Fluorochrome Dyes Research Articles

Optimal fixation period of a combined bioconstruction with buccal epithelial cells in limbal cell deficiency in experiment

BACKGROUND: The search for an effective method for the treatment of limbal stem cell deficiency of various etiologies, leading to intense clouding and vascularization of the cornea, followed by a significant decrease in visual acuity, remains an important and relevant topic in ophthalmology. The results of the studies showed that transplantation of buccal epithelial cells could significantly improve the prognosis of treatment in this category of patients.
 AIM: To determine the optimal fixation period of a combined bioconstruction with buccal epithelial cells for the treatment of limbal cell deficiency in experiment.
 MATERIALS AND METHODS: At the first stage, the corneal epithelium was removed with a scraper in the eyes of experimental animals (12 eyes), and the limb was excised along the entire circumference. Next, to isolate epithelial buccal cells and manufacture a combined bioconstruction consisting of buccal cells, a collagen carrier and a soft contact lens, a flap of the mucous membrane of 5 5 mm was taken from the cheeks of rabbits. At the second stage, after the formation of a fibrovascular pannus on the cornea, it was excised to transparent layers of the cornea, a combined bioconstruction was placed on top. Further, one U-shaped suture was applied at the border of the inner and outer third of the eyelids. Temporary blepharorraphia persisted for 3 days (6 eyes) and 5 days (6 eyes), after the specified time, stitches were removed from the eyelids, and bioconstructions were removed. The bioconstructions removed from the eyes were stained with a vital (lifetime) fluorochrome dye based on tripaflavin and acridine orange, followed by analysis in a fluorescent microscope. The structure of cell nuclei, the overall integrity of the cytoplasm, and the presence of secretory vesicles were evaluated. After 7 and 14 days after transplantation of the bioconstruction, the area of erosion, the extent of new vessels and the transparency of the cornea were evaluated. When the observation was completed, the rabbits were removed from the experiment, and the eyes were enucleated and subjected to histological examination.
 RESULTS: On the surface of all bioconstructions removed after 5 days, only leukocytes were detected. At the same time, on collagen matrix of bioconstructions removed after 3 days, in addition to leukocytes, there were also buccal epithelial cells with normal nuclear structure and chromatin topography in its composition, as well as with secretory vesicles. On days 714, both groups showed a decrease in the area of erosion, while the dynamics of ocular surface recovery processes was more pronounced in the group with fixation of the bioconstruction for 5 days.
 CONCLUSIONS: According to the results of in vivo staining with fluorochrome dye of combined bioconstructions removed from the cornea of experimental animals 3 and 5 days after superficial keratectomy, as well as the data of clinical and histological examination, the optimal period for bioconstruction fixation was established to be 5 days.

Direct Setting of Eastern Oyster (Crassostrea virginica) Larvae Confirmed with Calcein, a Fluorochrome Dye

Direct Setting of Eastern Oyster (Crassostrea virginica) Larvae Confirmed with Calcein, a Fluorochrome Dye

Effect of Cavity Disinfection Protocols on Microtensile Bond Strength of Universal Adhesive to Dentin

The purpose of this study was to compare the effect of different disinfection protocols of dentin on bond strength of an MDP-containing universal adhesive. Twelve extracted mandibular third molars were separated horizontally at the mid-coronal of crown to get smooth and sound dentin surfaces using low-speed diamond saw. The teeth were randomly fallen into four groups: chlorhexidine (CHX), ozone, Er,Cr:YSGG laser irradiation (LASER) and no treatment (control). After cavity disinfection application, a universal adhesive (G-Premio Bond) was applied to the surface of dentin according to self-etch mode as instructed by the manufacturer. After incremental built-up of composite resin (Charisma Smart), the specimens were immersed in distilled water at 37°C for 24h. Dentin/composite beams with 1 mm² cross sectional area were produced and micro-tensile bond strength (µTBS) was applied on these beams (n=20). Failure mods were determined under a stereomicroscope at ×40. The resin penetration of samples stained with Rhodamine B fluorochrome dye was examined with a confocal laser scanning microscope. Statistical analysis was performed with SPSS-22. Test results were analyzed using One-way Anova and Tukey HSD Post-Hoc tests (p<0.05). The µTBS value of control (35.13±6.20) was the highest statistically among the groups (p<0.05). The lowest µTBS were obtained by LASER (19.25±4.66) and CHX (23.07±7.01). There was no significant difference between CHX and LASER, and between CHX and ozone (p>0.5). All applications of cavity disinfection procedures decreased the µTBS of the resin-dentin interface.

