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Surface reconstruction determines the fate of catalytic sites on the near-surface during the oxygen evolution reaction. However, deciphering the conversion mechanism of various intermediate-states during surface reconstruction remains a challenge. Herein, we employed an optical imaging technique to draw the landscape of dynamic surface reconstruction on individual Co3O4 nanoparticles. By regulating the surface states of Co3O4 nanoparticles, we explored dynamic growth of the CoOx(OH)y sublayer on single Co3O4 nanoparticles and directly identified the conversion between two dynamics. Rich oxygen vacancies induced more active sites on the surface and prolonged surface reconstruction, which enhanced electrochemical redox and oxygen evolution. These results were further verified by in situ electrochemical extinction spectroscopy of single Co3O4 nanoparticles. We elucidate the heterogeneous evolution of surface reconstruction on individual Co3O4 nanoparticles and present a unique perspective to understand the fate of catalytic species on the nanosurface, which is of enduring significance for investigating the heterogeneity of multielectron-transfer events. [ABSTRACT FROM AUTHOR]

High-valent iron-oxo species (FeIV=O) has been a long-sought-after oxygen transfer reagent in biological and catalytic chemistry but suffers from a giant challenge in its gentle and selective synthesis. Herein, we propose a new strategy to synthesize surface FeIV=O (=FeIV=O) on nanoscale zero-valent iron (nZVI) using chlorite (ClO2 -) as the oxidant, which possesses an impressive =FeIV=O selectivity of 99%. =FeIV=O can be energetically formed from the ferrous (FeII) sites on nZVI through heterolytic Cl-O bond dissociation of ClO2 - via a synergistic effect between electron-donating surface =FeII and proximal electron-withdrawing H2O, where H2O serves as a hydrogen-bond donor to the terminal O atom of the adsorbed ClO2 - thereby prompting the polarization and cleavage of Cl-O bond for the oxidation of =FeII toward the final formation of =FeIV=O. With methyl phenyl sulfoxide (PMS16O) as the probe molecule, the isotopic labeling experiment manifests an exclusive 18O transfer from Cl18O2 - to PMS16O18O mediated by =FeIV=18O. We then showcase the versatility of =FeIV=O as the oxygen transfer reagent in activating the C-H bond of methane for methanol production and facilitating selective triphenylphosphine oxide synthesis with triphenylphosphine. We believe that this new =FeIV=O synthesis strategy possesses great potential to drive oxygen transfer for efficient high-value-added chemical synthesis. [ABSTRACT FROM AUTHOR]

Tuning and probing electrical states of molybdenum disulfide (MoS2) with single-unit-cell thickness are attractive for next-generation nanoelectronics by virtue of unique electronic and optical attributes. In this work, we first demonstrated a plasmonic-based phase imaging of individual metal nanoparticles to directly visualize the characteristic switching of the electron-doping regime and near-complete suppression of the conductivity regime for an MoS2 monolayer by modulating potential. Intermediate states of electrical switching for the MoS2 monolayer are directly imaged by ultrasensitive phase variation of a single Au nanoparticle, which is attributable to the reversible switching between classical electromag-netic plasmonic coupling and quantum plasmonic coupling. This model can be operated as a logical inverter converting a logical signal 0-1 and as a universal logical NOR gate electronic directly imaged by plasmonic technique, potentially finding applications in novel nanoelectronics and optoelectronics. [ABSTRACT FROM AUTHOR]

Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1- interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle. [ABSTRACT FROM AUTHOR]

