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Mesenchymal stem cells (MSCs) are essential in regenerative medicine. However, conventional expansion and harvesting methods often fail to maintain the essential extracellular matrix (ECM) components, which are crucial for their functionality and efficacy in therapeutic applications. Here, we introduce a bone marrow-inspired macroporous hydrogel designed for the large-scale production of MSC-ECM spheroids. Through a soft-templating approach leveraging liquid-liquid phase separation, we engineer macroporous hydrogels with customizable features, including pore size, stiffness, bioactive ligand distribution, and enzyme-responsive degradability. These tailored environments are conducive to optimal MSC proliferation and ease of harvesting. We find that soft hydrogels enhance mechanotransduction in MSCs, establishing a standard for hydrogel-based 3D cell culture. Within these hydrogels, MSCs exist as both cohesive spheroids, preserving their innate vitality, and as migrating entities that actively secrete functional ECM proteins. Additionally, we also introduce a gentle, enzymatic harvesting method that breaks down the hydrogels, allowing MSCs and secreted ECM to naturally form MSC-ECM spheroids. These spheroids display heightened stemness and differentiation capacity, mirroring the benefits of a native ECM milieu. Our research underscores the significance of sophisticated materials design in nurturing distinct MSC subpopulations, facilitating the generation of MSC-ECM spheroids with enhanced therapeutic potential. [ABSTRACT FROM AUTHOR]

The fine regulation of catalysts by the atomic-level removal of inactive atoms can promote the active site exposure for performance enhancement, whereas suffering from the difficulty in controllably removing atoms using current micro/nano-scale material fabrication technologies. Here, we developed a surface atom knockout method to promote the active site exposure in an alloy catalyst. Taking Cu3Pd alloy as an example, it refers to assemble a battery using Cu3Pd and Zn as cathode and anode, the charge process of which proceeds at about 1.1 V, equal to the theoretical potential difference between Cu2+/Cu and Zn2+/Zn, suggesting the electricity-driven dissolution of Cu atoms. The precise knockout of Cu atoms is confirmed by the linear relationship between the amount of the removed Cu atoms and the battery cumulative specific capacity, which is attributed to the inherent atom-electron-capacity correspondence. We observed the surface atom knockout process at different stages and studied the evolution of the chemical environment. The alloy catalyst achieves a higher current density for oxygen reduction reaction compared to the original alloy and Pt/C. This work provides an atomic fabrication method for material synthesis and regulation toward the wide applications in catalysis, energy, and others. [ABSTRACT FROM AUTHOR]

Intrathecal synthesis of central nervous system (CNS)-reactive autoantibodies is observed across patients with autoimmune encephalitis (AE), who show multiple residual neurobehavioral deficits and relapses despite immunotherapies. We leveraged two common forms of AE, mediated by leucine-rich glioma inactivated-1 (LGI1) and contactin-associated protein-like 2 (CASPR2) antibodies, as human models to comprehensively reconstruct and profile cerebrospinal fluid (CSF) B cell receptor (BCR) characteristics. We hypothesized that the resultant observations would both inform the observed therapeutic gap and determine the contribution of intrathecal maturation to pathogenic B cell lineages. From the CSF of three patients, 381 cognate-paired IgG BCRs were isolated by cell sorting and scRNA-seq, and 166 expressed as monoclonal antibodies (mAbs). Sixty-two percent of mAbs from singleton BCRs reacted with either LGI1 or CASPR2 and, strikingly, this rose to 100% of cells in clonal groups with ≥4 members. These autoantigen-reactivities were more concentrated within antibody-secreting cells (ASCs) versus B cells (P < 0.0001), and both these cell types were more differentiated than LGI1- and CASPR2-unreactive counterparts. Despite greater differentiation, autoantigen-reactive cells had acquired few mutations intrathecally and showed minimal variation in autoantigen affinities within clonal expansions. Also, limited CSF T cell receptor clonality was observed. In contrast, a comparison of germline-encoded BCRs versus the founder intrathecal clone revealed marked gains in both affinity and mutational distances (P = 0.004 and P < 0.0001, respectively). Taken together, in patients with LGI1 and CASPR2 antibody encephalitis, our results identify CSF as a compartment with a remarkably high frequency of clonally expanded autoantigen-reactive ASCs whose BCR maturity appears dominantly acquired outside the CNS. [ABSTRACT FROM AUTHOR]

