The contribution of the Drosophila model to lipid droplet research

Intracellular lipid droplets have long been misconceived as evolutionarily conserved but functionally frugal components of cellular metabolism. An ever-growing repertoire of functions has elevated lipid droplets to fully-fledged cellular organelles. Insights into the multifariousness of these organelles have been obtained from a range of model systems now employed for lipid droplet research including the fruit fly, Drosophila melanogaster. This review summarizes the progress in fly lipid droplet research along four main avenues: the role of lipid droplets in fat storage homeostasis, the control of lipid droplet structure, the lipid droplet surface as a dynamic protein-association platform, and lipid droplets as mobile organelles. Moreover, the research potential of the fruit fly model is discussed with respect to the prevailing general questions in lipid droplet biology.     

How do groups of red imported fire ants (Hymenoptera: Formicidae) feed on a droplet of sugar water?

Many previous studies have focused on the foraging behaviors and strategies of the red imported fire ants, Solenopsis invicta Buren on solid food or granular bait; little attention has been paid to how liquid sugar is fed upon. In the present study, behavioral responses of S. invicta to 25% sucrose water droplets were observed. Five foraging patterns were identified in S. invicta colonies under laboratory conditions: (i) no feeding, no sucrose water feeding was observed; (ii) surround feeding, ants surrounded and fed along the edge of the sucrose droplet; (iii) stacked feeding, ants stacked and fed along the edge of the sucrose droplet; (iv) droplet‐break feeding, ants broke the liquid droplet and sucked sucrose water that spread on surface of the substance or soil particles previously transported by ants; and (v) cover feeding, whole surface of the sucrose droplet was covered by layers of feeding ants. This is the first time cover feeding in S. invicta has been reported, which obviously requires more ants compared to the other patterns. In addition, individual ants were tracked in videos under laboratory conditions, and behavioral repertoires that led to stacking, covering and droplet‐breaking were identified and described. The field investigation showed that surround feeding was most frequently performed by S. invicta foragers; however, cover feeding was not observed under field conditions during this study. Both laboratory and field studies showed colony‐level variations in sugar‐water feeding.     

Development and Application of a Process Window for Achieving High-Quality Coating in a Fluidized Bed Coating Process

Next to the coating formulation, process conditions play important roles in determining coating quality. This study aims to develop an operational window that separates layering from agglomeration regimes and, furthermore, the one that leads to the best coating quality in a fluidized bed coater. The bed relative humidity and the droplet size of the coating aerosol were predicted using a set of engineering models. The coating quality was characterized using a quantitative image analysis method, which measures the coating thickness distribution, the total porosity, and the pore size in the coating. The layering regime can be achieved by performing the coating process at a certain excess of the viscous Stokes number (ΔSt _v). This excess is dependent on the given bed relative humidity and droplet size. The higher the bed relative humidity, the higher is the ΔSt _v required to keep the process in the layering regime. Further, it is shown that using bed relative humidity and droplet size alone is not enough to obtain constant coating quality. The changes in bed relative humidity and droplet size have been identified to correlate to the fractional area of particles sprayed per unit of time. This parameter can effectively serve as an additional parameter to be considered for a better control on the coating quality. High coating quality is shown to be achieved by performing the process close to saturation and spraying droplets small enough to obtain high spraying rate, but not too small to cause incomplete coverage of the core particles.     

Influence of ants (Hymenoptera: Formicidae) on honeydew excretion and escape behaviors in a myrmecophile,dalbulus quinquenotatus (Homoptera: Cicadellidae), and its congeners

