Fluorescent Dye Tracer Research Articles

On-Target Deposition from Two Engine-Powered Sprayers in Medium-Foliage-Density Citrus Canopies

Spray penetration into citrus canopies is critical for adequate coverage and deposition to ensure effective pest control. However, mismatch of air assistance to target canopy characteristics can lead to unintended spraying losses through overpenetration. To evaluate the effect of air assistance on on-target deposition, two sprayers (surrogates for airflow rates) were used to apply a fluorescent tracer dye solution @ a target concentration of 300 ppm to 16 medium-foliage-density tree blocks in a commercial mandarin orchard. The complete factorial experiment in three replications, also designed for validating a model-based spray decision support tool, comprised two forward travel speeds (1.6 and 4.8 km/h), two disc-core nozzles (TeeJet® D3-25 and D6-45), and either one or two nozzle rows to obtain a wide range of application rates (496 to 9719 L/ha). Dye deposition significantly decreased with canopy depth (p =< 0.001) by nearly seven times across the 3.4 m wide canopies but was not significant over 1.2 to 2.2 m sampling height (p = 0.867). Deposition obtained with the low-airflow-rate sprayer was significantly greater (p =< 0.001) than that obtained with the high-airflow-rate sprayer over the dose range likely due to too much air pushing out spray droplets. This study underscores the importance of matching the air assistance of orchard sprayers to the target canopy.

Comparing the Fate and Transport of MS2 Bacteriophage and Sodium Fluorescein in a Karstic Chalk Aquifer.

Groundwater flow and contaminant migration tracing is a vital method of identifying and characterising pollutant source-pathway-receptor linkages in karst aquifers. Bacteriophages are an attractive alternative tracer to non-reactive fluorescent dye tracers, as high titres (>1012 pfu mL-1) can be safely released into the aquifer, offering improved tracer detectability. However, the interpretation of bacteriophage tracer breakthrough curves is complicated as their fate and transport are impacted by aquifer physicochemical conditions. A comparative tracer migration experiment was conducted in a peri-urban catchment in southeast England to characterise the behaviour of MS2 bacteriophage relative to sodium fluorescein dye in a karstic chalk aquifer. Tracers were released into a stream sink and detected at two abstraction boreholes located 3 km and 10 km away. At both sites, the loss of MS2 phage greatly exceeded that of the solute tracer. In contrast, the qualitative shape of the dye and phage breakthrough curves were visually very similar, suggesting that the bacteriophage arriving at each site was governed by comparable transport parameters to the non-reactive dye tracer. The colloid filtration theory was applied to explain the apparent contradiction of comparable tracer breakthrough patterns despite massive phage losses in the subsurface. One-dimensional transport models were also fitted to each breakthrough curve to facilitate a quantitative comparison of the transport parameter values. The model results suggest that the bacteriophage migrates through the conduit system slightly faster than the fluorescent dye, but that the former is significantly less dispersed. These results suggest that whilst the bacteriophage tracer cannot be used to predict receptor concentrations from transport via karstic flow paths, it can provide estimates for groundwater flow and solute contaminant transit times. This study also provides insight into the attenuation and transport of pathogenic viruses in karstic chalk aquifers.

Methods for quantifying rain-splash dispersal of Neonectria ditissima conidia in apple canopies

