Method for Collecting Air-Water Interface Microbes Suitable for Subsequent Microscopy and Molecular Analysis in both Research and Teaching Laboratories

A method has been developed for collecting air-water interface (AWI) microbes and biofilms that enables analysis of the same sample with various combinations of bright-field and fluorescence light microscopy optics, scanning and transmission electron microscopy (TEM), and atomic force microscopy. The identical sample is then subjected to molecular analysis. The sampling tool consists of a microscope slide supporting appropriate substrates, TEM grids, for example, that are removable for the desired protocols. The slide with its substrates is then coated with a collodion polymer membrane to which in situ AWI organisms adhere upon contact. This sampling device effectively separates the captured AWI bacterial community from the bulk water community immediately subtending. Preliminary data indicate that the AWI community differs significantly from the water column community from the same sample site when both are evaluated with microscopy and with 16S ribosomal DNA sequence-based culture-independent comparisons. This microbe collection method can be used at many levels in research and teaching.     

Analysis of Low-temperature Tolerance of a Tomato (Lycopersicon esculentum) Cybrid with Chloroplasts from a more Chilling-tolerant L. hirsutum Accession

Growth and photosynthesis of an alloplasmic tomato (cybrid),i.e. line AH47, containing the nuclear genome of the chilling-sensitivecytoplasmic albino mutant of L. esculentum Mill. ‘LargeRed Cherry’ (LRC) and the plastome of a more chilling-toleranthigh-altitude accession of the related wild species L. hirsutumHumb. & Bonpl. LA 1777, were investigated at an optimal(25/20°C) and suboptimal (16/14°C) day/night temperatureregime and their performance compared with that of both euplasmicparents. The cybrid shoot had a similar biomass and developmentrate to the nuclear tomato (L. esculentum) parent at both temperatureregimes. Compared with the biomass production of shoots grownat optimal temperature, the reduction in shoot biomass at suboptimaltemperature was smaller for L. hirsutum than for L. esculentumand the cybrid. This difference was related to a stronger inhibitionof leaf area expansion in L. esculentum and the cybrid in thesuboptimal temperature regime than in L. hirsutum. Irrespectiveof the temperature regime under which the plants were grown,photosynthetic performance and leaf pigment, carbohydrate andsoluble-protein contents of the cybrid resembled those of thenuclear parent. No advantages of the alien L. hirsutum chloroplastwith respect to growth and photosynthesis-related characteristicswere observed in the cybrid in the suboptimal temperature regime,indicating that the temperature sensitivity of the photosyntheticapparatus is regulated by nuclear genes. An adverse consequenceof interspecific chloroplast transfer was the increased susceptibilityto chill-induced photoinhibition of the cybrid. It is concludedthat cybridization is not a useful tool for improving low-temperaturetolerance of tomato. Copyright 2000 Annals of Botany Company Alloplasmic tomato, chloroplast, cybrid(ization), growth, low-temperature tolerance, Lycopersicon esculentum, L. hirsutum, photosynthesis, plastome, tomato     

Composition of the essential oil of Mentha aquatica

The overall composition of the essential oil of a botanically described Mentha aquatica (2n = 96) has been determined. Limonene, caryophyllene and germacrene-D are the major hydrocarbons and bicyclogermacrene and viridiflorol, an oxygenated compound, are present in substantial amounts. The presence of small amounts of menthones, mentholes and menthyl acetates is indicated.     

Determination of the dissociation constants of ropinirole and some impurities and their quantification using capillary zone electrophoresis

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary zone electrophoresis (CZE) was used for the determination of the dissociation constants of ropinirole and five structurally related impurities, potentially formed during its synthesis and for separation and quantification of these substances. The dissociation constants obtained from the CZE measurements were confirmed by UV spectrophotometry for some of the test compounds, obtaining a good agreement between the values. Careful optimization of the running buffer composition permitted base-line resolution of the six compounds in a borate buffer containing acetonitrile and magnesium sulfate (a 100 mM borate buffer containing 30 mM MgSO₄ and 20 vol.% of acetonitrile). It was shown that CZE can determine the level of these impurities, down to a level of 0.05% of the main component within 15 min.     

Fast Skeletal Muscle Troponin Activation Increases Force of Mouse Fast Skeletal Muscle and Ameliorates Weakness Due to Nebulin-Deficiency

The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension–pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations <1 µM), CK-2066260 increased tension of NEB KO fibers to beyond that of WT fibers. Crossbridge cycling kinetics were studied by measuring k_tr (rate constant of force redevelopment following a rapid shortening/restretch). CK-2066260 greatly increased k_tr at submaximal activation levels in both WT and NEB KO fiber bundles. We also studied the sarcomere length (SL) dependence of the CK-2066260 effect (SL 2.1 µm and 2.6 µm) and found that in the NEB KO fibers, CK-2066260 had a larger effect on calcium sensitivity at the long SL. We conclude that fast skeletal muscle troponin activation increases force at submaximal activation in both wildtype and NEB KO fiber bundles and, importantly, that this troponin activation is a potential therapeutic mechanism for increasing force in NM and other skeletal muscle diseases with loss of muscle strength.     