In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult mouse central nervous system (CNS). Our work confirms and extends prior studies to demonstrate that astrocytes are the predominant cell type in the adult CNS that transduce Hh signaling, revealed by the expression of Gli1, a target gene of the canonical pathway that is activated in cells receiving Hh, and other key pathway transduction components. Gli1+ (Hh-responsive) astrocytes are distributed in specific regions of the CNS parenchyma, including layers 4/5/6 of the neocortex, hypothalamus, thalamus, and spinal cord, among others. Notably, although BBB properties in endothelial cells are normally regulated by both paracellular and transcellular mechanisms, conditional inactivation of Hh signaling in astrocytes results in transient, region-specific BBB defects that affect transcytosis but not paracellular diffusion. These findings stand in contrast to prior studies that implicated astrocytes as a source of Sonic hedgehog that limited extravasation via both mechanisms [J. I. Alvarez et al., Science 334, 1727-1731 (2011)]. Furthermore, using three distinct Cre driver lines as well as pharmacological approaches to inactivate Hh-pathway transduction globally in CNS astrocytes, we find that these specific BBB defects are only detected in the rostral hypothalamus and spinal cord but not the cortex or other regions where Gli1+ astrocytes are found. Together, our data show that Gli1+ Hh-responsive astrocytes have regionally distinct molecular and functional properties and that the pathway is required to maintain BBB properties in specific regions of the adult mammalian CNS. [ABSTRACT FROM AUTHOR]

The precise measurement of thermodynamic and kinetic properties for biomolecules provides the detailed information for a multitude of applications in biochemistry, biosensing, and health care. However, sensitivity in characterizing the thermodynamic binding affinity down to a single molecule, such as the Gibbs free energy (Gb), enthalpy (Hb), and entropy (Sb), has not materialized. Here, we develop a nanoparticle-based technique to probe the energetic contributions of single-molecule binding events, which introduces a focused laser of optical tweezer to an optical path of plasmonic imaging to accumulate and monitor the transient local heating. This single-molecule calorimeter uncovers the complex nature of molecular interactions and binding characterizations, which can be employed to identify the thermodynamic equilibrium state and determine the energetic components and complete thermodynamic profile of the free energy landscape. This sensing platform promises a breakthrough in measuring thermal effect at the single-molecule level and provides a thorough description of biomolecular specific interactions. [ABSTRACT FROM AUTHOR]

Ataxia telangiectasia and Rad3 related (ATR) activation after replication stress involves a cascade of reactions, including replication protein A (RPA) complex loading onto single-stranded DNA and ATR activator loading onto chromatin. The contribution of histone modifications to ATR activation, however, is unclear. Here, we report that H3K14 trimethylation responds to replication stress by enhancing ATR activation. First, we confirmed that H3K14 monomethylation, dimethylation, and trimethylation all exist in mammalian cells, and that both SUV39H1 and SETD2 methyltransferases can catalyze H3K14 trimethylation in vivo and in vitro. Interestingly, SETD2-mediated H3K14 trimethylation markedly increases in response to replication stress induced with hydroxyurea, a replication stress inducer. Under these conditions, SETD2- mediated H3K14me3 recruited the RPA complex to chromatin via a direct interaction with RPA70. The increase in H3K14me3 levels was abolished, and RPA loading was attenuated when SETD2 was depleted or H3K14 was mutated. Rather, the cells were sensitive to replication stress such that the replication forks failed to restart, and cell-cycle progression was delayed. These findings help us understand how H3K14 trimethylation links replication stress with ATR activation. [ABSTRACT FROM AUTHOR]

Stem cells with the capability to self-renew and differentiate into multiple cell derivatives provide platforms for drug screening and promising treatment options for a wide variety of neural diseases. Nevertheless, clinical applications of stem cells have been hindered partly owing to a lack of standardized techniques to characterize cell molecular profiles noninvasively and comprehensively. Here, we demonstrate that a label-free and noninvasive single-cell Raman microspectroscopy (SCRM) platform was able to identify neural cell lineages derived from clinically relevant human induced pluripotent stem cells (hiPSCs). By analyzing the intrinsic biochemical profiles of single cells at a large scale (8,774 Raman spectra in total), iPSCs and iPSC-derived neural cells can be distinguished by their intrinsic phenotypic Raman spectra. We identified a Raman biomarker from glycogen to distinguish iPSCs from their neural derivatives, and the result was verified by the conventional glycogen detection assays. Further analysis with a machine learning classification model, utilizing t-distributed stochastic neighbor embedding (t-SNE)-enhanced ensemble stacking, clearly categorized hiPSCs in different developmental stages with 97.5% accuracy. The present study demonstrates the capability of the SCRM-based platform to monitor cell development using high content screening with a noninvasive and label-free approach. This platform as well as our identified biomarker could be extensible to other cell types and can potentially have a high impact on neural stem cell therapy. [ABSTRACT FROM AUTHOR]

