅m⁻¹⋅K⁻¹ at conditions near the CMB (22, 23). Such a high thermal conductivity reduces the amount of heat that can be transported by convective flow (11) and raises a question as to what powered the convection prior to inner core growth over Earth’s history [the so-called new core paradox (24)]. Thus far, several hypotheses have been proposed to reconcile this paradox, including a possible large conductivity reduction due to nickel and light elements (25–28), a rapid core cooling rate (29), or exsolution of chemically saturated species from the core to the lowermost mantle, such as MgO, SiO₂, or FeO (e.g., refs. 30–32). The general consensus is that incorporation of light element(s) depresses high P-T thermal conductivity of iron by impurity scattering (12); this effect was assumed in our previous modeling of the high P-T transport properties of Fe-Ni alloyed with 1.8 wt % Si (25). The lowered thermal conductivity implies that thermal convection is easier to maintain. The rapid core cooling model would imply a young inner core and requires a hidden core heat source, such as radioactivity, which is not supported by geochemical evidence (29). The exsolution mechanism would offer an additional energy source to drive an early compositionally driven geodynamo (32), although some experiments find exsolution to be unlikely (33). The viability of each of these scenarios depends sensitively on the transport properties of iron alloyed with a significant amount of light element(s) (∼8 to 10 wt %) at core P-T conditions. Information on these electrical and thermal transport properties of iron alloys remain uncertain due to the sparsity of experimental and theoretical data.Here we focus on the geodynamic consequences of the transport properties of iron alloyed with 4 to 10 wt % silicon, which is considered to be one of the major light element candidates in the Earth’s core due to its geo- and cosmochemical abundance (5), high solubility in solid and liquid iron (34), and isotopic evidence (35). Fe-Si alloys have been the subject of previous studies focused on understanding the structural and physical properties of the core material, including its high P-T phase diagram (36–39), elasticity (40–44), melting behavior (36, 45, 46), and transport properties (25, 47–49). The observed density discontinuity of ∼4 to 5% across the inner-core boundary (ICB) indicates that excess light elements partition into the outer core during inner-core solidification (6, 50). We should note that the concentration of Si in the core remains uncertain. While some experiments have shown that Fe alloyed with ∼9 wt % Si can satisfy the density profile of the outer core and Fe alloyed with ∼4 wt % Si for the inner core, respectively (37, 40, 41, 51, 52), other studies indicate that a dominant Si light alloying component is unable to reproduce both the density and sound velocity distribution in the outer core (53, 54).High P-T diamond anvil cell (DAC) experiments had been previously conducted to constrain the electrical and thermal conductivity of Fe-Si alloys (28, 47, 55, 56), specifically their T-dependent resistivity and thermal conductivity at core pressures. The thermal conductivity of Fe-8 wt % Si (hereafter Fe-8Si) was measured using a high-P ultrafast optical pump probe and high P-T flash-heating methods (28). The results showed that 8 wt % silicon in solid hexagonal close-packed (hcp) Fe can strongly reduce the conductivity of pure iron by a factor of ∼2, i.e., giving ∼20 W⋅m⁻¹⋅K⁻¹ at ∼132 GPa and 3,000 K. However, the electrical resistivity of solid Fe-6.5Si at ∼99 GPa and 2,000 K was recently measured to be ∼73 µΩ⋅cm (56), which is higher than that of pure iron (22) by ∼60% at comparable conditions. The results imply a thermal conductivity of ∼66 W⋅m⁻¹⋅K⁻¹ using the Wiedemann–Franz law (TL = ρ_eκ) assuming an ideal Sommerfeld Lorentz number (L = L₀: 2.44 × 10⁻⁸ W⋅m⁻¹⋅K⁻²). Meanwhile, another recent study reported a moderate thermal conductivity of 50 to 70 W⋅m⁻¹⋅K⁻¹ for an Fe-5Ni-8Si alloy near CMB P-T conditions (∼140 GPa and 4,000 K) modeled from the measured resistivity of Fe-10Ni and Fe-1.8Si alloys using the four-probe van der Pauw method in laser-heated DACs (25). The results on Fe-10Ni and Fe-1.8Si alloys reveal a linear relationship between resistivity and temperature at a given high pressure, which is very similar to that of hcp Fe (22), over the range of measurements. In contrast, density functional theory (DFT)-based molecular dynamics simulations predict a small negative T dependence of the resistivity at high pressure when liquid Fe is alloyed with a significant amount of light elements (e.g., ∼13 wt % Si) (27). These experimental and computational results raise the possibility that the high P-T thermal transport behavior and its temperature dependence in Fe-Si alloys with a few wt % Si (e.g., 2 wt %) and a larger wt % Si (e.g., 8 to 10 wt %) can be quite different, making it difficult to evaluate the light element effects on the energetics of the core.In this study, we directly measured the electrical resistivities of polycrystalline hcp Fe-4.3 wt % Si (Fe-4.3Si, or Fe_0.92Si_0.08) and Fe-9 wt % Si (Fe-9Si, or Fe_0.84Si_0.16) alloys to ∼136 GPa and 3,000 K. We also computed the electrical resistivity and thermal conductivity of these Fe-Si alloys in solid and liquid phases using first-principles molecular dynamics (FPMD) and dynamical mean field theory (DMFT) calculations. The calculations include contributions from scattering off of Si as well as both electron–phonon (e-ph) and electron–electron (e-e) scattering. Our results are used to evaluate the Si impurity effects on the transport properties of Fe-Si alloy at P-T conditions of the topmost outer core. Assuming Si is the sole light element in the core, our results are used to constrain core thermal conductivity, which is in turn used to assess core heat flux, thermal state, and energy sources driving the geodynamo through geodynamical modeling.     

Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability

Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ^−/− mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ^−/− mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ^−/− mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper–tyrosine-phosphorylated in the PTPσ^−/− mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ^−/− mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.Protein tyrosine phosphatase (PTP)σ, encoded by PTPRS (1), consists of a cell adhesion molecule-like ectodomain containing three immunoglobulin (Ig)-like and three to eight fibronectin type III repeats, a transmembrane domain, and a cytosolic region with two PTPase domains, of which the first (D1) is catalytically active (2). PTPσ expression is developmentally regulated and found primarily in the nervous system and specific epithelia (3, 4). It was previously shown to play a role in axon growth and path finding (5–7), neuroregeneration (5, 8, 9), autophagy (10), and neuroendocrine development (11–13).To investigate the function of PTPσ in vivo, our group (11) and Tremblay and coworkers (12) generated PTPσ^−/− mice. These mice exhibited high neonatal mortality, various neurological and neuroendocrine defects, colitis, and cachexia (5, 11, 13, 14). Analysis of the intestinal tissue in surviving mice by our group revealed the presence of mucosal inflammation, intestinal crypt branching, and villus blunting: all features of colitis similar to the enteropathy associated with human inflammatory bowel disease (IBD) (15). Notably, PTPσ^−/− mice also showed increased susceptibility to chemical and infectious models of murine colitis, specifically treatment with dextran sodium sulfate (DSS) or infection with Citrobacter rodentium (15). The intestinal phenotype in the mice strongly inferred a connection between PTPσ and IBD.IBD is a chronic, idiopathic, relapsing disorder affecting the gastrointestinal tract, where Crohn disease and ulcerative colitis (UC) are the two major forms (16). In IBD pathogenesis, the presence of environmental factors together with polymorphisms in IBD-susceptibility genes cause an abnormal innate and adaptive host immune response to commensal gut bacteria, leading to sustained and deleterious inflammation (17). Chronic infection (18, 19), dysbiosis (19), defective mucosal barrier defense (20), and insufficient microbial clearance (19) have all been implicated as factors contributing to IBD pathogenesis. The disease is known to have a strong genetic component, as evidenced by specific populations exhibiting a disproportionately high incidence (21) and the high disease concordance between monozygotic twins (22). Genome-wide association studies and associated metaanalyses have implicated several genes and pathways in IBD, notably genes associated with intestinal barrier defense [MYO9B (23), PARD3 (24), MAGI2 (24), CDH1 (25, 26)].Through SNP analysis of IBD patients, we showed that PTPRS is genetically associated with UC (15). The identified SNP polymorphism leads to alternative splicing in the extracellular region of the epithelial isoform of PTPσ, causing loss of the third Ig domain (15). This splicing might potentially lead to altered ligand recognition or may affect receptor dimerization (27). In addition, through an interaction-trap assay, we identified the apical junction complex (AJC) proteins E-cadherin (CDH1) and β-catenin (CTNNB1) as colonic substrates of PTPσ (15). Interestingly, recent large-scale genetic studies have identified over 160 loci that affect risk of developing IBD, many of which involved in barrier regulation (24, 28, 29).The AJC confers polarity to epithelial cells and maintains intestinal barrier integrity (30). Defective regulation of AJC proteins creates disrupted epithelial barriers, permeability defects, and aberrant intestinal morphology (30, 31), similar to defects seen in IBD. The connection between the AJC and IBD is further demonstrated by our earlier SNP analysis, which revealed a haplotype polymorphism in CDH1 that is associated with Crohn disease, leading to a truncated E-cadherin protein that fails to localize to the plasma membrane (PM), as also observed in IBD patient biopsy samples (25). Thus, we postulate that PTPσ regulates epithelial barrier integrity through regulation of AJC proteins and that defective PTPσ function may contribute to IBD.In this report, we demonstrate an intestinal epithelial barrier defect in the PTPσ^−/− mice and identify the AJC protein ezrin as an in vivo colonic substrate for PTPσ. We further demonstrate that dephosphorylation of ezrin-Y353 or -Y145 by PTPσ leads to its redistribution from the PM to the cytosol, similar to its localization following induction of IBD in mice.     