Few leafhopper species are known to be ant-attended. Evidence is presented that unequivocally demonstrates that Dalbulus quinquenotatusis a myrmecophile. In a greenhouse study, the behavior of D. quinquenotatusand four Dalbulusspecies not associated with ants was observed in the absence and presence of the pavement ant, Tetramorium caespitum. D. quinquenotatusis readily contacted by tending ants and responds to stroking on the abdomen from ants' antennae by excreting and holding honeydew droplets until droplets are removed by ants. Nonattended Dalbulusspecies avoid contact with ants by walking,jumping, or flying away when approached. D. quinquenotatusexcretes three to six times the volume of honeydew as do two nonattended species, D. maidisand D. gelbus.Droplets of D. quinquenotatusare about 23% larger in diameter and excreted two to four times more frequently than for the other species. D. quinquenotatustakes about 0.15 s to form honeydew droplets on the anal tube, then holds the droplet an average of 0.28 s before expelling it when ants are absent. In the presence of ants, D. quinquenotatusholds the droplet an average of 1.31 s, which gives ants time to harvest the droplet. Nonattended species, however, immediately expel droplets from the anal tube after droplet formation. This ant-leafhopper mutualism apparently has evolved due to the ability of D. quinquenotatusto have extensive physical contact with ants and excrete large amounts of honeydew. D. chiapensis,a possible descendent of D. quinquenotatus,may secondarily have lost its mutualistic relationship with ants.     

Plasma membrane regeneration in the myxomyceteBadhamia utricularis

Summary Plasmodial strands of the myxomyceteBadhamia utricularis were injured and, after fixation and sectioning, examined using light and transmission electron microscopy. Injury results in the formation of a cytoplasmic droplet that rapidly regenerates a plasma membrane by the apparent fusion of vesicles at its surface. During the same time several structurally distinct layers form within the droplet. Some of these internal layers, which persist for several minutes after injury, may also arise from vesicle fusion. These results have differences from those reported for the relatedPhysarum polycephalum.     

A simple microcapsule generator design for islet encapsulation

Techniques for immunoisolation and immobilization of viable cells within semipermeable microcapsules have been developed using highly sophisticated droplet generator systems. We propose here an indigenously designed, simple and efficient droplet generator system for encapsulation of the pancreatic islets employing chitosanalginate matrix. The droplet generator system comprises of a needle assembly, a 3-way valve with extended rubber tubing and a filtration unit connected to a pressure pump. Microbeads of the size of around 400 μm diameter or even lesser (minimun attainable size 20.2 μm) could be easily generated using the droplet generator system proposed here. Islet microcapsules cultured in Roswell Park Memorial Institute (RPMI) 1640 with 10% fetal calf serum showed around 98% viability, comparable to that of the non-encapsulated islets. Transplantation of microencapsulated islets to streptozotocin (STZ)-induced diabetic mice, resulted in disappearance of hyperglycemia and restoration of normoglycaemia during a 30-day follow-up suggesting graft functionality. No graft failures were observed in any of the transplanted mice (n = 15) and none of them showed membrane associated fibrous overgrowth, which can be attributed to the fibroblast-growth inhibitory properties of chitosan. The proposed design appears to be superior in its simplicity and cost effectiveness and comparable in performance with the microcapsule generator designs proposed so far. The proposed system can be further exploited for encapsulation and immunoisolation of various cell types in alginate based matrices.     

In Vitro Characterization and Mosquito (Aedes aegypti) Repellent Activity of Essential-Oils-Loaded Nanoemulsions

The nanoemulsions composed of citronella oil, hairy basil oil, and vetiver oil with mean droplet sizes ranging from 150 to 220 nm were prepared and investigated both in vitro and in vivo. Larger emulsion droplets (195–220 nm) shifted toward a smaller size (150–160 nm) after high-pressure homogenization and resulted in higher release rate. We proposed that thin films obtained from the nanoemulsions with smaller droplet size would have higher integrity, thus increasing the vaporization of essential oils and subsequently prolonging the mosquito repellant activity. The release rates were fitted with Avrami’s equations and n values were in the same range of 0.6 to 1.0, implying that the release of encapsulated limonene was controlled by the diffusion mechanism from the emulsion droplet. By using high-pressure homogenization together with optimum concentrations of 5% (w/w) hairy basil oil, 5% (w/w) vetiver oil (5%), and 10% (w/w) citronella oil could improve physical stability and prolong mosquito protection time to 4.7 h due to the combination of these three essential oils as well as small droplet size of nanoemulsion.     