Rett syndrome (RTT) is a devastating neurodevelopmental disorder primarily caused by mutations in the methyl-CpG binding protein 2 (Mecp2) gene. Here, we found that inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 1 (RIPK1) kinase ameliorated progression of motor dysfunction after onset and prolonged the survival of Mecp2-null mice. Microglia were activated early in myeloid Mecp2-deficient mice, which was inhibited upon inactivation of RIPK1 kinase. RIPK1 inhibition in Mecp2-deficient microglia reduced oxidative stress, cytokines production and induction of SLC7A11, SLC38A1, and GLS, which mediate the release of glutamate. Mecp2-deficient microglia release high levels of glutamate to impair glutamate-mediated excitatory neurotransmission and promote increased levels of GluA1 and GluA2/3 proteins in vivo, which was reduced upon RIPK1 inhibition. Thus, activation of RIPK1 kinase in Mecp2-deficient microglia may be involved both in the onset and progression of RTT. [ABSTRACT FROM AUTHOR]

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures. [ABSTRACT FROM AUTHOR]

Like biological species, words in language must compete to survive. Previously, it has been shown that language changes in response to cognitive constraints and over time becomes more learnable. Here, we use two complementary research paradigms to demonstrate how the survival of existing word forms can be predicted by psycholinguistic properties that impact language production. In the first study, we analyzed the survival of words in the context of interpersonal communication. We analyzed data from a large-scale serial-reproduction experiment in which stories were passed down along a transmission chain over multiple participants. The results show that words that are acquired earlier in life, more concrete, more arousing, and more emotional are more likely to survive retellings. We reason that the same trend might scale up to language evolution over multiple generations of natural language users. If that is the case, the same set of psycholinguistic properties should also account for the change of word frequency in natural language corpora over historical time. That is what we found in two large historical-language corpora (Study 2): Early acquisition, concreteness, and high arousal all predict increasing word frequency over the past 200 y. However, the two studies diverge with respect to the impact of word valence and word length, which we take up in the discussion. By bridging micro-level behavioral preferences and macro-level language patterns, our investigation sheds light on the cognitive mechanisms underlying word competition. [ABSTRACT FROM AUTHOR]

Sodium-sulfur (Na-S) batteries are attracting intensive attention due to the merits like high energy and low cost, while the poor stability of sulfur cathode limits the further development. Here, we report a chemical and spatial dual-confinement approach to improve the stability of Na-S batteries. It refers to covalently bond sulfur to carbon at forms of C-S/N-C= S bonds with high strength for locking sulfur. Meanwhile, sulfur is examined to be S1-S2 small species produced by thermally cutting S8 large molecules followed by sealing in the confined pores of carbon materials. Hence, the sulfur cathode achieves a good stability of maintaining a high-capacity retention of 97.64% after 1000 cycles. Experimental and theoretical results show that Na+ is hosted via a coordination structure (N···Na···S) without breaking the C-S bond, thus impeding the formation and dissolution of sodium polysulfide to ensure a good cycling stability. This work provides a promising method for addressing the S-triggered stability problem of Na-S batteries and other S-based batteries. [ABSTRACT FROM AUTHOR]

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine. [ABSTRACT FROM AUTHOR]

LUBAC-mediated linear ubiquitination plays a pivotal role in regulation of cell death and inflammatory pathways. Genetic deficiency in LUBAC components leads to severe immune dysfunction or embryonic lethality. LUBAC has been extensively studied for its role in mediating TNF signaling. However, Tnfr1 knockout is not able to fully rescue the embryonic lethality of LUBAC deficiency, suggesting that LUBAC may modify additional key cellular substrates in promoting cell survival. GPx4 is an important selenoprotein involved in regulating cellular redox homeostasis in defense against lipid peroxidation-mediated cell death known as ferroptosis. Here we demonstrate that LUBAC deficiency sensitizes to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. LUBAC binds and stabilizes GPx4 by modulating its linear ubiquitination both in normal condition and under oxidative stress. Our findings identify GPx4 as a key substrate of LUBAC and a previously unrecognized role of LUBAC-mediated linear ubiquitination in regulating cellular redox status and cell death. [ABSTRACT FROM AUTHOR]