Robust high-temperature potassium-ion batteries enabled by carboxyl functional group energy storage

The popularly reported energy storage mechanisms of potassium-ion batteries (PIBs) are based on alloy-, de-intercalation-, and conversion-type processes, which inevitably lead to structural damage of the electrodes caused by intercalation/de-intercalation of K⁺ with a relatively large radius, which is accompanied by poor cycle stabilities. Here, we report the exploration of robust high-temperature PIBs enabled by a carboxyl functional group energy storage mechanism, which is based on an example of p-phthalic acid (PTA) with two carboxyl functional groups as the redox centers. In such a case, the intercalation/de-intercalation of K⁺ can be performed via surface reactions with relieved volume change, thus favoring excellent cycle stability for PIBs against high temperatures. As proof of concept, at the fixed working temperature of 62.5 °C, the initial discharge and charge specific capacities of the PTA electrode are ∼660 and 165 mA⋅h⋅g⁻¹, respectively, at a current density of 100 mA⋅g⁻¹, with 86% specific capacity retention after 160 cycles. Meanwhile, it delivers 81.5% specific capacity retention after 390 cycles under a high current density of 500 mA⋅g⁻¹. The cycle stabilities achieved under both low and high current densities are the best among those of high-temperature PIBs reported previously.

With the consumption of energy, advanced green energy systems with high specific capacity, long-term cycle stability, and high power/energy density are highly desired (1–3). In terms of the abundant reserves of sodium with similar chemical properties to Li, sodium ion batteries (SIBs) are expected to replace the currently popular lithium ion batteries (LIBs) (4, 5). Unfortunately, with respect to the higher negative redox potential of Na⁺ than that of Li⁺, the energy densities of SIBs are still relatively low (6). As a promising alternative, potassium-ion batteries (PIBs) have lower negative redox potential than SIBs (−2.93 vs. −2.71 V), implying their higher energy/power densities. Moreover, K⁺ has weaker Lewis acidity compared with both Li⁺ and Na⁺, indicating that the ionic conductivity of PIBs is better than that of LIBs and SIBs (7). What is more, PIBs can use aluminum instead of copper foil as the current collector to reduce costs (8, 9), representing their more exciting commercial applications, especially because K⁺ resources are much richer than Li⁺ (2.09 vs. 0.0017 wt%) (10, 11).Unfortunately, the intrinsically large K⁺ radius makes it highly difficult to intercalate/de-intercalate in electrode materials with sluggish kinetics (12, 13), which causes PIBs to often deliver low-rate performances and low specific capacities with poor cycle stabilities, especially under high-temperature and harsh working conditions with a significantly accelerated K⁺ intercalation/de-intercalation process. For instance, Zhang and coworkers (14) reported that KVPO₄ could enable PIBs to work up to 55 °C. Wang and coworkers (15) reported that an azo group as the redox center favored the stable charge/discharge of PIBs up to 60 °C. Nevertheless, the cycling stability was still poor for PIBs. The reported works suggested that PIBs could run for 50 cycles at 50 °C (14) and 80 (15) and 60 (16) cycles at 60 °C, respectively. This might be mainly attributed to conventional K⁺ storage mechanisms, namely, alloy-, deintercalation-, and conversion-type processes (7, 17–19), which can facilitate structural damage with rapid capacity degradation induced by the large volume change under high temperatures (15, 20, 21).So, to overcome the poor cycle stability of PIBs, it might fundamentally rely on the progress of K⁺ storage mechanisms. Here, we report a K⁺ storage mechanism toward the exploration of robust PIBs against high temperatures, which is based on a carboxyl functional group with two carboxyl functional groups as the redox centers. In such cases, the intercalation/de-intercalation of K⁺ can be performed via surface reactions between carboxyl functional groups and K⁺ with relieved volume change, thus favoring the specific capacity of PIBs against high temperatures. Correspondingly, as an example for PIB electrodes based on p-phthalic acid (PTA) materials, when operated at 62.5 °C, their initial discharge and charge specific capacities are ∼660 and 165 mA⋅h⋅g⁻¹, respectively, at a current density of 100 mA⋅g⁻¹, with 86% specific capacity retention after 160 cycles. Meanwhile, the specific capacity holds 81.5% retention after 390 cycles under a high current density of 500 mA⋅g⁻¹. Such achieved cycle stabilities under both low and high current densities are state of the art among those of PIBs reported previously.     

Proton exchange membrane fuel cells powered with both CO and H2

The CO electrooxidation is long considered invincible in the proton exchange membrane fuel cell (PEMFC), where even a trace level of CO in H₂ seriously poisons the anode catalysts and leads to huge performance decay. Here, we describe a class of atomically dispersed IrRu-N-C anode catalysts capable of oxidizing CO, H₂, or a combination of the two. With a small amount of metal (24 μg_metal⋅cm⁻²) used in the anode, the H₂ fuel cell performs its peak power density at 1.43 W⋅cm⁻². When operating with pure CO, this catalyst exhibits its maximum current density at 800 mA⋅cm⁻², while the Pt/C-based cell ceases to work. We attribute this exceptional catalytic behavior to the interplay between Ir and Ru single-atom centers, where the two sites act in synergy to favorably decompose H₂O and to further facilitate CO activation. These findings open up an avenue to conquer the formidable poisoning issue of PEMFCs.

The proton exchange membrane fuel cell (PEMFC) is one of the key enabling technologies for the transition to the upcoming hydrogen economy (1–9). However, PEMFCs have their Achilles’ heel, i.e., they are easily poisoned by carbon monoxide (10–12). The platinum catalysts, being used exclusively to drive the fuel cell anode, bind to CO preferentially (1 ppm CO in H₂ causes >90% surface blockage) and cease to catalyze hydrogen oxidation due to site blockage (13–16). This feature of Pt makes the use of cheap crude H₂ (∼US$1.5⋅kg⁻¹) from steam reforming unrealistic, and the fuel cell vehicles (FCVs) are now fed exclusively with pure or purified H₂ at ∼10 times higher price (∼US$13 to16⋅kg⁻¹).To prompt the widespread application of PEMFCs, the anode catalysts should possess certain antipoisoning behavior (17–21), as the H₂ quality may not always be fully assured in each time of refilling. The exploration for less CO-sensitive anode catalysts has been around since the emergence of the PEMFC technology (2, 3, 5). Various Pt-based alloys were explored to alleviate the CO poisoning through the Langmuir–Hinshelwood reaction mechanism (21, 22). However, impractically high voltage losses (0.2 to 0.5 V) are still evident in the presence of trace level CO (10 to 1,000 ppm), owning to the intrinsically too strong CO adsorption on the current metallic Pt catalysts. Therefore, designing catalytic sites with reduced CO adsorption energy is highly desirable for tackling such a problem. Meanwhile, as water is also the reactant for CO electrooxidation, specific catalytic sites capable of activating water at a sufficiently low potential is demanded (23, 24). However, these stringent requirements have not been met by the currently available catalysts yet. For instance, PtRu/C, the best CO tolerant catalysts to date, requires a high Pt usage (normally >0.4 mg⋅cm⁻²) to afford for a certain cell performance in a CO presence (25, 26), which runs in the opposite direction to the cost reduction demand of the technique.Herein, we report a class of IrRu-N-C catalysts, with Ir and Ru single atoms densely and uniformly populated in nitrogen–carbon composites. The catalyst represents an example of high-efficiency single-atom catalysis in an H₂ fuel cell with practical power density. Meanwhile, the Ir and Ru single sites act in synergy to fast catalyze the CO electrooxidation reaction (COOR), where both CO and H₂O are sufficiently activated at a low potential. Therefore, the IrRu-N-C catalyst at ultralow metal loading exhibits even better antipoisoning behavior (10 to 1,000 ppm CO) than the PtRu/C catalysts that is 17 times higher in mass loading (0.4 mg⋅cm⁻²). Our results thus suggest a promising bimetal center design strategy for producing active and antipoisoning hydrogen oxidation reaction (HOR) catalysts for PEMFC anodes.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

From the Cover: Structural differences in amyloid-β fibrils from brains of nondemented elderly individuals and Alzheimer's disease patients

Although amyloid plaques composed of fibrillar amyloid-β (Aβ) assemblies are a diagnostic hallmark of Alzheimer''s disease (AD), quantities of amyloid similar to those in AD patients are observed in brain tissue of some nondemented elderly individuals. The relationship between amyloid deposition and neurodegeneration in AD has, therefore, been unclear. Here, we use solid-state NMR to investigate whether molecular structures of Aβ fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aβ fibrils from AD tissue. Two-dimensional solid-state NMR spectra of isotopically labeled Aβ fibrils, prepared by seeded growth from frontal lobe tissue extracts, are similar in the two cases but with statistically significant differences in intensity distributions of cross-peak signals. Differences in solid-state NMR data are greater for 42-residue amyloid-β (Aβ42) fibrils than for 40-residue amyloid-β (Aβ40) fibrils. These data suggest that similar sets of fibril polymorphs develop in nondemented elderly individuals and AD patients but with different relative populations on average.