Many microorganisms can be dispersed by rain-splash, whereby spores become suspended in water and are spread via droplets. The resulting dispersal gradient is dependent on several factors including rainfall intensity, the nature of the plant canopy and its effects on splash, deposition, redistribution (secondary splash) and filtering. Gradients of spore dispersal with distance are important for understanding epidemics, and the primary dispersal gradient can shape an epidemic for several pathogen generations. However, microorganisms are difficult to trap, identify and enumerate efficiently. This makes it difficult to study the spread and dispersal of pathogens to aid in biosecurity responses and management of epidemics. We used macroconidia of Neonectria ditissima, the causal organism of apple canker, to explore patterns of rain-splash dispersal in tree canopies. We investigated the use of a fluorescent tracer dye, PTSA (1,3,6,8-pyrenetetrasulfonic acid), as a surrogate to conidia capture during natural and artificial rain events, and lens tissue as ‘surrogate leaves’ to recapture tracer dye. Conidia and dye were released from central point sources 2.5 m above the ground and recaptured in passive rainwater traps or artificial ‘leaves’. Quantile regression and exponential models were used to explore variation and dispersal gradients derived for both conidia and dye, with and without tree canopy and with natural or artificial precipitation. Estimated dispersal gradients were steeper with a flatter tail when no tree canopy was present, whereas presence of tree canopy resulted in more variation and shallower predicted dispersal gradients, with fatter tails, predicting potential dispersal to further distances from the source. The majority of conidia and dye were recaptured at less than 1 m from the source, but small concentrations of spores were detected up to 3 m and dye more than 6 m. High variation in natural conditions requires further investigation to fully quantify natural dispersal gradients. Nevertheless, these results show the merit of tracer dye, artificial leaves, and quantile regression as tools to estimate potential dispersal patterns of N. ditissima and other rain-splash dispersed microorganisms, considering rain-splash factors in real canopies and natural situations for predicting inoculum dispersal.

Bark water uptake through lenticels increases stem hydration and contributes to stem swelling.

Foliar water uptake can recharge water storage tissue and enable greater hydration than through access to soil water alone; however, few studies have explored the role of the bark in facilitating water uptake. We investigated pathways and dynamics of bark water uptake (BWU) in stems of the mangrove Avicennia marina. We provide novel evidence that specific entry points control dynamics of water uptake through the outer bark surface. Furthermore, using a fluorescent symplastic tracer dye we provide the first evidence that lenticels on the outer bark surface facilitate BWU, thus increasing stem water content by up to 3.7%. X-ray micro-computed tomography showed that BWU was sufficient to cause measurable swelling of stem tissue layers increasing whole stem cross-sectional area by 0.83 mm2 or 2.8%, implicating it as a contributor to the diel patterns of water storage recharge that buffer xylem water potential and maintain hydration of living tissue.

Effect of spray droplet spectra on control of Poa annua with pronamide

AbstractAnnual bluegrass is a troublesome weed in turfgrass, with reported resistance to at least 12 herbicide sites of action. The mitotic-inhibiting herbicide pronamide has both preemergence and postemergence activity on susceptible annual bluegrass populations. Previous studies suggest that postemergence activity may be compromised due to lack of root uptake, as well as target-site- and translocation-based mechanisms. Research was conducted to determine the effects of spray droplet spectra on spray coverage and control of annual bluegrass with pronamide, flazasulfuron, and a mixture of pronamide plus flazasulfuron. Herbicides were delivered to annual bluegrass plants having two to three leaves via five different spray spectra based on volume median diameters (VMD) of 200, 400, 600, 800, and 1,000 µm. Fluorescent tracer dye was added to each treatment solution to quantify the effects of herbicide and spray droplet spectra on herbicide deposition. In another experiment, the efficacy of 0.58, 1.16, and 2.32 kg pronamide ha−1; 0.022, 0.044, and 0.088 kg flazasulfuron ha−1, or a combination of the two, were assessed in iteration with droplet spectrum sprays of 400 and 1,000 µm on two pronamide-resistant and two pronamide-susceptible annual bluegrass populations. Spray droplet spectrum affected the deposition of pronamide and flazasulfuron, applied alone and in combination. Pronamide foliar deposition decreased with increasing droplet spectra. Pronamide efficacy was affected by droplet spectrum, with the largest (1,000 µm) exhibiting improved control. Flazasulfuron efficacy and pronamide plus flazasulfuron efficacy were not affected by droplet spectra. Pronamide plus flazasulfuron mixture controlled all four populations more effectively than pronamide alone, regardless of droplet spectra. A mixture of pronamide plus flazasulfuron applied with relatively large droplets may be optimal for annual bluegrass control, which offers valuable insights for optimizing herbicide application and combatting herbicide resistance. However, applications in this controlled-growth pot study may not mimic conditions in which thatch and turfgrass canopy limit the soil deposition of pronamide.

