[Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants].

Science.gov (United States)

Boronnikova, S V; Kalendar', R N

2010-01-01

Species-specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm' region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP-markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.

[Non-LTR retrotransposons: LINEs and SINEs in plant genome].

Science.gov (United States)

Cheng, Xu-Dong; Ling, Hong-Qing

2006-06-01

Retrotransposons are one of the drivers of genome evolution. They include LTR (long terminal repeat) retrotransposons, which widespread in Eukaryotagenomes, show structural similarity to retroviruses. Non-LTR retrotransposons were first discovered in animal genomes and then identified as ubiquitous components of nuclear genomes in many species across the plant kingdom. They constitute a large fraction of the repetitive DNA. Non-LTR retrotransposons are divided into LINEs (long interspersed nuclear elements) and SINEs (short interspersed nuclear elements). Transposition of non-LTR retrotransposons is rarely observed in plants indicating that most of them are inactive and/or under regulation of the host genome. Transposition is poorly understood, but experimental evidence from other genetic systems shows that LINEs are able to transpose autonomously while non-autonomous SINEs depend on the reverse transcription machinery of other retrotransposons. Phylogenic analysis shows LINEs are probably the most ancient class of retrotransposons in plant genomes, while the origin of SINEs is unknown. This review sums up the above data and wants to show readers a clear picture of non-LTR retrotransposons.

Genetic Diversity Assessment of Portuguese Cultivated Vicia faba L. through IRAP Markers

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Diana Tomás

2016-03-01

Full Text Available Faba bean have been grown in Portugal for a long time and locally adapted populations are still maintained on farm. The genetic diversity of four Portuguese faba bean populations that are still cultivated in some regions of the country was evaluated using the Inter Retrotransposons Amplified Polymorphism (IRAP technique. It was shown that molecular markers based on retrotransposons previously identified in other species can be efficiently used in the genetic variability assessment of Vicia faba. The IRAP experiment targeting Athila yielded the most informative banding patterns. Cluster analysis using the neighbor-joining algorithm generated a dendrogram that clearly shows the distribution pattern of V. faba samples. The four equina accessions are separated from each other and form two distinct clades while the two major faba bean accessions are not unequivocally separated by the IRAP. Fluorescent In Situ Hybridization (FISH analysis of sequences amplified by IRAP Athila revealed a wide distribution throughout V. faba chromosomes, confirming the whole-genome coverage of this molecular marker. Morphological characteristics were also assessed through cluster analysis of seed characters using the unweighted pair group method arithmetic average (UPGMA and principal component analysis (PCA, showing a clear discrimination between faba bean major and equina groups. It was also found that the seed character most relevant to distinguish accessions was 100 seed weight. Seed morphological traits and IRAP evaluation give similar results supporting the potential of IRAP analysis for genetic diversity studies.

How to measure separation and angles between inter-molecular fluorescent markers

DEFF Research Database (Denmark)

Flyvbjerg, Henrik

Structure and function of an individual biomolecule can be explored with minimum two fluorescent markers of different colors. Since the light of such markers can be spec- trally separated and imaged simultaneously, the markers can be colocalized. Here, we describe the method used for such two......-color colocalization microscopy. Then we extend it to fluorescent markers with fixed orientations and in intramolecular proximity. Our benchmarking of this extension produced two extra results: (a) we established short double-labeled DNA molecules as probes of 3D orientation of anything to which one can attach them...

Impact of Low-Energy Ion Beam Implantation on the Expression of Ty1-copia-like Retrotransposons in Wheat (Triticum aestivum)

International Nuclear Information System (INIS)

Ya Huiyuan; Jiao Zhen; Gu Yunhong; Wang Weidong; Qin Guangyong; Huo Yuping

2007-01-01

Retrotransposon-like elements are major constituents of most eukaryotic genomes. For example, they account for roughly 90% of the wheat (Triticum aestivum) genome. Previous study on a wheat strain treated by low-energy N + ions indicated the variations in AFLP (Amplified Fragment Length Polymorphism ) markers. One such variation was caused by the re-activation of Ty1-copia-like retrotransposons, implying that the mutagenic effects of low-energy ions might work through elevated activation of retrotransposons. In this paper an expression profile of Ty1-copia-like retrotransposons in wheat treated by low-energy N + ions is reported. The reverse transcriptase (RT) domains of these retrotransposons were amplified by reverse-transcriptional polymerase chain reaction (RT-PCR) and sequentially cloned. 42 and 65 clones were obtained from the treated (CL) and control materials (CK), respectively. Sequence analysis of each clone was performed by software. Phylogeny and classification were calculated responding to the sequences of the RT domains. All the results show that there is much difference in the RT domain between the control sample and the treated sample. Especially, the RT domains from the treated group encode significantly more functional ORF (open reading frames) than those from the control sample. This observation suggests that the treated sample has higher activation of retrotransposons, possibly as a consequence of low-energy ion beam irradiation. It also suggests that retrotransposons in the two groups impact the host gene expression in two different ways and carry out different functions in wheat cells

Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants

OpenAIRE

Lanciano, Sophie; Carpentier, M. C.; Llauro, C.; Jobet, E.; Robakowska-Hyzorek, D.; Lasserre, E.; Ghesquière, Alain; Panaud, O.; Mirouze, Marie

2017-01-01

Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposon...

Copia and Gypsy retrotransposons activity in sunflower (Helianthus annuus L.)

Science.gov (United States)

2009-01-01

Background Retrotransposons are heterogeneous sequences, widespread in eukaryotic genomes, which refer to the so-called mobile DNA. They resemble retroviruses, both in their structure and for their ability to transpose within the host genome, of which they make up a considerable portion. Copia- and Gypsy-like retrotransposons are the two main classes of retroelements shown to be ubiquitous in plant genomes. Ideally, the retrotransposons life cycle results in the synthesis of a messenger RNA and then self-encoded proteins to process retrotransposon mRNA in double stranded extra-chromosomal cDNA copies which may integrate in new chromosomal locations. Results The RT-PCR and IRAP protocol were applied to detect the presence of Copia and Gypsy retrotransposon transcripts and of new events of integration in unstressed plants of a sunflower (Helianthus annuus L.) selfed line. Results show that in sunflower retrotransposons transcription occurs in all analyzed organs (embryos, leaves, roots, and flowers). In one out of sixty-four individuals analyzed, retrotransposons transcription resulted in the integration of a new element into the genome. Conclusion These results indicate that the retrotransposon life cycle is firmly controlled at a post transcriptional level. A possible silencing mechanism is discussed. PMID:20030800

[Ulysses retrotransposon aspartate proteinase (Drosophila virilis)].

Science.gov (United States)

Volkov, D A; Savvateeva, L V; Dergousova, N I; Rumsh, L D

2002-01-01

Retrotransposones are mobile genetic elements occurring in genomes of bacteria, plants or animals. Retrotransposones were found to contain nucleotide sequences encoding proteins which are homological to retroviral aspartic proteinases. Our research has been focused on Ulysses which is mobile genetic element found in Drosophila virilis. We suggested a primary structure of Ulysses proteinase using comparative analysis of amino acid sequences of retroviral proteinases and proteinases from retrotransposones. The appropriate cDNA fragment has been cloned and expressed in E. coli. The purification of recombinant protein (12 kD) has been carried out by affinity chromatography using pepstatine-agarose. The obtained protein has proteolytic activity at optimum pH 5.5 like the majority of aspartic proteinases.

Efficient DNA Fingerprinting Based on the Targeted Sequencing of Active Retrotransposon Insertion Sites Using a Bench-Top High-Throughput Sequencing Platform

OpenAIRE

Monden, Yuki; Yamamoto, Ayaka; Shindo, Akiko; Tahara, Makoto

2014-01-01

In many crop species, DNA fingerprinting is required for the precise identification of cultivars to protect the rights of breeders. Many families of retrotransposons have multiple copies throughout the eukaryotic genome and their integrated copies are inherited genetically. Thus, their insertion polymorphisms among cultivars are useful for DNA fingerprinting. In this study, we conducted a DNA fingerprinting based on the insertion polymorphisms of active retrotransposon families (Rtsp-1 and LI...

Convergent evolution of ribonuclease h in LTR retrotransposons and retroviruses.

Science.gov (United States)

Ustyantsev, Kirill; Novikova, Olga; Blinov, Alexander; Smyshlyaev, Georgy

2015-05-01

Ty3/Gypsy long terminals repeat (LTR) retrotransposons are structurally and phylogenetically close to retroviruses. Two notable structural differences between these groups of genetic elements are 1) the presence in retroviruses of an additional envelope gene, env, which mediates infection, and 2) a specific dual ribonuclease H (RNH) domain encoded by the retroviral pol gene. However, similar to retroviruses, many Ty3/Gypsy LTR retrotransposons harbor additional env-like genes, promoting concepts of the infective mode of these retrotransposons. Here, we provide a further line of evidence of similarity between retroviruses and some Ty3/Gypsy LTR retrotransposons. We identify that, together with their additional genes, plant Ty3/Gypsy LTR retrotransposons of the Tat group have a second RNH, as do retroviruses. Most importantly, we show that the resulting dual RNHs of Tat LTR retrotransposons and retroviruses emerged independently, providing strong evidence for their convergent evolution. The convergent resemblance of Tat LTR retrotransposons and retroviruses may indicate similar selection pressures acting on these diverse groups of elements and reveal potential evolutionary constraints on their structure. We speculate that dual RNH is required to accelerate retrotransposon evolution through increased rates of strand transfer events and subsequent recombination events. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Identification and characterization of REC66, a Ty1-copia-like retrotransposon in the genome of red flower of Mirabilis jalapa L.

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Shunri Jiang

2017-01-01

Full Text Available Mirabilis jalapa Lis the most commonly grown ornamental species of Mirabilis and is available in a range of brilliant colors. However, genetic research on Mirabilis jalapa Lis limited. Using fluorescent differential display (FDD screening, we report the identification of a novel Ty1-copia-like retrotransposon in the genome of the red flower of Mirabilis jalapa L, and we named it REC66based on its sequence homology to the GAG protein from Ty1-copiaretrotransposon. Using degenerate primers based on the DNA sequence of REC66, a total of fourteen different variants in reverse transcriptase (RT sequence were recovered from the genomic DNA. These RT sequences show a high degree of heterogeneity characterized mainly by deletion mutation; they can be divided into three subfamilies, of which the majority encode defective RT. This is the first report of a Ty1-copiaretrotransposon in Mirabilis jalapa L. The finding could be helpful for the development of new molecular markers for genetic studies, particularly on the origin and evolutionary relationships of M. jalapa L, and the study of Ty1-copiaretrotransposons and plant genome evolution in the genus Mirabilisor family Nyctaginaceae.

Citrus and Prunuscopia-like retrotransposons.

Science.gov (United States)

Asíns, M J; Monforte, A J; Mestre, P F; Carbonell, E A

1999-08-01

Many of the world's most important citrus cultivars ("Washington Navel", satsumas, clementines) have arisen through somatic mutation. This phenomenon occurs fairly often in the various species and varieties of the genus.The presence of copia-like retrotransposons has been investigated in fruit trees, especially citrus, by using a PCR assay designed to detect copia-like reverse transcriptase (RT) sequences. Amplification products from a genotype of each the following species Citrus sinensis, Citrus grandis, Citrus clementina, Prunus armeniaca and Prunus amygdalus, were cloned and some of them sequenced. Southern-blot hybridization using RT clones as probes showed that multiple copies are integrated throughout the citrus genome, while only 1-3 copies are detected in the P. armeniaca genome, which is in accordance with the Citrus and Prunus genome sizes. Sequence analysis of RT clones allowed a search for homologous sequences within three gene banks. The most similar ones correspond to RT domains of copia-like retrotransposons from unrelated plant species. Cluster analysis of these sequences has shown a great heterogeneity among RT domains cloned from the same genotype. This finding supports the hypothesis that horizontal transmission of retrotransposons has occurred in the past. The species presenting a RT sequence most similar to citrus RT clones is Gnetum montanum, a gymnosperm whose distribution area coincides with two of the main centers of origin of Citrus spp. A new C-methylated restriction DNA fragment containing a RT sequence is present in navel sweet oranges, but not in Valencia oranges from which the former originated suggesting, that retrotransposon activity might be, at least in part, involved in the genetic variability among sweet orange cultivars. Given that retrotransposons are quite abundant throughout the citrus genome, their activity should be investigated thoroughly before commercializing any transgenic citrus plant where the transgene(s) is part

Retrotransposon Domestication and Control in Dictyostelium discoideum

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Marek Malicki

2017-10-01

Full Text Available Transposable elements, identified in all eukaryotes, are mobile genetic units that can change their genomic position. Transposons usually employ an excision and reintegration mechanism, by which they change position, but not copy number. In contrast, retrotransposons amplify via RNA intermediates, increasing their genomic copy number. Hence, they represent a particular threat to the structural and informational integrity of the invaded genome. The social amoeba Dictyostelium discoideum, model organism of the evolutionary Amoebozoa supergroup, features a haploid, gene-dense genome that offers limited space for damage-free transposition. Several of its contemporary retrotransposons display intrinsic integration preferences, for example by inserting next to transfer RNA genes or other retroelements. Likely, any retrotransposons that invaded the genome of the amoeba in a non-directed manner were lost during evolution, as this would result in decreased fitness of the organism. Thus, the positional preference of the Dictyostelium retroelements might represent a domestication of the selfish elements. Likewise, the reduced danger of such domesticated transposable elements led to their accumulation, and they represent about 10% of the current genome of D. discoideum. To prevent the uncontrolled spreading of retrotransposons, the amoeba employs control mechanisms including RNA interference and heterochromatization. Here, we review TRE5-A, DIRS-1 and Skipper-1, as representatives of the three retrotransposon classes in D. discoideum, which make up 5.7% of the Dictyostelium genome. We compile open questions with respect to their mobility and cellular regulation, and suggest strategies, how these questions might be addressed experimentally.

