The obligate intracellular parasite, Toxoplasma gondii, modulates host immunity in a variety of highly specific ways. Previous work revealed a polymorphic, injected parasite factor, ROP16, to be a key virulence determinant and regulator of host cell transcription. These properties were shown to be partially mediated by dysregulation of the host transcription factors STAT3 and STAT6, but the molecular mechanisms underlying this phenotype were unclear. Here, we use a Type I Toxoplasma strain deficient in ROP16 to show that ROP16 induces not only sustained activation but also an extremely rapid (within 1 min) initial activation of STAT6. Using recombinant wild-type and kinase-deficient ROP16, we demonstrate in vitro that ROP16 has intrinsic tyrosine kinase activity and is capable of directly phosphorylating the key tyrosine residue for STAT6 activation, Tyr 641 . Furthermore, ROP16 co-immunoprecipitates with STAT6 from infected cells. Taken together, these data strongly suggest that STAT6 is a direct substrate for ROP16 in vivo.Toxoplasma gondii, a ubiquitous, intracellular parasite of the phylum Apicomplexa, infects an estimated one-third of the human population as well as a broad range of warm-blooded animals. Infection is typically asymptomatic and chronic; however, severe and even fatal disease can result from acute infections of congenitally infected or immunocompromised hosts. Strikingly, Toxoplasma-induced inflammatory pathology in adult immunocompetent patients has also been reported (1, 2).Understanding the host/parasite interactions that underpin these various disease manifestations has become an area of active investigation, and recent work has implicated as key players a set of polymorphic proteins that are secreted from the apical organelles known as rhoptries (3). One of these, a putative serine-threonine kinase known as ROP16, was revealed as a parasite factor responsible for many changes in host gene expression (4). Much of the effect of ROP16 on transcription was found to depend on sustained activation of STAT3 and STAT6, two host transcription factors that can negatively regulate Th1 inflammatory responses. ROP16 is thus poised to mediate the inflammatory status of the host, an intriguing role for a parasite effector because Th1-driven immunopathology, not just uncontrolled parasite growth and dissemination, has been proposed to be a significant contributor to the disease outcome (5).Although the potential significance of the ROP16 effect on STATs is clear, the molecular mechanism by which it accomplishes this sustained activation was not apparent from the initial studies. STAT activation requires phosphorylation at specific, conserved tyrosine residues; this phosphorylation, which induces dimerization and nuclear translocation of the STATs, is canonically initiated and maintained in the cytosol by either receptorassociated tyrosine kinases (such as the JAKs) or non-receptor tyrosine kinases (e.g. Src family kinases) (6). The duration of STAT activation is usually limited by negative feedback beca...

Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligandbinding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved smallmolecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule. Biological systems use ligand-dependent proteins and nucleic acids as molecular switches to transduce inputs into appropriate cellular responses. Artificial molecular switches are of particular interest (1-5), because they enable biological functions to be controlled by small-molecule inputs chosen by the researcher rather than by nature. Protein-splicing elements (inteins) mediate profound changes in the structure and function of proteins and therefore are powerful starting points for the creation of artificial molecular switches. Inteins catalyze their own excision from within a polypeptide and the ligation of the flanking external sequences, or exteins (6). Extein function is typically disrupted by the presence of an intein but restored after protein splicing.Small-molecule-dependent inteins represent attractive molecular switches, because in principle they can be inserted into an arbitrary protein of interest to render protein function dependent on the small molecule. Because intein splicing is rapid (6) compared with transcription and translation, the accumulated prespliced protein may be activated after addition of the smallmolecule effector on time scales that cannot be achieved by activating transcription or translation. In addition, this posttranslational activation may depend on the concentration of small molecule added, in contrast with the more common binary behavior of ligand-induced transcription. Small-moleculeactivated inteins therefore may combine advantages of smallmolecule-based chemical genetic approaches with the target specificity and generality of classical genetic approach...

An advanced bilayer gate dielectric stack consisting of Sc2O3∕La2O3∕SiOx annealed in nitrogen at 300°C was studied by scanning tunneling microscopy using bias dependent imaging. By changing the sample bias, electrical properties of different layers of the dielectric stack can be studied. At a sample bias of +3.5V, the conduction band of the La2O3 layer is probed revealing a polycrystalline film with an average grain size of about 27nm, in good agreement with that determined from planar transmission electron microscopy. High conductivity at grain boundaries, due possibly to dangling bonds, can be observed in this layer, as also observed in grain boundary assisted current conduction in metal-oxide-silicon structures. Imaging at a sample bias of −4V probes the interfacial SiOx layer and an amorphouslike image of the interfacial layer is obtained.

The tunneling current versus voltage characteristic of the Sc2O3∕La2O3∕SiOx high-κ gate stack is examined using scanning tunneling microscopy in ultrahigh vacuum. Different measurement bias polarities allow information on the location (i.e., in the high-κ or interfacial SiOx layer) of the electronic traps to be extracted. Two types of localized leakage sites may be distinguished. Lowering of the electron barrier height and trap-assisted tunneling are proposed as the two leakage mechanisms.

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The impact of nitrogen incorporation on the physical and electrical characteristics of the HfO2 is examined. X-ray photoelectron spectroscopy shows that nitrogen can be incorporated into the HfO2 via a two-step thermal anneal—first in ultrahigh vacuum (UHV) and subsequently in N2. Following the N2 anneal, scanning tunneling microscopy in UHV reveals a marked reduction in the low-voltage leakage current under gate injection biasing. From band theory and existing first-principles simulation results, one may consistently attribute this improvement to the passivation of oxygen vacancies in the HfO2 by nitrogen. Improvement in the breakdown strength of the HfO2 subjected to ramp-voltage stress (substrate injection) is also observed after the N2 anneal. The local current-voltage curves acquired concurrently during the ramp-voltage stress exhibit “space-charge limited conduction,” which implies that the observed improvement in breakdown strength may be related to a limitation of the current flow through the gate stack in the high stress voltage regime.

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Through scanning tunneling microscopy, the rate of electrical stress induced trap generation in the vicinity of the tip/high-κ interface of the Sc2O3/La2O3/SiOx gate stack is observed to be much higher than that at the SiOx/Si interface, implying that the former is more susceptible to electrical stress induced wear out. This polarity dependence is discussed in the context of current trap generation models and is shown to be best explained by anode hot-hole effect induced by inherently lower surface plasmon threshold energy at the metal/high-κ interface and a higher hole trapping rate in the relatively thick high-κ.