Utility of Hooded Broadcast Sprayer in Reducing Herbicide Particle Drift in Cotton

Use of hooded sprayers to mitigate spray particle drift during pesticide applications in cotton has not been investigated. Therefore, experiments were conducted in cotton fields in 2021 and 2022 to compare particle drift of dicamba applied with open and hooded broadcast sprayers at six different spray qualities: Fine (F), Medium (M), Coarse (C), Very Coarse (VC), Extremely Coarse (EC), and Ultra Coarse (UC). A fluorescent tracer dye was mixed and applied with the dicamba solution to measure drift deposition at different downwind distances up to 105 m from the target area. Results showed particle drift for F and M spray qualities applied with a hooded sprayer were reduced up to 94% and 77%, respectively, out to 10 m downwind from the application area compared to the open boom sprayer. Hooded sprayer decreased particle drift for C and VC spray qualities as well but only for short distances downwind (≤ 5 m). Sprayer type did not affect the particle drift for EC and UC spray qualities and it was also significantly lower than other spray qualities across both sprayer types. From 20 to 60 m downwind, dicamba applications with hooded sprayer exhibited as much as 42% less drift than open boom sprayer applications regardless of the spray quality. These data suggested that hooded sprayers are effective in reducing particle drift in cotton and thus can be utilized as a viable spray drift management technique for herbicide applications in cotton.

Quantifying Mixing in Sewer Networks for Source Localization

There has been a recent increase of interest in sewer network water quality, both for pollutants and wastewater epidemiology. Of particular interest is the ability to perform cost-effective small-scale monitoring to understand the sewer network and perform source localization (the process of identifying the sources of materials of interest within the network), enabling prioritization of combined sewer overflow (CSO) interventions and targeted response to the detection of infectious diseases. Rhodamine WT fluorescent dye tracing was carried out in the combined sewer networks of four UK cities, for which network geometries were available. Over 100 dye concentration profiles were recorded, from which discharge, travel time (velocity), and dispersion were quantified. A simplified hydraulic and water quality (conservative solute transport) modeling approach was used to investigate dispersion further. A theoretical method for calculating dispersion over a reach with nonuniform properties was derived and used with the models and recorded data to develop a method for estimating the dispersion coefficient in sewers. Novel simultaneous injections into multiple manholes within one sewer network were conducted. Modeling of these injections validated the modeling approach and explained the measured concentration profiles, demonstrating both the potential of hydraulic and solute transport modeling and the new dispersion coefficient predictor for source localization. Such modeling can be used to develop sewer network “fingerprints” and source location probability plots based on residence time distribution (RTD) theory to maximize information from limited water quality monitoring. This will aid managers and operators in identifying potential intermittent sources of material within the network.

Balanced multiphase mixing through a narrow gap

Mixing of a two-phase flow through a gap connecting adjacent channels was investigated for a variety flow rates and two gap heights. The mixing was characterized by the amount of gross liquid and net gas mass transfer from one channel to the other. The liquid mixing through the gap was calculated based on the measured mass flow rates and dye tracer concentrations at the inlet and outlet of each channel. The gas net transfer, phase fraction distributions, bubble size distributions, and gas interface velocity were measured using dual-plane wire mesh conductivity sensors at both inlets and outlets. Additionally, imaging of the fluorescent tracer dye and air bubbles were used together with Spectral Proper Orthogonal Decomposition (SPOD) to further characterize the mixing mechanisms. The water inlet Reynolds number was varied from 4 to 10×104 and the volumetric gas quality from 0 to 0.35 which resulted in gas volume fractions up to 15%. All conditions were in the bubbly flow regime. Air was introduced by a needle array producing nominally monodispersed bubbles at lower gas flow rates with broader bubble size distributions at higher gas flow rates. The Sauter mean diameter varied from 3 to 8 mm, depending on flow rates. For these balanced flows large coherent structures dominated the mixing. However, even mere O(5%) volumetric quality gas injection could significantly suppress the coherent structures reducing mixing by over 50%.

Fluorescence Intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of Cell Differentiation in the Small Intestinal Organoids Using Cholera Toxin.