Amyloid plaques in brain tissue, containing fibrils formed by amyloid-β (Aβ) peptides, are one of the diagnostic pathological signatures of Alzheimer''s disease (AD). Clear genetic and biomarker evidence indicates that Aβ is key to AD pathogenesis (1). However, Aβ is present as a diverse population of multimeric assemblies, ranging from soluble oligomers to insoluble fibrils and plaques, and may lead to neurodegeneration by a number of possible mechanisms (2–7).One argument against a direct neurotoxic role for Aβ plaques and fibrils in AD is the fact that plaques are not uncommon in the brains of nondemented elderly people, as shown both by traditional neuropathological studies (8, 9) and by positron emission tomography (10–13). On average, the quantity of amyloid is greater in AD patients (10) and (at least in some studies) increases with decreasing cognitive ability (12, 14, 15) or increasing rate of cognitive decline (16). However, a high amyloid load does not necessarily imply a high degree of neurodegeneration and cognitive impairment (11, 13, 17).A possible counterargument comes from studies of the molecular structures of Aβ fibrils, which show that Aβ peptides form multiple distinct fibril structures, called fibril polymorphs (18–20). Polymorphism has been demonstrated for fibrils formed by both 40-residue amyloid-β (Aβ40) (19, 21–24) and 42-residue amyloid-β (Aβ42) (22, 25–29) peptides, the two main Aβ isoforms. Among people with similar total amyloid loads, variations in neurodegeneration and cognitive impairment may conceivably arise from variations in the relative populations of different fibril polymorphs. As a hypothetical example, if polymorph A was neurotoxic but polymorph B was not, then people whose Aβ peptides happened to form polymorph A would develop AD, while people whose Aβ peptides happened to form polymorph B would remain cognitively normal. In practice, brains may contain a population of different propagating and/or neurotoxic Aβ species, akin to prion quasispecies or “clouds,” and the relative proportions of these and their dynamic interplay may affect clinical phenotype and rates of progression (30).Well-established connections between molecular structural polymorphism and variations in other neurodegenerative diseases lend credence to the hypothesis that Aβ fibril polymorphism plays a role in variations in the characteristics of AD. Distinct strains of prions causing the transmissible spongiform encephalopathies have been shown to involve different molecular structural states of the mammalian prion protein PrP (30–32). Distinct tauopathies involve different polymorphs of tau protein fibrils (33–37). In the case of synucleopathies, α-synuclein has been shown to be capable of forming polymorphic fibrils (38–40) with distinct biological effects (41–43).Experimental support for connections between Aβ polymorphism and variations in characteristics of AD comes from polymorph-dependent fibril toxicities in neuronal cell cultures (19), differences in neuropathology induced in transgenic mice by injection of amyloid-containing extracts from different sources (44–46), differences in conformation and stability with respect to chemical denaturation of Aβ assemblies prepared from brain tissue of rapidly or slowly progressing AD patients (47), and differences in fluorescence emission spectra of structure-sensitive dyes bound to amyloid plaques in tissue from sporadic or familial AD patients (48, 49).Solid-state NMR spectroscopy is a powerful method for investigating fibril polymorphism because even small, localized changes in molecular conformation or structural environment produce measurable changes in ¹³C and ¹⁵N NMR chemical shifts (i.e., in NMR frequencies of individual carbon and nitrogen sites). Full molecular structural models for amyloid fibrils can be developed from large sets of measurements on structurally homogeneous samples (21, 25, 26, 29, 38, 50). Alternatively, simple two-dimensional (2D) solid-state NMR spectra can serve as structural fingerprints, allowing assessments of polymorphism and comparisons between samples from different sources (22, 51).Solid-state NMR requires isotopic labeling and milligram-scale quantities of fibrils, ruling out direct measurements on amyloid fibrils extracted from brain tissue. However, Aβ fibril structures from autopsied brain tissue can be amplified and isotopically labeled by seeded fibril growth, in which fibril fragments (i.e., seeds) in a brain tissue extract are added to a solution of isotopically labeled peptide (21, 22, 52). Labeled “daughter” fibrils that grow from the seeds retain the molecular structures of the “parent” fibrils, as demonstrated for Aβ (19, 21, 24, 53) and other (54, 55) amyloid fibrils. Solid-state NMR measurements on the brain-seeded fibrils then provide information about molecular structures of fibrils that were present in the brain tissue at the time of autopsy. Using this approach, Lu et al. (21) developed a full molecular structure for Aβ40 fibrils derived from one AD patient with an atypical clinical history (patient 1), showed that Aβ40 fibrils from a second patient with a typical AD history (patient 2) were qualitatively different in structure, and showed that the predominant brain-derived Aβ40 polymorph was the same in multiple regions of the cerebral cortex from each patient. Subsequently, Qiang et al. (22) prepared isotopically labeled Aβ40 and Aβ42 fibrils from frontal, occipital, and parietal lobe tissue of 15 patients in three categories, namely typical long-duration Alzheimer''s disease (t-AD), the posterior cortical atrophy variant of Alzheimer''s disease (PCA-AD), and rapidly progressing Alzheimer''s disease (r-AD). Quantitative analyses of 2D solid-state NMR spectra led to the conclusions that Aβ40 fibrils derived from t-AD and PCA-AD tissue were indistinguishable, with both showing the same predominant polymorph; that Aβ40 fibrils derived from r-AD tissue were more structurally heterogeneous (i.e., more polymorphic); and that Aβ42 fibrils derived from all three categories were structurally heterogeneous, with at least two prevalent Aβ42 polymorphs (22).In this paper, we address the question of whether Aβ fibrils that develop in cortical tissue of nondemented elderly individuals with high amyloid loads are structurally distinguishable from fibrils that develop in cortical tissue of AD patients. As described below, quantitative analyses of 2D solid-state NMR spectra of brain-seeded samples indicate statistically significant differences for both Aβ40 and Aβ42 fibrils. Differences in the 2D spectra are subtle, however, indicating that nondemented individuals and AD patients do not develop entirely different Aβ fibril structures. Instead, data and analyses described below suggest overlapping distributions of fibril polymorphs, with different relative populations on average.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:1H detection and dynamic nuclear polarization–enhanced NMR of Aβ1-42 fibrils

Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, ¹H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on ¹³C,¹⁵N-enriched, and fully protonated samples of M₀Aβ_1-42 fibrils by high-field ¹H-detected NMR at 23.4 T and 18.8 T, and ¹³C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M₀Aβ_1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than ¹H detection. These results suggest that ¹H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.