Oligothiophenes as Fluorescent Markers for Biological Applications

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Antonio Manetto

2012-01-01

Full Text Available This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (biomolecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described.

PpRT1: the first complete gypsy-like retrotransposon isolated in Pinus pinaster.

Science.gov (United States)

Rocheta, Margarida; Cordeiro, Jorge; Oliveira, M; Miguel, Célia

2007-02-01

We have isolated and characterized a complete retrotransposon sequence, named PpRT1, from the genome of Pinus pinaster. PpRT1 is 5,966 bp long and is closely related to IFG7 gypsy retrotransposon from Pinus radiata. The long terminal repeats (LTRs) have 333 bp each and show a 5.4% sequence divergence between them. In addition to the characteristic polypurine tract (PPT) and the primer binding site (PBS), PpRT1 carries internal regions with homology to retroviral genes gag and pol. The pol region contains sequence motifs related to the enzymes protease, reverse transcriptase, RNAseH and integrase in the same typical order known for Ty3/gypsy-like retrotransposons. PpRT1 was extended from an EST database sequence indicating that its transcription is occurring in pine tissues. Southern blot analyses indicate however, that PpRT1 is present in a unique or a low number of copies in the P. pinaster genome. The differences in nucleotide sequence found between PpRT1 and IFG7 may explain the strikingly different copy number in the two pine species genome. Based on the homologies observed when comparing LTR region among different gypsy elements we propose that the highly conserved LTR regions may be useful to amplify other retrotransposon sequences of the same or close retrotransposon family.

Retrotransposon hypomethylation in melanoma and expression of a placenta-specific gene.

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Erin C Macaulay

Full Text Available In the human placenta, DNA hypomethylation permits the expression of retrotransposon-derived genes that are normally silenced by methylation in somatic tissues. We previously identified hypomethylation of a retrotransposon-derived transcript of the voltage-gated potassium channel gene KCNH5 that is expressed only in human placenta. However, an RNA sequence from this placental-specific transcript has been reported in melanoma. This study examined the promoter methylation and expression of the retrotransposon-derived KCNH5 transcript in 25 melanoma cell lines to determine whether the acquisition of 'placental' epigenetic marks is a feature of melanoma. Methylation and gene expression analysis revealed hypomethylation of this retrotransposon in melanoma cell lines, particularly in those samples that express the placental KCNH5 transcript. Therefore we propose that hypomethylation of the placental-specific KCNH5 promoter is frequently associated with KCNH5 expression in melanoma cells. Our findings show that melanoma can develop hypomethylation of a retrotransposon-derived gene; a characteristic notably shared with the normal placenta.

Modeling the amplification dynamics of human Alu retrotransposons.

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Dale J Hedges

2005-09-01

Full Text Available Retrotransposons have had a considerable impact on the overall architecture of the human genome. Currently, there are three lineages of retrotransposons (Alu, L1, and SVA that are believed to be actively replicating in humans. While estimates of their copy number, sequence diversity, and levels of insertion polymorphism can readily be obtained from existing genomic sequence data and population sampling, a detailed understanding of the temporal pattern of retrotransposon amplification remains elusive. Here we pose the question of whether, using genomic sequence and population frequency data from extant taxa, one can adequately reconstruct historical amplification patterns. To this end, we developed a computer simulation that incorporates several known aspects of primate Alu retrotransposon biology and accommodates sampling effects resulting from the methods by which mobile elements are typically discovered and characterized. By modeling a number of amplification scenarios and comparing simulation-generated expectations to empirical data gathered from existing Alu subfamilies, we were able to statistically reject a number of amplification scenarios for individual subfamilies, including that of a rapid expansion or explosion of Alu amplification at the time of human-chimpanzee divergence.

Modeling the amplification dynamics of human alu retrotransposons.

Directory of Open Access Journals (Sweden)

2005-09-01

Full Text Available Retrotransposons have had a considerable impact on the overall architecture of the human genome. Currently, there are three lineages of retrotransposons (Alu, L1, and SVA that are believed to be actively replicating in humans. While estimates of their copy number, sequence diversity, and levels of insertion polymorphism can readily be obtained from existing genomic sequence data and population sampling, a detailed understanding of the temporal pattern of retrotransposon amplification remains elusive. Here we pose the question of whether, using genomic sequence and population frequency data from extant taxa, one can adequately reconstruct historical amplification patterns. To this end, we developed a computer simulation that incorporates several known aspects of primate Alu retrotransposon biology and accommodates sampling effects resulting from the methods by which mobile elements are typically discovered and characterized. By modeling a number of amplification scenarios and comparing simulation-generated expectations to empirical data gathered from existing Alu subfamilies, we were able to statistically reject a number of amplification scenarios for individual subfamilies, including that of a rapid expansion or explosion of Alu amplification at the time of human-chimpanzee divergence.

Evolutionary genomics revealed interkingdom distribution of Tcn1-like chromodomain-containing Gypsy LTR retrotransposons among fungi and plants

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Blinov Alexander

2010-04-01

Full Text Available Abstract Background Chromodomain-containing Gypsy LTR retrotransposons or chromoviruses are widely distributed among eukaryotes and have been found in plants, fungi and vertebrates. The previous comprehensive survey of chromoviruses from mosses (Bryophyta suggested that genomes of non-seed plants contain the clade which is closely related to the retrotransposons from fungi. The origin, distribution and evolutionary history of this clade remained unclear mainly due to the absence of information concerning the diversity and distribution of LTR retrotransposons in other groups of non-seed plants as well as in fungal genomes. Results In present study we preformed in silico analysis of chromodomain-containing LTR retrotransposons in 25 diverse fungi and a number of plant species including spikemoss Selaginella moellendorffii (Lycopodiophyta coupled with an experimental survey of chromodomain-containing Gypsy LTR retrotransposons from diverse non-seed vascular plants (lycophytes, ferns, and horsetails. Our mining of Gypsy LTR retrotransposons in genomic sequences allowed identification of numerous families which have not been described previously in fungi. Two new well-supported clades, Galahad and Mordred, as well as several other previously unknown lineages of chromodomain-containing Gypsy LTR retrotransposons were described based on the results of PCR-mediated survey of LTR retrotransposon fragments from ferns, horsetails and lycophytes. It appeared that one of the clades, namely Tcn1 clade, was present in basidiomycetes and non-seed plants including mosses (Bryophyta and lycophytes (genus Selaginella. Conclusions The interkingdom distribution is not typical for chromodomain-containing LTR retrotransposons clades which are usually very specific for a particular taxonomic group. Tcn1-like LTR retrotransposons from fungi and non-seed plants demonstrated high similarity to each other which can be explained by strong selective constraints and the

LINE retrotransposon RNA is an essential structural and functional epigenetic component of a core neocentromeric chromatin.

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Anderly C Chueh

2009-01-01

Full Text Available We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10 containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A-binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A-binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A-binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A-associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation.

Fluorescent sensors based on bacterial fusion proteins

International Nuclear Information System (INIS)

Mateu, Batirtze Prats; Pum, Dietmar; Sleytr, Uwe B; Toca-Herrera, José L; Kainz, Birgit

2014-01-01

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

SREBP controls oxygen-dependent mobilization of retrotransposons in fission yeast.

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Alfica Sehgal

2007-08-01

Full Text Available Retrotransposons are mobile genetic elements that proliferate through an RNA intermediate. Transposons do not encode transcription factors and thus rely on host factors for mRNA expression and survival. Despite information regarding conditions under which elements are upregulated, much remains to be learned about the regulatory mechanisms or factors controlling retrotransposon expression. Here, we report that low oxygen activates the fission yeast Tf2 family of retrotransposons. Sre1, the yeast ortholog of the mammalian membrane-bound transcription factor sterol regulatory element binding protein (SREBP, directly induces the expression and mobilization of Tf2 retrotransposons under low oxygen. Sre1 binds to DNA sequences in the Tf2 long terminal repeat that functions as an oxygen-dependent promoter. We find that Tf2 solo long terminal repeats throughout the genome direct oxygen-dependent expression of adjacent coding and noncoding sequences, providing a potential mechanism for the generation of oxygen-dependent gene expression.

Stress-induced rearrangement of Fusarium retrotransposon sequences.

Science.gov (United States)

Anaya, N; Roncero, M I

1996-11-27

Rearrangement of fusarium oxysporum retrotransposon skippy was induced by growth in the presence of potassium chlorate. Three fungal strains, one sensitive to chlorate (Co60) and two resistant to chlorate and deficient for nitrate reductase (Co65 and Co94), were studied by Southern analysis of their genomic DNA. Polymorphism was detected in their hybridization banding pattern, relative to the wild type grown in the absence of chlorate, using various enzymes with or without restriction sites within the retrotransposon. Results were consistent with the assumption that three different events had occurred in strain Co60: genomic amplification of skippy yielding tandem arrays of the element, generation of new skippy sequences, and deletion of skippy sequences. Amplification of Co60 genomic DNA using the polymerase chain reaction and divergent primers derived from the retrotransposon generated a new band, corresponding to one long terminal repeat plus flanking sequences, that was not present in the wild-type strain. Molecular analysis of nitrate reductase-deficient mutants showed that generation and deletion of skippy sequences, but not genomic amplification in tandem repeats, had occurred in their genomes.

High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers

International Nuclear Information System (INIS)

Schellenberger, Pascale; Kaufmann, Rainer; Siebert, C. Alistair; Hagen, Christoph; Wodrich, Harald; Grünewald, Kay

2014-01-01

Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell. - Highlights: • Vitrified mammalian cell were imaged by fluorescence and electron cryo microscopy. • TetraSpeck fluorescence markers were added to correct shifts between cryo fluorescence channels. • FluoSpheres fiducials were used as reference points to assign new coordinates to cryoEM images. • Adenovirus particles were localised with an average correlation precision of 63 nm

High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers

Energy Technology Data Exchange (ETDEWEB)

Schellenberger, Pascale [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Kaufmann, Rainer [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU (United Kingdom); Siebert, C. Alistair; Hagen, Christoph [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Wodrich, Harald [Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, University of Bordeaux SEGALEN, 146 rue Leo Seignat, 33076 Bordeaux (France); Grünewald, Kay, E-mail: kay@strubi.ox.ac.uk [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

2014-08-01

Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell. - Highlights: • Vitrified mammalian cell were imaged by fluorescence and electron cryo microscopy. • TetraSpeck fluorescence markers were added to correct shifts between cryo fluorescence channels. • FluoSpheres fiducials were used as reference points to assign new coordinates to cryoEM images. • Adenovirus particles were localised with an average correlation precision of 63 nm.

BARE retrotransposons are translated and replicated via distinct RNA pools.

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Wei Chang

Full Text Available The replication of Long Terminal Repeat (LTR retrotransposons, which can constitute over 80% of higher plant genomes, resembles that of retroviruses. A major question for retrotransposons and retroviruses is how the two conflicting roles of their transcripts, in translation and reverse transcription, are balanced. Here, we show that the BARE retrotransposon, despite its organization into just one open reading frame, produces three distinct classes of transcripts. One is capped, polyadenylated, and translated, but cannot be copied into cDNA. The second is not capped or polyadenylated, but is destined for packaging and ultimate reverse transcription. The third class is capped, polyadenylated, and spliced to favor production of a subgenomic RNA encoding only Gag, the protein forming virus-like particles. Moreover, the BARE2 subfamily, which cannot synthesize Gag and is parasitic on BARE1, does not produce the spliced sub-genomic RNA for translation but does make the replication competent transcripts, which are packaged into BARE1 particles. To our knowledge, this is first demonstration of distinct RNA pools for translation and transcription for any retrotransposon.

Activation of an endogenous retrotransposon associated with epigenetic changes in Lotus japonicus

DEFF Research Database (Denmark)

Fukai, Eigo; Stougaard, Jens; Hayashi, Makoto

2013-01-01

Long terminal repeat retrotransposons occupy a large portion of genomes in flowering plants. In spite of their abundance, the majority are silenced and rarely transpose. One of the examples of a highly active retrotransposon is Lotus Retrotransposon 1(LORE1), of the model legume Lotus japonicus...... significance of LORE1 as a member of chromovirus, a chromodomain containing clade of the Gypsy superfamily. Then we discuss possibilities and methodologies for using endogenous transposable elements as mutagens to generate gene tagging populations in plants...

LTRsift: a graphical user interface for semi-automatic classification and postprocessing of de novo detected LTR retrotransposons.

