Phylogenetic implications of the microbody-lipid globule complex in zoosporic fungi

Chytridiomycetous fungal zoospores contain a unique and intricate association of organelles, the 'microbody-lipid globule complex' (MLC). The spatial arrangement of organelles in the MLC appears important in the utilization of lipid globules for energy, but in addition, the structural association of organelles in the MLC reveals phylogenetic trends within this diverse group of organisms. Variations in the structure of the MLC correlate well with current phylogenetic concepts of aquatic fungi, yet suggest new relationships among these posteriorly uniflagellate zoospores. Based upon the organization of organelles in the MLC, 4 basic patterns of MLCs can be recognized, and these correspond to the 4 orders of Chytridiomycetes. The MLC in its simplest form consists of a microbody appressed to the edge of a lipid globule. In more highly organized MLCs, mitochondria subtend the microbody and a cisterna surmounts one side of the lipid globule. The organization and structure is still more complex in other MLCs where ER is elaborated into a tubular network of membranes or where small microbodies or mitochondria fuse into 'giant' organelles. The structural organization of the MLC provides an additional criterion by which the phylogeny of awuatic fungi can be evaluated.     

The use of33P-labelled riboprobes forin situ hybridizations: localization of myosin alkali light-chain mRNAs in adult human skeletal muscle

Summary ³³P-labelled probes were used to localize the mRNAs coding for the myosin alkali light-chain isoforms MLC 1f/3f and MLC 1sb in adult human muscles, which are distributed in characteristic fibre type specific patterns. In situ hybridizations of ³³P-labelled probes were compared with probes carrying ³⁵S or digoxigenin labels. Signals of equal strength were obtained with each of the three labels. The preferentially peripheral localization of these mRNAs in the muscle fibres could be clearly seen with all three probes, with digoxigenin probes providing the best resolution. ³³P can serve as a viable alternative in this type of experiment.These experiments with adult human muscles also showed that the post mortem stability of RNA in human muscle is better than generally assumed. We could detect no signs of degradation in RNA prepared from heart ventricle as well as skeletal muscle up to 24 hours post mortem. In situ hybridizations worked equally well in biopsy material as in post mortem samples.     

Design consideration and probes for fluorescence resonance energy transfer studies

Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-beta-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 A in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.     

A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics in Dictyostelium

Red fluorescent proteins (RFPs) combined with GFP are attractive probes for double-fluorescence labeling of proteins in live cells. However, the application of these proteins is restrained by stable oligomer formation and by their weak fluorescence in vivo. Previous attempts to eliminate these problems by mutagenesis of RFP from Discosoma (DsRed) resulted in the monomeric mRFP1 and in the tetrameric RedStar RFP, which is distinguished by its enhanced fluorescence in vivo. Based on these mutations, we have generated an enhanced monomeric RFP, mRFPmars, and report its spectral properties. Together with green fluorescent labels, we used mRFPmars to visualize filamentous actin structures and microtubules in Dictyostelium cells. This enhanced RFP proved to be suitable to monitor the dynamics of cytoskeletal proteins in cell motility, mitosis, and endocytosis using dual-wavelength fluorescence microscopy.     

Gold nanoclusters as novel optical probes for in vitro and in vivo fluorescence imaging

Fluorescent probes play an important role in the development of fluorescence-based imaging techniques for life sciences research. Gold nanoclusters (AuNCs) are a novel type of fluorescent nanomaterials which have attracted great interest in recent years. Composed of only a few atoms, these ultrasmall AuNCs exhibit quantum confinement effects and molecule-like properties. Fluorescent AuNCs have an attractive set of features including ultrasmall size, good biocompatibility and photostability, and tunable emission in the red to near-infrared spectral region, which make them promising as fluorescent labels for biological imaging. Examples of their application include live cell labeling, cancer cell targeting, cellular apoptosis monitoring, and in vivo tumor imaging. Here, we present a brief overview of recent advances in utilizing these emissive ultrasmall AuNCs as optical probes for in vitro and in vivo fluorescence imaging.     

