Nucleic Acid Stain Research Articles

Odontonema cuspidatum and Psychotria punctata, Two New Hosts of Cucumber mosaic virus in the United States.

Cucumber mosaic virus (CMV) has a reported host range of 750 to 1,200 species (2,3) that includes weeds, wild plants, crops, and ornamentals. Two new CMV hosts were recently identified in Florida. In July 2011, leaves of Odontonema cuspidatum (firespike), a member of the Acanthaceae, with virus-like symptoms were sent to FDACS-DPI. Firespike is an ornamental shrub native to Mexico with evergreen ovate leaves tapering to a pointed tip. Leaf symptoms included severe leaf distortion with some subtle yellowing or mosaic on younger leaves. Pink-red crystals were seen in leaf strips stained with the nucleic acid stain Azure A, indicating a viral infection. In January 2012, leaves of Psychotria punctata (dotted wild coffee), a member of the Rubiaceae, with virus-like symptoms were sent to FDACS-DPI. Dotted wild coffee is a small exotic tropical tree found in south Florida with many tiny leaf nodules inhabited by endosymbiotic bacteria. In addition to the nodules, these leaves had many large dark green ringspots surrounded with a yellow halo. Both samples were positive for CMV when tested with ImmunoStrips and/or by conventional ELISA using CMV antiserum (Agdia, Elkhart, IN). To confirm CMV infection, reverse transcriptase (RT)-PCR on total RNA from a leaf sample of each plant species was used with previously published cucumovirus primers (1). An expected ~940 bp product was amplified from each sample and cloned into pGEM-T (Promega, Madison, WI). Ten clones from each plant species were sequenced in both directions. After removal of primer sequences, the 906 bp products were 96.3% identical with each other and showed 96.8 to 98.9% nucleotide identity with CMV sequences from Hungary, the United States, and Austria (GenBank Accession Nos. AF517802, U20668, and HQ916354, respectively). Identification of CMV infection in these two species expands the known host range and therefore the reservoir of this plant virus. This has implications for the ornamental industry in general and Florida farmers in particular.

Analysis of Ago2 and RhoGDI Proteins in Morphologically Normal and Abnormal Porcine Sperm Using Digital Image Processing Software.

Advancements in computerized analysis of biological images allow for the examination of sperm features that would normally go undetected using standard semen evaluation procedures. The objectives of this study were to examine and measure the histological profiles of two peptide markers and nucleic acid staining within sperm heads using digital image processing and to also determine if these profiles were predictive of general sperm morphology. Semen samples were obtained from genetically similar boars (n=10) and were classified as morphologically normal (=75% normal; n=5) or abnormal (<75% normal; n=5) at the stud using SpermVision (Minitube, Verona, WI). Counts were performed to compare the proportions of abnormality type (head, midpiece, tail) between groups. The percentage of abnormal heads did not differ between groups (4% versus 4% for normal and abnormal respectively); therefore, morphology classification was primarily determined by midpiece and tail abnormalities. For immunofluorescent detection of Ago2 and RhoGDI, samples were fixed, smeared on slides, air dried, and rehydrated in PBST. Slides were incubated at 4°C overnight in primary antibody, polyclonal rabbit Ago2 or RhoGDI (Cell Signaling, Danover, MA). Slides were then washed and incubated in secondary antibody, Alexafluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) and counterstained with DAPI (Sigma, St Louis, MO). Slides were washed, mounted with PBS, coverslipped, and immediately examined using a Leica LMD6000 microscope and Epi Fluorescence Filter Module LMD-BGR (Bannockburn, IL). Multispectral imaging was performed using a Nuance VIS Flex system (Cambridge Research & Instrumentation, Woburn, MA). Green and blue spectral channels were extracted from all images which were unmixed using the same spectral protocol for each slide. Images were then loaded into Cell Profiler (www.cellprofiler.org) for automated feature identification, analysis, and quantification. A pipeline was constructed to identify sperm heads, nucleic acid staining, and protein label, while also exporting measurements which included size, shape, intensity, and texture. Data were analyzed, grouped by protein, in JMP using a mixed model analysis of variance to compare parameters between groups (normal/abnormal) while accounting for animal, slide, image, and cell variation. In both the Ago2/DAPI and RhoGDI/DAPI analyses, integrated DAPI intensity and mean DAPI intensity were higher in abnormal samples than normal (P<0.05), which indicates that DNA is more accessible for DAPI binding in abnormal sperm. Integrated RhoGDI intensity was greater in the normal group (P<0.05), while there were no differences in Ago2 intensity between groups. Logistic regression in JMP was used to develop a model in which the parameters predicted classification as normal or abnormal. Stepwise procedures were used to select the most significant parameters for logistic regression prediction. The significant set of Ago2/DAPI parameters were able to correctly classify 87% of the sperm into the appropriate group, while the significant set of RhoGDI/DAPI parameters correctly classified 84% of the sperm. These results show that digital imaging processing and the use of peptide markers are able to detect differences present in sperm heads that are not readily detectable using standard semen analysis procedures. Digital image processing may provide a means to allow for increased accuracy and precision in semen analysis.