Marking the shells of juvenile and adult eastern oysters, Crassostrea virginica, with the fluorochrome dye calcein and measuring growth and mortality after marking

ABSTRACT Techniques for positive re-identification of finfish are well developed and are used to estimate vital rates, migration patterns and stock structure. Fluorochrome dyes, like nontoxic calcein, have been used with success to mark hard parts of fish and the shells of some molluscs. Yet, despite the commercial and ecological importance of the eastern oyster Crassostrea virginica, a systematic evaluation of the effectiveness of calcein for marking this species does not exist. In this study, methods that have been used for marking freshwater mussels with calcein were tested on 2-week- and 2-month-old juvenile C. virginica and on 3-year-old adult C. virginica. Individuals were immersed in 250 mg l−1 of calcein for 24 h either once (single treatment) or three times (triple treatment), and then observed to determine whether marks were visible, how long they remained visible and whether calcein influenced growth and mortality of the different age classes. Results indicated that all marked individuals were recognizable under blue light, that multiple distinct marks were possible on juveniles and that marks on juveniles could remain visible for 3.75 years. Growth of juveniles and adults treated with calcein was not significantly different from controls after 11 months. Survivorship did not differ between treated and control individuals for all treatments and ages, except the 2-month-old juvenile oysters that were immersed three times. Results indicate that calcein is an effective and safe marking technique for juvenile oysters from robust cohorts, and could be used to enhance studies of growth, mortality, larval transport and settlement in situ.

Marking the Shells of Pediveliger Eastern Oysters Crassostrea Virginica, with a Calcein Fluorochrome Dye

The continued development of effective mark and recapture tools for marine invertebrates is required to better understand fundamental planktonic processes such as larval transport and mortality. Mass chemical marking methods provide researchers with the ability to mark large quantities of individuals at one time with reduced handling stress and costs. Although 3-day-old larvae of Crassostrea virginica (eastern oyster) have been marked with calcein (a fluorochrome dye) and recaptured, pediveligers have not been marked nor has mark retention through metamorphosis been verified. To track mark retention through metamorphosis, 12,000 pediveligers were marked for 24 and 48 h in calcein of 25 and 50 mg L–1 concentrations, and mark retention was tracked every 7 days for 4 wk. Results indicated that calcein did not affect initial settlement rates, that the distinct bands on larval shells were visible after metamorphosis, that individuals marked as larvae were positively identified up to 4 wk postsettlement without sacrificing individuals, and that lower calcein concentrations than previously documented were effective. This study demonstrates that calcein is a reliable method for marking C. virginica pediveligers and suggests that relatively low concentrations of calcein can produce marks and could reduce costs and potential harmful effects.

The probiotics Vibrio diabolicus (Ili), Vibrio hepatarius (P62), and Bacillus cereus sensu stricto (P64) colonize internal and external surfaces of Penaeus vannamei shrimp larvae and protect it against Vibrio parahaemolyticus