Science.gov (United States)

Steinbiss, Sascha; Kastens, Sascha; Kurtz, Stefan

2012-11-07

Long terminal repeat (LTR) retrotransposons are a class of eukaryotic mobile elements characterized by a distinctive sequence similarity-based structure. Hence they are well suited for computational identification. Current software allows for a comprehensive genome-wide de novo detection of such elements. The obvious next step is the classification of newly detected candidates resulting in (super-)families. Such a de novo classification approach based on sequence-based clustering of transposon features has been proposed before, resulting in a preliminary assignment of candidates to families as a basis for subsequent manual refinement. However, such a classification workflow is typically split across a heterogeneous set of glue scripts and generic software (for example, spreadsheets), making it tedious for a human expert to inspect, curate and export the putative families produced by the workflow. We have developed LTRsift, an interactive graphical software tool for semi-automatic postprocessing of de novo predicted LTR retrotransposon annotations. Its user-friendly interface offers customizable filtering and classification functionality, displaying the putative candidate groups, their members and their internal structure in a hierarchical fashion. To ease manual work, it also supports graphical user interface-driven reassignment, splitting and further annotation of candidates. Export of grouped candidate sets in standard formats is possible. In two case studies, we demonstrate how LTRsift can be employed in the context of a genome-wide LTR retrotransposon survey effort. LTRsift is a useful and convenient tool for semi-automated classification of newly detected LTR retrotransposons based on their internal features. Its efficient implementation allows for convenient and seamless filtering and classification in an integrated environment. Developed for life scientists, it is helpful in postprocessing and refining the output of software for predicting LTR

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

Science.gov (United States)

Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

2015-12-15

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Label free selective detection of estriol using graphene oxide-based fluorescence sensor

Science.gov (United States)

Kushwaha, H. S.; Sao, Reshma; Vaish, Rahul

2014-07-01

Water-soluble and fluorescent Graphene oxide (GO) is biocompatible, easy, and economical to synthesize. Interestingly, GO is also capable of quenching fluorescence. On the basis of its fluorescence and quenching abilities, GO has been reported to serve as an energy acceptor in a fluorescence resonance energy transfer (FRET) sensor. GO-based FRET biosensors have been widely reported for sensing of proteins, nucleic acid, ATP (Adenosine triphosphate), etc. GO complexes with fluorescent dyes and enzymes have been used to sense metal ions. Graphene derivatives have been used for sensing endocrine-disrupting chemicals like bisphenols and chlorophenols with high sensitivity and good reproducibility. On this basis, a novel GO based fluorescent sensor has been successfully designed to detect estriol with remarkable selectivity and sensitivity. Estriol is one of the three estrogens in women and is considered to be medically important. Estriol content of maternal urine or plasma acts as an important screening marker for estimating foetal growth and development. In addition, estriol is also used as diagnostic marker for diseases like breast cancer, osteoporosis, neurodegenerative and cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, etc. In this present study, we report for the first time a rapid, sensitive with detection limit of 1.3 nM, selective and highly biocompatible method for label free detection of estriol under physiological conditions using fluorescence assay.

Envelope-like retrotransposons in the plant kingdom: evidence of their presence in gymnosperms (Pinus pinaster).

Science.gov (United States)

Miguel, Célia; Simões, Marta; Oliveira, Maria Margarida; Rocheta, Margarida

2008-11-01

Retroviruses differ from retrotransposons due to their infective capacity, which depends critically on the encoded envelope. Some plant retroelements contain domains reminiscent of the env of animal retroviruses but the number of such elements described to date is restricted to angiosperms. We show here the first evidence of the presence of putative env-like gene sequences in a gymnosperm species, Pinus pinaster (maritime pine). Using a degenerate primer approach for conserved domains of RNaseH gene, three clones from putative envelope-like retrotransposons (PpRT2, PpRT3, and PpRT4) were identified. The env-like sequences of P. pinaster clones are predicted to encode proteins with transmembrane domains. These sequences showed identity scores of up to 30% with env-like sequences belonging to different organisms. A phylogenetic analysis based on protein alignment of deduced aminoacid sequences revealed that these clones clustered with env-containing plant retrotransposons, as well as with retrotransposons from invertebrate organisms. The differences found among the sequences of maritime pine clones isolated here suggest the existence of different putative classes of env-like retroelements. The identification for the first time of env-like genes in a gymnosperm species may support the ancestrality of retroviruses among plants shedding light on their role in plant evolution.

A 21 000-year record of fluorescent organic matter markers in the WAIS Divide ice core

Science.gov (United States)

D'Andrilli, Juliana; Foreman, Christine M.; Sigl, Michael; Priscu, John C.; McConnell, Joseph R.

2017-05-01

Englacial ice contains a significant reservoir of organic material (OM), preserving a chronological record of materials from Earth's past. Here, we investigate if OM composition surveys in ice core research can provide paleoecological information on the dynamic nature of our Earth through time. Temporal trends in OM composition from the early Holocene extending back to the Last Glacial Maximum (LGM) of the West Antarctic Ice Sheet Divide (WD) ice core were measured by fluorescence spectroscopy. Multivariate parallel factor (PARAFAC) analysis is widely used to isolate the chemical components that best describe the observed variation across three-dimensional fluorescence spectroscopy (excitation-emission matrices; EEMs) assays. Fluorescent OM markers identified by PARAFAC modeling of the EEMs from the LGM (27.0-18.0 kyr BP; before present 1950) through the last deglaciation (LD; 18.0-11.5 kyr BP), to the mid-Holocene (11.5-6.0 kyr BP) provided evidence of different types of fluorescent OM composition and origin in the WD ice core over 21.0 kyr. Low excitation-emission wavelength fluorescent PARAFAC component one (C1), associated with chemical species similar to simple lignin phenols was the greatest contributor throughout the ice core, suggesting a strong signature of terrestrial OM in all climate periods. The component two (C2) OM marker, encompassed distinct variability in the ice core describing chemical species similar to tannin- and phenylalanine-like material. Component three (C3), associated with humic-like terrestrial material further resistant to biodegradation, was only characteristic of the Holocene, suggesting that more complex organic polymers such as lignins or tannins may be an ecological marker of warmer climates. We suggest that fluorescent OM markers observed during the LGM were the result of greater continental dust loading of lignin precursor (monolignol) material in a drier climate, with lower marine influences when sea ice extent was higher and

Retrotransposons and non-protein coding RNAs

DEFF Research Database (Denmark)

Mourier, Tobias; Willerslev, Eske

2009-01-01

does not merely represent spurious transcription. We review examples of functional RNAs transcribed from retrotransposons, and address the collection of non-protein coding RNAs derived from transposable element sequences, including numerous human microRNAs and the neuronal BC RNAs. Finally, we review...

An evolutionary arms race between KRAB zinc-finger genes ZNF91/93 and SVA/L1 retrotransposons

NARCIS (Netherlands)

Jacobs, F.M.J.; Greenberg, D.; Nguyen, N.; Haeussler, M.; Ewing, A.D.; Katzman, S.; Paten, B.; Salama, S.R.; Haussler, D.

2014-01-01

Throughout evolution primate genomes have been modified by waves of retrotransposon insertions1, 2, 3. For each wave, the host eventually finds a way to repress retrotransposon transcription and prevent further insertions. In mouse embryonic stem cells, transcriptional silencing of retrotransposons

LTRsift: a graphical user interface for semi-automatic classification and postprocessing of de novo detected LTR retrotransposons

Directory of Open Access Journals (Sweden)

Steinbiss Sascha

2012-11-01

Full Text Available Abstract Background Long terminal repeat (LTR retrotransposons are a class of eukaryotic mobile elements characterized by a distinctive sequence similarity-based structure. Hence they are well suited for computational identification. Current software allows for a comprehensive genome-wide de novo detection of such elements. The obvious next step is the classification of newly detected candidates resulting in (super-families. Such a de novo classification approach based on sequence-based clustering of transposon features has been proposed before, resulting in a preliminary assignment of candidates to families as a basis for subsequent manual refinement. However, such a classification workflow is typically split across a heterogeneous set of glue scripts and generic software (for example, spreadsheets, making it tedious for a human expert to inspect, curate and export the putative families produced by the workflow. Results We have developed LTRsift, an interactive graphical software tool for semi-automatic postprocessing of de novo predicted LTR retrotransposon annotations. Its user-friendly interface offers customizable filtering and classification functionality, displaying the putative candidate groups, their members and their internal structure in a hierarchical fashion. To ease manual work, it also supports graphical user interface-driven reassignment, splitting and further annotation of candidates. Export of grouped candidate sets in standard formats is possible. In two case studies, we demonstrate how LTRsift can be employed in the context of a genome-wide LTR retrotransposon survey effort. Conclusions LTRsift is a useful and convenient tool for semi-automated classification of newly detected LTR retrotransposons based on their internal features. Its efficient implementation allows for convenient and seamless filtering and classification in an integrated environment. Developed for life scientists, it is helpful in postprocessing and refining

Workflow for high-content, individual cell quantification of fluorescent markers from universal microscope data, supported by open source software.

Science.gov (United States)

Stockwell, Simon R; Mittnacht, Sibylle

2014-12-16

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators. Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software(1) to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes.

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Sudhir Kumar Rai

2017-12-01

Full Text Available Retroviruses and Long Terminal Repeat (LTR-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes.

Science.gov (United States)

Rai, Sudhir Kumar; Sangesland, Maya; Lee, Michael; Esnault, Caroline; Cui, Yujin; Chatterjee, Atreyi Ghatak; Levin, Henry L

2017-12-01

Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.

Switching of dominant retrotransposon silencing strategies from posttranscriptional to transcriptional mechanisms during male germ-cell development in mice.

Directory of Open Access Journals (Sweden)

Kota Inoue

2017-07-01

Full Text Available Mammalian genomes harbor millions of retrotransposon copies, some of which are transpositionally active. In mouse prospermatogonia, PIWI-interacting small RNAs (piRNAs combat retrotransposon activity to maintain the genomic integrity. The piRNA system destroys retrotransposon-derived RNAs and guides de novo DNA methylation at some retrotransposon promoters. However, it remains unclear whether DNA methylation contributes to retrotransposon silencing in prospermatogonia. We have performed comprehensive studies of DNA methylation and polyA(+ RNAs (transcriptome in developing male germ cells from Pld6/Mitopld and Dnmt3l knockout mice, which are defective in piRNA biogenesis and de novo DNA methylation, respectively. The Dnmt3l mutation greatly reduced DNA methylation levels at most retrotransposons, but its impact on their RNA abundance was limited in prospermatogonia. In Pld6 mutant germ cells, although only a few retrotransposons exhibited reduced DNA methylation, many showed increased expression at the RNA level. More detailed analysis of RNA sequencing, nascent RNA quantification, profiling of cleaved RNA ends, and the results obtained from double knockout mice suggest that PLD6 works mainly at the posttranscriptional level. The increase in retrotransposon expression was larger in Pld6 mutants than it was in Dnmt3l mutants, suggesting that RNA degradation by the piRNA system plays a more important role than does DNA methylation in prospermatogonia. However, DNA methylation had a long-term effect: hypomethylation caused by the Pld6 or Dnmt3l mutation resulted in increased retrotransposon expression in meiotic spermatocytes. Thus, posttranscriptional silencing plays an important role in the early stage of germ cell development, then transcriptional silencing becomes important in later stages. In addition, intergenic and intronic retrotransposon sequences, in particular those containing the antisense L1 promoters, drove ectopic expression of nearby

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

Science.gov (United States)

Bradford, Jolene A; Buller, Gayle; Suter, Michael; Ignatius, Michael; Beechem, Joseph M

2004-10-01

Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon

Human Retrotransposon Insertion Polymorphisms Are Associated with Health and Disease via Gene Regulatory Phenotypes

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Lu Wang

2017-08-01

Full Text Available The human genome hosts several active families of transposable elements (TEs, including the Alu, LINE-1, and SVA retrotransposons that are mobilized via reverse transcription of RNA intermediates. We evaluated how insertion polymorphisms generated by human retrotransposon activity may be related to common health and disease phenotypes that have been previously interrogated through genome-wide association studies (GWAS. To address this question, we performed a genome-wide screen for retrotransposon polymorphism disease associations that are linked to TE induced gene regulatory changes. Our screen first identified polymorphic retrotransposon insertions found in linkage disequilibrium (LD with single nucleotide polymorphisms that were previously associated with common complex diseases by GWAS. We further narrowed this set of candidate disease associated retrotransposon polymorphisms by identifying insertions that are located within tissue-specific enhancer elements. We then performed expression quantitative trait loci analysis on the remaining set of candidates in order to identify polymorphic retrotransposon insertions that are associated with gene expression changes in B-cells of the human immune system. This progressive and stringent screen yielded a list of six retrotransposon insertions as the strongest candidates for TE polymorphisms that lead to disease via enhancer-mediated changes in gene regulation. For example, we found an SVA insertion within a cell-type specific enhancer located in the second intron of the B4GALT1 gene. B4GALT1 encodes a glycosyltransferase that functions in the glycosylation of the Immunoglobulin G (IgG antibody in such a way as to convert its activity from pro- to anti-inflammatory. The disruption of the B4GALT1 enhancer by the SVA insertion is associated with down-regulation of the gene in B-cells, which would serve to keep the IgG molecule in a pro-inflammatory state. Consistent with this idea, the B4GALT1 enhancer

Different histories of two highly variable LTR retrotransposons in sunflower species.

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Mascagni, Flavia; Cavallini, Andrea; Giordani, Tommaso; Natali, Lucia

2017-11-15

In the Helianthus genus, very large intra- and interspecific variability related to two specific retrotransposons of Helianthus annuus (Helicopia and SURE) exists. When comparing these two sequences to sunflower sequence databases recently produced by our lab, the Helicopia family was shown to belong to the Maximus/SIRE lineage of the Sirevirus genus of the Copia superfamily, whereas the SURE element (whose superfamily was not even previously identified) was classified as a Gypsy element of the Ogre/Tat lineage of the Metavirus genus. Bioinformatic analysis of the two retrotransposon families revealed their genomic abundance and relative proliferation timing. The genomic abundance of these families differed significantly among 12 Helianthus species. The ratio between the abundance of long terminal repeats and their reverse transcriptases suggested that the SURE family has relatively more solo long terminal repeats than does Helicopia. Pairwise comparisons of Illumina reads encoding the reverse transcriptase domain indicated that SURE amplification may have occurred more recently than that of Helicopia. Finally, the analysis of population structure based on the SURE and Helicopia polymorphisms of 32 Helianthus species evidenced two subpopulations, which roughly corresponded to species of the Helianthus and Divaricati/Ciliares sections. However, a number of species showed an admixed structure, confirming the importance of interspecific hybridisation in the evolution of this genus. In general, these two retrotransposon families differentially contributed to interspecific variability, emphasising the need to refer to specific families when studying genome evolution. Copyright © 2017 Elsevier B.V. All rights reserved.

