Real-Time Dynamics of the Formation of Hydrated Electrons upon Irradiation of Water Clusters with Extreme Ultraviolet Light.

Free electrons in a polar liquid can form a bound state via interaction with the molecular environment. This so-called hydrated electron state in water is of fundamental importance, e.g., in cellular biology or radiation chemistry. Hydrated electrons are highly reactive radicals that can either directly interact with DNA or enzymes, or form highly excited hydrogen (H^{*}) after being captured by protons. Here, we investigate the formation of the hydrated electron in real-time employing extreme ultraviolet femtosecond pulses from a free electron laser, in this way observing the initial steps of the hydration process. Using time-resolved photoelectron spectroscopy we find formation timescales in the low picosecond range and resolve the prominent dynamics of forming excited hydrogen states.

A table-top monochromator for tunable femtosecond XUV pulses generated in a semi-infinite gas cell: Experiment and simulations.

We present a new design of a time-preserving extreme-ultraviolet (XUV) monochromator using a semi-infinite gas cell as a source. The performance of this beamline in the photon-energy range of 20 eV-42 eV has been characterized. We have measured the order-dependent XUV pulse durations as well as the flux and the spectral contrast. XUV pulse durations of ≤40 fs using 32 fs, 800 nm driving pulses were measured on the target. The spectral contrast was better than 100 over the entire energy range. A simple model based on the strong-field approximation is presented to estimate different contributions to the measured XUV pulse duration. On-axis phase-matching calculations are used to rationalize the variation of the photon flux with pressure and intensity.

Attosecond beamline with actively stabilized and spatially separated beam paths.

We describe a versatile and compact beamline for attosecond spectroscopy. The setup consists of a high-order harmonic source followed by a delay line that spatially separates and then recombines the extreme-ultraviolet (XUV) and residual infrared (IR) pulses. The beamline introduces a controlled and actively stabilized delay between the XUV and IR pulses on the attosecond time scale. A new active-stabilization scheme combining a helium-neon-laser and a white-light interferometer minimizes fluctuations and allows to control delays accurately (26 as rms during 1.5 h) over long time scales. The high-order-harmonic-generation region is imaged via optical systems, independently for XUV and IR, into an interaction volume to perform pump-probe experiments. As a consequence of the spatial separation, the pulses can be independently manipulated in intensity, polarization, and frequency content. The beamline can be combined with a variety of detectors for measuring attosecond dynamics in gases, liquids, and solids.

Photoelectron spectrometer for attosecond spectroscopy of liquids and gases.

A new apparatus for attosecond time-resolved photoelectron spectroscopy of liquids and gases is described. It combines a liquid microjet source with a magnetic-bottle photoelectron spectrometer and an actively stabilized attosecond beamline. The photoelectron spectrometer permits venting and pumping of the interaction chamber without affecting the low pressure in the flight tube. This pressure separation has been realized through a sliding skimmer plate, which effectively seals the flight tube in its closed position and functions as a differential pumping stage in its open position. A high-harmonic photon spectrometer, attached to the photoelectron spectrometer, exit port is used to acquire photon spectra for calibration purposes. Attosecond pulse trains have been used to record photoelectron spectra of noble gases, water in the gas and liquid states as well as solvated species. RABBIT scans demonstrate the attosecond resolution of this setup.

Evaluation of primary care futures: a faculty development program for community health center preceptors.

PURPOSE: To assess the impact of a national series of faculty development workshops for community health center preceptors. METHOD: Two hundred and twenty-three community health center preceptors from a variety of disciplines attended one of five workshops conducted in different regions of the country. The workshops emphasized active learning using role-play to provide skills in educational planning, teaching styles, evaluation, and feedback. The preceptors were evaluated before and immediately after the workshop, and again three months later. They were also asked to assess the quality of the workshop. RESULTS: The preceptors demonstrated significant increases in the use of five of seven teaching concepts while analyzing a role-play interaction. In addition, there were significant positive, immediate changes in familiarity with nine of 11 concepts, which were retained for at least three months. The preceptors also reported that they continued to use six of the effective teaching behaviors they had learned three months after the workshop. They were extremely positive in their assessments of the workshops. CONCLUSION: This preliminary study suggests that preceptors from a variety of backgrounds can improve their teaching knowledge and skills by participating in a brief faculty development program that emphasizes active learning.