Melatonin protects against defects induced by malathion during porcine oocyte maturation

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.     

Nanoscale droplet vaporisation by molecular dynamics

Nanoscale droplet vaporisation was studied by molecular dynamics, which allows the calculation of properties for droplets statistically without considering the discontinuous interface between a liquid droplet and surrounding gas. An argon droplet was created and immersed inside its vapour. After equilibration, the periphery of the system was heated by a carrier gas to vaporise the droplet. Replications were conducted to check the variation in the phenomenon. Thermodynamic properties such as the density, pressure and temperature profiles were sampled for each interval. The evolution of the surface tension of the droplet undergoing vaporisation was investigated. Moreover, the vaporisation rate of nanodroplets was compared with the kinetic theory-based Hertz–Knudsen–Langmuir equation and two diffusion-based models, which are the D² evaporation law and Kincaid and Longley model [Kincaid DC, Longley TS. A water droplet evaporation and temperature model. Trans ASAE. 1989; 32(2):457–463]. The kinetic model underestimates the vaporisation rate by one order of magnitude whereas the two diffusion-based models overestimate the rate by one order of magnitude.     

Cryopreservation of<Emphasis Type="Italic"> Chrysanthemum morifolium</Emphasis> (<Emphasis Type="Italic">Dendranthema grandiflora</Emphasis> Ramat.) using different approaches

Chrysanthemum species are grown both as ornamentals and for the production of pyrethrum. Recent increased production and breeding efforts have raised the need for the conservation of valuable germplasm. Chrysanthemum has been cryopreserved by controlled-rate-freezing as early as 1990. We report here deep-freezing of shoot tips of C. morifolium var. Escort by different technical procedures: controlled-rate-freezing, encapsulation/dehydration, ultra-rapid-freezing by the droplet method and vitrification. While vitrification yielded the highest shoot regeneration rates, the very simple droplet method was also successful in this respect. Droplet freezing was successfully performed with nine cultivars. Our results open the door to the successful use of alternative methods if one method fails to cryopreserve a variety. Furthermore, it enables comparative investigations of genetic stability and cyro-injury to be carried out.Abbreviations BA 6-Benzylaminopurine - NAA -Naphthaleneacetic acidCommunicated by H. Lörz     

Paleorhodococcus dominicanus n. gen., n sp. (Actinobacteria) in a faecal droplet of Triatoma dominicana (Hemiptera: Reduviidae: Triatominae) in Dominican amber

Paleorhodococcus dominicanus n. gen., n sp. (Actinobacteria) is described from a faecal droplet of Triatoma dominicana (Hemiptera: Reduviidae: Triatominae) in Dominican amber. The fossil can be distinguished from the species of the closely related extant genus Rhodococcus Zopf 1891 by its spherical–ellipsoidal cocci forming substrate filaments with elementary branching, the clustering of coccoidal elements, the short filaments bearing reduced side branches and its occurrence in a faecal droplet of the extinct triatomine bug, P. dominicanus. This is the first fossil record of an Actinobacter, which shows that these organisms formed symbiotic associations with insects by the mid-Tertiary.     

The cone photoreceptors and visual pigments of chameleons

Visual pigments, oil droplets and photoreceptor types in the retinas of four species of true chameleons have been examined by microspectrophotometry. The species occupy different photic environments: two species of Chamaeleo are from Madagascar and two species of Furcifer are from Africa and the Arabian Peninsula. In addition to double cones, four spectrally distinct classes of single cone were identified. No rod photoreceptors were observed. The visual pigments appear to be mixtures of rhodopsins and porphyropsins. Double cones contained a pale oil droplet in the principle member and both outer segments contained a long-wave-sensitive visual pigment with a spectral maximum between about 555 nm and 610 nm, depending on the rhodopsin/porphyropsin mixture. Long-wave-sensitive single cones contained a visual pigment spectrally identical to the double cones, but combined with a yellow oil droplet. The other three classes of single cone contained visual pigments with maxima at about 480–505, 440–450 and 375–385 nm, combined with yellow, clear and transparent oil droplets respectively. The latter two classes were sparsely distributed. The transmission of the lens and cornea of C. dilepis was measured and found to be transparent throughout the visible and near ultraviolet, with a cut off at about 350 nm.     

