Evaluating the Role of Drone-Produced Chemical Signals in Mediating Social Interactions in Honey Bees (<Emphasis Type="Italic">Apis mellifera)</Emphasis>

Pheromones play a critical role in shaping societies of social insects, including honey bees, Apis mellifera. While diverse functions have been ascribed to queen- and worker-produced compounds, few studies have explored the identity and function of male-produced (drone) compounds. However, several lines of evidence suggest that drones engage in a variety of social interactions inside and outside of the colony. Here we elucidate the chemical composition of extracts of the drone mandibular gland, and test the hypothesis that compounds produced in these glands, or a synthetic blend consisting of the six main compounds, mediate drone social interactions in and out of the colony. Drone mandibular glands primarily produce a blend of saturated, unsaturated and methyl branched fatty acids ranging in chain length from nonanoic to docosanoic acids, and both gland extracts and synthetic blends of these chemicals serve to attract drones outside of the hive, but do not attract workers inside the hive. These studies shed light on the role drones and drone-produced chemicals have on mediating social interactions with other drones and highlight their potential importance in communicating with other castes.     

Silicalite nanoparticles that promote transgene expression

Here, we report on a new zeolite-based silicalite nanoparticle that can enhance the transfection efficiencies generated by poly ethylene imine-plasmid DNA (PEI-pDNA) complexes via a sedimentation mechanism and can enhance the transfection efficiencies of pDNA alone when surface functionalized with amine groups. The silicalite nanoparticles have a mean size of 55?nm. Functionalizing the silicalite nanoparticles with amine groups results in a clear transition in zeta potential from -25.9 ± 2.3?mV (pH 7.4) for unfunctionalized silicalite nanoparticles to 4.9 ± 0.7?mV (pH 7.4) for amine functionalized silicalite nanoparticles. We identify that silicalite nanoparticles used to promote non-viral vector acceleration to the cell surface are found in acidic vesicles or the cytoplasm but not the nucleus. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay showed that the silicalite nanoparticles were non-toxic at the concentrations tested for transfection. We show that surface functionalization of silicalite nanoparticles with amine groups results in a significant (230%) increase in transfection efficiency of pDNA when compared to unfunctionalized silicalite nanoparticles. Silicalite nanoparticles enhanced pDNA-PEI induced transfection of human embryonic kidney (HEK-293) cells by over 150%.     

Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.     

Contributing to Understand the Crosstalk between Brain and Periphery in Methylmercury Intoxication: Neurotoxicity and Extracellular Vesicles

Human exposure to methylmercury (MeHg) is currently high in regions such as the Amazon. Understanding the molecular changes associated with MeHg-induced neurotoxicity and the crosstalk with the periphery is essential to support early diagnoses. This work aimed to evaluate cellular and molecular changes associated with behavioral alterations in MeHg acute exposure and the possible changes in extracellular vesicles (EVs) number and S100β content. Adults male Wistar rats were orally treated with 5 mg/kg for four days. Behavioral performance, molecular and histological changes in the cerebellum, and plasma EVs were assessed. MeHg-intoxicated animals performed significantly worse in behavioral tests. MeHg increased the number of GFAP+ cells and GFAP and S100β mRNA expression in the cerebellum but no change in NeuN+ or IBA-1+ cells number was detected. The number of exosomes isolated from plasma were decreased by the metal. S100B mRNA was detected in circulating plasma EVs cargo in MeHg exposure. Though preliminary, our results suggest astrocytic reactivity is displaying a protective role once there was no neuronal death. Interestingly, the reduction in exosomes number could be a new mechanism associated with MeHg-induced neurotoxicity and plasma EVs could represent a source of future biomarkers in MeHg intoxication.     

A Protein-Adsorbent Hydrogel with Tunable Stiffness for Tissue Culture Demonstrates Matrix-Dependent Stiffness Responses

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, a novel hydrophobic hydrogel system is presented via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, it is shown that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, it is revealed that stiffness-mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. The material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.     

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples

Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 10⁶ cells/mL to 1.67 × 10⁶ cells/mL. Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 10⁶ cells/mL. Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.     

Field-deployable, high-resolution, time-of-flight aerosol mass spectrometer

The development of a new high-resolution time-of-flight aerosol mass spectrometer (HR-ToF-AMS) is reported. The high-resolution capabilities of this instrument allow the direct separation of most ions from inorganic and organic species at the same nominal m/z, the quantification of several types of organic fragments (CxHy, CxHyOz, CxHyNp, CxHyOzNp), and the direct identification of organic nitrogen and organosulfur content. This real-time instrument is field-deployable, and its high time resolution (0.5 Hz has been demonstrated) makes it well-suited for studies in which time resolution is critical, such as aircraft studies. The instrument has two ion optical modes: a single-reflection configuration offers higher sensitivity and lower resolving power (up to approximately 2100 at m/z 200), and a two-reflectron configuration yields higher resolving power (up to approximately 4300 at m/z 200) with lower sensitivity. The instrument also allows the determination of the size distributions of all ions. One-minute detection limits for submicrometer aerosol are <0.04 microg m(-3) for all species in the high-sensitivity mode and <0.4 microg m(-3) in the high-resolution mode. Examples of ambient aerosol data are presented from the SOAR-1 study in Riverside, CA, in which the spectra of ambient organic species are dominated by CxHy and CxHyOz fragments, and different organic and inorganic fragments at the same nominal m/z show different size distributions. Data are also presented from the MIRAGE C-130 aircraft study near Mexico City, showing high correlation with independent measurements of surrogate aerosol mass concentration.