Achieving Molecular Selectivity in Imaging Using Multiphoton Raman Spectroscopy Techniques

In the case of most optical imaging methods, contrast is generated either by physical properties of the sample (Differential Image Contrast, Phase Contrast), or by fluorescent labels that are localized to a particular protein or organelle. Standard Raman and infrared methods for obtaining images are based upon the intrinsic vibrational properties of molecules, and thus obviate the need for attached fluorophores. Unfortunately, they have significant limitations for live-cell imaging. However, an active Raman method, called Coherent Anti-Stokes Raman Scattering (CARS), is well suited for microscopy, and provides a new means for imaging specific molecules. Vibrational imaging techniques, such as CARS, avoid problems associated with photobleaching and photo-induced toxicity often associated with the use of fluorescent labels with live cells. Because the laser configuration needed to implement CARS technology is similar to that used in other multiphoton microscopy methods, such as two-photon fluorescence and harmonic generation, it is possible to combine imaging modalities, thus generating simultaneous CARS and fluorescence images. A particularly powerful aspect of CARS microscopy is its ability to selectively image deuterated compounds, thus allowing the visualization of molecules, such as lipids, that are chemically indistinguishable from the native species.     

GPI-anchored protein organization and dynamics at the cell surface

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite.     

Myosin light chain isoforms retain their species-specific electrophoretic mobility after processing, which enables differentiation between six species: 2DE analysis of minced meat and meat products made from beef, pork and poultry

Investigation of protein changes as well as authentication of meat is particularly difficult in processed meat products due to their different composition, complexity and very often inhomogeneity. The aim of this study was to check if the inter-species differences in the expression of myosin light chain (MLC) isoforms observed in raw meat were retained in meat products. MLCs from mixtures of minced meat (16 variants), frankfurters and sausages (15 products) made from cattle, pig, chicken, turkey, duck and goose were analysed by 2DE. Species-specific patterns of MLC isoforms were observed in all the mixtures and processed meat products. Relatively small degradation was observed in the MLCs after processing. Image analysis enabled species identification of the meat in all samples when the content of meat of one species was not lower than 10%. However, it was impossible to differentiate between all the six species under investigation on the basis of individual isoform. It was possible when the combination of all the three isoforms (myosin light chain 1 fast, myosin light chain 2 fast and myosin light chain 3 fast) was analysed. The results evidenced that MLCs have potential to be used as markers in authentication of meat products made from the analysed six species.     

Rotational dynamics of lipid and the Ca-ATPase in sarcoplasmic reticulum. The molecular basis of activation by diethyl ether

We have investigated the role of lipid and protein dynamics in the activation of the Ca2+-dependent ATPase in sarcoplasmic reticulum (SR) by diethyl ether. Conventional and saturation-transfer electron paramagnetic resonance (EPR) were used to probe rotational motions of spin labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase in SR. We confirm previous studies (Salama, G., and Scarpa, A. (1980) J. Biol. Chem. 255, 6525-6528; Salama, G., and Scarpa, A. (1983) Biochem. Pharmacol. 32, 3465-3477; Kidd, A., Scales, D., and Inesi, G. (1981) Biochem. Biophys. Acta 65, 124-131) reporting that addition of diethyl ether to SR results in an approximately 2-fold enzymatic activation, without loss of coupling. Diethyl ether progressively fluidizes the SR membrane with respect to lipid hydrocarbon chain dynamics probed at several depths in the bilayer. Digital substractions, used to analyze two-component lipid spin label spectra, reveal that a 2-fold mobilization occurs in the population of lipid probes motionally restricted by the protein, while the remaining more mobile population is less affected. The microwave saturation properties of lipid probes also indicate that restricted motions of these probes are mobilized in maximally activated SR membranes. Saturation-transfer EPR, applied to maleimide spin-labeled Ca-ATPase, demonstrates that a 2-fold increase in microsecond rotational motion of the Ca-ATPase correlates with the maximal enzymatic activation. Effects of diethyl ether on both the enzymatic activity and molecular dynamics are completely reversible by dilution with buffer. We propose that ether activates by selectively mobilizing lipid chains adjacent to the enzyme, thus facilitating protein motions that are essential for calcium transport.     

Dosimetric evaluation study of IMRT and VMAT techniques for prostate cancer based on different multileaf collimator designs