Sonic hedgehog (Shh) signaling plays a critical role in regulating cerebellum development by maintaining the physiological proliferation of granule neuron precursors (GNPs), and its dysregulation leads to the oncogenesis of medulloblastoma. O-GlcNAcylation (O-GlcNAc) of proteins is an emerging regulator of brain function that maintains normal development and neuronal circuitry. Here, we demonstrate that O-GlcNAc transferase (OGT) in GNPs mediate the cerebellum development, and the progression of the Shh subgroup of medulloblastoma. Specifically, OGT regulates the neurogenesis of GNPs by activating the Shh signaling pathway via O-GlcNAcylation at S355 of GLI family zinc finger 2 (Gli2), which in turn promotes its deacetylation and transcriptional activity via dissociation from p300, a histone acetyltransferases. Inhibition of OGT via genetic ablation or chemical inhibition improves survival in a medulloblastoma mouse model. These data uncover a critical role for O-GlcNAc signaling in cerebellar development, and pinpoint a potential therapeutic target for Shh-associated medulloblastoma. [ABSTRACT FROM AUTHOR]

A non-Hermitian Weyl equation indispensably requires a three-dimensional (3D) real/synthetic space, and it is thereby perceived that a Weyl exceptional ring (WER) will not be present in thermal diffusion given its purely dissipative nature. Here, we report a recipe for establishing a 3D parameter space to imitate thermal spinor field. Two orthogonal pairs of spatiotemporally modulated advections are employed to serve as two synthetic parameter dimensions, in addition to the inherent dimension corresponding to heat exchanges. We first predict the existence of WER in our hybrid conduction-advection system and experimentally observe the WER thermal signatures verifying our theoretical prediction. When coupling two WERs of opposite topological charges, the system further exhibits surface-like and bulk topological states, manifested as stationary and continuously changing thermal processes, respectively, with good robustness. Our findings reveal the long-ignored topological nature in thermal diffusion and may empower distinct paradigms for general diffusion and dissipation controls. [ABSTRACT FROM AUTHOR]

Shigella flexneri, a gram-negative bacterium, is the major culprit of bacterial shigellosis and causes a large number of human infection cases and deaths worldwide annually. For evading the host immune response during infection, S. flexneri secrets two highly similar E3 ligases, IpaH1.4 and IpaH2.5, to subvert the linear ubiquitin chain assembly complex (LUBAC) of host cells, which is composed of HOIP, HOIL-1L, and SHARPIN. However, the detailed molecular mechanism underpinning the subversion of the LUBAC by IpaH1.4/2.5 remains elusive. Here, we demonstrated that IpaH1.4 can specifically recognize HOIP and HOIL-1L through its leucine-rich repeat (LRR) domain by binding to the HOIP RING1 domain and HOIL-1L ubiquitin-like (UBL) domain, respectively. The determined crystal structures of IpaH1.4 LRR/HOIP RING1, IpaH1.4 LRR/HOIL-1L UBL, and HOIP RING1/UBE2L3 complexes not only elucidate the binding mechanisms of IpaH1.4 with HOIP and HOIL-1L but also unveil that the recognition of HOIP by IpaH1.4 can inhibit the E2 binding of HOIP. Furthermore, we demonstrated that the interaction of IpaH1.4 LRR with HOIP RING1 or HOIL-1L UBL is essential for the ubiquitination of HOIP or HOIL-1L in vitro as well as the suppression of NF-κB activation by IpaH1.4 in cells. In summary, our work elucidated that in addition to inducing the proteasomal degradation of LUBAC, IpaH1.4 can also inhibit the E3 activity of LUBAC by blocking its E2 loading and/or disturbing its stability, thereby providing a paradigm showing how a bacterial E3 ligase adopts multiple tactics to subvert the key LUBAC of host cells. [ABSTRACT FROM AUTHOR]

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa- SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process. [ABSTRACT FROM AUTHOR]

