Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time-and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.The intestinal protozoan parasite Entamoeba histolytica is the etiologic agent of amebic colitis and liver abscess, which cause high rates of morbidity and mortality worldwide (49). The mechanism by which Entamoeba histolytica is able to invade and damage the host's target tissues has been the subject of intense research. Several virulence factors have been identified, including secreted cysteine proteinases (39, 42). These amebic enzymes have been implicated in the in vitro cytopathology of cell monolayers (20, 23), which correlates with the observed separation of colonic epithelial cells before invasion (51). Other correlates with invasion include the ability of cysteine proteinases to degrade extracellular matrix components (19) and colonic mucin (31, 32). Furthermore, cysteine proteinases enable E. histolytica to evade the host's immune defenses by activating and locally depleting complement (43), and by degrading anaphylotoxins C3a and C5a (41), human immunoglobulin G (IgG) (53), human IgA (21), and interleukin-18 (IL-18) (37).The recent completion of the Entamoeba histolytica genome project has revealed the presence of at least 40 genes encoding cysteine proteinases (25). Of all the cysteine proteinase genes, only ehcp1 and ehcp5 are unique to E. histolytica, as their orthologs are either absent (ehcp1) or nonfuncti...

Multilevel programing and charge transport characteristics of intrinsic SiOx-based resistive switching memory are investigated using TaN/SiOx/n++Si (MIS) and TiW/SiOx/TiW (MIM) device structures. Current transport characteristics of high- and low-resistance states (HRS and LRS) are studied in both device structures during multilevel operation. Analysis of device thermal response demonstrates that the effective electron energy barrier is strongly dependent on the resistance of the programed state, with estimates of 0.1 eV in the LRS and 0.6 eV in the HRS. Linear data fitting and conductance analyses indicate Poole-Frenkel emission or hopping conductance in the low-voltage region, whereas Fowler-Nordheim (F-N) or trap-assisted tunneling (TAT) is indicated at moderate voltage. Characterizations using hopping transport lead to hopping distance estimates of ∼1 nm in the LRS for both device structures. Relative permittivity values (εr) were extracted using the Poole-Frenkel formulism and estimates of local filament temperature, where εr values were ∼80 in the LRS and ∼4 in the HRS, suggesting a strongly polarized medium in the LRS. The onset of F-N tunneling or TAT corresponds to an observed “overshoot” in the I-V response with an estimated threshold of 1.6 ± 0.2 V, in good agreement with reported electro-luminescence results for LRS devices. Resistive switching is discussed in terms of electrochemical reactions between common SiO2 defects, and specific defect energy levels are assigned to the dominant transitions in the I-V response. The overshoot response in the LRS is consistent with TAT through either the Eγ' oxygen vacancy or the hydrogen bridge defect, both of which are reported to have an effective bandgap of 1.7 eV. The SET threshold at ∼2.5 V is modeled as hydrogen release from the (Si-H)2 defect to generate the hydrogen bridge, and the RESET transition is modeled as an electrochemical reaction that re-forms (SiH)2. The results provide further insights into charge transport and help identify potential switching mechanisms in SiOx-based unipolar resistive switching memory.

The physical mechanisms of unipolar resistive switching (RS) in SiOx-based resistive memory are investigated using TaN/SiOx/n++Si and TiW/SiOx/TiW device structures. RS is independent of SiOx thickness and device area, confirming that RS occurs in a localized region along a filamentary pathway. Results from experiments varying electrode type, series resistance, and the oxygen content of SiOxNy materials show the potential to optimize switching performance and control device programming window. Device materials with stoichiometry near that of SiO2 are found to have better operating stability as compared to extrinsic, N-doped SiOxNy materials. The results provide further insight into the physical mechanisms of unipolar operation and lead to a localized switching model based on electrochemical transitions involving common SiOx defects. High-temperature data retention measurements for over 104 s in high- and low-resistance states demonstrate the potential for use of intrinsic SiOx RS devices in future nonvolatile memory applications.

BackgroundCruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite life cycle and has been validated as a viable target to treat Chagas' disease. As a proof-of-concept, K11777, a potent inhibitor of cruzain, was found to effectively eliminate T. cruzi infection and is currently a clinical candidate for treatment of Chagas' disease.Methodology/Principal FindingsWRR-483, an analog of K11777, was synthesized and evaluated as an inhibitor of cruzain and against T. cruzi proliferation in cell culture. This compound demonstrates good potency against cruzain with sensitivity to pH conditions and high efficacy in the cell culture assay. Furthermore, WRR-483 also eradicates parasite infection in a mouse model of acute Chagas' disease. To determine the atomic-level details of the inhibitor interacting with cruzain, a 1.5 Å crystal structure of the protease in complex with WRR-483 was solved. The structure illustrates that WRR-483 binds covalently to the active site cysteine of the protease in a similar manner as other vinyl sulfone-based inhibitors. Details of the critical interactions within the specificity binding pocket are also reported.ConclusionsWe demonstrate that WRR-483 is an effective cysteine protease inhibitor with trypanocidal activity in cell culture and animal model with comparable efficacy to K11777. Crystallographic evidence confirms that the mode of action is by targeting the active site of cruzain. Taken together, these results suggest that WRR-483 has potential to be developed as a treatment for Chagas' disease.

Aqueous extracts of ripe areca nut without husk (ripe ANE) and fresh and tender areca nut with husk (tender ANE) were examined for their effects on the defensive functions of human neutrophils. Exposure of peripheral blood neutrophils to ripe ANE and tender ANE inhibited their bactericidal activity against oral pathogens, including Actinobacillus actinomycetemcomitans and Streptococcus mutans, in a dose-dependent manner. At the concentrations tested, ripe and tender ANEs did not significantly affect the viability of neutrophils as verified by their ability to exclude trypan blue dye. However, both ANEs inhibited the production of bactericidal superoxide anion by neutrophils as measured by cytochrome c reduction. Moreover, the ripe ANE inhibited neutrophils more effectively than did tender ANE. Arecoline, a major alkaloid of areca nut, only exhibited an inhibitory effect on the functions of neutrophils when high concentrations were used. Therefore, arecoline could not be used to explain the inhibitory effects observed for ANEs. In conclusion, our results demonstrated that ripe and tender ANEs reduced the antibacterial activity and the superoxide anion production of neutrophils. This effect may contribute to a less efficient elimination of bacteria from the periodontal environment. Inhibition of the antimicrobial functions of neutrophils may alter the microbial ecology of the oral cavity, and this may be one possible mechanism by which areca nut compromises the oral health of users of areca nut products.