The vibrios carrying the pirA and pirB toxin genes cause acute hepatopancreatic necrosis disease (AHPND) in cultivated shrimp, significantly affecting the yield. The use of probiotic strains is a promising strategy to prevent vibriosis in shrimp cultures. The microbial consortium formulated with three strains, Vibrio diabolicus (Ili strain), Vibrio hepatarius (P62 strain), and Bacillus cereus sensu stricto (P64 strain), is used to control vibriosis. Despite the effectiveness of these probiotics, until now there is not a description of the cellular structures necessary to help shrimp colonization, an important process to exclude pathogenic vibrios. Initially, the three probiotic strains were characterized by scanning electron microscopy, which allowed us to observe that Ili has numerous flagella and P62 has numerous fimbriae, while P64 has both flagella and fimbriae. Next, the colonization capacity of the three probiotic strains in P. vannamei shrimp larvae was determined by direct exposure of the shrimp larvae to probiotics previously stained with fluorochrome dyes, orange acridine, Evans blue, and DAPI. The larvae were observed using epifluorescence and confocal microscopy, which showed the three probiotics adhered to the body cuticle and internal gut lining. Larvae exposed to AHPND-causing V. parahaemolyticus stained with acridine orange were quickly and completely colonized, however, the previous treatment of larvae with probiotic consortium limited the colonization of the pathogenic Vibrio. The ability of probiotic strains to exclude pathogenic vibrio from shrimp surfaces could be explained by the cell surface appendages observed in probiotic strains, thus limiting the free spaces for pathogenic vibrios. Finally, the probiotic consortium in P. vannamei larvae was evaluated in a larviculture (Nauplii 5 to postlarvae 8), where the three probiotics were applied daily. The trial included a control group without the application of the probiotic. After 16 days of culture, the larvae treated with the microbial consortium and the control group were challenged with a pathogenic, the AHPND-causing V. parahaemolyticus strain. We registered a significant (P < 0.05) higher survival in the larvae treated with the microbial consortium 75.33 ± 12.62% compared to the control group 32.27 ± 9.57%. These results indicate that the preventive use of probiotic strains with colonization capacity increase larvae protection. This may be a good strategy for the control of AHPND in shrimp larvae hatcheries.

Biosorption of organic dye Acridine orange from aqueous solution using dry biomass of Bacillus cereus M116

Acridine orange (AO), a basic carcinogenic fluorochrome dye, is used in the industry for staining. In this study, Gram-positive bacteria, Bacillus cereus M116 (MTCC 5521) dry biomass was tested as an eco-friendly, easily available, and cheap biosorbent for the AO dye removal. We obtained optimum biosorption of AO at a biomass concentration of 0.25g/L and initial dye concentrations of 50-400mg/L at neutralto basic pH within the 300min contact time. Kinetics analysis of the biosorption process was best fitted with the pseudo-second-order reaction type. We also performed the isotherm analysis to predict the nature of the reaction taking place, which was found to follow the Redlich Peterson isotherm model with high determination coefficients. The maximum sorption capacity was 210.46mg/g of dry biomass. The differential FTIR spectroscopic analysis of pristine and AO-treated Bacillus cereus M116 cells suggested the potential involvement of carbonyl, hydroxyl, and amine groups in the biosorption process. Also, the scanning electron microscopy of the cells after AO removal confirmed a gross surface alteration compared to the untreated cells. Furthermore, Response Surface Model (RSM) analysis with the three-way ANOVA test confirms statistically significant interactions between the dye concentration, pH, and temperature with the biosorption capacity (p < 0.001). Hence, the dry biomass of Bacillus cereus M116 was found to be an effective bio-remedial for the AO removal.

Cryopreserved Cd34 Cells: Measuring Viability Pre-Thaw and Viability over Time Post-Thaw

Common practices associated with the use of cryopreserved hematopoietic progenitor cells (HPC) include measuring the viability of the thawed cells from a sample derived from the thawed product, and thawing cells at the patient's bedside out of concern for possible toxicity of DMSO in the post-thaw liquid cell suspension. The former practice carries the risk of discovering excess cell death when it is too late to correct, and the concomitant risk of non-engraftment. The latter practice carries all of the risks associated with manipulating cells in a non-controlled environment including risk of contamination and lack of suitable containment in the event of failure of a cryostorage container. In an effort to reduce risks associated with cell cryostorage, we are currently examining the validity of measuring CD34 viability from cells frozen in retention vials concomitantly with product freezing. We are also examining the stability of HPC being held in cryostorage medium from 1 to 24 hours post thaw. We present here our results from a pilot study performed on six bags of cells, previously identified for discard due to death of the intended recipients. Six bags of HPC and associated retention vials, frozen for 5 to 12 years, were selected at random from the pool of discarded products and de-identified per guidelines for discard of medical waste. CD34+ cells were identified using a modification of the ISHAGE method, and dead cells were identified by infiltration with the fluorochrome dye 7-AAD. CD34+ cells stored in retention vials were 94±11 percent viable, and CD34+ cells stored in bags were 96±7 percent viable. A Wilcoxan matched pairs test yielded p=0.6, indicating that these results were not different. Percent viable CD34+ cell recovery, relative to the known number of CD34+ cells per mL originally frozen, was determined immediately following thawing, and following 1, 2, 4 and 24 hours remining in thawed cryostorage medium, with recoveries (mean ± std. dev.) of 88 ± 31, 76 ± 33, 62 ± 28, 88 ± 41, and 49 ± 26 percent respectively. These preliminary results suggest that viable CD34 percent as measured from a retention vial may serve as a predictor of viability at thawing of it's associated product, and also that HPC held in cryostorage medium post thaw retain viability for at least four hours post thawing. Based on these results we are continuing these studies.