Human Flexor Tendon Tissue Engineering: Revitalization of Biostatic Allograft Scaffolds

Cadaveric tendon allografts form a readily available and underutilized source of graft material. Because of their material properties, allografts are biomechanically and biologically superior to synthetic scaffolds. However, before clinical use, allografts must undergo decellularization to reduce immunogenicity and oxidation to increase porosity, leaving a nonvital biostatic scaffold. Ex vivo seeding, or revitalization, is thought to hasten graft incorporation and stimulate intrinsic tendon healing, permitting early mobilization and return to function. In this study, we examined physical and biochemical augmentation methods, including scaffold surface scoring (physical) and rehydration of lyophilized scaffolds in serum (biochemical). Scaffolds were divided into four groups: (1) scored scaffolds, (2) lyophilized scaffolds rehydrated in fetal calf serum (FCS), (3) scaffolds both scored and rehydrated in FCS, and (4) control scaffolds. Scaffolds were reseeded with adipose-derived stem cells (ADSCs). Reseeding efficacy was quantified by a live cell and total cell assays and qualified histologically with hematoxylin and eosin, live/dead and SYTO green nucleic acid stains, TUNEL apoptosis stains, procollagen stains, and transmission electron microscopy. Scaffold-seeded cell viability at up to 2 weeks in vitro and up to 4 weeks in vivo was demonstrated with bioluminescent imaging of scaffolds seeded with luciferase-positive ADSCs. The effect of seeding on scaffold biomechanical properties was demonstrated with evaluation of ultimate tensile stress (UTS) and an elastic modulus (EM). We found that scaffold surface scoring led to an increase in live and total cell attachment and penetration (MTS assay, p<0.001 and DNA assay, p=0.003, respectively). Histology confirmed greater total cell number in both construct core and surface in scored compared with unscored constructs. Cells reseeded on scored constructs displayed reduced apoptosis, persistent procollagen production, and had a similar ultrastructural relationship to the surrounding matrix as native tenocytes on transmission electron microscopy. Rehydration of lyophilized scaffolds in serum did not improve reseeding. Seeded constructs demonstrated greater UTS and EM than unseeded constructs. Scaffolds seeded with ADSC-luc2-eGFP demonstrated persistent viability for at least 2 weeks in vitro. In conclusion, tendon surface scoring increases surface and core reseeding in vitro and may be incorporated as a final step in allograft processing before clinical implantation.

A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)

The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage and Escherichia coli as a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2'-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detect E. coli cells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infected E. coli cells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.

Plant-associated bacterial populations on native and invasive plant species: comparisons between 2 freshwater environments