Live cell microscopies of in vitro, ex vivo, and in vivo experimental intestinal models enable visualizing cell proliferation, differentiation, and functional cellular status in response to intrinsic and extrinsic (e.g., in the presence of microbiota) factors. While the use of transgenic animal models expressing biosensor fluorescent proteins can be laborious and not compatible with clinical samples and patient-derived organoids, the use of fluorescent dye tracers is an attractive alternative. In this protocol, we describe how the differentiation-dependent intestinal cell membrane composition can be labeled using fluorescent cholera toxin subunit B (CTX) derivatives. By using the culture of mouse adult stem cell-derived smallintestinal organoids, we show that CTX can bind specific plasma membrane domains in differentiation-dependent manner. Green (Alexa Fluor 488) and red (Alexa Fluor 555) fluorescent CTX derivatives also display additional contrast in a fluorescence lifetime domain, when probed by the fluorescence lifetime imaging microscopy (FLIM), and can be used together with other fluorescent dyes and cell tracers. Importantly, CTX staining remains confined to specific regions in the organoids after fixation, which enables using it in both live cell and fixed tissue immunofluorescence microscopies.

Land-use impacts on the hydrology of the Hidden River groundwater subbasin, Horse Cave, Hart County, Kentucky

Hidden River Cave, located in the city of Horse Cave, Ky., forms one of the main tributaries of the Hidden River groundwater subbasin that spans multiple counties in south-central Kentucky. Hidden River Cave formed in Mississippian-aged carbonates and consists of a dendritic network of canyons and collapsed domes; a major trunk stream flows through the cave that supports myriad subsurface ecosystems and recharges the Mammoth Cave aquifer and the Green River, important water resources on which several communities depend. Poor land-use practices historically have contaminated the cave stream. As a result, the hydrology of the Hidden River groundwater subbasin has been extensively studied using fluorescent dye-tracing, and developments in groundwater resource management have improved cave conditions. However, land-use boundaries that intersect with areas of recharge still influence contaminant transport to groundwater. This study combined groundwater dye-tracing, high-resolution stage data collection, and supervised classification in a geographic information system (GIS) to assess land-use impacts on the hydrology of the Hidden River groundwater subbasin. Dye-tracing confirmed that stormwater infrastructure in Horse Cave discharges to Hidden River Cave, and, subsequently, the Hidden River groundwater subbasin. High-resolution stage data determined that the cave’s major trunk streams respond to precipitation within 40 minutes to 1.5 hours, while baselevel conditions, except after sustained precipitation, are met three to four days after precipitation ends. Supervised classification determined that development is concentrated in Horse Cave and has increased by approximately 7 % between 1989 and 2017. These results suggest opportunities for the implementation of karst-specific stormwater management regulations where such regulations are weak.

The visual pathway in sea spiders (Pycnogonida) displays a simple serial layout with similarities to the median eye pathway in horseshoe crabs

BackgroundPhylogenomic studies over the past two decades have consolidated the major branches of the arthropod tree of life. However, especially within the Chelicerata (spiders, scorpions, and kin), interrelationships of the constituent taxa remain controversial. While sea spiders (Pycnogonida) are firmly established as sister group of all other extant representatives (Euchelicerata), euchelicerate phylogeny itself is still contested. One key issue concerns the marine horseshoe crabs (Xiphosura), which recent studies recover either as sister group of terrestrial Arachnida or nested within the latter, with significant impact on postulated terrestrialization scenarios and long-standing paradigms of ancestral chelicerate traits. In potential support of a nested placement, previous neuroanatomical studies highlighted similarities in the visual pathway of xiphosurans and some arachnopulmonates (scorpions, whip scorpions, whip spiders). However, contradictory descriptions of the pycnogonid visual system hamper outgroup comparison and thus character polarization.ResultsTo advance the understanding of the pycnogonid brain and its sense organs with the aim of elucidating chelicerate visual system evolution, a wide range of families were studied using a combination of micro-computed X-ray tomography, histology, dye tracing, and immunolabeling of tubulin, the neuropil marker synapsin, and several neuroactive substances (including histamine, serotonin, tyrosine hydroxylase, and orcokinin). Contrary to previous descriptions, the visual system displays a serial layout with only one first-order visual neuropil connected to a bilayered arcuate body by catecholaminergic interneurons. Fluorescent dye tracing reveals a previously reported second visual neuropil as the target of axons from the lateral sense organ instead of the eyes.ConclusionsGround pattern reconstruction reveals remarkable neuroanatomical stasis in the pycnogonid visual system since the Ordovician or even earlier. Its conserved layout exhibits similarities to the median eye pathway in euchelicerates, especially in xiphosurans, with which pycnogonids share two median eye pairs that differentiate consecutively during development and target one visual neuropil upstream of the arcuate body. Given multiple losses of median and/or lateral eyes in chelicerates, and the tightly linked reduction of visual processing centers, interconnections between median and lateral visual neuropils in xiphosurans and arachnopulmonates are critically discussed, representing a plausible ancestral condition of taxa that have retained both eye types.