When a metal nanoparticle is brought near to a metal surface within electron tunneling distance (~1 nm), classical electromagnetic coupling between the nanoparticle and the metal is expected to transition to quantum coupling. We show that this transition can be observed as a drastic phase change in the surface plasmon resonance (SPR) images of the gold nanoparticles. We study the transition by controlling the distance between the nanoparticles and electrode surface, modeling the impact of the transition on the SPR image in terms of a phase shift and demonstrating detection of microRNA based on the transition from classical to quantum coupling. The work shows that the quantum coupling can be directly visualized in SPR, and the extremely sensitive dependence of the transition on distance leads to a biosensing principle with SPR. [ABSTRACT FROM AUTHOR]

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation. [ABSTRACT FROM AUTHOR]

Glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) plays a critical role in cancer metabolism by coordinating glycolysis and biosynthesis. A well-validated PGAM1 inhibitor, however, has not been reported for treating pancreatic ductal adenocarcinoma (PDAC), which is one of the deadliest malignancies worldwide. By uncovering the elevated PGAM1 expressions were statistically related to worse prognosis of PDAC in a cohort of 50 patients, we developed a series of allosteric PGAM1 inhibitors by structure-guided optimization. The compound KH3 significantly suppressed proliferation of various PDAC cells by down-regulating the levels of glycolysis and mitochondrial respiration in correlation with PGAM1 expression. Similar to PGAM1 depletion, KH3 dramatically hampered the canonic pathways highly involved in cancer metabolism and development. Additionally, we observed the shared expression profiles of several signature pathways at 12 h after treatment in multiple PDAC primary cells of which the matched patient-derived xenograft (PDX) models responded similarly to KH3 in the 2 wk treatment. The better responses to KH3 in PDXs were associated with higher expression of PGAM1 and longer/stronger suppressions of cancer metabolic pathways. Taken together, our findings demonstrate a strategy of targeting cancer metabolism by PGAM1 inhibition in PDAC. Also, this work provided "proof of concept" for the potential application of metabolic treatment in clinical practice. [ABSTRACT FROM AUTHOR]

Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibodydrug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn't impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance,without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies. [ABSTRACT FROM AUTHOR]

Trimeric intracellular cation (TRIC) channels are thought to provide counter-ion currents that facilitate the active release of Ca2+ from intracellular stores. TRIC activity is controlled by voltage and Ca2+ modulation, but underlying mechanisms have remained unknown. Here we describe high-resolution crystal structures of vertebrate TRIC-A and TRIC-B channels, both in Ca2+-bound and Ca2+-free states, and we analyze conductance properties in structureinspired mutagenesis experiments. The TRIC channels are symmetric trimers, wherein we find a pore in each protomer that is gated by a highly conserved lysine residue. In the resting state, Ca2+ binding at the luminal surface of TRIC-A, on its threefold axis, stabilizes lysine blockage of the pores. During active Ca2+ release, luminal Ca2+ depletion removes inhibition to permit the lysinebearing and voltage-sensing helix to move in response to consequent membrane hyperpolarization. Diacylglycerol is found at interprotomer interfaces, suggesting a role in metabolic control. [ABSTRACT FROM AUTHOR]

Electron transfer reactions can now be followed at the single-molecule level, but the connection between the microscopic and macroscopic data remains to be understood. By monitoring the conductance of a single molecule, we show that the individual electron transfer reaction events are stochastic and manifested as large conductance fluctuations. The fluctuation probability follows first-order kinetics with potential dependent rate constants described by the Butler-Volmer relation. Ensemble averaging of many individual reaction events leads to a deterministic dependence of the conductance on the external electrochemical potential that follows the Nernst equation. This study discloses a systematic transition from stochastic kinetics of individual reaction events to deterministic thermodynamics of ensemble averages and provides insights into electron transfer processes of small systems, consisting of a single molecule or a small number of molecules. [ABSTRACT FROM AUTHOR]

The metabolic regulation of cell death is sophisticated. A growing body of evidence suggests the existence of multiple metabolic checkpoints that dictate cell fate in response to metabolic fluctuations. However, whether microRNAs (miRNAs) are able to respond to metabolic stress, reset the threshold of cell death, and attempt to reestablish homeostasis is largely unknown. Here, we show that miR-378/378* KO mice cannot maintain normal muscle weight and have poor running performance, which is accompanied by impaired autophagy, accumulation of abnormal mitochondria, and excessive apoptosis in skeletal muscle, whereas miR-378 overexpression is able to enhance autophagy and repress apoptosis in skeletal muscle of mice. Our in vitro data show that metabolic stress-responsive miR-378 promotes autophagy and inhibits apoptosis in a cell-autonomous manner. Mechanistically, miR-378 promotes autophagy initiation through the mammalian target of rapamycin (mTOR)/unc-51-like autophagy activating kinase 1 (ULK1) pathway and sustains autophagy via Forkhead box class O (FoxO)-mediated transcriptional reinforcement by targeting phosphoinositide-dependent protein kinase 1 (PDK1). Meanwhile, miR-378 suppresses intrinsic apoptosis initiation directly through targeting an initiator caspase- Caspase 9. Thus, we propose that miR-378 is a critical component of metabolic checkpoints, which integrates metabolic information into an adaptive response to reduce the propensity of myocytes to undergo apoptosis by enhancing the autophagic process and blocking apoptotic initiation. Lastly, our data suggest that inflammationinduced down-regulation of miR-378 might contribute to the pathogenesis of muscle dystrophy. [ABSTRACT FROM AUTHOR]

Exosomes play an important role in numerous cellular processes. Fundamental study and practical use of exosomes are significantly constrained by the lack of analytical tools capable of physical and biochemical characterization. In this paper, we present an optical approach capable of imaging single exosomes in a label-free manner, using interferometric plasmonic microscopy. We demonstrate monitoring of the real-time adsorption of exosomes onto a chemically modified Au surface, calculating the image intensity, and determining the size distribution. The sizing capability enables us to quantitatively measure the membrane fusion activity between exosomes and liposomes. We also report the recording of the dynamic interaction between exosomes and antibodies at the single-exosome level, and the tracking of hit-stay-run behavior of exosomes on an antibody-coated surface. We anticipate that the proposed method will contribute to clinical exosome analysis and to the exploration of fundamental issues such as the exosome-antibody binding kinetics. [ABSTRACT FROM AUTHOR]

Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICEAc) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICEAc-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity. [ABSTRACT FROM AUTHOR]

The phenomenon of delayed flowering after the application of nitrogen (N) fertilizer has long been known in agriculture, but the detailed molecular basis for this phenomenon is largely unclear. Here we used a modified method of suppression-subtractive hybridization to identify two key factors involved in N-regulated flowering time control in Arabidopsis thaliana, namely ferredoxin-NADP+-oxidoreductase and the blue-light receptor cryptochrome 1 (CRY1). The expression of both genes is induced by low N levels, and their loss-offunction mutants are insensitive to altered N concentration. Low-N conditions increase both NADPH/NADP+ and ATP/AMP ratios, which in turn affect adenosine monophosphate-activated protein kinase (AMPK) activity. Moreover, our results show that the AMPK activity and nuclear localization are rhythmic and inversely correlated with nuclear CRY1 protein abundance. Low-N conditions increase but high-N conditions decrease the expression of several key components of the central oscillator (e.g., CCA1, LHY, and TOC1) and the flowering output genes (e.g., GI and CO). Taken together, our results suggest that N signaling functions as a modulator of nuclear CRY1 protein abundance, as well as the input signal for the central circadian clock to interfere with the normal flowering process. [ABSTRACT FROM AUTHOR]