Early diagnosis of cerebral thrombosis by EGFP-EGF1 protein conjugated ferroferric oxide magnetic nanoparticles.

Cerebral thrombosis disease is a worldwide problem, with high rates of morbidity, disability, and mortality. Magnetic resonance imaging diffusion-weighted imaging was used as an important early diagnostic method for cerebral thrombotic diseases; however, its diagnosis time is 2 h after onset. In this study, we designed EGFP-EGF1-NP-Fe3O4 for earlier diagnosis of cerebral thrombosis by taking advantage of EGFP-EGF1 fusion protein, in which EGF1 can bind with tissue factor and enhanced green fluorescent protein has previously been widely used as a fluorescent protein marker. EGFP-EGF1-NP-Fe3O4 or NP-Fe3O4 reaches the highest concentration in the infarction areas in 1 h. To evaluate the targeting ability of EGFP-EGF1-NP-Fe3O4, a fluorochrome dye, Dir, was loaded into the nanoparticle. As shown by the in vivo organ multispectral fluorescence imaging, Dir-loaded EGFP-EGF1-NP-Fe3O4 exhibited higher fluorescence than those of model rats treated with Dir-loaded NP-Fe3O4. Coronal frozen sections and transmission electron microscope further showed that EGFP-EGF1-NP-Fe3O4 was mainly accumulated in the tissue factor exposure region of brain. The data indicated that the EGFP-EGF1-NP-Fe3O4 targeted cerebral thrombosis and might be applied in the early diagnosis of intracranial thrombosis.

Development and evaluation of real-time loop-mediated isothermal amplification assay for rapid detection of cystic echinococcosis.

BackgroundCystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)-complex, is a neglected parasitic disease of public health importance. The disease is endemic in many African and Mediterranean countries including the Sudan. The objective of the present study was to develop and evaluate a real-time loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of CE in humans and domestic live stock in Sudan.MethodsA set of six LAMP primers, designed from the mitochondrial NADH-1 gene of EG cattle strain of genotype 5 (G5), was used as a target for LAMP assay. The assay was performed at a constant temperature (63 °C), with a real-time follow-up using a LightCycler and fluorochrome dye. Following amplification cycles in a simple water bath, LAMP products were observed for color change by naked eye and were visualized under UV light source using agarose gel electrophoresis.ResultsThe real-time LAMP assay identified a variety of hydatid cysts strains recovered in the Sudan, including Echinococcus canadenses (G6) and Echinococcus ortleppi (G5). Real-time LAMP positive results were detected by the presence of an amplification curve, whereas negative results were indicated by absence of fluorescence detection. Positive LAMP results appeared as a bluish-colored reaction as observed by naked eye, whereas negative LAMP results were observed as purple-colored reaction. The sensitivity studies indicated that the LAMP assay detected as little as a 10 fg of parasite DNA. There was 100 % agreement between results of the LAMP assay and our previously described nested PCR when testing 10-fold serial dilution of DNA extracted from EG-complex hydatid cyst. However, there was no cross-reactivity with other parasites including cysticercus bovis, Fasciola gigantica, and Schistosoma bovis and nucleic acid free samples.ConclusionThe developed LAMP assay would be expected to prove highly significant in epidemiological surveys of CE in developing countries or areas of resource-poor settings for both ease of use and cost.

Molecular cytogenetic studies in the ladybird beetle Henosepilachnaargus Geoffroy, 1762 (Coleoptera, Coccinellidae, Epilachninae).