Amyloid fibrils are highly stable protein deposits found in β-sheet conformations and are notoriously recognized as disruptive agents to cellular function in over 40 human diseases (1, 2). Alzheimer’s disease (AD) is the most pervasive of all known plaque-related diseases and is associated with the presence of amyloid-β (Aβ) peptides in the extracellular space of the brain (3–6). As of 2021, there are ∼6.2 million people in the United States living with Alzheimer’s dementia and ∼50 million worldwide (7), and there is as of yet no cure available for AD. In order to address this epidemic, it is essential that we learn as much as possible about the formation and structure of Aβ plaques, including the detailed features of their catalytic surface, in order to design and develop appropriate treatments to limit the propagation of aggregates and the generation of toxic forms.Aβ is derived from the C-terminal region of the amyloid precursor protein (APP), a membrane protein in neuronal cells, via proteolysis by β- and γ-secretase (8, 9). One of the principal challenges in rationalizing AD etiology is Aβ’s diversity in peptide length, mutations, and posttranslational modifications (10). Their low solubility renders solution NMR ineffective, and high-resolution diffraction analyses have thus far been restricted to shorter peptides with all or most residues being ordered in the fibril core structure (11). Cryogenic electron microscopy (cryo-EM) has made strides in resolution in fibril studies within the past decade (12–18), but faces challenges studying with atomic-level detail due to polymorphism and heterogeneity in the fibril macroassemblies. Studying the individual and collective roles of amyloids at atomic resolution therefore requires alternative, high-resolution, high-throughput techniques for structural analysis. Magic-angle spinning NMR (MAS-NMR) was introduced as a technique with the potential to address these problems (19, 20). Recent technical advances (21, 22) and progress in sample preparation (23) have vastly improved the sensitivity and resolution of the spectra (24). Accordingly, there are now publications describing high-resolution structures of Aβ (25–29) and other amyloid (12, 16, 30–35) fibrils based on distance and torsion angle constraints derived from MAS experiments.To date, all of the known NMR structures of amyloid fibrils were determined using constraints obtained from ¹³C/¹⁵N MAS spectra, which are inhomogeneously broadened and therefore feature well-resolved lines at low spinning frequencies (<25 kHz) (36). However, resolution often remains insufficient for in-depth analysis, and the experiments require relatively large amounts of peptide and extensive signal acquisition periods. Two relatively recent additions to the repertoire of MAS NMR experiments—namely, ¹H detection and dynamic nuclear polarization (DNP)—promise to circumvent these issues by reducing signal acquisition times or, alternatively, the amount of protein required for the experiment (37). ¹H detection offers a factor of (γ_H/γ_S)^3/2 gain in sensitivity, where S is usually a low γ-spin (38–40) such as ¹³C or ¹⁵N. In these two cases it is possible to achieve a factor of ∼8 or ∼32 gain in sensitivity, respectively. Importantly, ¹H detection also introduces an additional spectral dimension and therefore significantly increases the resolution. In parallel, DNP offers a general approach to enhancing sensitivity by factors of ∼100, dramatically reducing signal acquisition times (by ∼10⁴). It does so by exploiting the high spin polarization of unpaired electrons (of gyromagnetic ratio γ_e ∼660 times larger than γ_H) of a paramagnetic polarizing agent to enhance sensitivity by a theoretical factor of γ_e/γ_H. (41–44) Furthermore, DNP experiments are conducted at ∼100 K, thereby increasing the Boltzmann polarization and sensitivity by another factor of ∼3 over experiments conducted at ambient temperature (45).While these arguments are well established for MAS NMR in many systems, it is less obvious that they are applicable to amyloid samples because spectra of amyloids are known to be broad for a variety of reasons, such as sample purity and polymorphism. Furthermore, ¹H-detected NMR at moderate MAS frequencies (∼20 to 60 kHz) needs to be coupled to different levels of deuteration to ensure high sensitivity and narrow linewidths (42). Accordingly, deuteration with partial reprotonation of the amide or Hα sites has been implemented in pioneering studies on Aβ_1-40 at 20 kHz MAS (46), HET-s(218–289) (47), and D76N-β2m at 60 kHz MAS (48). In addition, selective protonation in Aβ_1-40 fibril methyl groups at 18 kHz MAS has led to highly resolved ¹H-detected ¹³C correlations (49). In deuterated samples, however, the amount of potentially available structural information is significantly reduced, which can impair high-resolution structure determinations. The advent of 0.7 mm MAS rotors that achieve ω_r/2π >110 kHz attenuates ¹H-¹H dipole couplings and allows direct acquisition of multidimensional ¹H data without requiring deuteration (50). Furthermore, the spectra provide assignments and structural information. While a proof-of-concept application of this approach was demonstrated on fully protonated highly regular prion fibrils (51, 52), it is not clear whether this methodology is generally applicable and extendable to the detection of resolved inter- and intramolecular contacts in complex amyloid assemblies.In parallel, our MAS DNP studies on M₀Aβ_1-42 (28, 32, 53) report significant broadening of the NMR lines at cryogenic temperatures, which was attributed to distributions of conformations trapped at low temperature and is therefore inhomogeneous in origin. The loss of resolution associated with the MAS DNP methodology is a major obstacle for the detailed structural study of uniformly labeled amyloid samples. Concurrently, reports of well-resolved spectra at high fields and spinning frequencies suggest that the broadening is homogeneous (54–56). The advent of DNP instrumentation operating at high magnetic fields (18.8 T) and faster MAS (ω_r/2π = 40 kHz) provides an approach to alleviate this limitation by attenuating homogeneous couplings (57). However, this comes at the expense of the enhancement factor, potentially compromising the capacity to carry out expeditious multidimensional and multinuclear correlations. Moreover, NMR spectra of amyloid fibrils are known to suffer from additional debilitating broadening associated with their heterogeneous character (sample purity, polymorphism, etc.), which may mitigate the benefits of high magnetic fields.In this work, we show that high resolution and sensitivity are possible for fibrils of M₀-Aβ_1-42. Notably, we demonstrate rapid resonance assignment and site-resolved detection of numerous site-specific internuclear proximities on submilligram sample quantities via ¹H-detected NMR at ω_r/2π ∼110 kHz and high field (23.4 T/1,000 MHz for ¹H) at room temperature and ¹³C-detected DNP MAS NMR at ω_r/2π = 40 kHz and high field (18.8 T/800 MHz for ¹H) at low temperature. While both ¹H detection and DNP afford increased sensitivity, we estimate, using approaches outlined by Ishii and Tycko (40), that DNP, with our current ε = 22, yields a factor of ∼6.5 higher sensitivity. These results therefore illuminate possible paths for the rapid structure elucidation of amyloid fibrils available in limited quantities.     

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-d-glucose 1-phosphate (βG1P) into d-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-d-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-d-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O–P bond orientation for binding the phosphate in the inert phosphate site differs by ∼30° between steps 1 and 2. By contrast, the orientations for the axial O–Mg–O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.Efficient enzyme catalysis of the manipulation of phosphates is one of the great achievements of evolution (1). Enzymes that operate on phosphate monoesters and anhydrides transfer the phosphoryl moiety, PO₃⁻, with rate accelerations approaching 10²¹ for monoesters, placing them among the most proficient of all enzymes (1). Phosphomutases, including α-phosphoglucomutase (αPGM) (2, 3) and β-phosphoglucomutase (βPGM) (4–6), phosphoglycerate mutase (7), α-phosphomannomutase (αPMM/PGM) (8), and N-acetylglucosamine-phosphate mutase (9), merit special attention because these enzymes have to be effective in donating a phosphoryl group to either of two hydroxyl groups that have intrinsically different reactivity. Only when both half-reactions of a phosphomutase are accessible to mechanistic analysis can the problem of how an enzyme accommodates two distinct chemistries within a single active site be resolved. Hexose 1-phosphate mutases, including enzymes central to glycolysis and other metabolic pathways, are well characterized (10, 11). They are generally activated by phosphorylation to form a covalent phosphoenzyme, which then donates its PO₃⁻ group to either of its substrates to deliver a common, transient, hexose 1,6-bisphosphate intermediate species. However, structural studies on phosphomutases are complicated by the rapid and often imbalanced equilibrium position between the substrates, and kinetic studies are problematic because of competitive, parallel pathways of enzyme activation and substrate inhibition (12, 13). As a result, transition states (TSs) for both half-reactions have not hitherto been accessible for mechanistic analysis.βPGM is the best-characterized hexose 1-phosphate mutase and is a member of the haloacid dehalogenase (HAD) superfamily (14), which has 58 HAD homologs in Homo sapiens (11). The key cellular role for βPGM is to support growth on maltose (14), which demands isomerization of β-d-glucose 1-phosphate (βG1P) via β-d-glucose 1,6-bisphosphate (βG16BP) into d-glucose 6-phosphate (G6P), a universal source of cellular energy. This interconversion is achieved via a transient, covalent phosphoenzyme intermediate involving an essential aspartic acid, Asp8, to conserve the phosphoryl group that migrates intermolecularly (Fig. 1). Mechanistically, this pathway demands the architecture of the catalytic site to be effective in promoting phosphoryl transfer from phospho-Asp8 to the 6-OH group of βG1P (step 1), followed by reverse phosphoryl transfer from 1β-OH of βG16BP to Asp8 (step 2).Open in a separate windowFig. 1.Reaction scheme and free energy profile for the conversion of βG1P into G6P via βG16BP catalyzed by βPGM. The phosphoryl transfer reaction between βG1P and the phosphoenzyme (βPGM^P) is step 1 (transferring phosphate is shown in blue), and the equivalent reaction between G6P and the phosphoenzyme is step 2 (transferring phosphate is shown in red). The two intermediate complexes are labeled βG16BP and βG61BP to indicate the two orientations of bound β-bisphosphoglucose. Intramolecular hydrogen bonds within the glucose phosphates are indicated in green. The PDB ID codes (shown in brown) for the structures of metal fluoride ground state analog (GSA) and TSA complexes are listed next to the corresponding steps. G6P is ca. 8 kJ⋅mol⁻¹ lower in free energy than βG1P at equilibrium (12). βG1P binds fivefold less tightly than G6P in an AlF₄⁻ TSA complex, corresponding to a binding energy difference of ca. 4 kJ⋅mol⁻¹. This places the TSA for step 1 (blue) ca. 12 kJ⋅mol⁻¹ (4 kJ⋅mol⁻¹ + 8 kJ⋅mol⁻¹) higher in free energy than the TSA for step 2 (red). The free energy levels of TS1 and TS2 are placed only approximately, using the assumption that the free energy difference (wavy arrows) between the TSA complex and the true TS is similar for both step 1 and step 2. The approximate relative free energy levels for the intermediate enzyme-bound states denoted with βG16BP and βG61BP are based on published data (13).Step 2 has been studied intensively, with analyses focused on structural studies of trifluoromagnesate (MgF₃⁻) and tetrafluoroaluminate (AlF₄⁻) transition state analogs (TSAs) and trifluoroberyllate ground state analogs for G6P complexes (4–6, 15). ¹⁹F NMR resonances for these complexes additionally have provided in situ probes for the electronic and protonic environment of the phosphate moiety in the active site (4–6, 15, 16). Such studies have confirmed a trigonal bipyramidal (tbp) TS associated with inline stereochemistry and general acid–base catalysis, following the rearrangement of near-attack conformers (6). By contrast, step 1, involving phosphorylation of the 6-OH group of βG1P, is not well understood. The corresponding TSA complexes hitherto have proved inaccessible; attempted crystallization of the mutase using βG1P with magnesium and fluoride provides the same MgF₃⁻ TSA complex as is formed directly with G6P because residual enzyme activity catalyzes mutation of βG1P into G6P at a rate competitive with crystallization of the complex (17). Similarly, although ¹⁹F NMR studies have identified a transient TSA complex for an AlF₄⁻ complex of βG1P, it readily isomerizes into the corresponding TSA complex of G6P (SI Appendix, Fig. S1). This impasse is resolved here by the synthesis and use of stable analogs of βG1P that resist mutase-catalyzed isomerization. Because it has been established that α-fluorination of 6-phosphonomethyl-6-deoxy-glucose (G6CP) can enhance or impair analog binding to glucose 6-phosphate dehydrogenase, depending on the stereochemistry of the α-fluorine substituent (18), we have synthesized both diastereoisomeric α-monofluoromethylenephosphonate analogs of βG1P, its methylenephosphonate analog, and the three corresponding phosphonate 1α-hydroxyl analogs. We have identified the two best-binding analogs by ¹⁹F NMR and measured their affinities with βPGM in TSA complexes using fluorescence titration. We have thereby obtained three high-resolution crystal structures of TSA complexes for step 1 of the mutase catalytic reaction. Their comparison with TSA complexes for step 2 establishes the substantially different binding modes for βG1P and G6P in their respective reactions.     

Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations

The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔS_Total = 1.4 kcal⋅mol⁻¹ per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (Ω_U/Ω_N). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔS_{helix−sheet} = 0.5 kcal⋅mol⁻¹), a property not fully reflected in the amide N-H and carbonyl C=O bond NMR order parameters. The results have implications for the thermodynamics of folding and binding, including estimates of solvent ordering and microscopic entropies obtained from NMR.An accurate determination of the loss of conformational entropy is critical for dissecting the energetics of reactions involving protein motions, including folding, conformational change, and binding (1–6). Given the difficulty of directly measuring the conformational entropy, most early estimates relied on computational approaches (2, 7–10), although, more recently, NMR methods have been used to measure site-resolved entropies (11). The computational methods often calculated the entropy of either the native state ensemble (NSE) or the denatured state ensemble (DSE) and invoked assumptions about the entropy of the other ensemble [e.g., assuming the NSE is a single state or that the DSE is a composite of all side-chain (SC) rotameric states in the Protein Data Bank (PDB)]. Most previous approaches focused on helices and omitted contributions from vibrations and correlated motions (12, 13), thereby partly accounting for the spectrum of calculated values.We address these issues by calculating the chain’s conformational entropy from the distributions of the backbone (BB) (ϕ,ψ) and SC rotametric angles, [χ_n], obtained from all-atom simulations of the NSE and DSE for mammalian ubiquitin (Ub). This study extends our previous calculation of the loss of BB entropy that used an experimentally validated DSE (14). The calculated angular distributions reflect both the Ramachandran (Rama) basin populations and the torsional vibrations. Correlated motions are accounted for through the use of joint probability distributions [e.g., P(ϕ,ψ,χ₁,χ₂)].The computed loss of BB entropy is 80% of the total entropy loss at 300 K. The BB entropy is independent of burial and residue type (excluding Pro, Gly, and pre-Pro residues) but depends on the secondary structure. Helical residues lose more BB entropy than sheet residues, TΔS_{helix−sheet} = 0.5 kcal⋅mol⁻¹ at 300 K, a difference not fully reflected by either amide N-H or carbonyl C=O bond NMR order parameters. The SC entropy loss, TΔS_SC ∼ 0.2 kcal⋅mol⁻¹⋅rotamer⁻¹, is largely independent of 2° structure and weakly correlated with burial. Combining this correlation with the average loss of BB entropy for each 2° structure type provides a site-resolved estimate of the entropy loss for an input structure (godzilla.uchicago.edu/cgi-bin/PLOPS/PLOPS.cgi). This estimate can assist in thermodynamic studies and coarse-grained modeling of protein dynamics and design.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

GABAρ subunits confer a bicuculline-insensitive component to GFAP+ cells of cerebellum

GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A subunits. Here we demonstrate that GFAP⁺ cells of the granular layer of cerebellum express GABAρ subunits during early postnatal development, thereby conferring peculiar pharmacologic characteristics to GABA responses. Electron microscopy revealed the presence of GABAρ in the plasma membrane of GFAP⁺ cells. In contrast, expression in the adult was restricted to Purkinje neurons and a subset of ependymal cells. Electrophysiological studies in vitro revealed that astrocytes express functional receptors with an EC₅₀ of 52.2 ± 11.8 μM for GABA. The evoked currents were inhibited by bicuculline (100 μM) and TPMPA (IC₅₀, 5.9 ± 0.6 μM), indicating the presence of a GABAρ component. Coimmunoprecipitation demonstrated protein–protein interactions between GABAρ1 and GABAα1, and double immunofluorescence showed that these subunits colocalize in the plasma membrane. Three populations of GABA-A receptors in astrocytes were identified: classic GABA-A, bicuculline-insensitive GABAρ, and GABA-A–GABAρ hybrids. Clusters of GABA-A receptors were distributed in the perinuclear space and along the processes of GFAP⁺ cells. Time-lapse microscopy showed GABAρ2-GFP accumulation in clusters located in the soma and along the processes. The clusters were relatively immobile, with mean displacement of 9.4 ± 0.9 μm and a net distance traveled of 1–2 μm, owing mainly to directional movement or simple diffusion. Modulation of GABAρ dynamics may be a novel mechanism of extrasynaptic transmission regulating GABAergic control of GFAP⁺ cells during early postnatal development.The role of GABAergic signaling is fundamental in the cerebellum, not only for influencing cell differentiation and neurotransmitter specification during early postnatal development, but also for controlling precise movements in the adult life (1–3). The expression of ionotropic GABA-A receptors with high affinity for the neurotransmitter is now well recognized, although the source of GABA involved in this process is controversial (4–6).GABA-A receptors mediating synaptic (phasic) or extrasynaptic (tonic) transmission are molecularly and functionally distinct. In contrast to neurons, the depolarizing effect of astrocytic GABA-A receptors persists through postnatal development, although the response may attenuate with age (7). In cerebellar astrocytes, the array of GABA-A subunits is heterogeneous, and modulation by benzodiazepines is different from that by neurons (8). Indeed, GABA responses of Bergmann cells and ependymal glial cells (EGCs) are insensitive to benzodiazepines or pentobarbital, owing to the assembly of receptors that include GABAδ or GABAρ subunits (9, 10).GABAρ subunits are part of the ionotropic GABA-A receptor family, which includes 19 identified genes that code for the same number of known proteins: α1–α6, β1–β3, γ1–γ3, δ, ε, Θ, π, and ρ1–ρ3 (11). GABA-A receptors are pentameric heterocomplexes composed of a combination of subunits, most commonly the α1β2γ2 combination, that gate a Cl⁻ channel on activation (11, 12). Other arrays may include δ, ρ, or ε subunits, which confer distinctive functional and pharmacologic properties. GABAρ subunits can combine in homopentameric arrangements that form receptors with high affinity for the neurotransmitter (GABA EC₅₀, 1–5 μM) and a low rate of desensitization, making them suitable for tonic transmission (11, 13, 14). GABAρ subunits are known to be expressed in the retina, where their presence in bipolar neurons controls the glutamatergic output (15, 16); they are present in other areas of the central nervous system as well, including striatum, hippocampus, and cerebellum, but their function there is not fully understood (17–19).The role of GABAρ in neuronal tonic (extrasynaptic) and phasic (synaptic) transmission has been demonstrated in the Purkinje neurons of the cerebellum (20); however, GABAρ subunits are also expressed in a large fraction of EGCs, specialized, ciliated GFAP⁺ cells that permit the flow of cerebrospinal fluid circulating in the fourth ventricle (10, 21). GABA-ionic currents in these cells are insensitive to pentobarbital and partially blocked by the GABA-A antagonist bicucculline as well as by (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), the first synthesized GABAρ antagonist (22). GABA-A receptors that include GABAρ subunits are also expressed in approximately 30% of the GFAP⁺ cells present in the striatum (19). Thus, it seems that cells of glial origin, such as EGCs and astrocytes, present a diverse array of ionotropic GABA receptors that include GABAρ subunits and whose functional role remains unidentified.In the course of recent work on assessing the presence of GABAρ subunits during early postnatal development of cerebellar EGCs, we corroborated their presence in this area but, unexpectedly, found that they are also widely distributed in a large proportion of GFAP⁺ cells of the granular layer (GL) that appear to be astrocytes. In this paper we report in detail the expression pattern of GABAρ subunits in GFAP⁺ cells of the cerebellum, the functional characterization of GABA responses of cerebellar astrocytes grown in vitro, and the participation of a GABAρ component in these responses. In addition, we provide evidence of the intracellular trafficking of GABAρ in astrocytes grown in vitro, and speculate about the possible synthesis of proteins in the processes of these cells.     

