The Use of Ureates as Activators for Samarium Diiodide.

A novel mode of SmI2 activation has been developed using ureates as reaction promoters. Several ureates formed by treatment of the corresponding ureas with n-BuLi have been shown to activate SmI2 to a substantial extent toward the reduction of 1-chlorodecane. Complexes formed from SmI2 and various ureates have been shown to be useful for the reduction of a variety of organohalides, including substrates of low reactivity such as aryl fluorides. Because of ease of synthesis and low molecular weight, the conjugate base of triethylurea (TEU(-)) was of primary focus. Visible spectroscopy and reactivity data are consistent with the hypothesis that the same complex is being formed when SmI2 is combined with either 2 or 4 equiv of TEU(-), in spite of the greater reactivity of SmI2/4 TEU(-) with some alkyl halides. We propose that the active reductant is an N,O chelate formed between SmI2 and 2 equiv of TEU(-).

Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis.

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V(H)H) library raised against TcdB, we identified two V(H)H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab.

Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.

The cutaneous bacterium Janthinobacterium lividum inhibits the growth of Trichophyton rubrum in vitro.

BACKGROUND: Tinea pedis (athlete's foot) is a fungal infection that is both widespread and challenging to treat. Standard treatments consist of topical and systemic therapies of antifungal agents, such as miconazole, itraconazole, and terbinafine. The extended nature of topical therapy and the toxicity of long-term systemic therapy limit the utility of current treatments. An alternate approach relies on an understanding of bacterial-fungal interactions. Specifically, a probiotic antifungal bacterium such as Janthinobacterium lividum can counter infection; Janthinobacterium is a major constituent of the human skin microbiota. Janthinobacterium lividum has been shown to ameliorate the effects of the cutaneous fungal disease chytridiomycosis in a vertebrate species (Rana muscosa). METHODS: Dual-culture plate challenge assays were performed using J. lividum and Trichophyton rubrum, the leading cause of athlete's foot. RESULTS: In all cases, T. rubrum colonies grew significantly smaller when co-cultured with J. lividum. CONCLUSION: These in vitro results suggest that J. lividum merits further investigation as a human cutaneous probiotic.

The invasive chytrid fungus of amphibians paralyzes lymphocyte responses.

The chytrid fungus, Batrachochytrium dendrobatidis, causes chytridiomycosis and is a major contributor to global amphibian declines. Although amphibians have robust immune defenses, clearance of this pathogen is impaired. Because inhibition of host immunity is a common survival strategy of pathogenic fungi, we hypothesized that B. dendrobatidis evades clearance by inhibiting immune functions. We found that B. dendrobatidis cells and supernatants impaired lymphocyte proliferation and induced apoptosis; however, fungal recognition and phagocytosis by macrophages and neutrophils was not impaired. Fungal inhibitory factors were resistant to heat, acid, and protease. Their production was absent in zoospores and reduced by nikkomycin Z, suggesting that they may be components of the cell wall. Evasion of host immunity may explain why this pathogen has devastated amphibian populations worldwide.

Synergistic inhibition of the lethal fungal pathogen Batrachochytrium dendrobatidis: the combined effect of symbiotic bacterial metabolites and antimicrobial peptides of the frog Rana muscosa.

A powerful mechanism for protection against disease in animals is synergy between metabolites present in the natural microbiota of the host and antimicrobial peptides (AMPs) produced by the host. We studied this method of protection in amphibians in regard to the lethal disease chytridiomycosis, which is caused by Batrachochytrium dendrobatidis (Bd). In this study, we show that the AMPs of Rana muscosa, as well as the metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) from Pseudomonas fluorescens, a bacterial species normally found on the skin of R. muscosa, were inhibitory to the growth of Bd in vitro. When both AMPs and 2,4-DAPG were used in growth inhibition assays, they worked synergistically to inhibit the growth of Bd. This synergy resulted in reduced minimum concentrations necessary for inhibition by either 2,4-DAPG or AMPs. This inhibitory concentration of AMPs did not inhibit the growth of a P. fluorescens strain that produced 2,4-DAPG in vitro, although its growth was inhibited at higher peptide concentrations. These data suggest that the AMPs secreted onto frog skin and the metabolites secreted by the resident beneficial bacteria may work synergistically to enhance protection against Bd infection on amphibian skin. These results may aid conservation efforts to augment amphibian skins' resistance to chytridiomycosis by introducing anti-Bd bacterial species that work synergistically with amphibian AMPs.

Amphibian immune defenses against chytridiomycosis: impacts of changing environments.

Eco-immunology is the field of study that attempts to understand the functions of the immune system in the context of the host's environment. Amphibians are currently suffering devastating declines and extinctions in nearly all parts of the world due to the emerging infectious disease chytridiomycosis caused by the chytrid fungus, Batrachochytrium dendrobatidis. Because chytridiomycosis is a skin infection and remains confined to the skin, immune defenses of the skin are critical for survival. Skin defenses include secreted antimicrobial peptides and immunoglobulins as well as antifungal metabolites produced by symbiotic skin bacteria. Low temperatures, toxic chemicals, and stress inhibit the immune system and may impair natural defenses against B. dendrobatidis. Tadpoles' mouth parts can be infected by B. dendrobatidis. Damage to the mouth parts can impair growth, and the affected tadpoles maintain the pathogen in the environment even when adults have dispersed. Newly metamorphosing frogs appear to be especially vulnerable to infection and to the lethal effects of this pathogen because the immune system undergoes a dramatic reorganization at metamorphosis, and postmetamorphic defenses are not yet mature. Here we review our current understanding of amphibian immune defenses against B. dendrobatidis and the ability of the pathogen to resist those defenses. We also briefly review what is known about the impacts of temperature, environmental chemicals, and stress on the host-pathogen interactions and suggest future directions for research.

Tripyrrolidinophosphoric acid triamide as an activator in samarium diiodide reductions.

The electrochemical and spectrophotometric characterization of the complex formed from samarium diiodide and 4 equiv of tripyrrolidinophosphoric acid triamide (TPPA) is presented. Kinetic studies indicate that the SmI(2)/TPPA complex possesses reactivity greater than the complex formed between samarium diiodide and 4 equiv of HMPA. Examples of the use of SmI(2)/TPPA in synthesis are presented.

Immune defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, in the South African clawed frog, Xenopus laevis.

Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.

Immune defenses of Xenopus laevis against Batrachochytrium dendrobatidis.

Amphibian populations are declining at an unprecedented rate worldwide. A number of declines have been linked to a pathogenic skin fungus, Batrachochytrium dendrobatidis. Although amphibians have robust immune defenses, many species seem to be very susceptible to infection by this fungus and to development of the lethal disease called chytridiomycosis. One species that is relatively resistant to B. dendrobatidis is Xenopus laevis. Because X. laevis has been used as a model for studies of immunity in amphibians and because it is relatively resistant to chytridiomycosis, it is a good model to examine immune defenses against B. dendrobatidis. Although much less is known about immune defenses in Bufo boreas, it serves as a second model species because it is very susceptible to B. dendrobatidis. Here we review what is known about innate antimicrobial peptide defenses in the skin and the development of immune responses following experimental immunization with heat-killed fungal cells. Development of an immunization protocol in X. laevis that induces effective defenses may suggest better strategies for protecting vulnerable species such as B. boreas.

Integrated microfluidic device for solid-phase extraction coupled to micellar electrokinetic chromatography separation.

An integrated microdevice was utilized for the autonomous coupling of solid-phase extraction (SPE) to micellar electrokinetic chromatography (MEKC). Porous plugs of polymethacrylate polymer approximately 200 microm in length) were fabricated by ultraviolet irradiation in microchannels. Microcolumns of hydrophobic beads packed against the polymethacrylate plugs were utilized for the quantitative extraction of rhodamine B, yielding preconcentration factors over 200 for a 90-s extraction. The calculated detection limit for this dye was 60 fM. A sample of coumarin dyes were concentrated by SPE, eluted in a nonaqueous solvent from a separate on-chip reservoir, and injected by a gated valve onto a separate column for MEKC analysis. Using the integrated device, a completely automated sequence of extraction, elution, injection, separation, and detection were performed in less than 5 min. Observed separation efficiencies were high, with plate heights below 2 microm. The analysis was at least 3 times faster than semiautomated, conventional, solid-phase extraction, while requiring no user intervention. The design, fabrication, and autonomous operation of the device is discussed.

Colorimetric detection of uranium(VI) on building surfaces after enrichment by solid phase extraction.

A method for detecting and quantifying uranium(VI) levels on building materials that include concrete, Plexiglas, glass and steel surfaces is presented. Uranium(VI) was extracted from building material surfaces using a pH 2.2 buffer rinse and, subsequently complexed by an organic chelating agent, arsenazo III. The application of a uranium-chelating molecule, arsenazo III, allows for concentration enhancement using C(18) solid phase extraction and colorimetric detection of the uranium complex using ultraviolet-visible spectroscopy at 654nm. The method has a detection limit (based on 3sigma) of 40ng/L (5ng/cm(2)) and an overall extraction efficiency greater than 80% for each surface type (concrete, Plexiglas, glass, steel). Methods to prevent interference by metal ions commonly found on building materials are discussed.

Compact, high voltage power supply for the lab-on-a-chip.

The design, construction and operation of a simple, inexpensive and compact high voltage power supply (HVPS) for use in conjunction with a simple cross capillary electrophoresis microchip is presented. The microchip HVPS utilizes a single high voltage power supply (15 kV), a voltage-divider network, to give the voltages necessary to operate a gated injection valve, and two high voltage relays for switching between the open and closed gate sequences of the injection. In order to accommodate the application of different simple cross microchip dimensions, a set of equations for defining the resistor network and ensuring proper gate performance are presented.

High-efficiency, two-dimensional separations of protein digests on microfluidic devices.

High-efficiency, two-dimensional separations of tryptic digests were achieved using glass microfluidic devices. Following micellar electrokinetic chromatography (MEKC) separations in a 19.6-cm-long serpentine channel, the peptides were rapidly sampled into a 1.3-cm-long second-dimension channel, where they were separated by capillary electrophoresis (CE). The turns in the serpentine channel were asymmetrically tapered to minimize geometrical contributions to band broadening and to provide ample channel length for high-efficiency chromatographic separations. Analysis of rhodamine B injections routinely produced plate numbers of 230000 and 40000 in the first (MEKC) and second (CE) dimensions, respectively, corresponding to plate heights of 0.9 and 0.3 microm. The electric field strengths were 200 V/cm for MEKC and 2400 V/cm for CE. In analysis times less than 15 min, two-dimensional separation of bovine serum albumin tryptic digest produced a peak capacity of 4200 (110 in the first dimension and 38 in the second dimension). The system was used to identify a peptide from a tryptic digest of ovalbumin using standard addition and to distinguish between tryptic digests of human and bovine hemoglobin.