The ladybird Henosepilachna argus Geoffroy, 1762 has been cytogenetically studied. In addition we have conducted a review of chromosome numbers and the chromosomal system of sex determination available in the literature in species belonging to the genus Henosepilachna and in its closely related genus Epilachna. Chromosome number of Henosepilachna argus was 2n=18, including the sex chromosome pair, a common diploid chromosome number within the tribe Epilachnini. The study of prophase I meiotic chromosomes showed the typical Xyp “parachute” bivalent as in the majority of species of Coccinellidae. C-banding and fluorescent staining with AT-specific DAPI fluorochrome dye have been carried out for the first time in H. argus. C-banding technique revealed that heterochromatic blocks are pericentromerically located and DAPI staining showed that this heterochromatin is AT rich.Fluorescence in situ hybridizations using rDNA and the telomeric TTAGG sequence as probes have been carried out. FISH using rDNA showed that the nucleolar organizing region is located on the short arm of the X chromosome. FISH with the telomeric sequence revealed that in this species telomeres of chromosomes are composed of the pentanucleotide TTAGG repeats. This is the first study on the telomeric sequences in Coccinellidae.

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria.

Examination of osteoarthritis and subchondral bone alterations within the stifle joint of an ovariectomised ovine model

The exact relationship between osteoporosis and osteoarthritis is still a matter for debate for many. The ovariectomised ewe is frequently used as a model for osteoporosis, resulting in significant alterations in bone morphometry and turnover in both trabecular and subchondral bone after 1 year. This study examines whether ovariectomy has any impact on development of osteoarthritis within the ovine stifle joint at the same time point. In addition, we investigate whether there are any significant correlations present between articular cartilage degeneration and alterations in microstructural parameters or turnover rates in the underlying bone. Twenty-two sheep were examined in this study; 10 of the sheep underwent ovariectomy and 12 were kept as controls. Five distinctive fluorochrome dyes were administered intravenously at 12-week intervals to both groups, to label sites of bone turnover. All animals were then sacrificed 12 months postoperatively. Although most specimens showed some evidence of osteoarthritis, no measurable difference between the two study groups was detected. Osteoarthritis was associated with a thinning of the subchondral plate, specifically the subchondral cortical bone; however, whereas previous studies have suggested a link between trabecular thinning and osteoarthritis, this was not confirmed. No correlation was found between osteoarthritis and bone turnover rates of either the subchondral trabecular bone or bone plate. In conclusion, despite the fact that ovariectomy results in marked morphological and structural changes in the ovine stifle joint at 1-year postoperatively, no evidence was found to suggest that it plays a direct role in the aetiology of osteoarthritis.

Chromosome analysis in the dioecious cucurbit Coccinia grandis (L.) Voigt

Karyotype analysis and meiotic behavior in a dioecious cucurbit Coccinia grandis, carrying heteromorphic sex chromosomes, has been carried out using conventional and fluorochrome dyes. Distinct differences in karyotype formula with 15m+4M+2sm+2m: sm+1m: st for male plant and 14m+6M+2sm+2m: st for female plant were observed with orcein staining. In both cases one autosome pair with two constrictions was present. Male plants are seen to possess a heteromorphic Y chromosome with two constrictions which is sufficiently larger than other small sized chromosomes. Giemsa followed by CMA/ DAPI staining through EMA method applied for the first time in this dioecious species, revealed CMApositive signals in different chromosomes. Detailed meiotic analysis has been carried out from diakinesis and metaphase I to establish genomic stability.

Subchondral osteopenia and accelerated bone remodelling post‐ovariectomy – a possible mechanism for subchondral microfractures in the aetiology of spontaneous osteonecrosis of the knee?