Intelligent Sprayer Spray Rates Optimization to Efficiently Apply Chemicals in Modern Apple Orchards

Highlights Intelligent sprayers require crop canopy-specific base spray rate (SR) adjustments. SRs can be estimated by Tree Row Volume (TRV) and Unit Canopy Row (UCR) methods. This study formulated TRV and UCR based SRs for modern apple orchard canopies. Field trials evaluated spray deposition and coverage variations for these rates. TRV and UCR based treatments had comparable spray performance to the grower-adapted application rate. Abstract. The efficiency of air-assisted sprayer-based chemical applications in horticultural crops is dependent on several factors, including that of the application rate as the amount of liquid per ground area (L ha-1 or gal acre-1 [GPA]). In contrast to the conventional GPA-based application rates, recently developed intelligent and precision sprayers use the input spray rate (SR) as the amount of liquid volume (L) required to spray one cubic meter of crop canopy (L m-3 or ounces per cubic foot). This input rate ranges between 0.06 and 0.13 L m-3 depending on the architecture and canopy size. However, the exact value of input SR is decided based on grower experience and manufacturer recommendation and can often be a broad estimation. Therefore, this study was conducted to explore methods that could be used to have a precise estimate of the input SR related to the canopy being sprayed for optimal spray applications. Tree row volume (TRV) and unit canopy row (UCR) methods were used to estimate input SRs and the sprayer field efficiency was evaluated in a vertical fruiting wall-trained commercial apple orchard, typical in the State of Washington, USA. Field spray trials were conducted in the full canopy growth stage, spraying a mix of fluorescent tracer dye of 2 g L-1 concentration at a calibration speed of 1.50 m s-1. Spray deposition and coverage were evaluated in replicated field trials using deposit samplers (mylar cards and screen) and water-sensitive papers, respectively. Test results demonstrated that both TRV (SR: 0.09 L m-3) and UCR (SR: 0.10 L m-3) were effective methods to calculate the spray rates since their spray deposition and coverage were comparable to that of the grower-adapted application rate (935 L ha-1). While the spray rates calculated by both methods provided better application efficiency, an SR of 0.09 L m-3 calculated by the TRV method would be preferable in spray applications of fully foliated fruiting wall-trained apple trees as it sprayed less liquid. Although the TRV method could potentially be applicable for estimating SR with an adjustment factor to compensate for the pruning level, crop-specific further evaluations are suggested for different canopy training systems. Keywords: Intelligent sprayer, Modern orchard systems, Spray rate, Tree row volume, Unit canopy row.

Toxicological Study of Fluorescent Hydrologic Tracer Dye Effects on Cave Bacteria.

Fluorescent dyes are commonly used as hydrologic tracers in a variety of surface and subsurface environments, including karst aquifers and caves, but the fragile nature of karstic groundwater ecosystems suggests a cautious approach to selecting dyes. This study tested the effects of four fluorescent dye tracers (uranine, eosin, pyranine, sulforhodamine B) on microorganisms from Fort Stanton Cave, New Mexico, United States. Toxicity of the dyes was tested on bacteria isolated from the cave and on a sediment sample collected adjacent to Snowy River in Fort Stanton Cave. The isolates showed minimal inhibition by the four dyes in an agar diffusions assay. Minimum inhibitory concentrations calculated from liquid culture assays of one isolate were 35 g/L for uranine, 3.5g/L for eosin, 0.1g/L for pyranine, and 10mg/L for sulforhodamine B. A 14 C-glucose radiotracer experiment showed zero inhibition of overall microbial activity in a sediment sample at all dye concentrations, except at 350 g/L eosin. Thus, there are no cave-specific findings to indicate that Fort Stanton's microbes are especially sensitive to these commonly used dyes. Moreover, a literature survey of mutagenicity tests on these dyes indicates they are safe for environmental use. These results corroborate previous dye toxicity tests and suggest that these four dyes are suitable for use at Fort Stanton Cave in the concentration ranges commonly used for groundwater tracing. While broader testing of dyes with microbes from other caves is advised, the results suggest the dyes may be safe for all karst aquifers.