Plant-microbial interactions have been well studied because of the ecological importance of such relationships in aquatic systems. However, general knowledge regarding the composition of these biofilm communities is still evolving, partly as a result of several confounding factors that are attributable to plant host properties and to hydrodynamic conditions in aquatic environments. In this study, the occurrences of various bacterial phylogenetic taxa on 2 native plants, i.e., mayapple (Podophyllum peltatum L.) and cow parsnip (Heracleum maximum Bartram), and on an invasive species, i.e., garlic mustard (Alliaria petiolata (M. Bieb.) Cavara & Grande), were quantitatively examined using nucleic acid staining and fluorescence in situ hybridization. The plants were incubated in triplicates for about a week within the Kalamazoo River and Pierce Cedar Creek as well as in microcosms. The bacterial groups targeted for enumeration are known to globally occur in relatively high abundance and are also ubiquitously distributed in freshwater environments. Fluorescence in situ hybridization analyses of the bacterioplankton assemblages revealed that the majority of bacterial cells that hybridized with the different probes were similar between the 2 sites. In contrast, the plant-associated populations while similar on the 3 plants incubated in Kalamazoo River, their representations were highest on the 2 native plants relative to the invasive species in Pierce Cedar Creek. Overall, our results further suggested that epiphytic bacterial assemblages are probably under the influences of and probably subsequently respond to multiple variables and conditions in aquatic milieus.

Abstract 2475: The abundance of SLC7a11 transporter in different tissues

Abstract The purpose of this study is to investigate the abundance of the SLC7A11 transporter in different tissues. The SLC7A11 is a plasma membrane transporter which mediates the transport of one cystine into the cell in exchange for one intracellular glutamate. We performed RT-PCR and immunohistochemistry to demonstrate the abundance of SLC7A11 transporter in different tissues. Tissue array slides were purchased from US Biomax Inc. The tissues were stained with or without SLC7A11 primary antibody. The images were captured using the software NIS elements connected to Nikon Eclipse series microscope. The images were then analyzed for staining intensity compared to their negative controls by using the software, Imagej. Panc-1, BxPC-3 and MIAPaCa-2 are human pancreatic cancer cell lines which were purchased from ATCC. The cells were cultured at 370C in humidified atmosphere of 5% CO2 using MEM or RPMI medium with low cystine levels. RNA was extracted from all the cell lines and converted into cDNA for Real Time PCR analysis. PCR reactions were prepared using SYBR green (which acts as a nucleic acid stain), forward and reverse SLC7A11 or GAPDH primers, and diluted cDNA. The reactions were added into 96-well PCR strips and sealed to prevent evaporation of the samples. The ABI 7300 machine was run after configuring the software and finally analyzed using 7300 system SDS software. The immunohistochemistry images demonstrate that most cancers especially pancreatic cancers have a high abundance of SLC7A11 transporter compared to normal tissues. The RT-PCR results showed that PANC-1 has significantly higher SLC7A11 expression compared to BxPC-3 and MIAPaCa-2 cell lines. The results indicate that SLC7A11 transporter is highly expressed in cancer tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2475. doi:1538-7445.AM2012-2475

Sensitivity of detecting environmental DNA

In a recent article from Jerde et al. 2011, a method is described to detect the presence of Asian carp at water sites using a PCR-based test termed environmental DNA (eDNA). In short, the procedure is to filter-collect fish cells that are presumably shed in water, isolate DNA from the filters, and amplify species-specific mitochondrial DNA (mtDNA) sequences by PCR. Gel electrophoresis, nucleic acid staining, and visualization of a band that migrates at the appropriate molecular weight are used to detect positive amplicons. The sensitivity of this procedure is stated to be 3.3 × 10−8 ng/μL for Bighead carp and 7.25 × 10−11 ng/μL for Silver carp where these numbers refer to total carp DNA. This claim is problematic. The value of volume used in stating sensitivity ng/μL is meaningless. Expression only relates to mass (ng). The reader is left asking did the investigator use 1 uL in the PCR reaction or more? For the calculations presented below, I assume that the meaning is 1 μL but the reported sensitivity is so far from being theoretically possible that changing this assumption even by multiple logs does not affect the outcome. If one takes a value of 5 pg of DNA/carp cell: (literature values for carp vary between 2 to 7 pg/cell depending on ploidy), then the number of carp cells in 1 ng is 200. Values for mtDNA copy number in fish skin cells vary according to age between 291 and 429 per cell (Hartmann et al. 2011). If a generous value of 1000 copies of mtDNA/cell is assumed then the product of the number of carp cells times mtDNA copy number is 2 × 105 copies/ng. The limit of detection (single copy of mtDNA) would be 5×10−6 ng. This theoretical value differs significantly from those experimentally reported, approximately 60 times or 80,000 times more for Bighead carp or Silver carp DNA, respectively. The assumptions used here to calculate copy numbers are heavily biased toward a liberal approximation of mtDNA copy number. Further, an assumption is made that single copy amplification could be attained with endpoint PCR. Even then, reported sensitivity cannot be reconciled with theoretical values. The vast overestimate of PCR sensitivity in this article raises questions about credibility of the eDNA assay. We thank Dr. Volker Vogt for his helpful discussions.