The hypofractionated radiotherapy modality was established to reduce treatment durations and enhance therapeutic efficiency, as compared to conventional fractionation treatment. However, this modality is challenging because of rigid dosimetric constraints. This study aimed to assess the impact of multi-leaf collimator (MLC) widths (10 mm and 5 mm) on plan quality during the treatment of prostate cancer. Additionally, this study aimed to investigate the impact of the MLC mode of energy on the Agility flattening filter (FF), MLC Agility-free flattening filter (FFF), and MLCi2 for patients receiving hypofractionated radiotherapy. Two radiotherapy techniques; Intensity Modulated Radiotherapy (IMRT) and Volumetric Modulated Arc Radiotherapy (VMAT), were used in this research. In the present study, computed tomography simulations of ten patients (six plans per patient) with localized prostate adenocarcinoma were analyzed. Various dosimetric parameters were assessed, including monitor units, treatment delivery times, conformity, and homogeneity indices. To evaluate the plan quality, dose-volume histograms (DVHs) were estimated for each technique. The results demonstrated that the determined dosimetric parameters of planning target volume (PTV)p (such as D mean, conformity, and homogeneity index) showed greater improvement with MLC Agility FF and MLC Agility FFF than with MLCi2. Additionally, the treatment delivery time was reduced in the MLC Agility FF (by 31%) and MLC Agility FFF (by 10.8%) groups compared to the MLCi2 group. It is concluded that for both the VMAT and IMRT techniques, the smaller width (5 mm) MLCs revealed better planning target volume coverage, improved the dosimetric parameters for PTV, reduced the treatment time, and met the constraints for OARs. It is therefore recommended to use 5 mm MLCs for hypofractionated prostate cancer treatment due to better target coverage and better protection of OARs.

     

The preparation of polyamide-oligonucleotide probes containing multiple non-radioactive labels.

Oligonucleotide probes containing multiple non-radioactive labels have been prepared by utilising and extending the methods used to prepare polyamide-oligonucleotide conjugates. The probes were prepared by incorporating suitable amino acid residues, such as lysines, in the polyamide, which were then used as sites for the attachment of the non-radioactive labels. The procedures developed give control over the distance of the label from the oligonucleotide, and also the inter-label distance. The labels can be conveniently introduced while the substrate is still on the solid support. Even though fluorescent oligonucleotide probes prepared in this way carrying multiple carboxyfluorescein labels gave low levels of fluorescence due to quenching, the probes containing ten biotin labels gave a detection sensitivity of approximately 5 attomole (3 million molecules).     

Fluorescence recovery spectroscopy as a probe of slow rotational motions.

Pump-and-probe techniques can be used to follow the slow rotational motions of fluorescent labels bound to macromolecules in solution. A strong pulse of polarized light initially anisotropically depletes the ground-state population. A continuous low-intensity beam of variable polarization then probes the anisotropic ground-state distribution. Using an additional emission polarizer, the generated fluorescence can be recorded as it rises towards its prepump value. A general theory of fluorescence recovery spectroscopy (FRS) is presented that allows for irreversible depletion processes like photobleaching as well as slowly reversible processes like triplet formation. In either case, rotational motions modulate recovery through cosine-squared laws for dipolar absorption and emission processes. Certain pump, probe, and emission polarization directions eliminate the directional dependence of either dipole and simplify the resulting expressions. Two anisotropy functions can then be constructed to independently monitor the rotations of either dipole. These functions are identical in form to the anisotropy used in fluorescence depolarization measurements and all rotational models developed there apply here with minor modifications. Several setups are discussed that achieve the necessary polarization alignments. These include right-angle detection equipment that is commonly available in laboratories using fluorescence methods.     

Monitoring Biophysical Properties of Lipid Membranes by Environment-Sensitive Fluorescent Probes

We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy

The discovery that the lipids constituting the plasma membrane are not randomly distributed, but instead are able to form laterally segregated lipid domains with different properties has given hints how the formation of such lipid domains influences and regulates many processes occurring at the plasma membrane. While in model systems these lipid domains can be easily accessed and their properties studied, it is still challenging to determine the properties of cholesterol rich lipid domains, the so called “Rafts”, in the plasma membrane of living cells due to their small size and transient nature. One promising technique to address such issues is fluorescence lifetime imaging (FLIM) microscopy, as spatially resolved images make the visualization of the lateral lipid distribution possible, while at the same time the fluorescence lifetime of a membrane probe yields information about the bilayer structure and organization of the lipids in lipid domains and various properties like preferential protein-protein interactions or the enrichment of membrane probes. This review aims to give an overview of the techniques underlying FLIM probes which can be applied to investigate the formation of lipid domains and their respective properties in model membrane and biological systems. Also a short technical introduction into the techniques of a FLIM microscope is given.     

