Colocalization Analysis of Lipo-Deoxyribozyme Consisting of DNA and Protic Catalysts in a Vesicle-Based Protocellular Membrane Investigated by Confocal Microscopy

The linkage between the self-reproduction of compartments and the replication of DNA in a compartment is a crucial requirement for cellular life. In our giant vesicle (GV)-based model protocell, this linkage is achieved through the action of a supramolecular catalyst composed of membrane-intruded DNA and amphiphilic acid catalysts (C@DNA) in a GV membrane. In this study, we examined colocalization analysis for the formation of the supramolecular catalyst using a confocal laser scanning fluorescence microscope with high sensitivity and resolution. Red fluorescence spots emitted from DNA tagged with Texas Red (Texas Red-DNA) were observed in a GV membrane stained with phospholipid tagged with BODIPY (BODIPY-HPC). To our knowledge, this is the first direct observation of DNA embedded in a GV-based model protocellular membrane containing cationic lipids. Colocalization analysis based on a histogram of frequencies of “normalized mean deviation product” revealed that the frequencies of positively correlated [lipophilic catalyst tagged with BODIPY (BODIPY-C) and Texas Red-DNA] were significantly higher than those of [BODIPY-HPC and Texas Red-DNA]. This result demonstrates the spontaneous formation of C@DNA in the GV membrane, which serves as a lipo-deoxyribozyme for producing membrane lipids from its precursor.

Nanoparticle-mediated transgene expression of insulin-like growth factor 1 in the guinea pig placenta differentially affects fetal liver gene expression depending on maternal nutrient status

Fetal growth restriction (FGR) occurs in up to 10% of pregnancies and is a leading cause of infant morbidity and mortality. Additionally, FGR has been implicated in contributing to the development of long-term health outcomes including increasing the risk for future cardiovascular and endocrine diseases. Currently, there is limited preventative strategies and no effective treatment options for FGR. To address this need, we are developing a therapeutic targeting the placenta to increase expression of human insulin-like growth factor 1 (hIGF-1) and enhance placental development and function, with the goal of correcting fetal growth trajectories. Methods Initially, an ultrasound-guided, transcutaneous, intra-placental injection of a non-viral, Texas-red conjugated polymer-based nanoparticle containing a plasmid with the green fluorescent protein (GFP) gene under the control of the placenta-specific promotors Plac1 or Cyp19a1 was performed to determine nanoparticle uptake and transgene expression in the guinea pig placenta. Subsequently, using the established maternal nutrient restriction (MNR) guinea pig model of FGR, placentas were treated with an unconjugated nanoparticle containing a plasmid with the hIGF-1 gene under the Cyp19a1 promotor at mid-pregnancy (gestational day (GD) 30-33). Five days after treatment placentas and fetal liver tissue was collected, weighed, and fixed for histology or snap-frozen for qPCR analysis of mRNA expression. Results Histological analysis of Texas-red and GFP fluorescence in placenta and fetal liver tissue confirmed nanoparticle uptake and transgene expression and that nanoparticle was unable to cross the placenta to fetal circulation. In situ hybridization for plasmid-specific mRNA confirmed sustained hIGF-1 expression five days after treatment. MNR resulted in 20-25% decreased fetal weight at mid-pregnancy (P<0.001) that was not changed with nanoparticle treatment (P>0.05). There was no effect of nanoparticle treatment on the volume densities of trophoblasts or fetal capillaries in the placenta (P>0.05 for both). However, treatment did reduce the interhaemal distance between the maternal blood space and fetal circulation in the MNR placentas compared to sham treated MNR placentas (P<0.001). In the fetuses, placental nanoparticle treatment increased circulating glucose by 38-50% (P<0.001) and was associated with differential changes to fetal liver mRNA expression of genes associated with gluconeogenesis. Gene expression changes were dependent on if the fetus was growth restricted or not; nanoparticle treatment: down-regulated gluconeogenesis gene expression in the normal growing fetuses but increased expression in the FGR fetuses. Conclusions The current study shows that treatment of the guinea pig placenta with a polymer-based nanoparticle causes expression of hIGF-1 and ultimately increases fetal glucose concentrations within five days of treatment. Furthermore, the data shows that the placenta and fetal liver respond differently to nanoparticle treatment depending on fetal growth conditions.

Optimisation of phagocytosis assay in rainbow trout (Oncorhynchus mykiss)

A phagocytosis assay is one of the most commonly used functional immunological methods. There are many possible ways of assessing leucocytes and their ability to ingest different particles. The aim of this study was to optimise the phagocytosis assay in rainbow trout (Oncorhynchus mykiss) using labelled zymosan particles (Alexa Fluor 488 and Texas Red conjugate). Whole blood was incubated with the particles under different conditions and leucocytes were subsequently isolated by haemolysis in a hypotonic environment. The effect of the different incubation time, temperature, blood volume and dilution on the phagocytic activity was evaluated by flow cytometry. Our experiments showed that the incubation for at least 2 h at 15 °C provided optimal results, while the blood volume and dilution had no significant effect. The optimised assay will be used for the examination of fish health and in further experimental studies.