Phagocytosis of Bacteria Adhering to a Biomaterial Surface in a Surface Thermodynamic Perspective

Bacterial biofilms can increase the pathogenicity of infection and constitute a major problem in modern health-care, especially on biomaterial implants and devices. Biofilms are difficult to eradicate by the host immune system, even with antibiotics, and have been the number one cause of biomaterial implant and device failure for decades. Therefore, it is important to understand how immune cells interact with adhering pathogens. This study firstly aims to develop a simple method to quantify phagocytosis of six different strains of staphylococci adhering on a surface with phase-contrast-microscopy. Phagocytosis of adhering staphylococci to a glass surface by phagocytes was quantified in a parallel plate flow chamber, and expressed as a phagocytosis rate, accounting for the number of adhering staphylococci initially present and for the duration of phagocytosis. Murine macrophages were more effective in clearing staphylococci from a surface than human phagocytes, which require differentiation from their monocyte or promyelocytic state during an experiment. Direct visualization of internalization of a GFP-modified S. aureus strain inside phagocytes confirmed the validity of the method proposed. As a second aim, the differences in phagocytosis rates observed were investigated on a surface thermodynamic basis using measured contact angles of liquids on macroscopic lawns of staphylococci and phagocytes, confirming that phagocytosis of adhering pathogens can be regarded as a surface phenomenon. In addition, surface thermodynamics revealed that phagocytosis of adhering pathogens is determined by an interplay of physical attraction between pathogens and phagocytes and the influence of chemo-attractants. For future studies, these results will help to place in vitro experiments and murine infection models in better perspective with respect to human ones.     

Adverse Drug Events in Older Hospitalized Patients: Results and Reliability of a Comprehensive and Structured Identification Strategy

Background

Older patients are at high risk for experiencing Adverse Drug Events (ADEs) during hospitalization. To be able to reduce ADEs in these vulnerable patients, hospitals first need to measure the occurrence of ADEs, especially those that are preventable. However, data on preventable ADEs (pADEs) occurring during hospitalization in older patients are scarce, and no ‘gold standard’ for the identification of ADEs exists.

Methodology

The study was conducted in three hospitals in the Netherlands in 2007. ADEs were retrospectively identified by a team of experts using a comprehensive and structured patient chart review (PCR) combined with a trigger-tool as an aid. This ADE identification strategy was applied to a cohort of 250 older hospitalized patients. To estimate the intra- and inter-rater reliabilities, Cohen’s kappa values were calculated.

Principal Findings

In total, 118 ADEs were detected which occurred in 62 patients. This ADE yield was 1.1 to 2.7 times higher in comparison to other ADE studies in older hospitalized patients. Of the 118 ADEs, 83 (70.3%) were pADEs; 51 pADEs (43.2% of all ADEs identified) caused serious patient harm. Patient harm caused by ADEs resulted in various events. The overall intra-rater agreement of the developed strategy was substantial (κ = 0.74); the overall inter-rater agreement was only fair (κ = 0.24).

Conclusions/Significance

The ADE identification strategy provided a detailed insight into the scope of ADEs occurring in older hospitalized patients, and showed that the majority of (serious) ADEs can be prevented. Several strategy related aspects, as well as setting/study specific aspects, may have contributed to the results gained. These aspects should be considered whenever ADE measurements need to be conducted. The results regarding pADEs can be used to design tailored interventions to effectively reduce harm caused by medication errors. Improvement of the inter-rater reliability of a PCR remains challenging.     

Pentavalent Single-Domain Antibodies Reduce Campylobacter jejuni Motility and Colonization in Chickens

Campylobacter jejuni is the leading cause of bacterial foodborne illness in the world, with symptoms ranging from acute diarrhea to severe neurological disorders. Contaminated poultry meat is a major source of C. jejuni infection, and therefore, strategies to reduce this organism in poultry, are expected to reduce the incidence of Campylobacter-associated diseases. We have investigated whether oral administration of C. jejuni-specific single-domain antibodies would reduce bacterial colonization levels in chickens. Llama single-domain antibodies specific for C. jejuni were isolated from a phage display library generated from the heavy chain IgG variable domain repertoire of a llama immunized with C. jejuni flagella. Two flagella-specific single-domain antibodies were pentamerized to yield high avidity antibodies capable of multivalent binding to the target antigen. When administered orally to C. jejuni-infected two-day old chicks, the pentabodies significantly reduced C. jejuni colonization in the ceca. In vitro, the motility of the bacteria was also reduced in the presence of the flagella-specific pentabodies, suggesting the mechanism of action is through either direct interference with flagellar motility or antibody-mediated aggregation. Fluorescent microscopy and Western blot analyses revealed specific binding of the anti-flagella pentabodies to the C. jejuni flagellin.