Structural and energetic analysis of metastable intermediate states in the E1P–E2P transition of Ca2+-ATPase

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase transports two Ca²⁺ ions from the cytoplasm to the SR lumen against a large concentration gradient. X-ray crystallography has revealed the atomic structures of the protein before and after the dissociation of Ca²⁺, while biochemical studies have suggested the existence of intermediate states in the transition between E1P⋅ADP⋅2Ca²⁺ and E2P. Here, we explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. The simulations suggest that a series of structural changes accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca²⁺ ions toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization. Free energy profiles of the transition assuming different protonation states suggest rapid exchanges between Ca²⁺ ions and protons when the Ca²⁺ ions are released toward the SR lumen.

Sarcoplasmic reticulum Ca²⁺-ATPase (SR Ca²⁺-ATPase or SERCA1a) is a representative P-type ATPase that transports Ca²⁺ ions against a 10⁴ times concentration gradient across the SR membrane. The transport mechanism was originally described by E1/E2 theory, whereby the protein alternates between Ca²⁺ high-affinity E1 and low-affinity E2 states. A more-detailed reaction cycle requires multiple steps, including the binding/dissociation of Ca²⁺, H⁺-counter transport, ATP-binding and hydrolysis, phosphorylation/dephosphorylation of Asp351, and the dissociation of ADP and Pi (1–3) (SI Appendix, Fig. S1). Structurally, the Ca²⁺-ATPase consists of three cytoplasmic domains (actuator; A, nucleotide-binding; N, and phosphorylation; P) and 10 transmembrane (TM) helices (M1-M10) (4, 5). Two Ca²⁺-binding sites are located in M4-M6 and M8 (6), while a nucleotide, ATP or ADP, is bound at the N–P domain interface (7). Functional interconnections between the cytoplasmic domains and TM helices are necessary in the reaction cycle (8, 9).Molecular mechanisms underlying Ca²⁺ uptake by the ATPase have been investigated in biochemical experiments as well as structural studies. In particular, crystal structures of the Ca²⁺-ATPase, which represent different physiological states in the cycle (SI Appendix, Fig. S1), have provided essential information for understanding structure–function relationships (8, 9). Each crystal structure well explains the results of mutagenesis (3, 10), limited proteolysis studies (11, 12), and other biochemical experiments. Comparisons between multiple crystal structures provide direct evidence on how conformational changes of the Ca²⁺-ATPase take place from one step to another in the cycle. For instance, crystal structures that represent E1P⋅ADP⋅2Ca²⁺ and E2P (Fig. 1 A and B) reveal important conformational changes to release Ca²⁺ toward the SR lumen: 1) the A-domain rotates ∼90°; 2) the threonine–glycine–glutamate–serine (TGES) loop in the A-domain reaches the phosphorylated Asp351 in the P-domain (13–15); 3) M1-M6 are rearranged to open the luminal gate for the dissociation of Ca²⁺. Despite the increasing structural information, there are still unresolved questions. A series of biochemical studies suggested the existence of two intermediate states, E1P⋅2Ca²⁺ and E2P⋅2Ca²⁺. However, atomistic structures and their energetics in these intermediate states would be required to understand their functional roles.Open in a separate windowFig. 1.Structures of SR Ca²⁺-ATPase embedded in a DOPC membrane in E1P⋅ADP⋅2Ca²⁺ (PDB ID: 2ZBD) (A) and E2P (PDB ID: 2ZBE) (B). The three cytoplasmic domains (A, N, and P) are colored in red, purple, and green, respectively. (C) A structural transition pathway predicted using the mean-force string method is projected onto a two-dimensional map along with the two distance RMSDs (dRMS) from the representative structures in MD simulations of E1P and E2P_dp. In the simulation, Glu908 at the Ca²⁺-binding sites is protonated to mimic the same protonation states of E1P. Only the atomic coordinates that are involved in the collective variables (CVs) are used for dRMS calculations. The five substates (SSs) are defined along the pathway via the fixed radius clustering method (1 to 17: red; 18 to 27: yellow; 28 to 35: green; 36 to 51: cyan; and 52 to 64: blue). Among the 64 images, 5 images (10, 23, 31, 44, and 57) are selected as five representative SSs.There are several computational tools to predict conformational transition pathways of proteins, such as morphing (16), normal mode analysis (17), or molecular dynamics (MD) simulation based on coarse-grained or atomistic models (18–21). However, large conformational changes of the Ca²⁺-ATPase happen on the milliseconds or slower time scales, which are not accessible in brute-force MD simulations (18–21) even when using MD-specialized supercomputers, such as Anton/Anton 2 (22, 23) or MDGRAPE-4A (24). In this study, we perform atomistic MD simulations with an enhanced conformational sampling method to investigate the conformational pathway and free energy profile in the transition between E1P⋅ADP⋅2Ca²⁺ and E2P. We utilize the mean-force string method (25) for obtaining one of the most probable transition pathways. The free energy profile along the pathway is then calculated with umbrella sampling (26). The same approach has been previously applied to adenylate kinase in solution (27) and multidrug transporter AcrB (28) and the ABC heme transporter (29) in biological membranes. A similar method, the string method with swarm trajectories (30), has been applied to several membrane proteins (31, 32). Das et al. applied the method to four conformational transitions of the Ca²⁺-ATPase, including the same step examined in the current study (33).In the current simulation study, we intend to compare the simulation results with those of existing structural and biochemical studies. In particular, the two intermediate states (E1P⋅2Ca²⁺ and E2P⋅2Ca²⁺) and the Ca²⁺-ATPase–lipid interactions in the E1P–E2P transition are examined using the simulation trajectories. The predicted interactions between phospholipids and basic sidechains of Ca²⁺-ATPase are compared with recent X-ray crystallography studies (34). We also investigate the effect of protonation states in the E1P–E2P transitions from atomistic MD simulations, which is difficult via experimental studies. By integrating structural, biochemical, and computational results, we shed light on the structural and energetic features of the E1P–E2P transition of the Ca²⁺-ATPase in unprecedented detail.     

A nitroaromatic cathode with an ultrahigh energy density based on six-electron reaction per nitro group for lithium batteries

Organic electrode materials have emerged as promising alternatives to conventional inorganic materials because of their structural diversity and environmental friendliness feature. However, their low energy densities, limited by the single-electron reaction per active group, have plagued the practical applications. Here, we report a nitroaromatic cathode that performs a six-electron reaction per nitro group, drastically improving the specific capacity and energy density compared with the organic electrodes based on single-electron reactions. Based on such a reaction mechanism, the organic cathode of 1,5-dinitronaphthalene demonstrates an ultrahigh specific capacity of 1,338 mAh⋅g⁻¹ and energy density of 3,273 Wh⋅kg⁻¹, which surpass all existing organic cathodes. The reaction path was verified as a conversion from nitro to amino groups. Our findings open up a pathway, in terms of battery chemistry, for ultrahigh-energy-density Li-organic batteries.