UV-microscopic studies on the vacuolar changes caused by the flavone “aglycone” isovitexin inSilene pratensis plants

Summary UV-microscopic and chromatographic studies have been performed on the variation in contents and configuration of the flavones present in epidermal cells of the petals, stem leaves, rosette leaves and cotyledons ofSilene pratensis plants. Most of the flavone contents is located in the vacuole of the upper epidermis cells, the concentration depending on the light intensity at which the plants were grown. In plants able to glycosylate isovitexin in the petals (genotypegG/. gl/gl fg/fg, accumulating isovitexin 7-O-glucoside) the vacuole is completely filled with the UV absorbing flavone. In plants which are unable to glycosylate isovitexin in their petals (genotypeg/g gl/gl fg/fg, accumulating only isovitexin) the upper epidermal cells of stem leaves and petals contain droplet like structures in their vacuoles. At high light intensities these structures increase in mass and become detectable in the visible light. These denser structures often condense to structures with radiating threads.As compared with the accumulation of isovitexin in upper epidermal cells of stem leaves and petals in genotypeg/g gl/gl fg/fg, the cotyledons and the rosette leaves contain two isovitexin glycosides. In the latter organs the upper epidermal cells are very similar to the upper epidermal cells fromgG/. gl/gl fg/fg plants, having a vacuole filled with UV absorbing material. It appears therefore that isovitexin itself causes the formation of the structurés in the cells. It was shown by varying the light intensity that a relative high concentration of isovitexin is necessary for the droplet like structures to appear. Still higher concentrations are needed for the formation of the structures with radiating threads. It is hypothesized that isovitexin interferes with the energy supply of the cells, which therefore are not able to maintain their turgor.     

Cytoplasmic droplets of painted turtle spermatozoa

Epididymal sperm from the painted turtle (Chrysemys picta) possess a cytoplasmic droplet which is located eccentrically on the sperm midpiece. The droplet contains a large quantity of lipid droplets in addition to hollow vesicles and degenerate mitochondrial fragments. Lipid droplets are closely associated with mitochondrial membranes and may function in the formation or degradation of mitochondria. Cytoplasmic droplets become detached from the sperm midpiece in a coordinated manner shortly before the commencement of fall mating and are not observed on sperm recovered from the oviduct of females. © 1992 Wiley-Liss, Inc.     

Characterization of the nematocidal toxocyst in <Emphasis Type="Italic">Pleurotus</Emphasis> subgen. <Emphasis Type="Italic">Coremiopleurotus</Emphasis>

Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.     

Review: Diagnosis of Helicobacter pylori infection

Endoscopic imaging of the stomach is improving. In addition to narrow band imaging, other methods, for example, blue light imaging and linked color imaging, are now available and can be combined with artificial intelligence systems to obtain information on the gastric mucosa and detect early gastric cancer. Immunohistochemistry is only recommended as an ancillary stain in case of chronic active gastritis without Helicobacter pylori detection by standard staining, and recommendations to exclude false negative H. pylori results have been made. Molecular methods using real‐time PCR, droplet digital PCR, or amplification refractory mutation system PCR have shown a high accuracy, both for detecting H. pylori and for clarithromycin susceptibility testing, and can now be used in clinical practice for targeted therapy. The most reliable non‐invasive test remains the ¹³C‐urea breath test. Large data sets show that DOB values are higher in women and that the cut‐off for positivity could be decreased to 2.74 DOB. Stool antigen tests using monoclonal antibodies are widely used and may be a good alternative to UBT, particularly in countries with a high prevalence of H. pylori infection. Attempts to improve serology by looking at specific immunodominant antigens to distinguish current and past infection have been made. The interest of Gastropanel^® which also tests pepsinogen levels was confirmed.     