The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-CI cotransporter (NCC) in the DCT. Immu-nostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj1(t+/+ mice (WT). This 40-pS K channel is absent in homozygous Kcnj10-/- (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline-alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline-alanine-rich kinase-dependent mechanism. [ABSTRACT FROM AUTHOR]

Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP–membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild-type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of β-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration–approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity. [ABSTRACT FROM AUTHOR]

To be successful pathogens, bacteria must often restrict the expression of virulence genes to host environments. This requires a physical or chemical marker of the host environment as well as a cognate bacterial system for sensing the presence of a host to appropriately time the activation of virulence. However, there have been remarkably few such signaksensor pairs identified, and the molecular mechanisms for host-sensing are virtually unknown. By directly applying a reporter strain of Vibrio cholerae, the causative agent of cholera, to a thin layer chromatography (TIC) plate containing mouse intestinal extracts, we found two host signals that activate virulence gene transcription. One of these was revealed to be the bile salt taurocholate. We then show that a set of bile salts cause dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulfide bonds between cysteine (C)-207 residues in its periplasmic domain. Various genetic and biochemical analyses led us to propose a model in which the other cysteine in the periplasmic domain, C21B, forms an inhibitory intramolecular disulfide bond with 007 that must be isomerized to form the active 007-007 intermolecular bond. We then found bile sah-dependent effects of these cysteine mutations on survival in vivo, correlating to our in vitro model. Our results are a demonstration of a mechanism for direct activation of the V. cho!erae virulence cascade by a host signal molecule. They further provide a paradigm for recognition of the host environment in pathogenic bacteria through periplasmic cysteine oxidation. [ABSTRACT FROM AUTHOR]

Our understanding of the mechanisms by which ducts and lobules develop is derived from model organisms and three-dimensional (3D) cell culture models wherein mammalian epithelial cells undergo morphogenesis to form multicellular spheres with a hollow central lumen. However, the mechanophysical properties associated with epithelial morphogenesis are poorly understood. We performed multidimensional live-cell imaging analysis to track the morphogenetic process starting from a single cell to the development of a multicellular, spherical structure composed of polarized epithelial cells surrounding a hollow lumen. We report that in addition to actively maintaining apicobasal polarity, the structures underwent rotational motions at rates of 15-20 μm/h and the structures rotated 360° every 4 h during the early phase of morphogenesis. Rotational motion was independent of the cell cycle, but was blocked by loss of the epithelial polarity proteins Scribble or Pard3, or by inhibition of dynein-based microtubule motors. Interestingly, none of the structures derived from human cancer underwent rotational motion. We found a direct relationship between rotational motion and assembly of endogenous basement membrane matrix around the 3D structures, and that structures that failed to rotate were defective in weaving exogenous laminin matrix. Dissolution of basement membrane around mature, nonrotating acini restored rotational movement and the ability to assemble exogenous laminin. Thus, coordinated rotational movement is a unique mechanophysical process observed during normal 3D morphogenesis that regulates laminin matrix assembly and is lost in cancer-derived epithelial cells. [ABSTRACT FROM AUTHOR]