Cultured heart cells from oyster : an experimental approach for evaluation of the toxicity of the marine pollutant tributyltin

European Community regulations on chemicals promote alternative methods to test substances presenting potential risks for the environment. In the present work, cultured atrial cells isolated from oyster (Crassostrea gigas) were used as an experimental model to investigate the toxicity of tributyltin (TBT) after short-time exposure at concen- trations representative of those that can be measured in seawater, marine sediments and/or bivalves bioaccumulating this pollutant. In vitro and in vivo assays produce values of the same order of magnitude for both animal/cell survival and heart/cardiomyocyte beating rate. The survival rate of whole animals decreased from 10 −6 M TBT after 3 days. For cultured cells, the viability, evaluated using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, significantly decreased after two days of treatment with 10 −6 M TBT, and after six days with 10 −10 M TBT .T he percentage of apoptotic cells, quantified by flow cytometry and YO-PRO r � -1 iodide, a nucleic acid stain that only permeates cells that are beginning to undergo apoptosis, increased significantly in these cases. Moreover, intracellu- lar concentration of Ca ++ had increased after 10 min of exposition to 10 −6 M, and could be associated with apoptotic processes. As patch clamp experiments showed that Ca ++ conductance was decreased, intracellular calcium increase could mainly be due to a release from internal stores. The decreases in beating rhythm could be explained by the de- crease in adenosine triphosphate (ATP) production revealed by 31 P nuclear magnetic resonance (NMR) spectroscopy and confirmed by the increase of the KATP channel conductance. The related hyperpolarization and the disturbances of the energetic metabolism were clearly related to the loss of the atrial cell contractility and viability.

Dead or alive? Viability assessment of micro- and mesoplankton

The rapid and efficient analysis of plankton samples (e.g. enumeration, identification, biomass determination) has been an important driver for recent technological developments in (semi-) automated analysis and imaging instruments. Most focus has been on identification and abundance estimates, while less attention has been given to viability, i.e. the assessment of whether the organisms are dead or alive. However, a wide spectrum of scientific applications requires accurate viability determinations, e.g. the monitoring of invasive species in ship ballast water. The transfer of species through ballast water forms a major threat to marine ecosystems, resulting in significant environmental and economic losses. A variety of viability stains and viability assessment methods are available, but there has been no systematic investigation how these methods perform for larger organisms (epsilon 50 m). We review the current procedures for viability determination for large plankton and present a cross-comparison of three methods: cell digestion assay (CDA), SYTOX Green nucleic acid staining and Neutral Red vital staining. The CDA and SYTOX Green methods did not perform well and gave various problems linked to the multicellular nature of zooplankton, autofluorescence and/or constraints set by the definition of cell death. Although some issues remain and there is no universal method, the Neutral Red vital stain proved the most robust viability method in this study and is broadly applicable to both phytoplankton and zooplankton larger than 50 m.

Molecular detection of plant pathogenic bacteria using polymerase chain reaction single-strand conformation polymorphism

The application of polymerase chain reaction (PCR) technology to molecular diagnostics holds great promise for the early identification of agriculturally important plant pathogens. Ralstonia solanacearum, Xanthomoans axonopodis pv. vesicatoria, and Xanthomonas oryzae pv. oryzae are phytopathogenic bacteria, which can infect vegetables, cause severe yield loss. PCR-single-strand conformation polymorphism (PCR-SSCP) is a simple and powerful technique for identifying sequence changes in amplified DNA. The technique of PCR-SSCP is being exploited so far, only to detect and diagnose human bacterial pathogens in addition to plant pathogenic fungi. Selective media and serology are the commonly used methods for the detection of plant pathogens in infected plant materials. In this study, we developed PCR-SSCP technique to identify phytopathogenic bacteria. The PCR product was denatured and separated on a non-denaturing polyacrylamide gel. SSCP banding patterns were detected by silver staining of nucleic acids. We tested over 56 isolates of R. solanacearum, 44 isolates of X. axonopodis pv. vesicatoria, and 20 isolates of X. oryzae pv. oryzae. With the use of universal primer 16S rRNA, we could discriminate such species at the genus and species levels. Species-specific patterns were obtained for bacteria R. solanacearum, X. axonopodis pv. vesicatoria, and X. oryzae pv. oryzae. The potential use of PCR-SSCP technique for the detection and diagnosis of phytobacterial pathogens is discussed in the present paper.