Environmental conditions are key factors in the progression of plant disease epidemics. Light affects the outbreak of plant diseases, but the underlying molecular mechanisms are not well understood. Here, we report that the light-harvesting complex II protein, LHCB5, from rice is subject to light-induced phosphorylation during infection by the rice blast fungus Magnaporthe oryzae. We demonstrate that single-nucleotide polymorphisms (SNPs) in the LHCB5 promoter control the expression of LHCB5, which in turn correlates with the phosphorylation of LHCB5. LHCB5 phosphorylation enhances broadspectrum resistance of rice to M. oryzae through the accumulation of reactive oxidative species (ROS) in the chloroplast. We also show that LHCB5 phosphorylation-induced resistance is inheritable. Our results uncover an immunity mechanism mediated by phosphorylation of light-harvesting complex II. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) accounts for a disproportionately high number of deaths due to a lack of targeted therapies and an increased likelihood of distant recurrence. Estrogen receptor beta (ERβ), a well-characterized tumor suppressor, is expressed in 30% of TNBCs, and its expression is associated with improved patient outcomes. We demonstrate that therapeutic activation of ERβ elicits potent anticancer effects in TNBC through the induction of a family of secreted proteins known as the cystatins, which function to inhibit canonical TGFβ signaling and suppress metastatic phenotypes both in vitro and in vivo. These data reveal the involvement of cystatins in suppressing breast cancer progression and highlight the value of ERβ-targeted therapies for the treatment of TNBC patients. [ABSTRACT FROM AUTHOR]

RIPK1 is a critical mediator of cell death and inflammation downstream of TNFR1 upon stimulation by TNFα, a potent proinflammatory cytokine involved in a multitude of human inflammatory and degenerative diseases. RIPK1 contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The kinase activity of RIPK1 promotes cell death and inflammation. Here, we investigated the involvement of RIPK1-DD in the regulation of RIPK1 kinase activity. We show that a charge-conserved mutation of a lysine located on the surface of DD (K599R in human RIPK1 or K584R in murine RIPK1) blocks RIPK1 activation in necroptosis and RIPK1-dependent apoptosis and the formation of complex II. Ripk1K584R/K584R knockin mutant cells are resistant to RIPK1 kinasedependent apoptosis and necroptosis. The resistance of K584R cells, however, can be overcome by forced dimerization of RIPK1. Finally, we show that the K584R RIPK1 knockin mutation protects mice against TNFα-induced systematic inflammatory response syndrome. Our study demonstrates the role of RIPK1-DD in mediating RIPK1 dimerization and activation of its kinase activity during necroptosis and RIPK1-dependent apoptosis. [ABSTRACT FROM AUTHOR]

Apoptosis and necroptosis are two distinct cell death mechanisms that may be activated in cells on stimulation by TNFα. It is still unclear, however, how apoptosis and necroptosis may be differentially regulated. Here we screened for E3 ubiquitin ligases that could mediate necroptosis. We found that deficiency of Pellino 1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner during necroptosis. Ubiquitination of RIPK1 by PELI1 promotes the formation of necrosome and execution of necroptosis. Although PELI1 is not directly involved in mediating the activation of RIPK1, it is indispensable for promoting the binding of activated RIPK1 with its downstream mediator RIPK3 to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in necroptosis. However, we show that PELI1 deficiency sensitizes cells to both RIPK1-dependent and RIPK1-independent apoptosis as a result of down-regulated expression of c-FLIP, an inhibitor of caspase-8. Finally, we show that Peli1-/- mice are sensitized to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of RIPK1 that differentially controls the activation of necroptosis and apoptosis. [ABSTRACT FROM AUTHOR]

The troposphere is the region of the atmosphere characterized by low static stability, vigorous diabatic mixing, and widespread condensational heating in clouds. Previous research has argued that in the tropics, the upper bound on tropospheric mixing and clouds is constrained by the rapid decrease with height of the saturation water vapor pressure and hence radiative cooling by water vapor in clear-sky regions. Here the authors contend that the same basic physics play a key role in constraining the vertical structure of tropospheric mixing, tropopause temperature, and cloud-top temperature throughout the globe. It is argued that radiative cooling by water vapor plays an important role in governing the depth and amplitude of large-scale dynamics at extratropical latitudes. [ABSTRACT FROM AUTHOR]