Tandem repeats (TRs) have extremely high mutation rates and are often considered to be neutrally evolving DNA. However, in coding regions, TR copy number mutations can significantly affect phenotype and may facilitate rapid adaptation to new environments. In several human genes, TR copy number mutations that expand polyglutamine (polyQ) tracts beyond a certain threshold cause incurable neurodegenerative diseases. PolyQ-containing proteins exist at a considerable frequency in eukaryotes, yet the phenotypic consequences of natural variation in polyQ tracts that are not associated with disease remain largely unknown. Here, we use Arabidopsis thaliana to dissect the phenotypic consequences of natural variation in the polyQ tract encoded by EARLY FLOWERING 3 (ELF3), a key developmental gene. Changing ELF3 polyQ tract length affected complex ELF3-dependent phenotypes in a striking and nonlinear manner. Some natural ELF3 polyQ variants phenocopied elf3 loss-of-function mutants in a common reference background, although they are functional in their native genetic backgrounds. To test the existence of background-specific modifiers, we compared the phenotypic effects of ELF3 polyQ variants between two divergent backgrounds. Col and Ws, and found dramatic differences. In fact, the Col-ELF3 allele, encoding the shortest known ELF3 polyQ tract, was haploinsufficient in Ws x Col F1 hybrids. Our data support a model in which variable polyQ tracts drive adaptation to internal genetic environments. [ABSTRACT FROM AUTHOR]

Hydrogen-rich compounds hold promise as high-temperature superconductors under high pressures. Recent theoretical hydride structures on achieving high-pressure superconductivity are composed mainly of H2 fragments. Through a systematic investigation of Ca hydrides with different hydrogen contents using particleswam optimization structural search, we show that in the stoichiometry CaH6 a body-centered cubic structure with hydrogen that forms unusual "sodalite" cages containing enclathrated Ca stabilizes above pressure 150 GPa. The stability of this structure is derived from the acceptance by two H2 of electrons donated by Ca forming an "H4" unit as the building block in the construction of the three-dimensional sodalite cage. This unique structure has a partial occupation of the degenerated orbitals at the zone center. The resultant dynamic Jahn-Teller effect helps to enhance electronphonon coupling and leads to superconductivity of CaH6. A superconducting critical temperature (Tc) of 220-235 K at 150 GPa obtained from the solution of the Eliashberg equations is the highest among all hydrides studied thus far. [ABSTRACT FROM AUTHOR]

We recently implicated two recurrent somatic mutations in an adrenal potassium channel, KCNJ5, as a cause of aldosterone-producing adrenal adenomas (APAs) and one inherited KCNJ5 mutation in a Mendelian form of early severe hypertension with massive adrenal hyperplasia. The mutations identified all altered the channel selectivity filter, producing increased Na+ conductance and membrane depolarization, the signal for aldosterone production and proliferation of adrenal glomerulosa cells. We report herein members of four kindreds with early onset primary aldosteronism of unknown cause. Sequencing of KCNJ5 revealed that affected members of two kindreds had KCNJ5G151R mutations, identical to one of the prevalent recurrent mutations in APAs. These individuals had severe progressive aldosteronism and hyperplasia requiring bilateral adrenalectomy in childhood for blood pressure control. Affected members of the other two kindreds had KCNJ5G151E mutations, which are not seen in APAs. These subjects had easily controlled hypertension and no evidence of hyperplasia. Surprisingly, electrophysiology of channels expressed in 293T cells demonstrated that KCNJ5G151E was the more extreme mutation, producing a much larger Na+ conductance than KCNJ5G151R, resulting in rapid Na+-dependent cell lethality. We infer that this increased lethality limits adrenocortical cell mass and the severity of aldosteronism in vivo, accounting for the milder phenotype among these patients. These findings demonstrate striking variations in phenotypes and clinical outcome resulting from different mutations of the same amino acid in KCNJ5 and have implications for the diagnosis and pathogenesis of primary aldosteronism with and without adrenal hyperplasia. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin. [ABSTRACT FROM AUTHOR]