Interlaboratory assessment of mitotic index by flow cytometry confirms superior reproducibility relative to microscopic scoring

A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose-dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland-Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations.

Rapid detection of sacbrood virus (SBV) by one-step reverse transcription loop-mediated isothermal amplification assay

BackgroundSacbrood virus (SBV) primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required.FindingsA one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green.ConclusionsThe current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.

Single-cell electroporation using proton beam fabricated biochips

We report the design and fabrication of a novel single cell electroporation biochip featuring high aspect ratio nickel micro-electrodes with smooth side walls between which individual cells are attached. The biochip is fabricated using Proton Beam Writing (PBW), a new direct write lithographic technique capable of fabricating high quality high-aspect-ratio nano and microstructures. By applying electrical impulses across the biochip electrodes, SYTOX® Green nucleic acid stain is incorporated into mouse neuroblastoma (N2a) cells and observed via green fluorescence when the stain binds with DNA inside the cell nucleus. Three parameters; electric field strength, pulse duration, and numbers of pulses have been investigated for the single cell electroporation process. The results indicate high transfection rates as well as cell viability of 82.1 and 86.7% respectively. This single cell electroporation system may represent a promising method for the introduction of a wide variety of fluorophores, nanoparticles, quantum dots, DNAs and proteins into cells.

Disruption of Staphylococcus epidermidis biofilm formation using a targeted cationic peptide

This study reports the use of a targeted cationic peptide with the ability to disrupt Staphylococcus epidermidis biofilm formation. Complications due to nosocomial infections of implanted medical devices pose a significant health risk to patients, with Staphylococcus epidermidis often implicated in the case of blood-contacting biomaterials. S. epidermidis virulence relies mainly on its ability to form a biofilm, the main component of which is polysaccharide intercellular adhesin (PIA). We utilized the synthetic β6-20 peptide, known to specifically bind S. epidermidis, in order to deliver a cationic polylysine peptide (G(3)K(6)) to the bacterial surface and disrupt the charge-charge interactions needed for PIA retention and biofilm stability. The effects of the β6-20-G(3)K(6) peptide on biofilm formation were assessed using optical density, fluorescently labeled wheat germ agglutinin, nucleic acid stain (SYTO 9), and a metabolic assay (XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt). Biofilms formed in the presence of β6-20-G(3)K(6) peptide (100 μM) resulted in a 37.9% reduction in PIA content and a 17.5% reduction of adherent bacteria relative to biofilms grown in the absence of peptide. These studies demonstrate the targeting ability of the β6-20 peptide towards biomaterial-adherent S. epidermidis, and highlight the potential for disrupting the early stages of biofilm formation.

Groundwater Biofilm Dynamics Grown In Situ Along a Nutrient Gradient

This paper describes the in situ response of groundwater biofilms in an alluvial gravel aquifer system on the Canterbury Plains, New Zealand. Biofilms were developed on aquifer gravel, encased in fine mesh bags and suspended in protective columns in monitoring wells for at least 20 weeks. Four sites were selected in the same groundwater system where previous analyses indicated a gradient of increasing nitrate down the hydraulic gradient from Sites 1 to 4. Measurements during the current study classified the groundwater as oligotrophic. Biofilm responses to the nutrient gradients were assessed using bioassays, with biomass determined using protein and cellular and nucleic acid staining and biofilm activity using enzyme assays for lipid, carbohydrate, phosphate metabolism, and cell viability. In general, biofilm activity decreased as nitrate levels increased from Sites 1 to 4, with the opposite relationship for carbon and phosphorus concentrations. These results showed that the groundwater system supported biofilm growth and that the upper catchment supported efficient and productive biofilms (high ratio of activity per unit biomass).