Biodegradation and Micro- Scale Treatability Pattern of Loblolly Pine Heartwood Bioincised by Bacillus Subtilis and Physisporinus Vitreus

One strategy for improving the treatability of refractory wood species is biological incising, and its efficiency depends on how the microorganisms modify the porous structure of the wood. Evaluation of the bioincised wood treatability on a micro-scale can thus help to better understand the treatability enhancing mechanisms. In the present study, the biodegradation pattern and micro-scale treatability of Loblolly pine (Pinus taeda L.) heartwood were determined after bioincising with the white-rot fungus Physisporinus vitreus (Pers.: Fr.) P. Karsten isolate 136 and bacterium Bacillus subtilis UTB22. Oven-dried specimens with dimensions of 50 mm × 25 mm × 15 mm (L × T × R) were incubated with the microorganisms at (23±2) °C and (65±5) % relative humidity for six weeks. The control and exposed wood blocks were then pressure treated by 1 % fluorescent dye (fluorescein)-containing water to study the treatability pattern under a fluorescence microscope. The longitudinal and tangential air permeability and compression strength parallel to the grain of the specimens were also determined at the end of the incubation period. Scanning electron microscopic (SEM) studies showed that degradation by B. subtilis UTB22 was limited to the pit membranes, but the cell walls were also degraded to some extent by P. vitreus. The fungus caused a higher mass loss compared to the bacterium, whereas the permeability enhancing ability of the bacterium was more pronounced. The fluorescent dye tracer also showed that higher treatability with more uniformity was obtained by B. subtilis UTB22. The improvement in treatability by both microorganisms was mainly due to the degradation of the earlywood tracheids.

Reducing dermal exposure to agrochemical carcinogens using a fluorescent dye-based intervention among subsistence farmers in rural Honduras.

Occupational exposure to agrochemicals, some of which are known or suspected carcinogens, is a major health hazard for subsistence agricultural workers and their families. These impacts are more prevalent in low-and-middle income countries (LMIC) due to weak regulations, lack of awareness of the risks of contamination, predominant use of handheld backpack style spraying equipment, general lack of personal protective equipment (PPE), and low literacy about proper agrochemical application techniques. Reducing exposure to agrochemicals was identified as a paramount concern by rural Hondurans working with a community-engaged research initiative. Fluorescent tracer dyes have been described as a means of visualizing and quantifying dermal exposure to agricultural chemicals, and exposure models adapted for LMIC have been developed previously. Tracer dyes have also been used in educational simulations to promote pesticide safety. However, studies evaluating the effectiveness of these educational dye interventions in reducing future exposure have been lacking. To evaluate whether observing one's own chemical contamination after applying agrochemicals changed the amount of occupational dermal exposure during a subsequent chemical application. We employed a multi-modal community intervention in a rural village in Honduras that incorporated chemical safety education and use of a fluorescent tracer dye during pesticide application on two consecutive occasions, and compared dermal exposure between the intervention group (previous dye experience and safety education, n=6) and the control group (safety education only, n=7). Mean total visual score (TVS) of the tracer dye, which accounts for both extent and intensity of whole-body contamination, was lower among those who had previously experienced the dye intervention (mean TVS=41.3) than among participants who were dye-naïve (mean TVS=78.4), with a difference between means of -37.10 (95% CI [-66.26, -7.95], p=0.02). Stratifying by body part, contamination was significantly lower for the anterior left lower extremity and bilateral feet for the dye-experienced group vs. dye-naïve, with most other segments showing a trend toward decreased contamination as well. Participants who had previously experienced the dye intervention were significantly less contaminated than the dye-naïve control group during a subsequent spraying event. The findings of this small pilot study suggest that a multi-modal, community-based approach that utilizes fluorescence-augmented contamination for individualized learning (FACIL) may be effective in reducing dermal exposure to carcinogenic agrochemicals among subsistence farmers in Honduras and other LMIC.

Evolutionary modification of gastrulation in Parvulastra exigua, an asterinid seastar with holobenthic lecithotrophic development.

Gastrulation is a fundamental morphogenetic process in development. In echinoderms with ancestral-type development through feeding larvae, gastrulation involves radially symmetrical invagination of cells around the blastopore. Gastrulation in the seastar Parvulastra exigua, a species with non-feeding larvae deviates from this pattern. Microinjection of cells with fluorescent lineage tracer dye revealed that early blastomeres contribute unequally to ectoderm and endoderm. In embryos injected at the two-cell stage, asymmetry was evident in the fluorescence at the top of the archenteron and animal pole ectoderm. Archenteron elongation is driven by asymmetrical involution of cells with more cells crossing the blastopore on one side. Lineages of cells injected at the four-cell stage also differed in allocation to endoderm and ectoderm. In embryos injected at the eight-cell stage ectodermal and endodermal fates were evident reflecting the animal and vegetal fates determined by third cleavage as typical of echinoderms. Modification of gastrulation associated with evolution of development in P. exigua shows that this foundational morphogenetic process can be altered despite its importance for subsequent development. However, observations of slight asymmetry in the lineage fates of blastomeres in asterinids with planktotrophic development indicates that gastrulation by asymmetrical involution in P. exigua may be a hypertrophic elaboration of a pre-existing state in ancestral-type development. As for echinoids with lecithotrophic development, involution as a mechanism to contribute to archenteron elongation may be associated with the impact of extensive maternal nutritive reserves on the mechanics of cell movement and a novel innovation to facilitate early development of the adult rudiment.

Method for detecting and quantitating capture of organic molecules in hypervelocity impacts

Enceladus is a prime candidate in the solar system for in-depth astrobiological studies searching for habitability and life because it has a liquid water ocean with significant organic content and ongoing cryovolcanic activity. The presence of ice plumes that jet up through fissures in the ice crust covering the sub-surface ocean, enables remote sampling and in-situ analysis via a fly-by mission. However, capture and transport of organic materials to organic analyzers presents distinctive challenges as it is unknown whether, and to what extent, organic molecules imbedded in ice particles can be captured and survive hypervelocity impacts. This manuscript provides a fluorescence microscopic method to parametrically determine the amount of an organic fluorescent tracer dye, Pacific Blue™ (PB) deposited on a metallic surface. This method can be used to measure the capture and survival outcomes of terrestrial hypervelocity impact experiments where an ice projectile labeled with Pacific Blue impacts a soft metal surface. This work is an important step in the advancement of instruments like the Enceladus Organic Analyzer for detecting biosignatures in an Enceladus plume fly-by mission.An apparatus consisting of a substrate humidification shroud coupled with an epifluorescence microscope with CCD detector is developed to measure the intensity of quantitatively deposited Pacific Blue droplets under controlled humidity.Calibration curves are produced that relate the integrated fluorescence intensity of humidified PB droplets on metal foils to the number of PB molecules deposited.To demonstrate the utility of this method, our calibrations are used to analyze and quantitate organic capture and survival (up to 11% capture efficiency) following ice particle impacts at a velocity of 1.7 km/s on an aluminum substrate.

Micelles self-assembled by 3-O-\u03b2-d-glucopyranosyl latycodigenin enhance cell membrane permeability, promote antibiotic pulmonary targeting and improve anti-infective efficacy

BackgroundNanoparticle-based pulmonary drug delivery systems are commonly developed and applied for drug-targeted delivery. They exhibit significant advantages compared to traditional pulmonary drug delivery systems. However, developing the formulation of each drug is a time-consuming and laborious task.ResultsIn this study, a universal lung-targeting nanoparticle was designed and constructed. The self-assembled micelles were composed of a platycodon secondary saponin, 3-O-β-d-glucopyranosyl platycodigenin 682 (GP-682), based on its specific amphiphilic structure. The GP-682 micelles exhibited a relatively stable zeta potential with a particle size between 60 and 90 nm, and the critical micelle concentration (CMC) value was approximately 42.3 μg/mL. Preincubation of GP-682 micelles markedly enhanced their cell membrane permeability and improved drug uptake in vitro. The results were visualized using fluorescent dye tracing, transmission electron microscopy (TEM) observations and the lactate dehydrogenase (LDH) release assay. The obtained benefits enhanced the distribution of levofloxacin (Lev) in mouse lung tissue and reduced antibiotics overdosing. The acute lung injury mouse model induced by the Pseudomonas aeruginosa PA 14 strain demonstrated that preinjection of GP-682 micelles before antibiotic administration resulted in a higher survival rate and anti-infective efficacy in vivo. It also caused reductions in pulmonary injury, bacterial invasion and cytokine expression compared with treatment with Lev alone.ConclusionsGP-682 micelles are another nanoparticle-based pulmonary drug delivery system and provide a new lung-targeting therapy option.

The Development of an Environmental Surveillance Protocol to Detect Candida auris and Measure the Adequacy of Discharge Room Cleaning Performed by Different Methods

Background: Contaminated surfaces within patient rooms and on shared equipment is a major driver of healthcare-acquired infections (HAIs). The emergence of Candida auris in the New York City metropolitan area, a multidrug-resistant fungus with extended environmental viability, has made a standardized assessment of cleaning protocols even more urgent for our multihospital academic health system. We therefore sought to create an environmental surveillance protocol to detect C. auris and to assess patient room contamination after discharge cleaning by different chemicals and methods, including touch-free application using an electrostatic sprayer. Surfaces disinfected using touch-free methods may not appear disinfected when assessed by fluorescent tracer dye or ATP bioluminescent assay. Methods: We focused on surfaces within the patient zone which are touched by the patient or healthcare personnel prior to contact with the patient. Our protocol sampled the over-bed table, call button, oxygen meter, privacy curtain, and bed frame using nylon-flocked swabs dipped in nonbacteriostatic sterile saline. We swabbed a 36-cm2 surface area on each sample location shortly after the room was disinfected, immediately inoculated the swab on a blood agar 5% TSA plate, and then incubated the plate for 24 hours at 36°C. The contamination with common environmental bacteria was calculated as CFU per plate over swabbed surface area and a cutoff of 2.5 CFU/cm2 was used to determine whether a surface passed inspection. Limited data exist on acceptable microbial limits for healthcare settings, but the aforementioned cutoff has been used in food preparation. Results: Over a year-long period, terminal cleaning had an overall fail rate of 6.5% for 413 surfaces swabbed. We used the protocol to compare the normal application of either peracetic acid/hydrogen peroxide or bleach using microfiber cloths to a new method using sodium dichloroisocyanurate (NaDCC) applied with microfiber cloths and electrostatic sprayers. The normal protocol had a fail rate of 9%, and NaDCC had a failure rate of 2.5%. The oxygen meter had the highest normal method failure rate (18.2%), whereas the curtain had the highest NaDCC method failure rate (11%). In addition, we swabbed 7 rooms previously occupied by C. auris–colonized patients for C. auris contamination of environmental surfaces, including the mobile medical equipment of the 4 patient care units that contained these rooms. We did not find any C. auris, and we continue data collection. Conclusions: A systematic environmental surveillance system is critical for healthcare systems to assess touch-free disinfection and identify MDRO contamination of surfaces.Funding: NoneDisclosures: None

Hydrometeorological factors determining the development of water table cave patterns in high alpine zones. The Ordesa and Monte Perdido National Park, NE Spain

This study is focussed on the geomorphological characterization and the processes driving the evolution of the highest karst system in Western Europe, which is located in the Ordesa and Monte Perdido National Park (PNOMP), in the central-southern Pyrenees. The karst system does not seem to have a well-developed epikarst. The studied area shows a karst network of polygenic branchwork type in the vadose zone. Additionally, the explored karst conduits in the epiphreatic zone show a water table cave pattern that is different to the looping one, which is the expected cave pattern development for a karst located in a mountain zone where a high irregular recharge is expected. We have designed a conceptual recharge model through a multidisciplinary approach, which has included the making of a new detailed geological and hydrogeological map of the study area, morphometric analysis of cavities, tests with fluorescent dye tracers and hydrometeorological monitoring of the karst system associated with the Garcés Spring. This spring, together with the Font Blanca Spring, constitutes the main water discharge point of the hydrogeological system. The conceptual recharge model explains how the observed unexpected cave pattern has developed in this karst.