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of “lab-on-a-chip” platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.     

The rates of fungal development and lesion formation in leaves of Vicia faba during infection by Botrytis cinerea and Botrytis fabae

Loss of the water droplet above inoculation sites during the first day after inoculation inhibited lesion formation by Botrytis cinerea and prevented the development of spreading lesions of B. fabae. With droplets present two general patterns of infection by B. cinerea were determined; in one, few or no symptoms were produced and in the other, limited lesions developed with marked browning of the inoculation site. Where few or no symptoms were produced, germination and germ-tube growth were inhibited on the leaf surface. B. cinerea was inhibited within the leaf at sites bearing limited lesions; invading hyphae were restricted to brown epidermal cells. Fungal growth on the leaf surface was greatest at sites with most browning beneath the droplet area. Variation in lesion development by B. cinerea could not be related to droplet position or leaf damage during normal preparation for inoculation. Plants differed in their susceptibility to lesion formation by B. cinerea. B. fabae, with droplet present, was not inhibited on the leaf surface and spread inter- and intra-cellularly beneath the inoculum drop and then into surrounding tissues. Delay in spread until the inoculation site was completely necrotic and colonized suggested that B. fabae is partially inhibited during the initial phase of infection. The rate of lesion spread varied in different plants and was most rapid in the youngest leaves.     

Tetrachromatic colour vision in the duck (Anas platyrhynchos L.):microspectrophotometry of visual pigments and oil droplets

Summary Microspectrophotometric examination of the visual receptors of the duck,Anas platyrhynchos, revealed four types of single cone containing visual pigments absorbing maximally at about 420 nm, 452 nm, 502 nm and 570 nm. A single population of double cones contained the P570 in both members. Rods absorbed maximally at 505 nm.Within the single cones, three types of oil droplet, acting as cut-off filters, were identified by the wavelength at which 50% transmission occurred, approximately 580, 515 and 450 nm. A further droplet, transparent throughout the visible spectrum, was also found in a small population of single cones. A fifth droplet type with a variable cutoff between 475–500 nm was located in the principal member of the double cones.The optical density of the anterior half of the eye, established by spectrophotometry, was used, in conjunction with the visual pigment and oil droplet combinations found within intact cones, to estimate the relative spectral sensitivities of the major cone types within the retina.     

Isolation of chick retina cones and study of their diversity based on oil droplet colour and nucleus position

The chick retina has four morphological cone types that differ not only in shape, but also in the visual pigment in the outer segment, in the colour of the oil droplet in the inner segment and in synaptic connectivity. Neither the type of droplet nor the visual pigment has been definitively established for the four cone types. The main aim of the present work has been the isolation of entire live photoreceptors in order to study the oil droplet colour in each cone type and to quantify each type. We have improved an earlier retinal cell isolation method and obtained large numbers of entire cones. Principal cones (27% of the cones) possess a yellow or colourless droplet. Accessory cones (27% of the cones) all contain a small pale green droplet. Straight cones (44% of the cones) have a red, orange, yellow, or colourless droplet. Oblique cones (1.66% of the cones) all have a colourless droplet. We have found that straight cones with a red, orange, or yellow droplet differ in terms of the position of the nucleus and their percentage and conclude that they are distributed in three rows in the outer nuclear layer (ONL) of the central retina. Our study of 4,6-diamidino-2-phenylindole-stained retinal sections has revealed three rows of nuclei instead of the two currently thought to form the ONL. Together, our results show a larger cone diversity than previously known, suggest a larger functional diversity and provide an efficient method for isolating entire chick photoreceptors. This work was supported by grants from the Dirección General de Investigación, Ministerio de Educación y Ciencia of Spain (no. BFU2005-08786-C02-01) and from the Comunidad de Madrid, Spain (no. 920648/2006).