Investigations of line scanning proton therapy with dynamic multi-leaf collimator

PurposeScanning proton therapy has dosimetric advantage over passive treatment, but has a large penumbra in low-energy region. This study investigates the penumbra reduction when multi-leaf collimators (MLCs) are used for line scanning proton beams and secondary neutron production from MLCs.MethodsScanning beam plans with and without MLC shaping were devised. Line scanning proton plan of 36 energy layers between 71.2 and 155.2 MeV was generated. The MLCs were shaped according to the cross-sectional target shape for each energy layer. The two-dimensional doses were measured through an ion-chamber array, depending on the presence of MLC field, and Monte Carlo (MC) simulations were performed. The plan, measurement, and MC data, with and without MLC, were compared at each depth. The secondary neutron dose was simulated with MC. Ambient neutron dose equivalents were computed for the line scanning with 10 × 10 × 5 cm³ volume and maximum proton energy of 150 MeV, with and without MLCs, at lateral distances of 25–200 cm from the isocenter. The neutron dose for a wobbling plan with 10 × 10 × 5 cm³ volume was also evaluated.ResultsThe lateral penumbra width using MLC was reduced by 23.2% on average, up to a maximum of 32.2%, over the four depths evaluated. The ambient neutron dose equivalent was 18.52% of that of the wobbling beam but was 353.1% larger than the scanning open field.ConclusionsMLC field shaping with line scanning reduced the lateral penumbra and should be effective in sparing normal tissue. However, it is important to investigate the increase in neutron dose.     

Interaction of peptides with binary phospholipid membranes: application of fluorescence methodologies

The application of fluorescence methodologies to obtain information about the extent, dynamics and topology of peptide interaction with binary phospholipid (mainly zwitterionic/anionic) mixtures is reviewed. First, general approaches based on peptide (tryptophan residues) fluorescence properties that give information about its partition, location and dynamics will be presented. Then, methodologies based on membrane probes fluorescence that report the influence of peptide binding and/or incorporation on the lateral organization (phase separation) of membrane phospholipids will be described. Specific examples taken from the literature that illustrate both situations are presented as well as formalisms for data analysis. It is shown that steady-state and time-resolved fluorescence data (particularly important in the case of fluorescence resonance energy transfer studies) give complementary information, allowing a molecular picture of peptide interaction with biphasic systems to be drawn.     

Reducing treatment machine downtime with a preventative MLC maintenance procedure

The persistent use of MLCs through VMAT and IMRT is causing additional wear and tear on these mechanical parts, leading to an increase in MLC interlocks, breakdowns and failures. This study investigates the effect of an MLC clean and service procedure on MLC performance and positional accuracy demonstrated through in-house service logbook reports, Varian MLC backlash test results, daily MLC position QC results and Varian TrueBeam trajectory log file data.A service and clean of each individual MLC leaf was carried out on 3 standard MLC and 2 high definition MLC (HDMLC) Varian TrueBeam linacs. In-house service logbook reports, Varian backlash test results, daily MLC picket fence QC results and beam hold data from patient delivery trajectory log files were analysed for up to 6 months pre and 2 years post the MLC service/clean to assess the impact on unplanned MLC maintenance work and MLC performance.The median (range) of hardware faults reduced from 12 (1–17) pre clean to 0 (0–1) and 4 (1–11) at 12 and 24 months respectively for all linacs with the exception of a HDMLC linac where faults increased from 4 pre-clean to 13 and 20 again at 12 and 24 months respectively. The reduction in faults in the alternative 4 linacs was consistent with the reduction in the number of MLCs reaching the 0.3 mm and the 0.4 mm backlash recommendations in the first 12 and 24 months following the service/clean. The increase in faults in the HDMLC linac was also consistent with the increase in MLCs reaching the 0.3 mm backlash recommendation in the first 12 and 24 months. The median (range) of MLCs reaching a daily picket fence QC position tolerance of 0.25 mm reduced from 14 (1–40) pre clean to 0 (0) at both 12 and 24 months post-clean. This demonstrates the improvement in MLC performance caused by the MLC service/clean but also reveals factors other than MLC position accuracy influence hardware faults. Additionally, the number of beam hold-offs determined from patient delivery trajectory log files were found to have no correlation with the MLC service/clean.The MLC service/clean improves MLC performance and MLC position accuracy, reducing reactive repair work for engineering and physics staff. The results were maintained for 1 year post the MLC service/clean, with a trend back towards pre-clean levels in the subsequent 12–24 months. This suggests this preventative maintenance work could be performed at a frequency of > 2 years. This period of reduced faults and improved performance is significant given the 10 year expected lifespan of a linac.     

Chromosome-specific DNA repeat probes.

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.     

Temporally and spectrally resolved imaging microscopy of lanthanide chelates.

The combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal. Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and rotational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope.