Solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells. QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO(2), but also ZnO and others including CuO. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative; in others it is the opposite. These studies have been well reviewed. In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents. CdTe QDs have anti-microbial effects alone or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water. In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent. The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.     

How emergence and death assumptions affect count-based estimates of butterfly abundance and lifespan

Transect count data form the basis of many butterfly and other insect monitoring programs worldwide. A clear understanding of the limitations of such datasets, including the potential for biases in the statistical methods used to analyze them, is therefore crucial. The classical Zonneveld model (CZ) can extract estimates of a suite of demographic parameters from transect count datasets, and has also been used in theoretical analyses of protandry and reproductive asynchrony. The CZ relies on strong assumptions about the emergence and death processes underlying observed transect count datasets. Though reasonable as a starting place, a growing body of empirical evidence suggests these assumptions will, in many cases, not hold. Here, I explore how violations of these assumptions bias CZ-based estimates of two key population parameters: total population size and mean individual lifespan. To do this, I generalize the Zonneveld model by relaxing the symmetrical emergence distribution and constant death rate assumptions such that the generalized models contain the CZ as a special case. Using the generalized models as data generating processes, I then show that the CZ is able to closely mimic the shape of the abundance time course produced by either variant of the generalized model under a wide range of conditions, but produces highly biased estimates of population size and mean lifespan in doing so. My analysis therefore demonstrates both that the CZ is not robust to violations of its emergence and death assumptions, and that a good observed fit to transect count data does not mean these assumptions are satisfied.     

SDS‐PAGE‐free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes

Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS‐PAGE‐free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1‐butyl‐3‐methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D‐SCX‐RPLC‐ESI‐MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.     

含氯消毒饮用水对微生态制剂使用的影响

目的了解临床使用消毒饮用水稀释益生菌产品,对益生菌活菌数的影响。方法配制不同有效氯水溶液,测定不同放置时间四个菌种活菌数变化情况。结果微囊包被屎肠球菌、蜡样芽胞杆菌在有效氯5 ppm和10ppm中使用有效活菌数不受任何影响;微囊包被粪肠球在有效氯5 ppm中浸泡2 h内使用,不影响其有效活菌;而在10ppm中1 h之内使用有效活菌数不受影响;微囊包被枯草芽胞杆菌在有效氯5 ppm中1 h之内活菌数不受影响,而增加浸泡时间及有效氯浓度都会影响其有效活菌数。结论在临床使用微生态产品可以使用含氯消毒饮用水稀释但是需尽快用完不要超过否则影响部分菌株的使用效果。     

Haemocyte response associated with induction of shell regeneration in the deep-sea vent mussel Bathymodiolus azoricus (Bivalvia: Mytilidae)

This study reports on the haemocyte responses after induction of shell regeneration in the hydrothermal mussel Bathymodiolus azoricus. Haemolymph was drawn from live mussels collected at Menez Gwen hydrothermal vent site (850 m depth) at the Mid Atlantic Ridge (MAR) and was compared with those collected following laboratory acclimatisation (1 atm and Ca-rich algal diet) and also with induced specimen for up to 30 days. Simultaneously, histological changes in mantle micro-morphology with the histochemical detection of Ca mobilisation in tissues were conducted.On the basis of light- and transmission electron microscopy, it is concluded that the physiological equipment involved in shell regeneration in the deep sea bivalve closely resembles that in littoral mytilids, a group that B. azoricus is closely related. This in spite of previously alleged molecular and cellular adaptations to extreme conditions typical at deep sea hydrothermal vents. Three types of blood cells were identified sharing various morphological similarities with those in many non-vent bivalves. Significant increase in the number of circulating haemocytes was detected from day 5 after induction shell regeneration. It is suggested that the increase may be a result of migration of haemocytes from the connective tissue, probably to the shell growth frontline. It is alleged that a first peak in haemocyte number is a non-specific immune response related wound healing, which renders changes in the pallial fluid that are favourable for CaCO₃ deposition. The conspicuous presence of an unidentified, acid soluble, highly refractive structure in the haemolymph of induced mussels was detected, which may play a role in Ca nucleation.This study has set the stage for investigations underway on the influence of hydrostatic pressure on shell biomineralisation in B. azoricus subjected to post-capture hyperbaric simulations.     

Conservation of Britain's biodiversity: the case of Hieracium cyathis (Asteraceae) and its relation to other apomictic taxa

Hieracium cyathis (Ley) W. R. Linton (Asteraceae), Chalice Hawkweed, is a rare species endemic to Britain. It is a distinct member of Hieracium Section Oreadea , characterized by a short acladium and out-curved tips to the phyllaries. Historic records and current field survey indicate it has occurred in two localities in SW England and seven in South Wales. It is still present in all sites except for one Welsh locality, is significantly threatened at Cheddar Gorge, and two Welsh sites have only three plants each. Comparative morphology, cultivation experiments and amplified fragment length polymorphism (AFLP) analysis do not support the reported differences between material from SW England and Wales. AFLP analysis showed that every plant in the field was genetically similar but different, and minor genetic variation was found in one progeny array. It is a triploid (2 n  = 27) with apomictic seed production, and 26% potentially viable pollen. It qualifies as 'Near Threatened' under the 2001 IUCN threat criteria. General guidelines for conservation of microspecies of Hieracium are discussed.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 669–680.     

Screening method for detection of hydrocarbon-oxidizing bacteria in oil-contaminated water and soil specimens

Toxic action of crude oil on the living world and ecosystems in general is a global problem of both aquatic and terrestrial environments. Bearing in mind the possibility of biodegradation of this toxicant, the procedures of determining counts and activity of cultivable microorganisms, and especially of bacteria responsible for degradation processes, are of great significance. The aim of this work was to study the possibility of modifying some solid media by adding triphenyltetrazolium chloride reagent as an indicator of the dehydrogenase activity, to develop a simple screening method for a simultaneous assessment of the count and activity of cultivable hydrocarbon-oxidizing bacteria in the oil-contaminated environments. The modified method appeared to be rapid and very suitable for the intended purposes.     

Variation in the herd composition counts of sika deer

Herd composition counts (HCC) are commonly used to assess population status in deer. We evaluated the reliability of HCC of sika deer (Cervus nippon Temminck) using repeated counts and by comparing estimated sex ratios and calf-to-female ratios of marked deer on Nakanoshima Island, Hokkaido, Japan between April 1999 and October 2000. Although both total counts and sex and age ratios fluctuated greatly by month, seasonal changes showed a relatively small variance. This suggested that seasonal changes in behavior within sex and age classes contributed to biased ratio estimates obtained from HCC. Route counts should be used as a relative population abundance index with estimates of detection probability, especially for females. Adult sex ratios and age ratios from HCC were unbiased during the rutting season (October–November), and age ratios in spring could be used if yearlings are counted as adults.