Osteopenia and subchondral microfractures are implicated in the aetiology of spontaneous osteonecrosis of the knee (SPONK). The ovine tibia shows significant alterations of the trabecular architecture within the subchondral bone of the medial tibial plateau post-ovariectomy (OVX), including reduced trabecular bone volume fraction. We hypothesise that accelerated subchondral bone resorption may also play a role in increasing microfracture risk at this site. Twenty-two sheep were examined in this study; 10 of the sheep underwent OVX, while the remainder (n = 13) were kept as controls (CON). Five fluorochrome dyes were administered intravenously at 12-week intervals via the jugular vein to both groups, to label sites of bone turnover. These animals were then killed at 12 months post-operatively. Bone turnover was significantly increased in the OVX group in both trabecular bone (2.024 vs. 1.047 no. mm(-2) ; P = 0.05) and within the subchondral bone plate (4.68 vs. 0.69 no. mm(-2) ; P < 0.001). In addition to the classically described turnover visible along trabecular surfaces, we also found visual evidence of intra-trabecular osteonal remodelling. In conclusion, this study shows significant alterations in bone turnover in both trabecular bone and within the subchondral bone plate at 1 year post-OVX. Remodelling of trabecular bone was due to both classically described hemi-osteonal and intra-trabecular osteonal remodelling. The presence of both localised osteopenia and accelerated bone remodelling within the medial tibial plateau provide a possible mechanism for subchondral microfractures in the aetiology of SPONK. Further utilisation of the OVX ewe may be useful for further study in this field.

Femoral growth plate is sensitive to loss of cardiac shunt in the American alligator

Longitudinal growth rate (LGR) has been shown to correlate with calcified cartilage column height (CCC) in the growth plates of birds. In order to infer limb bone growth patterns in non‐avian dinosaurs, this correlation needs to be documented in crocodilians. We tested the LGR‐CCC correlation in femora of juvenile alligators raised for two years under laboratory conditions, and injected with fluorochrome dyes (alizarin, calcein). Some alligators were constrained to in‐series circulation by left aortic ablation (n=24), whereas sham‐operated controls retained in‐parallel circulation (n=36). Animals either remained sedentary or were subjected to rigorous walking or swimming exercise (n=20). We found CCC correlated with LGR and was significantly augmented (+36%) in alligators with in‐series circulation compared to those with in‐parallel circulation. Exhaustive exercise reduced thickness (−15%) of the distal, but not proximal, growth plate, suggesting local effect of biomechanic loading. We propose loss of the pulmonary bypass shunt results in higher systemic arterial oxygen tension, which allows for greater oxygen diffusion distance through the growth plate. Among archosaur ancestors, thicker growth plates may have appeared concurrently with the origin of in‐series circulation, and thus set the stage for later acquisition of fast growth and endothermic metabolism. Supported by NSF IOB 04445680 to JWH.

Abstract 1511: Macrophage aggressiveness in the lung tumor microenvironment

Abstract Macrophages are highly versatile phagocytes active in multiple roles in the immune system of vertebrates. However, within the tumor microenvironment, macrophages initiate and are exposed to a variety of cell signals. Some of these signals are involved in tumor growth, angiogenesis, and metastasis, and have been shown to alter macrophage function. These tumor associated macrophages display characteristics that indicate polarization from M1 to M2 phenotypes, and have been shown to promote angiogenesis and metastasis in cancer patients. In addition, clinical trials have shown that a high macrophage count in breast cancer patients is associated with a poor prognosis. We hypothesize that the engulfment ability and aggressiveness of macrophages incubated with lung cancer cells will be down regulated. Human mononuclear phagocytotic U937 cells were differentiated to macrophages by exposure to phorbol-12-myristate-13-acetate (PMA). These cells were then marked with APC fluorochrome dye for identification, and phagocytic activity was measured using PE conjugated fluorescent beads. Macrophages were incubated with two lung cancer cell lines (A549, H460) for various times and phagocytic indices were quantified using flow cytometry. Preliminary results indicated no change in phagocytic activity after 1 hour of exposure, and a 15% down regulation of overall engulfment after 24 hours of contact. These results were compared to cells incubated with non-cancerous lymphocytes, where there was no change in overall phagocytic activity. These data also indicated that the majority of normal macrophages were highly aggressive (engulfing 3 or more beads when analyzed by flow cytometry), but macrophages that were incubated with cancer cell lines experienced a statistically significant down regulation in aggressiveness, where the number of macrophages which engulfed 3 or more beads was reduced by 30% (p = 0.044). These findings indicate that exposure of U937-derived macrophages to lung cancer cell lines resulted in a possible shift towards the M2 phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1511. doi:10.1158/1538-7445.AM2011-1511

Dietary Calcein Marking of Shovelnose Sturgeon and the Effect of Sunlight on Mark Retention