Cloning and analysis of a peptide synthetase gene of the balhimycin producer Amycolatopsis mediterranei DSM5908 and development of a gene disruption/replacement system.

A gene cloning system for Amycolatopsis mediterranei DSM5908, the producer of the glycopeptide antibiotic balhimycin, was developed for analysis of peptide synthetase genes. A modified direct transformation procedure was used to introduce DNA. The efficiency of DNA uptake depended on the age of the culture: mycelium of early stationary phase (52-55 h) cultures resulted in optimal transformation frequencies. Using the novel non-replicative integration vector pSP1, gene disruption plasmids were constructed. Highest integration frequencies were observed when the DNA was isolated from the dam/dcm Escherichia coli strain JM110. The efficiency of integration depended directly on the size of the cloned insert. Plasmids with fragments smaller than 1 kilobase (kb) were difficult to integrate. In gene replacement experiments a high double cross-over rate (31%) was demonstrated. Oligonucleotides derived from conserved regions of peptide synthetases were designed to identify balhimycin biosynthesis genes. Using these gene probes in plaque hybridization experiments, we identified peptide synthetase homologous DNA fragments in a lambda library of A. mediterranei. One peptide synthetase gene fragment was characterized by DNA sequencing and the results revealed a complete amino acid activating domain of a peptide synthetase gene, designated aps. The disruption of aps neither influenced balhimycin biosynthesis nor generated another apparent phenotype.

Adhesion of different bifidobacteria strains to human enterocyte-like Caco-2 cells and comparison with in vivo study.

The validity of the in vitro adhesion tests performed with cultured cell lines, was determined in this study by comparison with results obtained in vivo, in a previous study. To make this experiment the in vitro adhesion tests were performed during a long period by utilization of an appropriate medium, to determine the capacity of the adhered strain to colonize the intestinal tract. It was demonstrated that the ability of the strain to adhere and colonize the intestinal cell in vivo or the cultured intestinal cells in intro was similar.

The natural history of coccidioidal meningitis: VA-Armed Forces cooperative studies, 1955-1958.

From records on the clinical course of 699 military and veteran patients who had coccidioidomycosis before the advent of effective antifungal therapy, we identified 25 cases in which the manifestations of meningeal dissemination were described. Of 21 patients who had an identifiable initial infection, 16 developed meningeal symptoms (most frequently headache, vomiting, and nuchal rigidity) within the next 6 months. Associated pulmonary lesions were significantly more frequent in the right than in the left lung, as was also the case among 47 matched control patients who had coccidioidomycosis but not meningitis. Of 17 patients whose meningeal symptoms developed during the period studied, all died within 31 months. However, four of eight patients whose onset of symptoms preceded the study period survived for 55-146 months. Patients whose extrapulmonary dissemination involved only the meninges survived significantly longer than did those with more extensive infections. Even without therapy, the white blood cell count in cerebrospinal fluid markedly decreased during the course of the infection. The quantitative analysis provided herein offers a basis for meaningful comparisons with patients enrolled in current and future trials of therapy for coccidioidal meningitis.

Ketoconazole treatment of coccidioidal meningitis.

Fifteen patients with coccidioidal meningitis were treated with high doses of ketoconazole for up to 4 years. Five patients were treated with ketoconazole alone. One clinically failed, one developed hepatotoxicity, and three achieved remission of meningitis. One patient received intrathecal AMB in addition to ketoconazole for only 2 weeks before continuing on ketoconazole alone. He improved, but discontinued ketoconazole because of nausea and vomiting, and suffered a lethal relapse. Nine patients received ketoconazole in combination with prolonged courses of intrathecal AMB. Two patients were failures from nausea and vomiting, and the remaining seven either improved or experienced remission. The clinical responses appeared to be similar in patients receiving high-dose ketoconazole, either alone or combined with AMB, suggesting that there is no clinically significant antagonism of the drugs. Nausea and vomiting are significant limitations of high-dose ketoconazole. Ketoconazole alone is effective in some patients with coccidioidomycotic meningitis.

Comparison and diagnostic value of the coccidioidin heat-stable (HS and tube precipitin) antigens in immunodiffusion.