DIRS1-like retrotransposons are widely distributed among Decapoda and are particularly present in hydrothermal vent organisms

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Bonnivard Eric

2009-04-01

allowed for revealing for the first time a widespread distribution of these elements among a large phylum, here the order Decapoda. They also suggest some peculiar features of these retrotransposons in hydrothermal organisms where a great diversity of elements is already observed. Finally, this paper constitutes the first essential step which allows for considering further studies based on the dynamics of the DIRS1-like retrotransposons among several genomes.

Analysis of Hopi/Osr27 and Houba/Tos5/Osr13 retrotransposons in rice

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Gozde Yuzbasioglu

2016-03-01

Full Text Available We investigated Hopi/Osr27 (gypsy and Houba/Tos5/Osr13 (copy retrotransposon movements in 10-day-old roots and leaves of Oryza sativa cvs. Ipsala, Beser and Osmancik-97. Seeds from these three cultivars were germinated between filter papers in Petri dishes for 10 days. Three biologically independent (nonrelated seeds were germinated for each cultivar. Then, roots and leaves grown from the same rice plant were harvested and used for genomic DNA isolation. Inter-retrotransposon amplified polymorphism–polymerase chain reaction with suitable primers was performed with each DNA template to analyze the movements of Hopi/Osr27 and Houba/Tos5/Osr13 retrotransposons. Polymorphism ratios were evaluated both among cultivars and among roots and leaves from the same cultivar. The polymorphism ratios ranged from 0% to 17% for Hopi/Osr27 and from 10% to 87% for Houba/Tos5/Osr13. The obtained results at retrotransposon and varietal levels indicated that the retrotransposon type and genotype dependence are responsible for the occurrence of different variations. Transposable elements are very important for understanding the relationship between cultivars and evolution. Our findings are expected to contribute to the understanding of spontaneous genomic insertion events and their effects on the genetic and epigenetic changes during rice development.

Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

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Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

2005-04-01

This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

SERS and fluorescence-based ultrasensitive detection of mercury in water.

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Makam, Pandeeswar; Shilpa, Rohilla; Kandjani, Ahmad Esmaielzadeh; Periasamy, Selvakannan R; Sabri, Ylias Mohammad; Madhu, Chilakapati; Bhargava, Suresh Kumar; Govindaraju, Thimmaiah

2018-02-15

The development of reliable and ultrasensitive detection marker for mercury ions (Hg 2+ ) in drinking water is of great interest for toxicology assessment, environmental protection and human health. Although many Hg 2+ detection methods have been developed, only few offer sensitivities below 1pM. Herein, we describe a simple histidine (H) conjugated perylene diimide (PDI) bolaamphiphile (HPH) as a dual-responsive optical marker to develop highly selective and sensitive probe as visible (sol-to-gel transformation), fluorescence and SERS-based Hg 2+ sensor platform in the water. Remarkably, HPH as a SERS marker supported on Au deposited monodispersed nanospheres monolayers (Au-MNM) of polystyrene offers an unprecedented selectivity and the best ever reported detection limit (LOD) of 60 attomolar (aM, 0.01 parts-per-quadrillion (ppq)) for Hg 2+ in water. This is ten orders of magnitude lower than the United States Environmental Protection Agency (USEPA) tolerance limit of Hg 2+ in drinking water (10nM, 2 ppb). This simple and effective design principle of host-guest interactions driven fluorescence and SERS-based detection may inspire the future molecular engineering strategies for the development of ultrasensitive toxic analyte sensor platforms. Copyright © 2017 Elsevier B.V. All rights reserved.

Invisible marker based augmented reality system

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Park, Hanhoon; Park, Jong-Il

2005-07-01

Augmented reality (AR) has recently gained significant attention. The previous AR techniques usually need a fiducial marker with known geometry or objects of which the structure can be easily estimated such as cube. Placing a marker in the workspace of the user can be intrusive. To overcome this limitation, we present an AR system using invisible markers which are created/drawn with an infrared (IR) fluorescent pen. Two cameras are used: an IR camera and a visible camera, which are positioned in each side of a cold mirror so that their optical centers coincide with each other. We track the invisible markers using IR camera and visualize AR in the view of visible camera. Additional algorithms are employed for the system to have a reliable performance in the cluttered background. Experimental results are given to demonstrate the viability of the proposed system. As an application of the proposed system, the invisible marker can act as a Vision-Based Identity and Geometry (VBIG) tag, which can significantly extend the functionality of RFID. The invisible tag is the same as RFID in that it is not perceivable while more powerful in that the tag information can be presented to the user by direct projection using a mobile projector or by visualizing AR on the screen of mobile PDA.

Assessment of genetic diversity in Chinese eared pheasant using fluorescent-AFLP markers

DEFF Research Database (Denmark)

Li, Xiujuan; Zhu, Yaohong; Liu, Panqi

2010-01-01

on the list of the world’s threatened species. In this paper, 74 individuals from the four eared pheasant species were assessed for population genetic diversity by means of fluorescent-AFLP markers. A total of 429 AFLP peaks were amplified by 11 pairs of fluorescent EcoRI/TaqI primer combinations. Out of all...... using Jaccard’s similarity coefficients (SC) and the corresponding dendrogram. It was found that there was a moderate genetic distance between the four species (SC = 0.674–0.832). Brown eared pheasant was genetically closely related to blue eared pheasant (SC = 0.832), while white eared pheasant...

How a retrotransposon exploits the plant's heat stress response for its activation.

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Vladimir V Cavrak

2014-01-01

Full Text Available Retrotransposons are major components of plant and animal genomes. They amplify by reverse transcription and reintegration into the host genome but their activity is usually epigenetically silenced. In plants, genomic copies of retrotransposons are typically associated with repressive chromatin modifications installed and maintained by RNA-directed DNA methylation. To escape this tight control, retrotransposons employ various strategies to avoid epigenetic silencing. Here we describe the mechanism developed by ONSEN, an LTR-copia type retrotransposon in Arabidopsis thaliana. ONSEN has acquired a heat-responsive element recognized by plant-derived heat stress defense factors, resulting in transcription and production of full length extrachromosomal DNA under elevated temperatures. Further, the ONSEN promoter is free of CG and CHG sites, and the reduction of DNA methylation at the CHH sites is not sufficient to activate the element. Since dividing cells have a more pronounced heat response, the extrachromosomal ONSEN DNA, capable of reintegrating into the genome, accumulates preferentially in the meristematic tissue of the shoot. The recruitment of a major plant heat shock transcription factor in periods of heat stress exploits the plant's heat stress response to achieve the transposon's activation, making it impossible for the host to respond appropriately to stress without losing control over the invader.

Transcriptionally active LTR retrotransposons in Eucalyptus genus are differentially expressed and insertionally polymorphic.

Science.gov (United States)

Marcon, Helena Sanches; Domingues, Douglas Silva; Silva, Juliana Costa; Borges, Rafael Junqueira; Matioli, Fábio Filippi; Fontes, Marcos Roberto de Mattos; Marino, Celso Luis

2015-08-14

In Eucalyptus genus, studies on genome composition and transposable elements (TEs) are particularly scarce. Nearly half of the recently released Eucalyptus grandis genome is composed by retrotransposons and this data provides an important opportunity to understand TE dynamics in Eucalyptus genome and transcriptome. We characterized nine families of transcriptionally active LTR retrotransposons from Copia and Gypsy superfamilies in Eucalyptus grandis genome and we depicted genomic distribution and copy number in two Eucalyptus species. We also evaluated genomic polymorphism and transcriptional profile in three organs of five Eucalyptus species. We observed contrasting genomic and transcriptional behavior in the same family among different species. RLC_egMax_1 was the most prevalent family and RLC_egAngela_1 was the family with the lowest copy number. Most families of both superfamilies have their insertions occurring Eucalyptus species. Using EST analysis and qRT-PCRs, we observed transcriptional activity in several tissues and in all evaluated species. In some families, osmotic stress increases transcript values. Our strategy was successful in isolating transcriptionally active retrotransposons in Eucalyptus, and each family has a particular genomic and transcriptional pattern. Overall, our results show that retrotransposon activity have differentially affected genome and transcriptome among Eucalyptus species.

Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker.

Directory of Open Access Journals (Sweden)

Jan M Bruder

Full Text Available Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1 localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin's physiological response to UV radiation.

Mammalian-specific genomic functions: Newly acquired traits generated by genomic imprinting and LTR retrotransposon-derived genes in mammals.

Science.gov (United States)

Kaneko-Ishino, Tomoko; Ishino, Fumitoshi

2015-01-01

Mammals, including human beings, have evolved a unique viviparous reproductive system and a highly developed central nervous system. How did these unique characteristics emerge in mammalian evolution, and what kinds of changes did occur in the mammalian genomes as evolution proceeded? A key conceptual term in approaching these issues is "mammalian-specific genomic functions", a concept covering both mammalian-specific epigenetics and genetics. Genomic imprinting and LTR retrotransposon-derived genes are reviewed as the representative, mammalian-specific genomic functions that are essential not only for the current mammalian developmental system, but also mammalian evolution itself. First, the essential roles of genomic imprinting in mammalian development, especially related to viviparous reproduction via placental function, as well as the emergence of genomic imprinting in mammalian evolution, are discussed. Second, we introduce the novel concept of "mammalian-specific traits generated by mammalian-specific genes from LTR retrotransposons", based on the finding that LTR retrotransposons served as a critical driving force in the mammalian evolution via generating mammalian-specific genes.

Repetitive DNA and Plant Domestication: Variation in Copy Number and Proximity to Genes of LTR-Retrotransposons among Wild and Cultivated Sunflower (Helianthus annuus) Genotypes.

Science.gov (United States)

Mascagni, Flavia; Barghini, Elena; Giordani, Tommaso; Rieseberg, Loren H; Cavallini, Andrea; Natali, Lucia

2015-11-24

The sunflower (Helianthus annuus) genome contains a very large proportion of transposable elements, especially long terminal repeat retrotransposons. However, knowledge on the retrotransposon-related variability within this species is still limited. We used next-generation sequencing (NGS) technologies to perform a quantitative and qualitative survey of intraspecific variation of the retrotransposon fraction of the genome across 15 genotypes--7 wild accessions and 8 cultivars--of H. annuus. By mapping the Illumina reads of the 15 genotypes onto a library of sunflower long terminal repeat retrotransposons, we observed considerable variability in redundancy among genotypes, at both superfamily and family levels. In another analysis, we mapped Illumina paired reads to two sets of sequences, that is, long terminal repeat retrotransposons and protein-encoding sequences, and evaluated the extent of retrotransposon proximity to genes in the sunflower genome by counting the number of paired reads in which one read mapped to a retrotransposon and the other to a gene. Large variability among genotypes was also ascertained for retrotransposon proximity to genes. Both long terminal repeat retrotransposon redundancy and proximity to genes varied among retrotransposon families and also between cultivated and wild genotypes. Such differences are discussed in relation to the possible role of long terminal repeat retrotransposons in the domestication of sunflower. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Microprocessor controls the activity of mammalian retrotransposons

DEFF Research Database (Denmark)

Heras, Sara R.; Macias, Sara; Plass, Mireya

2013-01-01

RNA biogenesis, also recognizes and binds RNAs derived from human long interspersed element 1 (LINE-1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions...

340nm UV LED excitation in time-resolved fluorescence system for europium-based immunoassays detection

DEFF Research Database (Denmark)

Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter

2017-01-01

In immunoassay analyzers for in-vitro diagnostics, Xenon flash lamps have been widely used as excitation light sources. Recent advancements in UV LED technology and its advantages over the flash lamps such as smaller footprint, better wall-plug efficiency, narrow emission spectrum......, and no significant afterglow, have made them attractive light sources for gated detection systems. In this paper, we report on the implementation of a 340 nm UV LED based time-resolved fluorescence system based on europium chelate as a fluorescent marker. The system performance was tested with the immunoassay based...... on the cardiac marker, TnI. The same signal-to-noise ratio as for the flash lamp based system was obtained, operating the LED below specified maximum current. The background counts of the system and its main contributors were measured and analyzed. The background of the system of the LED based unit was improved...

Genome-wide analysis of LTR-retrotransposons in oil palm.

Science.gov (United States)

Beulé, Thierry; Agbessi, Mawussé Dt; Dussert, Stephane; Jaligot, Estelle; Guyot, Romain

2015-10-15

The oil palm (Elaeis guineensis Jacq.) is a major cultivated crop and the world's largest source of edible vegetable oil. The genus Elaeis comprises two species E. guineensis, the commercial African oil palm and E. oleifera, which is used in oil palm genetic breeding. The recent publication of both the African oil palm genome assembly and the first draft sequence of its Latin American relative now allows us to tackle the challenge of understanding the genome composition, structure and evolution of these palm genomes through the annotation of their repeated sequences. In this study, we identified, annotated and compared Transposable Elements (TE) from the African and Latin American oil palms. In a first step, Transposable Element databases were built through de novo detection in both genome sequences then the TE content of both genomes was estimated. Then putative full-length retrotransposons with Long Terminal Repeats (LTRs) were further identified in the E. guineensis genome for characterization of their structural diversity, copy number and chromosomal distribution. Finally, their relative expression in several tissues was determined through in silico analysis of publicly available transcriptome data. Our results reveal a congruence in the transpositional history of LTR retrotransposons between E. oleifera and E. guineensis, especially the Sto-4 family. Also, we have identified and described 583 full-length LTR-retrotransposons in the Elaeis guineensis genome. Our work shows that these elements are most likely no longer mobile and that no recent insertion event has occurred. Moreover, the analysis of chromosomal distribution suggests a preferential insertion of Copia elements in gene-rich regions, whereas Gypsy elements appear to be evenly distributed throughout the genome. Considering the high proportion of LTR retrotransposon in the oil palm genome, our work will contribute to a greater understanding of their impact on genome organization and evolution

Identification of retrotransposon-like sequences in Iranian river buffalo

African Journals Online (AJOL)

ONOS

2010-03-29

% of a genome (Waterston et al., 2002). Mobile elements can be divided into two classes: Class I includes retrotransposons and class II includes DNA tran- sposons ... including dog, cat, horse, cattle, donkey, kangaroo, etc.

Long Terminal Repeat Retrotransposon Content in Eight Diploid Sunflower Species Inferred from Next-Generation Sequence Data

Science.gov (United States)

Tetreault, Hannah M.; Ungerer, Mark C.

2016-01-01

The most abundant transposable elements (TEs) in plant genomes are Class I long terminal repeat (LTR) retrotransposons represented by superfamilies gypsy and copia. Amplification of these superfamilies directly impacts genome structure and contributes to differential patterns of genome size evolution among plant lineages. Utilizing short-read Illumina data and sequence information from a panel of Helianthus annuus (sunflower) full-length gypsy and copia elements, we explore the contribution of these sequences to genome size variation among eight diploid Helianthus species and an outgroup taxon, Phoebanthus tenuifolius. We also explore transcriptional dynamics of these elements in both leaf and bud tissue via RT-PCR. We demonstrate that most LTR retrotransposon sublineages (i.e., families) display patterns of similar genomic abundance across species. A small number of LTR retrotransposon sublineages exhibit lineage-specific amplification, particularly in the genomes of species with larger estimated nuclear DNA content. RT-PCR assays reveal that some LTR retrotransposon sublineages are transcriptionally active across all species and tissue types, whereas others display species-specific and tissue-specific expression. The species with the largest estimated genome size, H. agrestis, has experienced amplification of LTR retrotransposon sublineages, some of which have proliferated independently in other lineages in the Helianthus phylogeny. PMID:27233667

Evolutionary characterization of Ty3/gypsy-like LTR retrotransposons in the parasitic cestode Echinococcus granulosus.

Science.gov (United States)

Bae, Young-An

2016-11-01

Cyclophyllidean cestodes including Echinococcus granulosus have a smaller genome and show characteristics such as loss of the gut, a segmented body plan, and accelerated growth rate in hosts compared with other tissue-invading helminths. In an effort to address the molecular mechanism relevant to genome shrinkage, the evolutionary status of long-terminal-repeat (LTR) retrotransposons, which are known as the most potent genomic modulators, was investigated in the E. granulosus draft genome. A majority of the E. granulosus LTR retrotransposons were classified into a novel characteristic clade, named Saci-2, of the Ty3/gypsy family, while the remaining elements belonged to the CsRn1 clade of identical family. Their nucleotide sequences were heavily corrupted by frequent base substitutions and segmental losses. The ceased mobile activity of the major retrotransposons and the following intrinsic DNA loss in their inactive progenies might have contributed to decrease in genome size. Apart from the degenerate copies, a gag gene originating from a CsRn1-like element exhibited substantial evidences suggesting its domestication including a preserved coding profile and transcriptional activity, the presence of syntenic orthologues in cestodes, and selective pressure acting on the gene. To my knowledge, the endogenized gag gene is reported for the first time in invertebrates, though its biological function remains elusive.

The RNAPII-CTD Maintains Genome Integrity through Inhibition of Retrotransposon Gene Expression and Transposition.

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Maria J Aristizabal

2015-10-01

Full Text Available RNA polymerase II (RNAPII contains a unique C-terminal domain that is composed of heptapeptide repeats and which plays important regulatory roles during gene expression. RNAPII is responsible for the transcription of most protein-coding genes, a subset of non-coding genes, and retrotransposons. Retrotransposon transcription is the first step in their multiplication cycle, given that the RNA intermediate is required for the synthesis of cDNA, the material that is ultimately incorporated into a new genomic location. Retrotransposition can have grave consequences to genome integrity, as integration events can change the gene expression landscape or lead to alteration or loss of genetic information. Given that RNAPII transcribes retrotransposons, we sought to investigate if the RNAPII-CTD played a role in the regulation of retrotransposon gene expression. Importantly, we found that the RNAPII-CTD functioned to maintaining genome integrity through inhibition of retrotransposon gene expression, as reducing CTD length significantly increased expression and transposition rates of Ty1 elements. Mechanistically, the increased Ty1 mRNA levels in the rpb1-CTD11 mutant were partly due to Cdk8-dependent alterations to the RNAPII-CTD phosphorylation status. In addition, Cdk8 alone contributed to Ty1 gene expression regulation by altering the occupancy of the gene-specific transcription factor Ste12. Loss of STE12 and TEC1 suppressed growth phenotypes of the RNAPII-CTD truncation mutant. Collectively, our results implicate Ste12 and Tec1 as general and important contributors to the Cdk8, RNAPII-CTD regulatory circuitry as it relates to the maintenance of genome integrity.

Chemosensors and biosensors based on polyelectrolyte microcapsules containing fluorescent dyes and enzymes.

Science.gov (United States)

Kazakova, Lyubov I; Shabarchina, Lyudmila I; Anastasova, Salzitsa; Pavlov, Anton M; Vadgama, Pankaj; Skirtach, Andre G; Sukhorukov, Gleb B

2013-02-01

The concept of enzyme-assisted substrate sensing based on use of fluorescent markers to detect the products of enzymatic reaction has been investigated by fabrication of micron-scale polyelectrolyte capsules containing enzymes and dyes in one entity. Microcapsules approximately 5 μm in size entrap glucose oxidase or lactate oxidase, with peroxidase, together with the corresponding markers Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (Ru(dpp)) complex and dihydrorhodamine 123 (DHR123), which are sensitive to oxygen and hydrogen peroxide, respectively. These capsules are produced by co-precipitation of calcium carbonate particles with the enzyme followed by layer-by-layer assembly of polyelectrolytes over the surface of the particles and incorporation of the dye in the capsule interior or in the multilayer shell. After dissolution of the calcium carbonate the enzymes and dyes remain in the multilayer capsules. In this study we produced enzyme-containing microcapsules sensitive to glucose and lactate. Calibration curves based on fluorescence intensity of Ru(dpp) and DHR123 were linearly dependent on substrate concentration, enabling reliable sensing in the millimolar range. The main advantages of using these capsules with optical recording is the possibility of building single capsule-based sensors. The response from individual capsules was observed by confocal microscopy as increasing fluorescence intensity of the capsule on addition of lactate at millimolar concentrations. Because internalization of the micron-sized multi-component capsules was feasible, they could be further optimized for in-situ intracellular sensing and metabolite monitoring on the basis of fluorescence reporting.

Retrotransposon Proliferation Coincident with the Evolution of Dioecy in Asparagus.

Science.gov (United States)

Harkess, Alex; Mercati, Francesco; Abbate, Loredana; McKain, Michael; Pires, J Chris; Sala, Tea; Sunseri, Francesco; Falavigna, Agostino; Leebens-Mack, Jim

2016-09-08

Current phylogenetic sampling reveals that dioecy and an XY sex chromosome pair evolved once, or possibly twice, in the genus Asparagus Although there appear to be some lineage-specific polyploidization events, the base chromosome number of 2n = 2× = 20 is relatively conserved across the Asparagus genus. Regardless, dioecious species tend to have larger genomes than hermaphroditic species. Here, we test whether this genome size expansion in dioecious species is related to a polyploidization and subsequent chromosome fusion, or to retrotransposon proliferation in dioecious species. We first estimate genome sizes, or use published values, for four hermaphrodites and four dioecious species distributed across the phylogeny, and show that dioecious species typically have larger genomes than hermaphroditic species. Utilizing a phylogenomic approach, we find no evidence for ancient polyploidization contributing to increased genome sizes of sampled dioecious species. We do find support for an ancient whole genome duplication (WGD) event predating the diversification of the Asparagus genus. Repetitive DNA content of the four hermaphroditic and four dioecious species was characterized based on randomly sampled whole genome shotgun sequencing, and common elements were annotated. Across our broad phylogenetic sampling, Ty-1 Copia retroelements, in particular, have undergone a marked proliferation in dioecious species. In the absence of a detectable WGD event, retrotransposon proliferation is the most likely explanation for the precipitous increase in genome size in dioecious Asparagus species. Copyright © 2016 Harkess et al.

In vivo quantification of fluorescent molecular markers in real-time by ratio Imaging for diagnostic screening and image-guided surgery

NARCIS (Netherlands)

Bogaards, A.; Sterenborg, H. J. C. M.; Trachtenberg, J.; Wilson, B. C.; Lilge, L.

2007-01-01

Future applications of "molecular diagnostic screening" and "molecular image-guided surgery" will demand images of molecular markers with high resolution and high throughput (similar to >= 30 frames/second). MRI, SPECT, PET, optical fluorescence tomography, hyper-spectral fluorescence imaging, and

Retrotransposons. An RNA polymerase III subunit determines sites of retrotransposon integration.

Science.gov (United States)

Bridier-Nahmias, Antoine; Tchalikian-Cosson, Aurélie; Baller, Joshua A; Menouni, Rachid; Fayol, Hélène; Flores, Amando; Saïb, Ali; Werner, Michel; Voytas, Daniel F; Lesage, Pascale

2015-05-01

Mobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host. Copyright © 2015, American Association for the Advancement of Science.

The Influence of LINE-1 and SINE Retrotransposons on Mammalian Genomes.

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Richardson, Sandra R; Doucet, Aurélien J; Kopera, Huira C; Moldovan, John B; Garcia-Perez, José Luis; Moran, John V

2015-04-01

Transposable elements have had a profound impact on the structure and function of mammalian genomes. The retrotransposon Long INterspersed Element-1 (LINE-1 or L1), by virtue of its replicative mobilization mechanism, comprises ∼17% of the human genome. Although the vast majority of human LINE-1 sequences are inactive molecular fossils, an estimated 80-100 copies per individual retain the ability to mobilize by a process termed retrotransposition. Indeed, LINE-1 is the only active, autonomous retrotransposon in humans and its retrotransposition continues to generate both intra-individual and inter-individual genetic diversity. Here, we briefly review the types of transposable elements that reside in mammalian genomes. We will focus our discussion on LINE-1 retrotransposons and the non-autonomous Short INterspersed Elements (SINEs) that rely on the proteins encoded by LINE-1 for their mobilization. We review cases where LINE-1-mediated retrotransposition events have resulted in genetic disease and discuss how the characterization of these mutagenic insertions led to the identification of retrotransposition-competent LINE-1s in the human and mouse genomes. We then discuss how the integration of molecular genetic, biochemical, and modern genomic technologies have yielded insight into the mechanism of LINE-1 retrotransposition, the impact of LINE-1-mediated retrotransposition events on mammalian genomes, and the host cellular mechanisms that protect the genome from unabated LINE-1-mediated retrotransposition events. Throughout this review, we highlight unanswered questions in LINE-1 biology that provide exciting opportunities for future research. Clearly, much has been learned about LINE-1 and SINE biology since the publication of Mobile DNA II thirteen years ago. Future studies should continue to yield exciting discoveries about how these retrotransposons contribute to genetic diversity in mammalian genomes.

LTR retrotransposon landscape in Medicago truncatula: more rapid removal than in rice

Directory of Open Access Journals (Sweden)

Liu Jin-Song

2008-08-01

Full Text Available Abstract Background Long terminal repeat retrotransposons (LTR elements are ubiquitous Eukaryotic TEs that transpose through RNA intermediates. Accounting for significant proportion of many plant genomes, LTR elements have been well established as one of the major forces underlying the evolution of plant genome size, structure and function. The accessibility of more than 40% of genomic sequences of the model legume Medicago truncatula (Mt has made the comprehensive study of its LTR elements possible. Results We use a newly developed tool LTR_FINDER to identify LTR retrotransposons in the Mt genome and detect 526 full-length elements as well as a great number of copies related to them. These elements constitute about 9.6% of currently available genomic sequences. They are classified into 85 families of which 64 are reported for the first time. The majority of the LTR retrotransposons belong to either Copia or Gypsy superfamily and the others are categorized as TRIMs or LARDs by their length. We find that the copy-number of Copia-like families is 3 times more than that of Gypsy-like ones but the latter contribute more to the genome. The analysis of PBS and protein-coding domain structure of the LTR families reveals that they tend to use only 4–5 types of tRNAs and many families have quite conservative ORFs besides known TE domains. For several important families, we describe in detail their abundance, conservation, insertion time and structure. We investigate the amplification-deletion pattern of the elements and find that the detectable full-length elements are relatively young and most of them were inserted within the last 0.52 MY. We also estimate that more than ten million bp of the Mt genomic sequences have been removed by the deletion of LTR elements and the removal of the full-length structures in Mt has been more rapid than in rice. Conclusion This report is the first comprehensive description and analysis of LTR retrotransposons in the

Colour and surface fluorescence development and their relationship with Maillard reaction markers as influenced by structural changes during cornflakes production.

Science.gov (United States)

Farroni, Abel; Buera, María Del Pilar

2012-12-01

The aim of this work was to study colour and surface fluorescence development in relation to the chemical markers for the Maillard reaction at the cooking, flaking and toasting stages of cornflake production process. Colour was measured by a calibrated computer vision system. Surface fluorescence was measured on compressed samples. Aqueous extracted Maillard reaction markers (hydroxymethylfurfural, carboxymethyl-lysine, absorbance at 420nm and total fluorescence) were measured on protease hydrolyzed samples. Sample microstructure was observed by scanning electron microscopy. During cooking the colour coordinates L(∗) and b(∗) decreased and a(∗) increased. After flaking, the samples appeared lighter, while the pigment concentration, fluorescence and hydroxymethylfurfural did not change. Toasting generated bubbles in the matrix and L(∗) apparently increased, although brown pigment concentration increased. Pigment concentration did not correlate with surface colour due to the destruction or generation of interfaces. Surface and microstructure effects can be avoided by milling and compressing the samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

Assessment of genetic variation for the LINE-1 retrotransposon from next generation sequence data

Directory of Open Access Journals (Sweden)

Ramos Kenneth

2010-10-01

Full Text Available Abstract Background In humans, copies of the Long Interspersed Nuclear Element 1 (LINE-1 retrotransposon comprise 21% of the reference genome, and have been shown to modulate expression and produce novel splice isoforms of transcripts from genes that span or neighbor the LINE-1 insertion site. Results In this work, newly released pilot data from the 1000 Genomes Project is analyzed to detect previously unreported full length insertions of the retrotransposon LINE-1. By direct analysis of the sequence data, we have identified 22 previously unreported LINE-1 insertion sites within the sequence data reported for a mother/father/daughter trio. Conclusions It is demonstrated here that next generation sequencing data, as well as emerging high quality datasets from individual genome projects allow us to assess the amount of heterogeneity with respect to the LINE-1 retrotransposon amongst humans, and provide us with a wealth of testable hypotheses as to the impact that this diversity may have on the health of individuals and populations.

Effects of As2O3 on DNA methylation, genomic instability, and LTR retrotransposon polymorphism in Zea mays.

Science.gov (United States)

Erturk, Filiz Aygun; Aydin, Murat; Sigmaz, Burcu; Taspinar, M Sinan; Arslan, Esra; Agar, Guleray; Yagci, Semra

2015-12-01

Arsenic is a well-known toxic substance on the living organisms. However, limited efforts have been made to study its DNA methylation, genomic instability, and long terminal repeat (LTR) retrotransposon polymorphism causing properties in different crops. In the present study, effects of As2O3 (arsenic trioxide) on LTR retrotransposon polymorphism and DNA methylation as well as DNA damage in Zea mays seedlings were investigated. The results showed that all of arsenic doses caused a decreasing genomic template stability (GTS) and an increasing Random Amplified Polymorphic DNAs (RAPDs) profile changes (DNA damage). In addition, increasing DNA methylation and LTR retrotransposon polymorphism characterized a model to explain the epigenetically changes in the gene expression were also found. The results of this experiment have clearly shown that arsenic has epigenetic effect as well as its genotoxic effect. Especially, the increasing of polymorphism of some LTR retrotransposon under arsenic stress may be a part of the defense system against the stress.

Young, intact and nested retrotransposons are abundant in the onion and asparagus genomes.

Science.gov (United States)

Vitte, C; Estep, M C; Leebens-Mack, J; Bennetzen, J L

2013-09-01

Although monocotyledonous plants comprise one of the two major groups of angiosperms and include >65 000 species, comprehensive genome analysis has been focused mainly on the Poaceae (grass) family. Due to this bias, most of the conclusions that have been drawn for monocot genome evolution are based on grasses. It is not known whether these conclusions apply to many other monocots. To extend our understanding of genome evolution in the monocots, Asparagales genomic sequence data were acquired and the structural properties of asparagus and onion genomes were analysed. Specifically, several available onion and asparagus bacterial artificial chromosomes (BACs) with contig sizes >35 kb were annotated and analysed, with a particular focus on the characterization of long terminal repeat (LTR) retrotransposons. The results reveal that LTR retrotransposons are the major components of the onion and garden asparagus genomes. These elements are mostly intact (i.e. with two LTRs), have mainly inserted within the past 6 million years and are piled up into nested structures. Analysis of shotgun genomic sequence data and the observation of two copies for some transposable elements (TEs) in annotated BACs indicates that some families have become particularly abundant, as high as 4-5 % (asparagus) or 3-4 % (onion) of the genome for the most abundant families, as also seen in large grass genomes such as wheat and maize. Although previous annotations of contiguous genomic sequences have suggested that LTR retrotransposons were highly fragmented in these two Asparagales genomes, the results presented here show that this was largely due to the methodology used. In contrast, this current work indicates an ensemble of genomic features similar to those observed in the Poaceae.

Novel fluorescent sequence-related amplified polymorphism(FSRAP markers for the construction of a genetic linkage map of wheat(Triticum aestivum L.

Directory of Open Access Journals (Sweden)

Zhao Lingbo

2017-01-01

Full Text Available Novel fluorescent sequence-related amplified polymorphism (FSRAP markers were developed based on the SRAP molecular marker. Then, the FSRAP markers were used to construct the genetic map of a wheat (Triticum aestivumL. recombinant inbred line population derived from a Chuanmai 42×Chuannong 16 cross. Reproducibility and polymorphism tests indicated that the FSRAP markers have repeatability and better reflect the polymorphism of wheat varieties compared with SRAP markers. A total of 430 polymorphic loci between Chuanmai 42 and Chuannong 16 were detected with 189 FSRAP primer combinations. A total of 281 FSARP markers and 39 SSR markers re classified into 20 linkage groups. The maps spanned a total length of 2499.3cM with an average distance of 7.81cM between markers. A total of 201 markers were mapped on the B genome and covered a distance of 1013cM. On the A genome, 84 markers were mapped and covered a distance of 849.6cM. On the D genome, however, only 35 markers were mapped and covered a distance of 636.7cM. No FSRAP markers were distributed on the 7D chromosome. The results of the present study revealed that the novel FSRAP markers can be used to generate dense, uniform genetic maps of wheat.

Links between human LINE-1 retrotransposons and hepatitis virus-related hepatocellular carcinoma

Science.gov (United States)

Honda, Tomoyuki

2016-05-01

Hepatocellular carcinoma (HCC) accounts for approximately 80% of liver cancers, the third most frequent cause of cancer mortality. The most prevalent risk factors for HCC are infections by hepatitis B or hepatitis C virus. Findings suggest that hepatitis virus-related HCC might be a cancer in which LINE-1 retrotransposons, often termed L1, activity plays a potential role. Firstly, hepatitis viruses can suppress host defense factors that also control L1 mobilization. Secondly, many recent studies also have indicated that hypomethylation of L1 affects the prognosis of HCC patients. Thirdly, endogenous L1 retrotransposition was demonstrated to activate oncogenic pathways in HCC. Fourthly, several L1 chimeric transcripts with host or viral genes are found in hepatitis virus-related HCC. Such lines of evidence suggest a linkage between L1 retrotransposons and hepatitis virus-related HCC. Here, I briefly summarize current understandings of the association between hepatitis virus-related HCC and L1. Then, I discuss potential mechanisms of how hepatitis viruses drive the development of HCC via L1 retrotransposons. An increased understanding of the contribution of L1 to hepatitis virus-related HCC may provide unique insights related to the development of novel therapeutics for this disease.

340nm UV LED excitation in time-resolved fluorescence system for europium-based immunoassays detection

Science.gov (United States)

Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Pedersen, Christian

2017-02-01

In immunoassay analyzers for in-vitro diagnostics, Xenon flash lamps have been widely used as excitation light sources. Recent advancements in UV LED technology and its advantages over the flash lamps such as smaller footprint, better wall-plug efficiency, narrow emission spectrum, and no significant afterglow, have made them attractive light sources for gated detection systems. In this paper, we report on the implementation of a 340 nm UV LED based time-resolved fluorescence system based on europium chelate as a fluorescent marker. The system performance was tested with the immunoassay based on the cardiac marker, TnI. The same signal-to-noise ratio as for the flash lamp based system was obtained, operating the LED below specified maximum current. The background counts of the system and its main contributors were measured and analyzed. The background of the system of the LED based unit was improved by 39% compared to that of the Xenon flash lamp based unit, due to the LEDs narrower emission spectrum and longer pulse width. Key parameters of the LED system are discussed to further optimize the signal-to-noise ratio and signal-to-background, and hence the sensitivity of the instrument.

Small supernumerary marker chromosome derived from proximal p-arm of chromosome 2: identification by fluorescent in situ hybridization.

Science.gov (United States)

Lasan Trcić, Ruzica; Hitrec, Vlasta; Letica, Ljiljana; Cuk, Mario; Begović, Davor

2003-08-01

Conventional cytogenetics detected an interstitial deletion of proximal region of p-arm of chromosome 2 in a 6-month-old boy with a phenotype slightly resembling Down's syndrome. The deletion was inherited from the father, whose karyotype revealed a small ring-shaped marker chromosome, in addition to interstitial deletion. Fluorescence in situ hybridization identified the marker, which consisted of the proximal region of the p-arm of chromosome 2, including a part of its centromere. This case shows that molecular cytogenetic analysis can reveal the mechanism of the formation of the marker chromosome.

Infection-Induced Retrotransposon-Derived Noncoding RNAs Enhance Herpesviral Gene Expression via the NF-κB Pathway.

Directory of Open Access Journals (Sweden)

John Karijolich

Full Text Available Short interspersed nuclear elements (SINEs are highly abundant, RNA polymerase III-transcribed noncoding retrotransposons that are silenced in somatic cells but activated during certain stresses including viral infection. How these induced SINE RNAs impact the host-pathogen interaction is unknown. Here we reveal that during murine gammaherpesvirus 68 (MHV68 infection, rapidly induced SINE RNAs activate the antiviral NF-κB signaling pathway through both mitochondrial antiviral-signaling protein (MAVS-dependent and independent mechanisms. However, SINE RNA-based signaling is hijacked by the virus to enhance viral gene expression and replication. B2 RNA expression stimulates IKKβ-dependent phosphorylation of the major viral lytic cycle transactivator protein RTA, thereby enhancing its activity and increasing progeny virion production. Collectively, these findings suggest that SINE RNAs participate in the innate pathogen response mechanism, but that herpesviruses have evolved to co-opt retrotransposon activation for viral benefit.

Forward and reverse genetics: The LORE1 retrotransposon insertion mutants

DEFF Research Database (Denmark)

Fukai, Eigo; Malolepszy, Anna; Sandal, Niels Nørgaard

2014-01-01

The endogenous Lotus retrotransposon 1 (LORE1) transposes in the germ line of Lotus japonicus plants that carry an active element. This feature of LORE1 has been exploited for generation of a large non-transgenic insertion mutant population, where insertions have been annotated using next-generat...

Plant centromeric retrotransposons: a structural and cytogenetic perspective

Czech Academy of Sciences Publication Activity Database

Neumann, Pavel; Navrátilová, Alice; Koblížková, Andrea; Kejnovský, Eduard; Hřibová, Eva; Hobza, Roman; Widmer, A.; Doležel, Jaroslav; Macas, Jiří

2011-01-01

Roč. 2, č. 4 (2011), s. 1-16 ISSN 1759-8753 R&D Projects: GA AV ČR KJB500960802; GA MŠk(CZ) LC06004; GA ČR GA522/09/0083 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z50380511 Keywords : plant chromosomes * retrotransposons * cytogenetic perspective Subject RIV: EB - Genetics ; Molecular Biology

Fluorescence and colour as markers for the Maillard reaction in milk-cereal based infants foods during storage

OpenAIRE

Bosch, Lourdes; Alegría, Amparo; Farre, Rosaura; Clemente Marín, Gonzalo

2007-01-01

[EN] Free and total fluorescence compounds and color formation were measured in three different milk-cereal based infant foods stored at 25, 30 and 37 degrees C for 9 months to evaluate the advanced and final stages of the Maillard reaction. Milk-cereal infant foods containing honey (13) or fruits (C) had fluorescent values higher than sample (A) without them. This difference could be ascribed to the higher monosaccharide (fructose and/or glucose) content of (B) and (C), which could increase ...

Retrotransposon-Encoded Reverse Transcriptase in the Genesis, Progression and Cellular Plasticity of Human Cancer

International Nuclear Information System (INIS)

Sinibaldi-Vallebona, Paola; Matteucci, Claudia; Spadafora, Corrado

2011-01-01

LINE-1 (Long Interspersed Nuclear Elements) and HERVs (Human Endogenous Retroviruses) are two families of autonomously replicating retrotransposons that together account for about 28% of the human genome. Genes harbored within LINE-1 and HERV retrotransposons, particularly those encoding the reverse transcriptase (RT) enzyme, are generally expressed at low levels in differentiated cells, but their expression is upregulated in transformed cells and embryonic tissues. Here we discuss a recently discovered RT-dependent mechanism that operates in tumorigenesis and reversibly modulates phenotypic and functional variations associated with tumor progression. Downregulation of active LINE-1 elements drastically reduces the tumorigenic potential of cancer cells, paralleled by reduced proliferation and increased differentiation. Pharmacological RT inhibitors (e.g., nevirapine and efavirenz) exert similar effects on tumorigenic cell lines, both in culture and in animal models. The HERV-K family play a distinct complementary role in stress-dependent transition of melanoma cells from an adherent, non-aggressive, to a non-adherent, highly malignant, growth phenotype. In synthesis, the retrotransposon-encoded RT is increasingly emerging as a key regulator of tumor progression and a promising target in a novel anti-cancer therapy

Molecular characterization of Fagaceae species using inter-primer binding site (iPBS) markers.

Science.gov (United States)

Coutinho, João Paulo; Carvalho, Ana; Martín, Antonio; Lima-Brito, José

2018-04-01

Retrotransposons (RTNs) contribute for genome evolution, influencing its size and structure. We investigated the utility of the RTN-based markers inter-primer binding site (iPBS) for the molecular characterization of 25 Fagaceae species from genera Castanea, Fagus and Quercus. The assessment of genetic diversity, relationships and structure, as well as taxonomic classification of Fagaceae based on molecular data is important for definition of conservation, forestry management strategies and discrimination among natural hybrids and their parents since natural hybridization may increase with the climate changes. Here, iPBS primers designed by other authors were tested alone and combined. Some of them were discriminative, revealed polymorphism within and among taxa allowing the production of a total of 150 iPBS markers. In addition, several monomorphic iPBS markers were also amplified in each taxon. The UPGMA dendrogram based on the pooled iPBS data revealed 27% of genetic similarity among species. The individuals were clustered per genus and most of the oaks per infrageneric group corroborating the adopted taxonomy. Globally, the iPBS markers demonstrated suitability for DNA fingerprinting, determination of phylogenies and taxonomic discrimination in Fagaceae, and could constitute a useful and alternative tool for germplasm characterization, and for definition of conservation strategies and forestry management. Moreover, these markers would be useful for fingerprinting natural hybrids that share morphological similarities with their parents. Since iPBS markers could also enable insights about RTNs evolution, an eventual correlation among iPBS polymorphism, variability of RTN insertions and/or genome size in Fagaceae is discussed.

Advances in plant gene-targeted and functional markers: a review

Directory of Open Access Journals (Sweden)

Poczai Péter

2013-02-01

Full Text Available Abstract Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and β-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information

Advances in plant gene-targeted and functional markers: a review

Science.gov (United States)

2013-01-01

Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and β-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information. Such techniques have the

Use of the IRAP marker to study genetic variability in Pseudocercospora fijiensis populations.

Science.gov (United States)

de Queiroz, Casley Borges; Santana, Mateus Ferreira; da Silva, Gilvan Ferreira; Mizubuti, Eduardo Seiti Gomide; de Araújo, Elza Fernandes; de Queiroz, Marisa Vieira

2014-03-01

Pseudocercospora fijiensis is the etiological agent of black Sigatoka, which is currently considered as one of the most destructive banana diseases in all locations where it occurs. It is estimated that a large portion of the P. fijiensis genome consists of transposable elements, which allows researchers to use transposon-based molecular markers in the analysis of genetic variability in populations of this pathogen. In this context, the inter-retrotransposon-amplified polymorphism (IRAP) was used to study the genetic variability in P. fijiensis populations from different hosts and different geographical origins in Brazil. A total of 22 loci were amplified and 77.3 % showed a polymorphism. Cluster analysis revealed two major groups in Brazil. The observed genetic diversity (H E) was 0.22, and through molecular analysis of variance, it was determined that the greatest genetic variability occurs within populations. The discriminant analysis of principal components revealed no structuring related to the geographical origin of culture of the host. The IRAP-based marker system is a suitable tool for the study of genetic variability in P. fijiensis.

Identification of a non-LTR retrotransposon from the gypsy moth

Science.gov (United States)

K.J. Garner; J.M. Slavicek

1999-01-01

A family of highly repetitive elements, named LDT1, has been identified in the gypsy moth, Lymantria dispar. The complete element is 5.4 kb in length and lacks long-terminal repeats, The element contains two open reading frames with a significant amino acid sequence similarity to several non-LTR retrotransposons. The first open reading frame contains...

Determinants of Genomic RNA Encapsidation in the Saccharomyces cerevisiae Long Terminal Repeat Retrotransposons Ty1 and Ty3

Directory of Open Access Journals (Sweden)

Katarzyna Pachulska-Wieczorek

2016-07-01

Full Text Available Long-terminal repeat (LTR retrotransposons are transposable genetic elements that replicate intracellularly, and can be considered progenitors of retroviruses. Ty1 and Ty3 are the most extensively characterized LTR retrotransposons whose RNA genomes provide the template for both protein translation and genomic RNA that is packaged into virus-like particles (VLPs and reverse transcribed. Genomic RNAs are not divided into separate pools of translated and packaged RNAs, therefore their trafficking and packaging into VLPs requires an equilibrium between competing events. In this review, we focus on Ty1 and Ty3 genomic RNA trafficking and packaging as essential steps of retrotransposon propagation. We summarize the existing knowledge on genomic RNA sequences and structures essential to these processes, the role of Gag proteins in repression of genomic RNA translation, delivery to VLP assembly sites, and encapsidation.

Identification of SSR and retrotransposon-based molecular markers ...

Indian Academy of Sciences (India)

SOLEIMANI GEZELJEH ALI

2018-03-13

Mar 13, 2018 ... A 10 × 10 simple lattice design with two replications was employed to measure the ..... was applied for the analysis population structure by using a software package of ... polygenic system (figures 1 and 2). The minimum, max-.

Ancient Origin of the U2 Small Nuclear RNA Gene-Targeting Non-LTR Retrotransposons Utopia.

Science.gov (United States)

Kojima, Kenji K; Jurka, Jerzy

2015-01-01

Most non-long terminal repeat (non-LTR) retrotransposons encoding a restriction-like endonuclease show target-specific integration into repetitive sequences such as ribosomal RNA genes and microsatellites. However, only a few target-specific lineages of non-LTR retrotransposons are distributed widely and no lineage is found across the eukaryotic kingdoms. Here we report the most widely distributed lineage of target sequence-specific non-LTR retrotransposons, designated Utopia. Utopia is found in three supergroups of eukaryotes: Amoebozoa, SAR, and Opisthokonta. Utopia is inserted into a specific site of U2 small nuclear RNA genes with different strength of specificity for each family. Utopia families from oomycetes and wasps show strong target specificity while only a small number of Utopia copies from reptiles are flanked with U2 snRNA genes. Oomycete Utopia families contain an "archaeal" RNase H domain upstream of reverse transcriptase (RT), which likely originated from a plant RNase H gene. Analysis of Utopia from oomycetes indicates that multiple lineages of Utopia have been maintained inside of U2 genes with few copy numbers. Phylogenetic analysis of RT suggests the monophyly of Utopia, and it likely dates back to the early evolution of eukaryotes.

Study on fluorescence of Maillard reaction compounds in breakfast cereals.

Science.gov (United States)

Delgado-Andrade, Cristina; Rufián-Henares, José A; Morales, Francisco J

2006-09-01

During the advanced stage of the Maillard reaction (MR) in food processing and cooking, Amadori rearrangement products undergo dehydration and fission and fluorescent substances are formed. Free and total (free + linked to the protein backbone) fluorescence (FIC) due to Maillard compounds in 60 commercial breakfast cereals was evaluated. Pronase was used for efficient release of linked fluorescent Maillard compounds from the protein backbone. Results were correlated with some heat-induced markers of the extent of the MR or sugar caramelisation during cereal processing, such as hydroxymethylfurfural, furfural, glucosilisomaltol and furosine. The effect of sample composition (dietary-fibre added, protein, etc.) on levels of FIC, expressed as fluorescence intensity (FI) per milligram of sample, is discussed. FIC is significantly correlated to the protein content of the sample and fluorescent Maillard compounds are mainly linked to the protein backbone. The ratio of total-FIC to free-FIC was 10.4-fold for corn-based, wheat-based and multicereal-based breakfast cereals but significantly higher in rice-based samples. Addition of dietary fibre or honey increased the FIC values. Data support the usefulness of FIC measurement as an unspecific heat-induced marker in breakfast cereals.

Retrotransposon-associated long non-coding RNAs in mice and men

Czech Academy of Sciences Publication Activity Database

Ganesh, Sravya; Svoboda, Petr

2016-01-01

Roč. 468, č. 6 (2016), s. 1049-1060 ISSN 0031-6768 R&D Projects: GA ČR(CZ) GBP305/12/G034; GA MŠk LO1419 EU Projects: European Commission 647403; European Commission 607720 Institutional support: RVO:68378050 Keywords : lncRNA * Retrotransposon * line * sine * ltr * MaLR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.156, year: 2016

Fickian-Based Empirical Approach for Diffusivity Determination in Hollow Alginate-Based Microfibers Using 2D Fluorescence Microscopy and Comparison with Theoretical Predictions

Directory of Open Access Journals (Sweden)

Maryam Mobed-Miremadi

2014-12-01

Full Text Available Hollow alginate microfibers (od = 1.3 mm, id = 0.9 mm, th = 400 µm, L = 3.5 cm comprised of 2% (w/v medium molecular weight alginate cross-linked with 0.9 M CaCl2 were fabricated to model outward diffusion capture by 2D fluorescent microscopy. A two-fold comparison of diffusivity determination based on real-time diffusion of Fluorescein isothiocyanate molecular weight (FITC MW markers was conducted using a proposed Fickian-based approach in conjunction with a previously established numerical model developed based on spectrophotometric data. Computed empirical/numerical (Dempiricial/Dnumerical diffusivities characterized by small standard deviations for the 4-, 70- and 500-kDa markers expressed in m2/s are (1.06 × 10−9 ± 1.96 × 10−10/(2.03 × 10−11, (5.89 × 10−11 ± 2.83 × 10−12/(4.6 × 10−12 and (4.89 × 10−12 ± 3.94 × 10−13/(1.27 × 10−12, respectively, with the discrimination between the computation techniques narrowing down as a function of MW. The use of the numerical approach is recommended for fluorescence-based measurements as the standard computational method for effective diffusivity determination until capture rates (minimum 12 fps for the 4-kDa marker and the use of linear instead of polynomial interpolating functions to model temporal intensity gradients have been proven to minimize the extent of systematic errors associated with the proposed empirical method.

Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.

Science.gov (United States)

Locati, Mauro D; Pagano, Johanna F B; Ensink, Wim A; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Zhu, Kongju; Spaink, Herman P; Girard, Geneviève; Rauwerda, Han; Jonker, Martijs J; Dekker, Rob J; Breit, Timo M

2017-04-01

5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci. © 2017 Locati et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Handheld Fluorescence Microscopy based Flow Analyzer.

Science.gov (United States)

Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

2016-03-01

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

Radiative transport-based frequency-domain fluorescence tomography

International Nuclear Information System (INIS)

Joshi, Amit; Rasmussen, John C; Sevick-Muraca, Eva M; Wareing, Todd A; McGhee, John

2008-01-01

We report the development of radiative transport model-based fluorescence optical tomography from frequency-domain boundary measurements. The coupled radiative transport model for describing NIR fluorescence propagation in tissue is solved by a novel software based on the established Attila(TM) particle transport simulation platform. The proposed scheme enables the prediction of fluorescence measurements with non-contact sources and detectors at a minimal computational cost. An adjoint transport solution-based fluorescence tomography algorithm is implemented on dual grids to efficiently assemble the measurement sensitivity Jacobian matrix. Finally, we demonstrate fluorescence tomography on a realistic computational mouse model to locate nM to μM fluorophore concentration distributions in simulated mouse organs

Comparative genomic analysis reveals multiple long terminal repeats, lineage-specific amplification, and frequent interelement recombination for Cassandra retrotransposon in pear (Pyrus bretschneideri Rehd.).

Science.gov (United States)

Yin, Hao; Du, Jianchang; Li, Leiting; Jin, Cong; Fan, Lian; Li, Meng; Wu, Jun; Zhang, Shaoling

2014-06-04

Cassandra transposable elements belong to a specific group of terminal-repeat retrotransposons in miniature (TRIM). Although Cassandra TRIM elements have been found in almost all vascular plants, detailed investigations on the nature, abundance, amplification timeframe, and evolution have not been performed in an individual genome. We therefore conducted a comprehensive analysis of Cassandra retrotransposons using the newly sequenced pear genome along with four other Rosaceae species, including apple, peach, mei, and woodland strawberry. Our data reveal several interesting findings for this particular retrotransposon family: 1) A large number of the intact copies contain three, four, or five long terminal repeats (LTRs) (∼20% in pear); 2) intact copies and solo LTRs with or without target site duplications are both common (∼80% vs. 20%) in each genome; 3) the elements exhibit an overall unbiased distribution among the chromosomes; 4) the elements are most successfully amplified in pear (5,032 copies); and 5) the evolutionary relationships of these elements vary among different lineages, species, and evolutionary time. These results indicate that Cassandra retrotransposons contain more complex structures (elements with multiple LTRs) than what we have known previously, and that frequent interelement unequal recombination followed by transposition may play a critical role in shaping and reshaping host genomes. Thus this study provides insights into the property, propensity, and molecular mechanisms governing the formation and amplification of Cassandra retrotransposons, and enhances our understanding of the structural variation, evolutionary history, and transposition process of LTR retrotransposons in plants. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays

OpenAIRE

Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Petersen, Paul Michael; Pedersen, Christian

2016-01-01

We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng/L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flas...

8-oxo-7,8-dihydroguanine level - the DNA oxidative stress marker - recognized by fluorescence image analysis in sporadic uterine adenocarcinomas in women.

Science.gov (United States)

Postawski, Krzysztof; Przadka-Rabaniuk, Dorota; Piersiak, Tomasz

2013-01-01

In the case of carcinogenesis in human endometrium no information exists on tissue concentration of 8-oxo-7,8-dihydroguanine, the DNA oxidative stress marker This was the main reason to undertake the investigation of this DNA modification in human uterine estrogen-dependent tissue cancers. In order to estimate the level of oxidative damage, 8-oxo-7,8-dihydroguanine was determined directly in cells of tissue microscope slides using OxyDNA Assay Kit, Fluorometric. Cells were investigated under confocal microscope. Images of individual cells were captured by computer-interfaced digital photography and analyzed for fluorescence intensities (continuous inverted 8-bit gray-scale = 0 [black]-255 [white]). Fluorescence scores were calculated for each of 13 normal endometrial samples and 31 uterine adenocarcinoma specimens. Finally the level of the oxidative stress marker was also analyzed according to histological and clinical features of the neoplasms. The obtained data revealed that: 8-oxo-7,8-dihydroguanine levels were higher in uterine adenocarcinomas than in normal endometrial samples (48,32 vs. 38,64; p<0,001); in contrast to normal endometrium there was no correlation between age and DNA oxidative modification content in uterine cancer; highest mean fluorescence intensity was recognized in G2 endometrial adenocarcinomas; level of 8-oxo-7,8-dihydroguanine does not depend on Body Mass Index (BMI) and cancer uterine wall infiltration or tumor FIGO stage. Our study indicates that accumulation of the oxidized DNA base may contribute to the development of endometrial neoplasia, however oxidative DNA damage does not seem to increase with tumor progression.