Analysis of tissue dependent DNA yield for optimal sampling of micro-moths in large-scale biodiversity surveys

DNA barcoding surveys of small insects usually extract DNA from either a complete insect or a leg. Little is known about how to optimize DNA quantity and quality from different insect parts while preserving a morphological voucher. Here, we quantify DNA yield from different body parts (antenna, hind leg, forewing, hind wing and abdomen) of the micro-moth Cameraria ohridella (Lepidoptera: Gracillariidae) using fluorescent nucleic acid stain (PicoGreen). Samples were preserved in 100% ethanol or dried for three weeks. Our experiment was designed to encompass practical sampling options during fieldwork. DNA quality was assessed by PCR amplification of the mitochondrial COI barcode fragment. In addition, we compared PCR amplification using Platinum® Taq and Qiagen DNA Polymerase and quantified sequence success of amplified DNA. We show that overall, dry parts showed higher eluted DNA yields. PCR and sequencing success rate were slightly higher for dry tissue than ethanol-preserved parts. We also show that Platinum® Taq yielded the highest PCR success rate and that all dry tissues are sequenceable. The optimal strategy for DNA barcoding surveys is therefore to mount micro-Lepidoptera specimens in the field for morphological analysis and sample tissues (hind legs are favoured) from dried samples at a later time (several weeks) in the lab for DNA barcoding using preferentially Platinum® Taq. If larger amounts of DNA are required (i.e. for nuclear gene sequencing), several legs from one side of the specimen or the abdomen should be preserved in pure ethanol.

A phase 1 dose‐escalation study of ARRY‐520, a kinesin spindle protein inhibitor, in patients with advanced myeloid leukemias

ARRY-520 selectively inhibits the mitotic kinesin spindle protein (KSP), which leads to abnormal monopolar spindle formation and apoptosis. A phase 1 trial was conducted to establish the safety and the maximum tolerated dose (MTD) of ARRY-520 given as a 1-hour infusion in either a single dose or on a day 1, 3, and 5 divided-dose schedule per cycle in patients with advanced or refractory myeloid leukemias. Additional objectives were to characterize pharmacokinetics, assess preliminary clinical activity, and explore biomarkers of KSP inhibition with ARRY-520. A total of 36 patients with acute myelogenous leukemia (n = 34) or myelodysplastic syndromes (n = 2) with a median age of 66 years (range, 21-88 years) were enrolled: 15 in the single-dose schedule (dose levels: 2.5, 3.75, 4.5, and 5.6 mg/m(2)) and 21 in the divided-dose schedule (dose levels: 0.8, 1.2, 1.5, and 1.8 mg/m(2)/day). The MTD was 4.5 mg/m(2) total dose per cycle for both dose schedules. Dose-limiting toxicities included mucositis, exfoliative rash, hand-foot syndrome, and hyperbilirubinemia. Grades 3 or 4 reversible drug-related myelosuppression were observed in 33 of 36 patients. Plasma pharmacokinetic analyses revealed low clearance of ARRY-520 (~3 L/hour), a volume of distribution of ~450 L, and a median terminal half-life of >90 hours. Monopolar spindles were observed in blood mononuclear cells, through use of 4',6-diamidino-2-phenylindole nucleic acid stain and antitubulin antibodies. On the basis of the relative lack of clinical activity, further development of ARRY-520 as an antileukemic agent was halted. (Clinicaltrials.gov identifier NCT00637052).

Shared and Divergent Stimulatory Effects of S100A4 and Uric Acid on Proliferation and Chemotaxis of Mesenchymal Stem Cells (MSCs). Interplay Between Necrotic Tumor Material and Immunosuppressive MSCs within Tumor Microenvironment,