Coccidioides immitis produces two unrelated heat-stable antigens which are diagnostically useful in immunodiffusion tests. One, the tube precipitin antigen, is valuable for specifically detecting antibody and diagnosing early primary cases of coccidioidomycosis. The other heat-stable antigen, designated HS, is the most useful coccidioidin antigen for specifically immunoidentifying C. immitis cultures. Both of the antigens were compared and evaluated for their usefulness in exoantigen and serologic immunodiffusion tests. Our studies indicated that the detection of tube precipitin antigen is of limited value for immunoidentifying C. immitis isolates because the antigen is common to certain gymnoascaceous saprophytes, such as Arachniotus, Auxarthron, and Malbranchea species, that form alternate arthroconidia. Antibodies to HS antigens are infrequently found in human sera from patients with coccidioidomycosis and are thus of little serodiagnostic value.

Characterization of protein and mannan polysaccharide antigens of yeasts, moulds, and actinomycetes.

Antigens in coccidioidin were compared with purified subfractions via tandem immunoelectrophoresis (IEP) and by a combination of advancing line and crossed IEP. Rocket IEP was suitable for titrating the reactions and showing the relationship between column fractions. These techniques required multicomponent antisera produced by hyperimmunization over many months and by the use of known standard migration pairs. The IEP variations were used to chart the development of antisera against coccidioidin factors, to monitor antigen purifications, and to test the immunochemical homogeneity of an isolated antigen. Mannose-based heteroglycans of Cryptococcus neoformans were recovered from the culture filtrate. After precipitation of the major viscous glucuronoxylomannan (GXM) with ethanol or cetyltrimethylammonium bromide, the supernate is reserved because it contains a galactoxylomannan (GalXM). After removal of glucuronic acid from the GXM, the resulting xylomannan of serotype A was amenable to 13C-nuclear magnetic resonance (NMR) spectrometry; it revealed nonreducing xylose, alpha-1,3-mannose, and alpha-1,2/1,3 disubstituted mannose, thus confirming by an independent means what was previously known. The characterization sequence of GalXM included: (1) gas-liquid chromatography (GLC) of neutral sugars as peracetylated aldononitriles; (2) methylation-fragmentation GLC mass spectrometry to determine the glycosidic linkages; and (3) 13C-NMR showing similarities to mannan of Saccharomyces cerevisiae. Affinity chromatography of the GalXM on concanavalin A separated the galactoxylo component from an adsorbed mannoprotein. Selection of monoclonal antibodies (MAbs) relies on presumptive enzyme immunoassays (EIAs) or radioimmunoassays for rapid screening of clones and for determination of isotypes; however, higher resolution confirmatory tests are needed to obtain MAbs of desired specificity. MAbs against Candida tropicalis mannan were labeled with horseradish peroxidase to use for detecting mannan in serum. MAbs against the partially purified "m" factor of histoplasmin were characterized by the enzyme-linked immunoelectro-transfer blot technique (EITB), revealing unsuspected complexity in the antigen. Secreted proteins of Nocardia asteroides were isoelectrically focused; three proteins, identified by EITB as promising to be specific for that actinomycete, were cut out of gels and used to immunize mice for production of MAbs. The fimbriae of Actinomyces viscosus and A. naeslundii that mediate lactose-reversible coagglutination with Streptococcus sanguis have been used to evoke MAbs.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of five commercially available immunodiffusion kits for detection of Coccidioides immitis and Histoplasma capsulatum antibodies.

Five commercial test kits for the serodiagnosis of coccidioidomycosis and histoplasmosis based upon immunodiffusion were evaluated. The correlation of results with the test kits in the Clinical Laboratory varied from 71 to 100% for coccidioidomycosis. The correlation for coccidioidomycosis immunodiffusion testing varied from 57 to 83% when results from the test kits and the Mycology Research Laboratory were compared. Only 81% correlation was noted between the two laboratories when the same reference system was used. Results with the test kits for Histoplasma serodiagnosis and results from the Mycology Research Laboratory showed a correlation of 52 to 75%. There were no false-positive results with any system. All of the commercial kits were 100% specific for the diagnosis of both coccidioidomycosis and histoplasmosis, but the sensitivity of the immunodiffusion tests varied with the system used.

The critical role of CO2 in the morphogenesis of Coccidioides immitis in cell-free subcutaneous chambers.

In order to ascertain the factors controlling conversion of Coccidioides immitis arthroconidia to endosporulating spherules, studies were conducted with the use of subcutaneously implanted dialysis chambers in mice. The dialysate had the following characteristics: pH approximately 7.36; CO2 partial pressure, approximately 50 mm Hg; O2 partial pressure, approximately 140 mm Hg; protein content, approximately 20 mg/ml; and no cells. When chambers were inoculated with arthroconidia, endosporulating spherules developed. Introduction of syngeneic phagocytes had no effect. When dialysate or autologous serum was studied in vitro in room air, arthroconidia converted to mycelia unless CO2 supplementation (CO2 partial pressure, 20-80 mm Hg) was provided, in which case endosporulating spherules developed. The effect of CO2 could not be reproduced with either NaHCO3 or other buffers. These studies indicate that CO2 at a partial pressure found in normal host tissues is essential for formation of endosporulating spherules and that host phagocytes are not needed for such conversion in tissue fluids, either in vivo or in vitro.

Reactivity of alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis with anti-Coccidioides immunoglobulin M precipitin antibody.

The alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis (C-ASWS) mycelia and spherules was shown to react with anti-Coccidioides immunoglobulin M (IgM) precipitin antibody, both in the classical tube precipitin test and in the immunodiffusion assay for tube precipitin antibody (IDTP). The reactions obtained between C-ASWS and reference IgM precipitin antibody were identical to the reaction obtained when reference coccidioidin (CDN) was used. Definitive proof that C-ASWS extracts contain antigenic determinants that are reactive with IgM tube precipitin antibody was obtained by solid-phase immunoadsorption. Elution of reference IDTP antiserum over a column containing mycelium C-ASWS coupled to Sepharose 4B completely adsorbed precipitin antibody; i.e., reactivity in the IDTP was demonstrable in the column eluate but not in the column effluent fraction. The antigenic composition of C-ASWS extracts was evaluated and compared with that of CDN by two-dimensional immunoelectrophoresis against burro anti-CDN. The results established that both mycelium and spherule C-ASWS contain antigenic determinants in common with only one antigen present in CDN. The latter, designated antigen 2, is a large polymer which is predominant among the antigenic components in CDN. On a dry weight comparison, antigen 2 determinants were most concentrated in spherule C-ASWS, followed by mycelium C-ASWS and reference IDTP antigen. The finding that C-ASWS extracts are reactive with IgM tube precipitin antibody and are antigenically identical to antigen 2 in CDN suggests that antigen 2 is the biologically active component of CDN in tube precipitin assays.

Adherence and penetration of vascular endothelium by Candida yeasts.

Metastatic infection after hematogenous dissemination of Candida species is presumably dependent on the fungus traversing the vascular endothelium. An in vitro model of the earliest events of metastatic Candida infection was developed with whole vascular strips. Freshly obtained porcine blood vessels were secured in a perforated Lucite template that allowed the application of yeasts directly to the endothelial surface. Multiple wells allowed experimental and control observations on the same vascular segments. Adherence to endothelium was greatest with Candida albicans and Candida tropicalis, less with Candida Krusei, and least with Candida parapsilosis, Candida pseudotropicalis, and Torulopsis glabrata. This hierarchy of adherence parallels that in other in vitro systems employing mucosal epithelial cells or fibrin-platelet matrixes and reflects the known virulence of the respective species and their potential for hematogenous dissemination. C. albicans and C. tropicalis yeasts that adhered were capable of directly traversing the endothelial surface before the production of germ tubes. Heat or Formalin-killed yeasts and viridans group streptococci, although adherent, were incapable of vascular penetration, a process presumably attributable to enzymatic digestion of host tissue. Loss of integrity of penetrated endothelial tissue was verified by loss of dye exclusion, lactic dehydrogenase release, and ultramicroscopic changes. These two steps, adherence and penetration, provide direct insight into the earliest events in hematogenous Candida species dissemination and suggest that C. albicans and C. tropicalis yeasts are capable of initiating tissue invasion before germ tubes have had the opportunity to form and participate in the invasive process.

Relationship of progesterone- and estradiol-binding proteins in Coccidioides immitis to coccidioidal dissemination in pregnancy.