Innovative therapeutic strategy using prostaglandin I2 agonist (ONO1301) combined with nano drug delivery system for pulmonary arterial hypertension

Clinical outcomes of pulmonary arterial hypertension (PAH) may be improved using targeted delivery system. We investigated the efficacy of ONO1301 (prostacyclin agonist) nanospheres (ONONS) in Sugen5416/hypoxia rat models of PAH. The rats were injected with saline (control) or ONONS (n = 10, each) on days 21 and 28, respectively. Hepatocyte growth factor (HGF)-expressing fibroblasts and inflammatory cytokines were measured. Cardiac performance was assessed and targeted delivery was monitored in vivo, using Texas red-labeled nanoparticles. Compared with control, HGF-expressing fibroblasts and HGF expression levels were significantly higher in the ONONS group, while the levels of interleukin-6, interleukin-1β, transforming growth factor-β, and platelet-derived growth factor were lower. Histological assessment revealed significant amelioration of the percent medial wall thickness in pulmonary vasculature of rats in the ONONS group. Rats in the ONONS group showed decreased proliferating cell nuclear antigen-positive smooth muscle cells and improved right ventricle pressure/left ventricle pressure. No difference was seen in the accumulation of Texas red-labeled nanoparticles in the brain, heart, liver, and spleen between PAH and normal rats. However, a significant area of nanoparticles was detected in the lungs of PAH rats. ONONS effectively ameliorated PAH, with selective delivery to the damaged lung.

PSII-35 Melatonin supplementation and restricted nutrition do not affect chorionic somatomammotropin (CSH) concentration in ovine placenta from mid- to late- gestation

Melatonin plays a role as a vasodilator. Vasoactive and angiogenic factors are expressed by placental binucleate cells (BNC) and produce chorionic somatomammotropin (CSH), known to impact fetal and placental growth. We hypothesized that melatonin supplementation and restricted nutrition from mid- to late-gestation would alter CSH concentration and some characteristics of BNC in placenta. At day 50 of gestation, ewes carrying singletons were randomly assigned to a 2 × 2 factorial design and were fed either an adequate (ADQ; 100% NRC; n = 15) or restricted (RES; 60% NRC; n = 15) diet supplemented with 0 (CON, n = 14) or 5 mg of melatonin (MEL; n = 16). Placentomes were collected on day 130 of gestation and preserved in formalin for histological analysis. Cotyledon (COT) were snap frozen for western immunoblotting analyses. Tissue sections were stained using biotinylated Dolichos Biflurus (DBA; a marker of fetal membrane) lectin and fluorescein labeled Texas red-avidin and fluorescein labeled Griffonia Simplifolica (BS) lectin (a marker of BNC). The number, area, and diameter of BNC in COT were determined by image analysis. For immunoblotting, protein was extracted from COT in SDS phosphate buffer, loaded equally, and separated on 12.5% polyacrylamide gels. Protein was transferred to PVDF membranes and incubated with rabbit anti-CSH. Bands were visualized and imaged. Data were analyzed using Proc Mixed procedure of SAS. Melatonin supplementation and restricted nutrition did not affect BNC number, area, or diameter, or CSH protein expression. While we reject our hypothesis that melatonin supplementation and nutrient restriction would alter the CSH concentration and BNC characteristics in COT, we continue to evaluate if the BNC produce angiogenic or vasoactive factors that may influence placental and mammary gland functions in sheep.

Drainage basin checklists and dichotomous keys for inland fishes of Texas

Species checklists and dichotomous keys are valuable tools that provide many services for ecological studies and management through tracking native and non-native species through time. We developed nine drainage basin checklists and dichotomous keys for 196 inland fishes of Texas, consisting of 171 native fishes and 25 non-native fishes. Our checklists were updated from previous checklists and revised using reports of new established native and non-native fishes in Texas, reports of new fish occurrences among drainages, and changes in species taxonomic nomenclature. We provided the first dichotomous keys for major drainage basins in Texas. Among the 171 native inland fishes, 6 species are considered extinct or extirpated, 13 species are listed as threatened or endangered by U.S. Fish and Wildlife Service, and 59 species are listed as Species of Greatest Conservation Need (SGCN) by the state of Texas. Red River drainage basin was the most speciose with 120 fishes. Rio Grande &amp; Pecos drainage basin had the greatest number of threatened or endangered fishes (N = 7) and the greatest number of SGCN fishes (N = 28). We revised drainage basin occurrences for 77 species. Drainage basin checklists and dichotomous keys provide finer resolution of species distributions within the geopolitical boundaries of Texas and can reduce probability of errors in fish identification errors by removing species not occurring within a natural boundary.

Detecting and controlling dye effects in single-virus fusion experiments

Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, while Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.Statement of SignificanceFluorescent dyes are powerful tools for labeling membranes and tracking subcellular objects, and fluorescence dequenching has further been used as a sensitive assay for membrane fusion. Here we show how incorporation of membrane dyes can perturb membrane fusion by influenza virus in a light-dependent manner. We provide a strategy to mitigate this by minimizing dye and light exposure. Finally, we show how in some cases these effects can be due to covalent reaction of some dyes with viral proteins upon illumination. These phenomena may be general and should be carefully controlled for in experiments using such labels.

Proteoliposomes as energy transferring nanomaterials: enhancing the spectral range of light-harvesting proteins using lipid-linked chromophores

Self-assembled proteoliposomes allow highly efficient energy transfer from the spectrally-complementary chromophore Texas Red to the plant light-harvesting protein LHCII, increasing the effective absorption range of this bio-hybrid system.