Abstract 3239 Background:Necrotic cell death is a characteristic feature of advanced solid tumor. S100 proteins and uric acid (UA) are released from necrotic (tumor) tissue regardless of tumor's origin. Both of these factors are known to influence immune response. Mesenchymal stem cells (MSCs) are often found within tumor microenvironment and are associated with poor prognosis of cancer patients in terms of metastasis and survival. MSCs seem to play a crucial role within tumor microenvironment probably due to their immunosuppressive capacity interfering with the specific anti-tumor immune response. Underlying mechanisms for MSC accumulation and stimulation in tumor tissue are not well characterized yet. S100A4 is known to promote metastasis in certain tumors such as colorectal cancer, but the exact mechanism of this effect still remains unclear. UA not only induces an acute inflammatory response but is also known to maintain chronic inflammation. In our previous experiments, we could already show that necrotic tumor material is capable of inducing chemotaxis and proliferation of MSCs. Focusing on S100A4 and UA, we now sought to characterize individual known factors within necrotic material which may be responsible for described effects. Materials and Methods:Proliferation assays were performed using CYQUANT Assay (Invitrogen) based on fluorescent staining of nucleic acid. Migration of fluorescent-labeled MSCs was assessed using FluoroBlok (BD) two-chamber chemotaxis plates, fluorescence within the bottom chamber of migration plates was measured with PolarStar plate reader (BMG). Results:We tested the influence of recombinant human S100A4 at concentrations between 50 and 1000 ng/ml on MSC proliferation and could demonstrate divergent effects depending on the chosen S100A4 concentration: S100A4 at concentrations not higher than 100 ng/ml enhanced the proliferation of MSCs in a dose dependent manner up to 50%, whereas concentrations above 100 ng/ml inhibited MSC proliferation down to 50% compared to medium containing 5% human serum without S100A4. We tested the chemotactic activity of recombinant human S100A4 at concentrations between 0.01 and 1000 ng/ml on MSCs and could demonstrate a dose dependent chemotactic activity up to 80% of the response which was achieved by positive control (50% FBS). By adding UA to suboptimal concentration of S100A4 (100ng/ml), we could enhance the chemotactic activity of S100A4 on MSCs. In contrast to its enhancing effect on MSC migration, UA inhibited dose dependently the proliferation inducing effect of S100A4 with strongest effect at 300μg UA/ml resulting in about 50% inhibition of proliferation compared to medium containing 100ng/ml S100A4 without additional UA. ELISA assays confirmed the presence of S100A4 in the necrotic tumor material which we had used in our previous experiments with similar stimulatory effects on MSCs in terms of proliferation and chemotaxis. Conclusions:MSCs are often found within tumor tissue and may influence the biologic behavior of tumor and host's immune response to tumor. We could characterize UA and S100A4 as bioactive factors within necrotic (tumor) material. Whereas S100A4 stimulated both proliferation as well as chemotaxis of MSCs, UA enhanced the chemotactic acitivity of S100A4 while inhibiting its proliferation inducing effect. Comparing our new data with our previous observations using tumor lysates to induce chemotaxis and proliferation of MSCs the overall effect of necrotic material seems to be rather proliferative and chemotactic even though some factors like UA may interfere with mentioned overall effects. Keeping in mind that MSCs can act as potent immunosuppressive cells, we conclude that tumor necrosis rather inhibits an effective anti-tumor immune response by favoring accumulation and proliferation of MSCs within tumor microenvironment. Our observations shed some light into the biology of MSCs within tumor microenvironment and open new questions concerning the interplay between MSCs, necrotic tumor cells and tumor progression. Possible strategies to break the described vicious circle by rather inducing an apoptotic tumor cell death or by reducing bioactive factors released from necrotic tissue within tumor microenvironment may be considered. Additionally, clinical investigations using specific antibodies to S100A4 or influencing serum concentrations of UA in tumor patients may be performed. Disclosures:No relevant conflicts of interest to declare.

The Sesquiterpene Oil α-Bisabolol Induces Apoptosis of B-Chronic Lymphocytic Leukemia Primary Cells

Abstract 1319We have recently demonstrated that the sesquiterpene oil α-bisabolol is cytotoxic against primary acute leukemia cells ex vivo and in chronic myeloid leukemia cell lines. It enters cells via lipid rafts and activates the mitochondrial-dependent intrinsic pathway of apoptosis, exerting a preferential toxicity against malignant vs normal cells probably due to their higher content in lipid rafts. Here we investigated the in vitro activity of α-bisabolol in primary cells from patients with B-Chronic Lymphocytic Leukemia (B-CLL).Twenty-six patients with newly diagnosed B-CLL gave their informed consent to the study. Cells were collected before any treatment, purified and cultured for 24 hours with serial dilutions of α-bisabolol. Citotoxicity was quantified in flow cytometry by the BD Trucount™ technology to allow comparison between neoplastic and normal residual lymphocytes. B-CLL cells (IC50 42±15 μM) were significantly more sensitive towards α-bisabolol than normal B- (IC50 82±34 μM, p=.005) and T-cells (IC50 120±35 μM, p<.001). Citotoxicity was similar between the IgVH mutated (n=11) and the IgVH unmutated samples (n=7), as well as between the Binet stage A (n=20) and B-C (n=6) patients.To investigate the mechanisms of α-bisabolol-induced toxicity we treated B-CLL cells with 40 μM α-bisabolol for up to 3 hours. We observed a time-dependent increase in fluorescence of cells treated with the membrane-impermeant nucleic acid stain TO-PRO-3, already detactable after 30 minutes. When cells were loaded with the Ca2+ indicator Fluo-4 AM, an increase of Ca2+ influx was revealed already after 15 minutes. These early events indicate that α-bisabolol induces the loss of cellular membrane integrity, so triggering the apoptotic cascade.Then we assessed the mitochondrial transmembrane potential (ΔΨm) with the fluorochrome JC-1 to confirm that a mitochondrial damage is a concurrent mechanism in the apoptotic process induced by α-bisabolol. By flow cytometry we demonstrated that, after 3-hour incubation with 40 μM α-bisabolol, ΔΨm dissipation was already detectable in leukemic cells, while T-lymphocytes, evaluated as internal control in the same samples, stayed vital. To investigate the mitochondrial target of α-bisabolol we examined the function of the mitochondrial permeability transition pore (mPTP). After 5-hour incubation with 40 μM α-bisabolol we loaded cells with the calcein AM dye and added CoCl2 to distinguish between intact and damaged mitochondria, confirming that the function of mPTP was compromised in B-CLL cells but not in normal controls. Finally, to determine whether α-bisabolol affects the oxydative state of treated cells, we evaluated the intracellular concentration of reactive oxygen species (ROS) by measuring the fluorescent signal of CM-H2DCFDA loaded cells. When B-CLL cells were exposed to 40 μM a-bisabolol for 3 hours, they exhibited a clear fluorescence increase, indicating the striking generation of ROS: this was completely abrogated by the addition of N-acetylcysteine, a scavenger of intracellular ROS. Clues about the molecular mechanistics of α-bisabolol have also emerged from in vitro models based on treating cells previously transfected with BH3-only molecules. In this setting, α-bisabolol exposed cells seem to undergo detrimental, non-selective autophagy-like phenomena.Our data indicate that α-bisabolol exerts a level of cytotoxicity against B-CLL cells at concentrations that only partially affect normal B- and T-cells. Moreover, a brief exposure (3–5 hours) to α-bisabolol is sufficient to elicit multiple pro-apoptotic signals independently of the patients’ mutational status. Disclosures:No relevant conflicts of interest to declare.

Antifungal Activity of Resveratrol against Botrytis cinerea Is Improved Using 2-Furyl Derivatives

The antifungal effect of three furyl compounds closely related to resveratrol, (E)-3,4,5-trimethoxy-β-(2-furyl)-styrene (1), (E)-4-methoxy-β-(2-furyl)-styrene (2) and (E)-3,5-dimethoxy-β-(2-furyl)-styrene (3) against Botrytis cinerea was analyzed. The inhibitory effect, at 100 µg ml−1 of compounds 1, 2, 3 and resveratrol on conidia germination, was determined to be about 70%, while at the same concentration pterostilbene (a dimethoxyl derivative of resveratrol) produced complete inhibition. The title compounds were more fungitoxic towards in vitro mycelial growth than resveratrol and pterostilbene. Compound 3 was the most active and a potential explanation of this feature is given using density functional theory (DFT) calculations on the demethoxylation/demethylation process. Compound 3 was further evaluated for its effects on laccase production, oxygen consumption and membrane integrity of B. cinerea. An increase of the laccase activity was observed in the presence of compound 3 and, using Sytox Green nucleic acid stain, it was demonstrated that this compound altered B. cinerea membrane. Finally, compound 3 partially affected conidia respiration.