Pregnancy is a major risk factor for coccidioidal dissemination. Because rates of Coccidioides immitis growth and endospore release are stimulated in vitro by levels of unbound progesterone and 17 beta-estradiol that are achievable, in vivo, in the sera of pregnant women (i.e., 10(-9) to 10(-8) M), a specific-hormone-binding system in C. immitis was sought. Fungal cytosols were incubated with tritiated steroids plus or minus radioinert steroids to identify specific binding systems. All five strains of C. immitis tested exhibited specific saturable binding for progestin, estrogen, androgen, and (to a lesser extent) corticosterone and glucocorticoid hormone classes. Only low or inconsistent estrogen or androgen binding was found in Blastomyces dermatitidis and Torulopsis glabrata. Cryptococcus neoformans, Paracoccidioides brasiliensis, and non-albicans Candida species showed no binding. Scatchard analysis of progestin and estrogen binding in C. immitis revealed a high-capacity, low-affinity binding system that was unaffected by RNase and DNase, but 40 to 60% degraded by trypsin or heating. Ammonium sulfate precipitation resolved a high-affinity, low-capacity binding system (Kd = 1.24 X 10(-9) to 3.60 X 10(-8) M; number of binding sites = 0.014 to 0.20 pmol/mg of protein). The Kd of this system is sufficient to compete for unbound hormone in the sera of pregnant women. The high-capacity, low-affinity system may serve as a repository for hormone before its attachment to the specific binder. These studies suggest that the effects of nanomolar concentrations of sex hormones on C. immitis may be mediated by a specific cytosol protein-binding system and that stimulatory events observed in vitro may have relevance for the mechanism of coccidioidal dissemination in pregnancy.

Coccidioidomycosis: factors affecting the host-parasite interaction.

The inhaled arthroconidia of Coccidioides immitis mature to form large (30-80 micron) endosporulating spherules. Each spherule releases hundreds of endospores, taxing immunologic reserves. Polymorphonuclear neutrophils (PMNs) are prominent in the lesions of coccidioidomycosis, but their effectiveness is questionable. Arthroconidia possess an antiphagocytic surface derived from the original hyphal outer wall layer. Only 20%-30% of arthroconidia or endospores that are ingested by PMNs are killed. PMNs can digest the outer wall layer of spherules but may not induce lethal injury. Cell-mediated immunity is central to host defense. Macrophages ingest arthroconidia and endospores but may be unable to kill them unless lymphokines stimulate phagolysosomal fusion. Whether spherules can be killed by macrophages is unclear. C. immitis is stimulated directly by serum levels of estradiol and progesterone achieved in pregnant women. This, together with the depressed cell-mediated immunity of pregnancy, may account for the virulent nature of coccidioidomycosis in pregnant women.

Pityrosporum folliculitis. Its potential for confusion with skin lesions of systemic candidiasis.

Development of an erythematous, papulonodular to papulopustular skin eruption in four febrile, immunocompromised patients raised the possibility of a systemic mycosis when Gram's stains of unroofed lesions disclosed budding yeasts. Candidiasis, torulopsosis, sporotrichosis, and cryptococcosis were considered in the differential diagnosis, and therapy with amphotericin B was begun. Skin biopsy specimens disclosed fungi to be located exclusively within intact and ruptured hair follicles. Inability of the fungi to grow on media that were not supplemented with lipid and their structure and location all suggested the presence of Pityrosporum sp. When hematogenous dissemination of a mycosis to the skin is suspected, the diagnosis must be based on biopsy specimen demonstration of dermal invasion, ideally with positive cultures. Pityrosporum sp, common skin saprophytes, may produce folliculitis, and be mistaken for pathogenic yeasts.

Morphogenesis throughout saprobic and parasitic cycles of Coccidioides immitis.

The fungus, Coccidioides immitis, differs from other dimorphic pathogens in that its parasitic stage is a complex morphogenic cycle, raising the question that changes and composition during morphogenesis might influence host responses. As a prelude to examining the interaction of fungal morphogenesis and host responses, the life cycle of this fungus has been examined in greater detail than previously accomplished. During saprobic development, alternating enterothallic arthroconidia are formed as infectious propagules. The outer wall is broken and loosely adherent. Under in vitro conditions supporting the parasitic cycle, multinucleate arthroconidia transform into uninucleate round cells. Rapid, synchronous, nuclear replication is initiated, accompanied by increase in cell mass and deposition of new cell wall substance. As karyokinesis ceases, morphologic differentiation begins with invagination of the inner layers of the spherule wall and then is progressive, eventually segmenting the protoplasm into uninucleate endospores grouped in clusters within a hyaline membrane. Endospores, escaping through a break in the spherule wall, are held in aggregates by fibrils which are stretched and broken as endospores separate. It would seem that rapid production of hundreds of progeny from an original single cell, protected during development by an enclosing spherule wall and then released in clusters, should favor establishment of the fungus in a host, and dynamic changes in the cell wall during morphogenesis should influence the host response.