With the rapid development of social economy, the demand for high-energy-density storage systems is ever increasing, particularly in the fields of military, aerospace, medical, and civilian applications. Lithium-ion batteries (LIBs) have been extensively explored as high-energy-density storage devices (1–4). Commercial LIBs use crystalline transition metal oxide cathodes, such as LiCoO₂, LiMnO₂, and LiNi_xMn_yCo_1-x-yO₂ (5–12). They store energy via insertion/extraction of Li ions, which highly depends on the crystal structure and limits their capacities and energy densities (<300 mAh⋅g⁻¹ with energy density of <1,000 Wh⋅kg⁻¹). Some new battery systems with conversion reaction cathodes, such as Li-S (2,600 Wh⋅kg⁻¹) and Li-O₂ (3,450 Wh⋅kg⁻¹) batteries (13–19), have been extensively investigated due to their high theoretical energy densities. However, they suffer from serious dissolution, interfacial stability, and/or side reaction issues, and the practical energy densities are much lower than their theoretical values.Compared with the aforementioned rechargeable batteries, lithium primary batteries can provide remarkably higher energy densities. For example, lithium-fluorinated carbon (Li-CF_x) batteries use a CF_x cathode that has a high theoretical energy density of 2,180 Wh⋅kg⁻¹, which is the highest value among all commercial cathode materials in lithium batteries (20–22). Recently, it is reported that, different from the formation of crystal LiF in liquid electrolytes, amorphous LiF was produced and uniformly distributed on the carbon matrix when a solid-state electrolyte was used (23). This may enable a reversible electrochemical reactivity of CF_x with lithium and provide a possibility for high-energy-density batteries. Nevertheless, CF_x must be synthesized in harsh conditions with precise control, leading to high cost and hindering the wide application (20). Some liquid and gas cathodes, such as SO₂, SOCl₂, and SF₆ (24–27), can also offer high energy densities, but they tend to be volatile and induce severe safety hazards, especially on the occasion of thermal runaway. Therefore, it is highly desired to develop feasible and reliable battery systems with high energy density.Organic electrode materials offer many merits compared with inorganic electrode materials, including structure diversity and designability, abundance of raw materials, relatively easy synthesis, and environmental friendliness (28–35). Their energy storage processes rely on the uptake of cations or anions on the active groups, such as carbonyl group, quaternary nitrogen, and nitroxyl radical (36–40). Most of them undergo one electron reaction, leading to limited specific capacity and energy density (Fig. 1). For example, one carbonyl group (C=O) can be converted into C-OLi structure via accepting one electron and one lithium ion (36), providing a theoretical specific capacity of 957 mAh⋅g⁻¹ (based on the mass of functional group as marked by dashed circle in Fig. 1). If considering the inactive components, insulating property, and high solubility of organic molecules, most organic electrode materials exhibit inferior specific capacities. Although a high specific capacity of 902 mAh⋅g⁻¹ was gained for cyclohexanehexone in LIBs, its high solubility induces significant challenges in practical applications (41). Recently, a Li-containing organic compound, dilithium 1,4-phenylenebis ((methylsulfonyl) amide) (Li₂-p-PDSA) was synthesized, offering a new design for organic electrode materials, while it still follows the single-electron reaction path per electroactive group and shows a low specific capacity of 194 mAh⋅g⁻¹ (42). Therefore, new battery system needs to be developed to take full advantage of organic electrode materials for high-energy-density LIBs.Open in a separate windowFig. 1.Reaction mechanisms of organic groups in batteries.In this work, we report a battery chemistry of six-electron reduction on nitro group (−NO₂) of nitroaromatic cathode. Based on such a reaction, 1,5-dinitronaphthalene (1,5-DNN; Fig. 2A) as a cathode in LIBs achieves an ultrahigh specific capacity of 1,338 mAh⋅g⁻¹ and energy density of 3,273 Wh⋅kg⁻¹. This is a record for organic electrode materials, even higher than inorganic electrode materials. It is identified that nitro groups are reduced to amino groups through a six-electron transfer reaction via multiple characterization techniques. Our findings provide a path for achieving high-energy-density Li-organic batteries.Open in a separate windowFig. 2.Electrochemical performance of 1,5-DNN. (A) Chemical structure of 1,5-DNN. (B and C) Galvanostatic charge/discharge profiles of 1,5-DNN. (D) Performance comparison of 1,5-DNN with reported organic/inorganic electrode materials in terms of energy density, specific capacity and voltage. Cathodes for comparison (references): 1, p-DNB (43); 2, Li₂C₆O₆ (49); 3, P14AQ (50); 4, PTO (51); 5, 3Q (31); 6, AQ (44); 7, C₆O₆ (41); 8, o-DNB (43); 9, m-DNB (43); 10, 4,5-PhenQ (51); 11, C4Q (52); 12, LiCoO₂ (53); 13, LiFePO₄ (54); 14, NCA (55); 15, NCM-811 (56); 16, Li-rich (57); 17, CF_x (21); 18, MnO₂ (58); 19 SOCl₂ (59), 20 SO₂ (24); 21, S (60).     

Dietary ω-3 polyunsaturated fatty acids are protective for myopia

Myopia is a leading cause of visual impairment and blindness worldwide. However, a safe and accessible approach for myopia control and prevention is currently unavailable. Here, we investigated the therapeutic effect of dietary supplements of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on myopia progression in animal models and on decreases in choroidal blood perfusion (ChBP) caused by near work, a risk factor for myopia in young adults. We demonstrated that daily gavage of ω-3 PUFAs (300 mg docosahexaenoic acid [DHA] plus 60 mg eicosapentaenoic acid [EPA]) significantly attenuated the development of form deprivation myopia in guinea pigs and mice, as well as of lens-induced myopia in guinea pigs. Peribulbar injections of DHA also inhibited myopia progression in form-deprived guinea pigs. The suppression of myopia in guinea pigs was accompanied by inhibition of the “ChBP reduction–scleral hypoxia cascade.” Additionally, treatment with DHA or EPA antagonized hypoxia-induced myofibroblast transdifferentiation in cultured human scleral fibroblasts. In human subjects, oral administration of ω-3 PUFAs partially alleviated the near-work–induced decreases in ChBP. Therefore, evidence from these animal and human studies suggests ω-3 PUFAs are potential and readily available candidates for myopia control.

The myopia epidemic is now becoming a significant public health concern to modern society (1, 2). The percentage of the global population having myopia was predicted to increase from 28.3% in 2010 to 49.8% in 2050 (3). In East Asia, this percentage could even reach 90%, with up to 20% of the cases potentially developing into high myopia (refraction ≤−6 diopters [D]) (4, 5), which is one of the leading causes of irreversible blindness (6). The dramatic increase in the incidence of myopia, due in part to COVID-19 home confinement and increased online-viewing time, further highlights the importance of identifying a safe and effective approach to myopia control (7, 8).Behavioral, pharmacological, and optical interventions are the current approaches for myopia control, all of which have unique limitations. Behavioral intervention (such as increasing the time spent outdoors) retards the progression of myopia into high myopia (9). However, competitive educational systems, as well as lifestyles that incorporate increasingly more electronic products (the prevalence of which tends to reduce the amount of time spent outdoors), are hard to avoid. Pharmacological intervention, such as the use of atropine drops, is effective in limiting myopia progression. Its use, however, is off-label in most areas, and widespread acceptance is restricted because of the potential side effects such as chronic pupillary dilatation, loss of accommodation, and declining long-term effectiveness for sustained myopia control (10–12). Mori et al. reported that the dietary intake of crocetin, a naturally occurring apocarotenoid dicarboxylic acid found in crocus and other plants, could prevent myopia development in a mouse model and in children (13, 14), but more clinical studies are needed to prove the efficacy and safety of this agent. Optical corrections such as orthokeratology and peripheral defocusing lenses suffer from risks of infectious keratitis (15) and the requirement of professional support (16). Such limitations are critical, considering the implications of employing these approaches in a larger population, especially where the availability of medical services is limited.Thus, targeting multiple signaling cascades that promote the development of myopia, from retinal image processing to scleral growth, may be an effective strategy for myopia control. Previous studies have highlighted the significance of the cascade of events wherein the reduction of choroidal blood perfusion (ChBP, refers to the amount of choroidal blood flow) induces scleral hypoxia and myopia (17–20). The cascade begins when visual stimulus–induced myopic blur causes a reduction of choroidal thickness (ChT) and ChBP (20). In turn, these physiological changes induce hypoxia in the sclera, which is dependent on oxygen delivered by the choroidal vasculature (21). Hypoxia activates the hypoxia-induced factor-1α (HIF-1α) signaling pathway, which subsequently promotes scleral fibroblast-myofibroblast transdifferentiation and extracellular matrix (ECM) remodeling and in turn the development and progression of myopia (17).In the absence of effective treatments, and given the additional safety considerations when administering therapeutic agents to children (who are most vulnerable to myopia development), investigation into a dietary supplement for a safe and accessible approach for myopia control and prevention becomes compelling. However, research in this area is still new. Because of the beneficial effect of supplemental dietary omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on cardiovascular health, daily supplements have been recommended for intake by the Food and Agriculture Organization of the United Nations (2010), which endorses the use of two types of ω-3 PUFAs, videlicet, 250 mg/day each of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) for adults (22). Mammalian brains are invariably rich in DHA (23), and numerous studies have defined the importance of ω-3 PUFAs, particularly DHA, in human neuronal development in different developmental stages (24–26). Recently, an untargeted mass spectrometric assay reported that the amounts of serum fatty acid metabolites were reduced in myopic human subjects compared to nonmyopic subjects (27). In particular, the levels of serum DHA were significantly lower in the myopic group (27). DHA and EPA can promote relaxation of vascular smooth muscle cells and vasodilation (28–31), inhibit cancerous cell growth and survival by reducing HIF-1α expression (32–34), and suppress transforming growth factor (TGF)-β1–mediated myofibroblast transdifferentiation (35–37). These effects of DHA and EPA are also known to be associated with the inhibition of myopia development.The effects of ω-3 PUFAs on systemic conditions and on myopic subjects, combined with their involvement in mediating blood perfusion, HIF-1α, and cellular proliferation, prompted us to investigate if ω-3 PUFAs could suppress myopia development through modulating ChBP and scleral hypoxia. Thus, in this study, we first assessed the ability of ω-3 PUFAs supplementation to suppress myopia development in different animal models. We then examined the effects of DHA and EPA on ChBP and scleral hypoxia. We also determined if DHA or EPA could antagonize the effects of hypoxia on cultured human scleral fibroblasts (HSFs). Finally, we administered ω-3 PUFAs supplements to humans and observed their influence on near-work–induced ChBP reduction.     