AbstractCalcein, a fluorochrome dye, is a potential fish‐marking agent that has not been evaluated in sturgeon. Shovelnose sturgeon Scaphirhynchus platorynchus (average weight, 9.7 g) were fed calcein, immersed in a calcein bath, or left unmarked to determine calcein mark intensity. In the first study, six treatments were evaluated in a two‐by‐three factorial arrangement. Feed was formulated with 2.0 g of SE‐MARK/kg either as powder or in an encapsulated form. Sturgeon were fed the test diets for 5, 10, or 15 d. They readily ate feed containing powdered or encapsulated calcein. Sturgeon fed powdered calcein had more brilliant marks than those fed encapsulated calcein (8.27 versus 4.66 lm; P &lt; 0.03) 6 months postexposure. Fish fed calcein for 15 d (11.26 lm) were more brilliant (P &lt; 0.002) than fish fed for either 5 d (3.02 lm) or 10 d (5.11 lm). Post hoc comparison of the three treatment groups showed that sturgeon fed powdered calcein for 15 d (14.06 lm) were brighter (P &lt; 0.01) than fish fed encapsulated calcein (8.46 lm) or fish immersed in calcein (9.68 lm). In the second study, previously marked sturgeon were exposed to sunlight for 14 months to determine their retention of calcein marks. Dorsal marks were no longer visible on fish exposed to 100% sunlight after 8 weeks. Most but not all fish exposed to 20% sunlight had no discernable dorsal marks after 8 weeks, but ventral marks at the pectoral fin girdle were present on all fish in the 0% and 20% sunlight exposure treatments. Feeding calcein for 15 d appears to have excellent potential for practical application, such as distinguishing hatchery‐reared from wild fish. Ventral calcein marks remained visible after 14 months of exposure to 20% sunlight when sturgeon were reared in clear water.Received April 2, 2010; accepted September 22, 2010

EGFP-EGF1 protein-conjugated PEG–PLA nanoparticles for tissue factor targeted drug delivery

In a strategy for anti-thrombotic therapy, we have expressed EGFP-EGF1 fusion protein, in which EGF1 can bind with tissue factor (TF). EGFP has previously been widely used as a fluorescent protein marker. EGFP-EGF1 protein was thiolated and conjugated to the malemide covering on the pegylated nanoparticles (NP) to form the EGFP-EGF1-NP. The EGFP-EGF1-NP was characterized in terms of morphology, size and zeta potential. In vitro cell viability experiment confirmed that the biodegradable EGFP-EGF1-NP was safe. To evaluate the delivering ability of EGFP-EGF1-NP, a fluorochrome dye, Dir, was incorporated into the nanoparticle, and the loading capacity and release property of the particle were examined. In vitro results showed that the binding ability of EGFP-EGF1-NP with TF-expressing cells was significantly stronger than that of non-conjugated NP. In vivo multispectral fluorescent imaging demonstrated that EGFP-EGF1-NP had high specificity and sensitivity in targeting thrombi. Our study demonstrated that EGFP-EGF1-NP is a promising TF-targeting drug delivery system for thrombolytic treatment.

Structural adaptation and intracortical bone turnover in an ovine model of osteoporosis

Compact bone makes up approximately 80% of the human skeletal mass. This study examines the effect of estrogen deficiency on compact bone turnover and associated geometrical structural adaptation over a 31-month period in a large animal model. Twenty-seven skeletally mature sheep were divided into control (n = 16) and ovariectomy group (OVX, n = 11). Animals were administered five different fluorochrome dyes to label intracortical bone turnover, and sacrificed at 31 months. Compact bone samples were analyzed for cortical geometry, intracortical turnover at five time points, resorption cavities, porosity, and compressive strength. Intracortical bone turnover was significantly increased in OVX, which demonstrated seasonal variation. Cross-sectional area in OVX was significantly greater than control and was associated with an increased section modulus. Intracortical porosity was significantly increased in OVX, however, there was no significant difference in ultimate compressive strength between the groups. Our results demonstrate increased intracortical bone turnover, resportion spaces, and porosity in OVX, without adversely affecting compressive strength. Our results also support the hypothesis of geometrical adaptation of compact bone in response to estrogen deficiency. These results suggest an early structural compensatory response in compact bone, despite increased intracortical turnover.