Pseudogenes have been shown to acquire unique regulatory roles from more and more organisms. We report the observation of a cluster of siRNAs derived from pseudogenes of African Trypanosoma brucei using high through-put analysis. We show that these pseudogene-derived siRNAs suppress gene expression through RNA interference. The discovery that siRNAs may originate from pseudogenes and regulate gene expression in a unicellular eukaryote provides insights into the functional roles of pseudogenes and into the origin of noncoding small RNAs. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3.4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin αl (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (lP4)-bound conformations, and the CENTA1/ KIF13B-FHA/lP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3. while the second binds to both PIP3 and phosphatidylinositol 3.4-biphosphate (Pl(3,4)P2). The FHA domain of KIF13B interacts with the PHi domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule. [ABSTRACT FROM AUTHOR]

Germline mutations in the tumor suppressor gene PTEN(phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan-Riley-Ruvalcaba (BRR) syndromes, two dominantly. inherited disorders characterized by mental retardation, multiple hamartomas. and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense PtenΔ4-5 and missense PtenC124R and PtenG129E alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for PtenΔ4-5, hypomorphic function for PtenC124R, and gain of function for PtenG129E. These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms. [ABSTRACT FROM AUTHOR]

There is great interest in the exploration of hydrogen-rich compounds upon strong compression where they can become superconductors. Stannane (SnH4) has been proposed to be a potential high-temperature superconductor under pressure, but its high-pressure crystal structures, fundamental for the understanding of superconductivity, remain unsolved. Using an ab initio evolutionary algorithm for crystal structure prediction, we propose the existence of two unique high-pressure metallic phases having space groups Ama2 and P63/mmc, which both contain hexagonal layers of Sn atoms and semimolecular (perhydride) H2 units. Enthalpy calculations reveal that the Ama2 and P63/mmc structures are stable at 96-180 GPa and above 180 GPa, respectively, while below 96 GPa SnH4 is unstable with respect to elemental decomposition. The application of the Allen-Dynes modified McMillan equation reveals high superconducting temperatures of 15-22 K for the Ama2 phase at 120 GPa and 52-62 K for the P63/mmc phase at 200 GPa. [ABSTRACT FROM AUTHOR]

WNK4 (with no lysine kinase 4) inhibits ROMK channel activity in the distal nephron by stimulating clathrin-dependent endocytosis, an effect attenuated by SGK1 (serum-glucocorticoids-induced kinase)-mediated phosphorylation. It has been suggested that increased ROMK activity because of SGK1-mediated inhibition of WNK4 plays a role in promoting renal K secretion in response to elevated serum K or high K (HK) intake. In contrast, intravascular volume depletion also increases SGK1 activity but fails to stimulate ROMK channels and K secretion. Because HK intake decreases Src family protein tyrosine kinase (PTK) activity an inhibitor of ROMK channels, it is possible that Src family PTK may modulate the effects of SGK1 on WNK4. Here, we show that c-Src prevents SGK1 from attenuating WNK4's inhibition of ROMK activity. This effect of c-Src was WNK4-dependent because c-Src had no effect on ROMK harboring mutation at the site of c-Src phosphorylation (R1Y337A) in the absence of WNK4. Moreover, expression c-Src diminished the SGK1-mediated increase in serine phosphorylation of WNK4, suggesting that c-Src enhances WNK4-mediated inhibition of ROMK channels by suppressing the SGK1-induced phosphorylation. This notion is also supported by the observation that c-Src was not able to modulate the interaction between SGK1 and WNK4 mutants (WNK4S1169A or WNK4S1169D) in which an SGK1-phosphorylation site (serine 1169) was mutated by alanine or aspartate. We conclude that c-Src inhibits SGK1-mediated phosphorylation hereby restoring the WNK4-mediated inhibition of ROMK channels thus suppressing K secretion. [ABSTRACT FROM AUTHOR]

Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3lactin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca2+ and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca2~ locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed. [ABSTRACT FROM AUTHOR]