Microbes that synthesize minerals, a process known as microbial biomineralization, contributed substantially to the evolution of current planetary environments through numerous important geochemical processes. Despite its geological significance, the origin and evolution of microbial biomineralization remain poorly understood. Through combined metagenomic and phylogenetic analyses of deep-branching magnetotactic bacteria from the Nitrospirae phylum, and using a Bayesian molecular clock-dating method, we show here that the gene cluster responsible for biomineralization of magnetosomes, and the arrangement of magnetosome chain(s) within cells, both originated before or near the Archean divergence between the Nitrospirae and Proteobacteria. This phylogenetic divergence occurred well before the Great Oxygenation Event. Magnetotaxis likely evolved due to environmental pressures conferring an evolutionary advantage to navigation via the geomagnetic field. Earth’s dynamo must therefore have been sufficiently strong to sustain microbial magnetotaxis in the Archean, suggesting that magnetotaxis coevolved with the geodynamo over geological time. [ABSTRACT FROM AUTHOR]

Plant diversity in experimental systems often enhances ecosystem productivity, but the mechanisms causing this overyielding are only partly understood. Intercropping faba beans (Vicia faba L.) and maize (Zea mays L.) result in overyielding and also, enhanced nodulation by faba beans. By using permeable and impermeable root barriers in a 2-y field experiment, we show that root-root interactions between faba bean and maize significantly increase both nodulation and symbiotic N2 fixation in intercropped faba bean. Furthermore, root exudates from maize promote faba bean nodulation, whereas root exudates from wheat and barley do not. Thus, a decline of soil nitrate concentrations caused by intercropped cereals is not the sole mechanism for maize promoting faba bean nodulation. Intercropped maize also caused a twofold increase in exudation of flavonoids (signaling compounds for rhizobia) in the systems. Roots of faba bean treated with maize root exudates exhibited an immediate 11-fold increase in the expression of chalcone- flavanone isomerase (involved in flavonoid synthesis) gene together with a significantly increased expression of genes mediating nodulation and auxin response. After 35 d, faba beans treated with maize root exudate continued to show up-regulation of key nodulation genes, such as early nodulin 93 (ENOD93), and promoted nitrogen fixation. Our results reveal a mechanism for how intercropped maize promotes nitrogen fixation of faba bean, where maize root exudates promote flavonoid synthesis in faba bean, increase nodulation, and stimulate nitrogen fixation after enhanced gene expression. These results indicate facilitative root-root interactions and provide a mechanism for a positive relationship between species diversity and ecosystem productivity. [ABSTRACT FROM AUTHOR]

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead we report that these genes comprise transient TF-target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIPI targets are uniquely relevant to rapid N signaling in planta enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIPI at 1-5 min, but not at later time points following TF perturbation Moreover, promoters of these transient targets are uniquely enriched with os-regulatory motifs coinherited with bZIPI binding sites, suggesting a recruitment role for bZIPL This transient mode of TF action supports a classic, but forgotten, "hit-and-run" transcription model, which enables a "catalyst TF" to activate a large set of targets within minutes of signal perturbation. [ABSTRACT FROM AUTHOR]

Epithelial cadherin (E-cadherin), a member of the classical cadherin family, mediates calcium-dependent homophilic cell-cell adhesion. Crystal structures of classical cadherins reveal an adhesive dimer interface featuring reciprocal exchange of N-terminal β-strands between two protomers. Previous work has identified a putative intermediate (called the "X-dimer") in the dimerization pathway of wild-type E-cadherin EC1-EC2 domains, based on crystal structures of mutants not capable of strand swapping and on deceleration of binding kinetics by mutations at the X-dimer interface. In the present work, NMR relaxation dispersion spectroscopy is used to directly observe and characterize intermediate states without the need to disrupt the strand-swapped binding interface by mutagenesis. The results indicate that E-cadherin forms strand-swapped dimers predominantly by a mechanism in which formation of a weak and short-lived X-dimer-like state precedes the conformational changes required for formation of the mature strand-swapped dimeric structure. Disruption of this intermediate state through mutation reduces both association and dissociation rates by factors of ~104, while minimally perturbing affinity. The X-dimer interface lowers the energy barrier associated with strand swapping and enables E-cadherins to form strand-swapped dimers at a rate consistent with residence times in adherens junctions. [ABSTRACT FROM AUTHOR]

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluo-roethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-ß (Aß) and other amyloid aggregates present in Alzheimer's disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aß aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aß aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aß peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. [ABSTRACT FROM AUTHOR]

Recognition of viral nucleic acids by pattern recognition receptors initiates type 1 IFN induction and Innate antiviral Immune response. Here we show that LSm14A, a member of the LSm family Involved in RNA processing in the processing bodies, binds to synthetic or viral RNA and DNA and mediates IRF3 activation and IFN-β Induction. Knockdown of LSm14A Inhibits cytosollc RNA- and DNA-trlgger type I IFN production and cellular antiviral response. Moreover, LSm14A Is essential for early-phase Induction of IFN-β after either RNA or DNA virus infection. We further found that LSm14A-medlated IFN-β Induction requires RIG-l-VISA or MITA after RNA or DNA virus Infection, respectively, and viral Infection causes translocation of LSm14A to peroxisomes, where RIG-l, VISA, and MITA are located. These findings suggest that LSm14A Is a sensor for both viral RNA and DNA and plays an Important role In Initiating IFN-ßβinduction In the early phase of viral Infection [ABSTRACT FROM AUTHOR]

Inflammation is a significant player in the progression of heart failure and has detrimental effects on cardiac function. Prostaglandin (PG)E2, a major proinflammatory prostanoid in the cardiovascular system, is a potent stimulus in inducing intracellular CAMP but minimally affects cardiac contractile function. Here, we show that the PGE2 stimulation attenuates the adrenergic-induced cardiac coiitractile response in animal hearts. Stimulation with PGE2 leads to stimulatory G protein (Gs)-dependent production of CAMP. However, the induced CAMP is spatially restricted because of its degradation by phosphodiesterase (PDE)4 and cannot access the intracellular sarcoplasmic reticulum (SR) for increasing calcium signaling and myocyte contraction. Moreover, pretreatment with PGE2 significantly inhibits PKA activities at the SR induced by a β-adrenergic agonist, isoproterenol, and subsequently blocks isoproterenol-induced PKA phosphorylation of phospholamban and contractile responses in myocytes. Further analysis reveals that the PGE2-induced cAMP/PKA is sufficient to phosphorylate and activate PDE4D isoforms, which, in turn, spatially inhibits the diffusion of adrenergic-induced CAMP from the plasma membrane to the SR. Inhibition of PDE4 rescues the adrenergic-induced increase in cAMP/PKA activities at the SR. PKA phosphorylation of phospholamban, and contractile responses in PGE2-pretreated myocytes. Thus, this offers an example that one Gs-coupled receptor is able to inhibit the intracellular signaling transduction initiated by another Gs-coupled receptor via controlling the diffusion of CAMP, presenting a paradigm for G protein-coupled receptor (GPCR) signal transduction. It also provides a mechanism for the integration of signaling initiated by different neurohormonal stimuli, as well as long-term effects of chronically circulating proinflammatory factors in myocardium. [ABSTRACT FROM AUTHOR]

In the brains of patients with fetal Minamata disease (FMD), which is caused by exposure to methylmercury (MeHg) during development, many neurons are hypoplastic, ectopic, and disoriented, indicating disrupted migration, maturation, and growth. MeHg affects a myriad of signaling molecules, but little is known about which signals are primary targets for MeHg-induced deficits in neuronal development. In this study, using a mouse model of FMD, we examined how MeHg affects the migration of cerebellar granule cells during early postnatal development. The cerebellum is one of the most susceptible brain regions to MeHg exposure, and profound loss of cerebellar granule cells is detected in the brains of patients with FMD. We show that MeHg inhibits granule cell migration by reducing the frequency of somal Ca2+ spikes through alterations in Ca2+, cAMP, and insulin-like growth factor 1 (IGF1) signaling. First, MeHg slows the speed of granule cell migration in a dose-dependent manner, independent of the mode of migration. Second, MeHg reduces the frequency of spontaneous Ca2+ spikes in granule cell somata in a dose-dependent manner. Third, a unique in vivo live-imaging system for cell migration reveals that reducing the inhibitory effects of MeHg on somal Ca2+ spike frequency by stimulating internal Ca2+ release and 2+ influxes, inhibiting cAMP activity, or activating IGF1 receptors ameliorates the inhibitory effects of MeHg on granule cell migration. These results suggest that alteration of Ca2+ spike frequency and Ca2+, cAMP, and IGF1 signaling could be potential therapeutic targets for infants with MeHg intoxication. [ABSTRACT FROM AUTHOR]

The role of genetic inheritance in brain development has been well characterized, but little is known about the contributions of natural environmental stimuli, such as the effect of light-dark cycles, to brain development. In this study, we determined the role of light stimuli in neuronal cell migration to elucidate how environmental factors regulate brain development. We show that in early postnatal mouse cerebella, granule cell migration accelerates during light cycles and decelerates during dark cycles. Furthermore, cerebellar levels of insulin-like growth factor 1 (IGF-1) are high during light cycles and low during dark cycles. There are causal relationships between light-dark cycles, speed of granule cell migration, and cerebellar IGF-1 levels. First, changes in light-dark cycles result in corresponding changes in the fluctuations of both speed of granule cell migration and cerebellar IGF-1 levels. Second, in vitro studies indicate that exogenous IGF-1 accelerates the migration of isolated granule cells through the activation of IGF-1 receptors. Third, in vivo studies reveal that inhibiting the IGF-1 receptors decelerates granule cell migration during light cycles (high IGF-1 levels) but does not alter migration during dark cycles (low IGF-1 levels). In contrast, stimulating the IGF-1 receptors accelerates granule cell migration during dark cycles (low IGF-1 levels) but does not alter migration during light cycles (high IGF-1 levels). These results suggest that during early postnatal development light stimuli control granule cell migration by altering the activity of IGF-1 receptors through modification of cerebellar IGF-1 levels. [ABSTRACT FROM AUTHOR]

Angiotensin-converting enzyme 2 (ACE2) is a newly discovered ho- molog of ACE whose actions oppose those of angiotensin II (AngII). However, the underlying mechanisms by which ACE2 effectively suppresses early atherosclerotic lesions remain poorly understood. Here, we show, both in vitro and in vivo, that ACE2 inhibited the development of early atherosclerotic lesions by suppressing the growth of vascular smooth muscle cells (VSMCs) and improving endothelial function. In a relatively large cohort animal study (66 rabbits), aortic segments transfected by Ad-ACE2 showed significantly attenuated fatty streak formation, neointimal macrophage infiltration, and alleviation of impaired endothelial function. Segments also showed decreased expression of monocyte chemoattractant protein 1, lectin-like oxidized low-density lipoprotein receptor 1, and proliferating cell nuclear antigen, which led to the delayed onset of atherosclerotic lesions. At the cellular level, ACE2 significantly modulated AngII-induced growth and migration in human umbilical vein endothelial cells and VSMCs. The antiathero- sclerotic effect of ACE2 involved down-regulation of the ERK- p38, JAK-STAT, and AngII-ROS-NF-κB signaling pathways and up- regulation of the PI3K-Akt pathway. These findings revealed the molecular mechanisms of the antiatheroscierotic activity of ACE2 and suggested that modulation of ACE2 could offer a therapeutic option for treating atherosclerosis. [ABSTRACT FROM AUTHOR]

Viral infection causes activation of the transcription factors NF-κB and IRF3, which collaborate to induce type I interferons (IFNs) and cellular antiviral response. The mitochondrial outer membrane protein VISA acts as a critical adapter for assembling a virus-induced complex that signals NF-κB and IRF3 activation. Using a biochemical purification approach. we identified the WD repeat protein WDR5 as a VISA-associated protein. WDR5 was recruited to VISA in a viral infection dependent manner. Viral infection also caused translocation of WDR5 from the nucleus to mitochondria. Knockdown of WDR5 impaired the formation of virusinduced VISA-associated complex. Consistently, knockdown of WDR5 inhibited virus-triggered activation of IRF3 and NF-κB as well as transcription of the IFNBI gene. These findings suggest that WDR5 is essential in assembling a virus-induced VISA-associated complex and plays an important role in virus-triggered induction of type I IFNs. [ABSTRACT FROM AUTHOR]

Drug addiction is mediated by complex neuronal processes that converge on the shell of the nucleus accumbens (NAcSh). The NAcSh receives inputs from the lateral hypothalamus (LH), where selfstimulation can be induced. Melanin-concentrating hormone (MCH) is produced mainly in the LH, and its receptor (MCH1R) is highly expressed in the NAcSh. We found that, in the NAcSh, MCH1R is coexpressed with dopamine receptors (DiR and D2R), and that MCH increases spike firing when both DiR and D2R are activated. Also, injecting MCH potentiates cocaine-induced hyperactivity in mice. Mice lacking MCH1R exhibit decreased cocaine-induced conditioned place preference, as well as cocaine sensitization. Using a specific MCH1 R antagonist, we further show that acute blockade of the MCH system not only reduces cocaine self-administration, but also attenuates cueand cocaine-induced reinstatement. Thus, the MCH system has an important modulatory role in cocaine reward and reinforcement by potentiating the dopaminergic system in the NAcSh, which may provide a new rationale for treating cocaine addiction. [ABSTRACT FROM AUTHOR]

Helioxanthin is a natural product that inhibits the replication of a number of viruses. We found that a previously undescribed helioxanthin analogue, 8-1, exhibited potent anti-hepatitis B virus (HBV) activity with little cytotoxicity. 8-1 suppressed both HBV RNA and protein expression, as well as DNA replication of both wild- type and 3TC-resistant virus. Time-course analyses revealed that RNA expression was blocked first after treatment with 8-1, followed by viral proteins, and then DNA. 8-1 inhibited the activity of all HBV promoters by decreasing the binding of hepatocyte nuclear factor 4 (HNF-4), HNF-3, and fetoprotein factor to the precore/core promoter enhancer II region. The amount of HNF-4 and HNF-3 was decreased posttranscriptionally by 8-1 in HBV-producing cells, but not in HBV-negative cells. Therefore, 8-1 suppresses HBV replication by posttranscriptional down-regulation of critical transcription factors in HBV-producing cells, thus diminishing HBV promoter activity and blocking viral gene expression and replication. This mechanism is unique and different from other anti-HBV compounds previously described. [ABSTRACT FROM AUTHOR]

Using cell-based aptamer selection, we have developed a strategy to use the differences at the molecular level between any two types of cells for the identification of molecular signatures on the surface of targeted cells. A group of aptamers have been generated for the specific recognition of leukemia cells. The selected aptamers can bind to target cells with an equilibrium dissociation constant (Kd) in the nanomolar-to-picomolar range. The cell-based selection process is simple, fast, straightforward, and reproducible, and, most importantly, can be done without prior knowledge of target molecules. The selected aptamers can specifically recognize target leukemia cells mixed with normal human bone marrow aspirates and can also identify cancer cells closely related to the target cell line in real clinical specimens. The cell-based aptamer selection holds a great promise in developing specific molecular probes for cancer diagnosis and cancer biomarker discovery. [ABSTRACT FROM AUTHOR]

Equilibrium Fourier transform infrared (FTIR) and temperature-jump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L F5IL. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WI (3.23 μs at 60.2°C) and double phenylalanine mutant (3.01 μs at 49.9°C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 μs at 49.9°C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant however, unfolds faster with an unfolding time of 3.01 ,is compared with 6.97 μs for the WT at 49.9°C. [ABSTRACT FROM AUTHOR]

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001 , re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402 , 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390 , 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor signaling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that G s -biased signaling, but not G i activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-G i , EP4-G s , and EP4-G i in complex with endogenous prostaglandin E 2 (PGE 2 )or two synthetic agonists and comparing with PGE 2 -EP2-G s structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the G s /G i transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cytoplasmic structural features. In particular, the orientation of the PGE 2 ω-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their G s /G i coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for G s /G i coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.