Propagation of prions causing synucleinopathies in cultured cells

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)–YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson’s disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.James Parkinson first described a progressive deterioration of the nervous system in 1817 and called it “shaking palsy” (1). Almost one century later, Friederich Heinrich Lewy described the neuropathological hallmark now known as Lewy bodies (LBs) (2). Progress toward discerning the etiology of Parkinson’s disease (PD) was achieved 85 years later when the first of several studies identified mutations in or multiplications of the gene encoding α-synuclein, SNCA, in inherited cases of PD (3–5). These studies were corroborated by immunostaining for α-synuclein in brain sections from PD patients (6) and subsequently from dementia with Lewy bodies (DLB) cases (7, 8), which found that LBs are surrounded by a halo of α-synuclein polymers.Along with point mutations in SNCA (3), and duplication and triplication of the gene (4, 5) as causes of inherited PD, meta-analysis of genome-wide association studies (9) have identified common variations in SNCA as a risk factor for sporadic PD cases. Combined, these data strongly support an etiological role for α-synuclein in the pathogenesis of both the inherited and sporadic forms of PD.In 1998, brain sections from cases classified as multiple system atrophy (MSA) were analyzed for α-synuclein. Although no LBs were found, abundant immunostaining in the cytoplasm of glial cells was identified (8, 10, 11). A decade earlier, these large immunopositive deposits of α-synuclein were called glial cytoplasmic inclusions (GCIs) based on silver staining (12); they are primarily found in oligodendrocytes but have been occasionally observed in astrocytes and neurons. Limited ultrastructural studies performed on GCIs suggest that they are collections of poorly organized bundles of α-synuclein fibrils (8).In addition to the accumulation of α-synuclein into LBs in PD and GCIs in MSA, depigmentation of the substantia nigra pars compacta is a hallmark of both PD and the majority of MSA cases (13). This loss of dopaminergic neurons results in diminished input to the basal ganglia that is reflected in the motor deficits exhibited by patients. In the 1990s, fetal tissue transplants into the substantia nigra of PD patients were performed in an attempt to counteract the effects of dopamine loss. Strikingly, upon autopsy of patients that survived at least 10 years posttransplant, LBs were found in the grafted fetal tissue. Because these grafts were no more than 16 years old, the findings argued for host-to-graft transmission of LBs (14, 15). The results of these transplant studies offered evidence supporting the hypothesis that PD is a prion disease, characterized by a misfolded protein that self-propagates and gives rise to progressive neurodegeneration (16, 17). Additional support for this hypothesis came from studies on the spread of α-synuclein deposits from the substantia nigra to other regions of the CNS in PD patients (18).Even more convincing support for α-synuclein prions came from animal studies demonstrating the transmissibility of an experimental synucleinopathy. The first report used transgenic (Tg) mice expressing human α-synuclein containing the A53T mutation found in familial PD; the mice were designated TgM83 (19). Homozygous mice (TgM83^+/+) were found to develop spontaneous motor deficits along with increased amounts of insoluble phosphorylated α-synuclein throughout the brain between 8–16 months of age. Ten years later, Mougenot et al. (20) intracerebrally inoculated brain homogenates from sick TgM83^+/+ mice into ∼2-months-old TgM83^+/+ mice and found a substantial reduction in the survival time with incubation periods of ∼130 days. Similar observations were reported from two other groups using either homozygous TgM83^+/+ (21) or hemizygous TgM83^+/− (22) mice.Although our initial attempts to transmit PD to TgM83^+/− mice failed (23), the transmission of MSA to the same mouse line was the first demonstration of α-synuclein prions in human brain (22). The TgM83^+/− mice, which differ from their homozygous counterparts by not developing spontaneous disease, exhibited progressive CNS dysfunction ∼120 days following intrathalamic inoculation of brain homogenates from two MSA patients. Inoculation of brain fractions enriched for LBs from PD patients into wild-type (WT) mice and macaque monkeys induced aberrant α-synuclein deposits, but neither species developed neurological disease (24). In a similar approach, inoculation of WT mice with the insoluble protein fraction isolated from DLB patients also induced phosphorylated α-synuclein pathology after 15 months, but it failed to induce neurological disease characteristic of DLB (25).Because α-synuclein prions from MSA patients were transmissible to TgM83^+/− mice, we asked whether a more rapid cell-based bioassay could be developed to characterize the MSA prions. With the cell bioassay for progressive supranuclear palsy (PSP) in mind (26, 27), we began by constructing WT and mutant α-synuclein cDNAs fused to yellow fluorescent protein (YFP) (28–30) and expressed these in human embryonic kidney (HEK) cells. By testing the cells with full-length recombinant mutant human α-syn140*A53T fibrils, we induced aggregate formation in HEK cells expressing WT and mutant human SNCA transgenes. To expand these findings beyond synthetic prions and to examine natural prions, we report here that phosphotungstic acid (PTA) (31) can be used to selectively precipitate α-synuclein from MSA patients. Screening PTA-precipitated brain homogenate with our cellular bioassay, we detected MSA prions in all six of the cases examined. By measuring the distribution of prions in the substantia nigra, basal ganglia, cerebellum, and temporal gyrus, we found evidence to suggest that at least three different strains of α-synuclein prions may give rise to MSA. We also found that after enrichment by PTA precipitation, ∼6 million α-synuclein molecules comprised an infectious unit of MSA prions in cell culture. Importantly, we transmitted neurodegenerative disease to TgM83^+/− mice using PTA-precipitated brain homogenate from an MSA patient, confirming that the aggregate isolation methods used successfully purify prions from patient samples.     

Air pollutant emissions from Chinese households: A major and underappreciated ambient pollution source

As part of the 12th Five-Year Plan, the Chinese government has developed air pollution prevention and control plans for key regions with a focus on the power, transport, and industrial sectors. Here, we investigate the contribution of residential emissions to regional air pollution in highly polluted eastern China during the heating season, and find that dramatic improvements in air quality would also result from reduction in residential emissions. We use the Weather Research and Forecasting model coupled with Chemistry to evaluate potential residential emission controls in Beijing and in the Beijing, Tianjin, and Hebei (BTH) region. In January and February 2010, relative to the base case, eliminating residential emissions in Beijing reduced daily average surface PM_2.5 (particulate mater with aerodynamic diameter equal or smaller than 2.5 micrometer) concentrations by 14 ± 7 μg⋅m⁻³ (22 ± 6% of a baseline concentration of 67 ± 41 μg⋅m⁻³; mean ± SD). Eliminating residential emissions in the BTH region reduced concentrations by 28 ± 19 μg⋅m⁻³ (40 ± 9% of 67 ± 41 μg⋅m⁻³), 44 ± 27 μg⋅m⁻³ (43 ± 10% of 99 ± 54 μg⋅m⁻³), and 25 ± 14 μg⋅m⁻³ (35 ± 8% of 70 ± 35 μg⋅m⁻³) in Beijing, Tianjin, and Hebei provinces, respectively. Annually, elimination of residential sources in the BTH region reduced emissions of primary PM_2.5 by 32%, compared with 5%, 6%, and 58% achieved by eliminating emissions from the transportation, power, and industry sectors, respectively. We also find air quality in Beijing would benefit substantially from reductions in residential emissions from regional controls in Tianjin and Hebei, indicating the value of policies at the regional level.Over the past 30 years, China has experienced rapid economic growth, accompanied by accelerating urbanization, which has increased consumption of fossil fuels and worsened air quality. Although considerable efforts have been made to control air pollution, the focus has largely been on the power, transport, and, to a lesser extent, industry sectors, and reduction per unit activity has been offset by economic growth and increasing fossil fuel use (1). An air pollution control approach that prioritizes reductions from sources that create the highest pollutant exposures would be more effective in reducing the health impacts of air pollution. As the largest coal consumer, the power sector receives priority in efforts to reduce air pollutant emissions, and has significantly reduced emissions of sulfur dioxide (SO₂) and particulate matter (PM) in recent years (2). Industry and transportation emissions have also received attention (3), but the contribution of residential emissions to ambient air pollution has been relatively neglected. The residential sector is the largest emitter of carbonaceous aerosols (4, 5), which are formed by the inefficient combustion of fossil fuel and biomass in unregulated cooking and heating devices. Household combustion of coal also emits SO₂, a precursor to secondary PM_2.5 (particulate matter with aerodynamic diameter equal or smaller than 2.5 micrometer). In 2010, the residential sector accounted for around 18% of total energy consumption in China, but contributed 10%, 50%, and 69% of anthropogenic SO₂, black carbon (BC), and organic carbon (OC) emissions, respectively (5).Although not the focus of this paper, use of solid fuels (coal and biomass) for heating and cooking in households contributes directly to exposures in and around residences and is a major source of ill health in China. The Global Burden of Disease study found that direct household exposure to air pollution from solid fuels was responsible for ∼0.8 million premature deaths in China in 2013, about equal to the number of premature deaths from ambient particle pollution. Together, they make up the second largest risk factor in the country, ranked between high blood pressure and smoking (6–8). In addition to exposure within households, these emissions contribute to ambient air pollution, and thus affect populations over wide areas. To achieve the National Air Pollution Prevention and Control Action Plan (2013–2017) targets (hereafter the “Action Plan”) efficiently, regional data are needed to prioritize modifications to the structure of the energy sector to reduce health-damaging emissions from all sectors, including households. There have been estimates of the contribution of household emissions to ambient pollution in China based on global databases and models (9, 10). These analyses use coarse resolution models and have not been informed by local measurements, and are thus inadequate by themselves to guide local actions.Here, we use the Weather Research and Forecasting model with Chemistry (WRF-Chem) (11) to analyze the benefits of two residential emission mitigation scenarios during the heating season on PM_2.5 concentrations in Beijing and in the Beijing, Tianjin, and Hebei (BTH) region of northern China. In 2010, the population of this region was ∼104 million people, representing about 15% of the national population living in areas with significant household space-heating needs in winter (mid-November to mid-March). These needs are largely met using coal in simple devices with high emission factors in many households. This study provides a basis for further discussion of alternative emission control strategies across energy demand sectors in China.