Identification and chromosomal localization of the monkey retrotransposon in Mesa sp

Czech Academy of Sciences Publication Activity Database

Balint-Kurti, P.; Clendennen, S.; Doleželová, Marie; Valárik, Miroslav; Doležel, Jaroslav; Beetham, G. M.

2000-01-01

Roč. 263, č. 6 (2000), s. 908-915 ISSN 0026-8925 R&D Projects: GA ČR GV521/96/K117; GA AV ČR IAA5020803; GA MŠk ME 376 Institutional research plan: CEZ:AV0Z5038910 Keywords : In situ hybridization * chromosomal localization * monkey retrotransposon Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.462, year: 2000

LTR retrotransposon dynamics in the evolution of the olive (Olea europaea) genome

Czech Academy of Sciences Publication Activity Database

Barghini, E.; Natali, L.; Giordani, T.; Cossu, R.M.; Scalabrin, S.; Cattonaro, F.; Šimková, Hana; Vrána, Jan; Doležel, Jaroslav; Morgante, M.; Cavallini, A.

2015-01-01

Roč. 22, č. 1 (2015), s. 91-100 ISSN 1340-2838 R&D Projects: GA ČR GBP501/12/G090; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : LTR retrotransposons * next-generation sequencing * olive Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.267, year: 2015

Genetic barcoding with fluorescent proteins for multiplexed applications.

Science.gov (United States)

Smurthwaite, Cameron A; Williams, Wesley; Fetsko, Alexandra; Abbadessa, Darin; Stolp, Zachary D; Reed, Connor W; Dharmawan, Andre; Wolkowicz, Roland

2015-04-14

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded 'indefinitely'. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Recent expansion of heat-activated retrotransposons in the coral symbiont Symbiodinium microadriaticum

KAUST Repository

Chen, Jit Ern

2017-10-20

Rising sea surface temperature is the main cause of global coral reef decline. Abnormally high temperatures trigger the breakdown of the symbiotic association between corals and their photosynthetic symbionts in the genus Symbiodinium. Higher genetic variation resulting from shorter generation times has previously been proposed to provide increased adaptability to Symbiodinium compared to the host. Retrotransposition is a significant source of genetic variation in eukaryotes and some transposable elements are specifically expressed under adverse environmental conditions. We present transcriptomic and phylogenetic evidence for the existence of heat stress-activated Ty1-copia-type LTR retrotransposons in the coral symbiont Symbiodinium microadriaticum. Genome-wide analyses of emergence patterns of these elements further indicate recent expansion events in the genome of S. microadriaticum. Our findings suggest that acute temperature increases can activate specific retrotransposons in the Symbiodinium genome with potential impacts on the rate of retrotransposition and the generation of genetic variation under heat stress.The ISME Journal advance online publication, 20 October 2017; doi:10.1038/ismej.2017.179.

The role of retrotransposons in gene family expansions: insights from the mouse Abp gene family.

Science.gov (United States)

Janoušek, Václav; Karn, Robert C; Laukaitis, Christina M

2013-05-29

Retrotransposons have been suggested to provide a substrate for non-allelic homologous recombination (NAHR) and thereby promote gene family expansion. Their precise role, however, is controversial. Here we ask whether retrotransposons contributed to the recent expansions of the Androgen-binding protein (Abp) gene families that occurred independently in the mouse and rat genomes. Using dot plot analysis, we found that the most recent duplication in the Abp region of the mouse genome is flanked by L1Md_T elements. Analysis of the sequence of these elements revealed breakpoints that are the relicts of the recombination that caused the duplication, confirming that the duplication arose as a result of NAHR using L1 elements as substrates. L1 and ERVII retrotransposons are considerably denser in the Abp regions than in one Mb flanking regions, while other repeat types are depleted in the Abp regions compared to flanking regions. L1 retrotransposons preferentially accumulated in the Abp gene regions after lineage separation and roughly followed the pattern of Abp gene expansion. By contrast, the proportion of shared vs. lineage-specific ERVII repeats in the Abp region resembles the rest of the genome. We confirmed the role of L1 repeats in Abp gene duplication with the identification of recombinant L1Md_T elements at the edges of the most recent mouse Abp gene duplication. High densities of L1 and ERVII repeats were found in the Abp gene region with abrupt transitions at the region boundaries, suggesting that their higher densities are tightly associated with Abp gene duplication. We observed that the major accumulation of L1 elements occurred after the split of the mouse and rat lineages and that there is a striking overlap between the timing of L1 accumulation and expansion of the Abp gene family in the mouse genome. Establishing a link between the accumulation of L1 elements and the expansion of the Abp gene family and identification of an NAHR-related breakpoint in

A widespread occurrence of extra open reading frames in plant Ty3/gypsy retrotransposons

Czech Academy of Sciences Publication Activity Database

Steinbauerová, Veronika; Neumann, Pavel; Novák, Petr; Macas, Jiří

2011-01-01

Roč. 139, 11-12 (2011), s. 1543-1555 ISSN 0016-6707 Institutional research plan: CEZ:AV0Z50510513 Keywords : Additional ORFs * LTR retrotransposons * Repetitive DNA * Plant genome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.148, year: 2011

Facilitating in vivo tumor localization by principal component analysis based on dynamic fluorescence molecular imaging

Science.gov (United States)

Gao, Yang; Chen, Maomao; Wu, Junyu; Zhou, Yuan; Cai, Chuangjian; Wang, Daliang; Luo, Jianwen

2017-09-01

Fluorescence molecular imaging has been used to target tumors in mice with xenograft tumors. However, tumor imaging is largely distorted by the aggregation of fluorescent probes in the liver. A principal component analysis (PCA)-based strategy was applied on the in vivo dynamic fluorescence imaging results of three mice with xenograft tumors to facilitate tumor imaging, with the help of a tumor-specific fluorescent probe. Tumor-relevant features were extracted from the original images by PCA and represented by the principal component (PC) maps. The second principal component (PC2) map represented the tumor-related features, and the first principal component (PC1) map retained the original pharmacokinetic profiles, especially of the liver. The distribution patterns of the PC2 map of the tumor-bearing mice were in good agreement with the actual tumor location. The tumor-to-liver ratio and contrast-to-noise ratio were significantly higher on the PC2 map than on the original images, thus distinguishing the tumor from its nearby fluorescence noise of liver. The results suggest that the PC2 map could serve as a bioimaging marker to facilitate in vivo tumor localization, and dynamic fluorescence molecular imaging with PCA could be a valuable tool for future studies of in vivo tumor metabolism and progression.

Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors

DEFF Research Database (Denmark)

Sempere, Lorenzo F; Preis, Meir; Yezefski, Todd

2010-01-01

of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine clinical...

New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis.

Science.gov (United States)

Volkov, D A; Dergousova, N I; Rumsh, L D

2004-06-01

This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.

The Sinbad retrotransposon from the genome of the human blood fluke, Schistosoma mansoni, and the distribution of related Pao-like elements

Directory of Open Access Journals (Sweden)

Morales Maria E

2005-02-01

Full Text Available Abstract Background Of the major families of long terminal repeat (LTR retrotransposons, the Pao/BEL family is probably the least well studied. It is becoming apparent that numerous LTR retrotransposons and other mobile genetic elements have colonized the genome of the human blood fluke, Schistosoma mansoni. Results A proviral form of Sinbad, a new LTR retrotransposon, was identified in the genome of S. mansoni. Phylogenetic analysis indicated that Sinbad belongs to one of five discreet subfamilies of Pao/BEL like elements. BLAST searches of whole genomes and EST databases indicated that members of this clade occurred in species of the Insecta, Nematoda, Echinodermata and Chordata, as well as Platyhelminthes, but were absent from all plants, fungi and lower eukaryotes examined. Among the deuterostomes examined, only aquatic species harbored these types of elements. All four species of nematode examined were positive for Sinbad sequences, although among insect and vertebrate genomes, some were positive and some negative. The full length, consensus Sinbad retrotransposon was 6,287 bp long and was flanked at its 5'- and 3'-ends by identical LTRs of 386 bp. Sinbad displayed a triple Cys-His RNA binding motif characteristic of Gag of Pao/BEL-like elements, followed by the enzymatic domains of protease, reverse transcriptase (RT, RNAseH, and integrase, in that order. A phylogenetic tree of deduced RT sequences from 26 elements revealed that Sinbad was most closely related to an unnamed element from the zebrafish Danio rerio and to Saci-1, also from S. mansoni. It was also closely related to Pao from Bombyx mori and to Ninja of Drosophila simulans. Sinbad was only distantly related to the other schistosome LTR retrotransposons Boudicca, Gulliver, Saci-2, Saci-3, and Fugitive, which are gypsy-like. Southern hybridization and bioinformatics analyses indicated that there were about 50 copies of Sinbad in the S. mansoni genome. The presence of ESTs

Retrotransposons as regulators of gene expression.

Science.gov (United States)

Elbarbary, Reyad A; Lucas, Bronwyn A; Maquat, Lynne E

2016-02-12

Transposable elements (TEs) are both a boon and a bane to eukaryotic organisms, depending on where they integrate into the genome and how their sequences function once integrated. We focus on two types of TEs: long interspersed elements (LINEs) and short interspersed elements (SINEs). LINEs and SINEs are retrotransposons; that is, they transpose via an RNA intermediate. We discuss how LINEs and SINEs have expanded in eukaryotic genomes and contribute to genome evolution. An emerging body of evidence indicates that LINEs and SINEs function to regulate gene expression by affecting chromatin structure, gene transcription, pre-mRNA processing, or aspects of mRNA metabolism. We also describe how adenosine-to-inosine editing influences SINE function and how ongoing retrotransposition is countered by the body's defense mechanisms. Copyright © 2016, American Association for the Advancement of Science.

Development of Thinopyrum ponticum-specific molecular markers and FISH probes based on SLAF-seq technology.

Science.gov (United States)

Liu, Liqin; Luo, Qiaoling; Teng, Wan; Li, Bin; Li, Hongwei; Li, Yiwen; Li, Zhensheng; Zheng, Qi

2018-05-01

Based on SLAF-seq, 67 Thinopyrum ponticum-specific markers and eight Th. ponticum-specific FISH probes were developed, and these markers and probes could be used for detection of alien chromatin in a wheat background. Decaploid Thinopyrum ponticum (2n = 10x = 70) is a valuable gene reservoir for wheat improvement. Identification of Th. ponticum introgression would facilitate its transfer into diverse wheat genetic backgrounds and its practical utilization in wheat improvement. Based on specific-locus-amplified fragment sequencing (SLAF-seq) technology, 67 new Th. ponticum-specific molecular markers and eight Th. ponticum-specific fluorescence in situ hybridization (FISH) probes have been developed from a tiny wheat-Th. ponticum translocation line. These newly developed molecular markers allowed the detection of Th. ponticum DNA in a variety of materials specifically and steadily at high throughput. According to the hybridization signal pattern, the eight Th. ponticum-specific probes could be divided into two groups. The first group including five dispersed repetitive sequence probes could identify Th. ponticum chromatin more sensitively and accurately than genomic in situ hybridization (GISH). Whereas the second group having three tandem repetitive sequence probes enabled the discrimination of Th. ponticum chromosomes together with another clone pAs1 in wheat-Th. ponticum partial amphiploid Xiaoyan 68.

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting.

Directory of Open Access Journals (Sweden)

Shunsuke Suzuki

2007-04-01

Full Text Available Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10 is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii, but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus, suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5' region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

Assessing diversity among traditional Greek and foreign eggplant cultivars using molecular markers and morphometrical descriptors

International Nuclear Information System (INIS)

Augustinos, A.A.; Petropoulos, C.; Karasoulou, V.; Bletsos, F.; Papasotiropoulos, V.

2016-01-01

Eggplant is a widely cultivated vegetable crop of great economic importance. Its long lasting history of domestication, selection and breeding has led to the development of numerous cultivars with variable traits. In the present study, we assessed the diversity levels within and among eleven Greek and foreign cultivars, using 22 morphological descriptors and two different classes of molecular markers (retrotransposon microsatellite amplified polymorphism-REMAP markers and nuclear microsatellites). Our results, in accordance with other studies in the field showed: a) the limited levels of genetic polymorphism within the cultivars; b) the high morphological and genetic divergence existing among them as indicated by the genetic distance values calculated, which could be attributed to selection, inbreeding and bottleneck effects; and c) the lack of concordance among morphological descriptors and molecular markers. Despite these, our analysis showed that the utilization of combinations of markers is an effective method for the characterization of plant material providing also useful diagnostic tools for the identification and authentication of the selected Greek cultivars.

Assessing diversity among traditional Greek and foreign eggplant cultivars using molecular markers and morphometrical descriptors

Energy Technology Data Exchange (ETDEWEB)

Augustinos, A.A.; Petropoulos, C.; Karasoulou, V.; Bletsos, F.; Papasotiropoulos, V.

2016-07-01

Eggplant is a widely cultivated vegetable crop of great economic importance. Its long lasting history of domestication, selection and breeding has led to the development of numerous cultivars with variable traits. In the present study, we assessed the diversity levels within and among eleven Greek and foreign cultivars, using 22 morphological descriptors and two different classes of molecular markers (retrotransposon microsatellite amplified polymorphism-REMAP markers and nuclear microsatellites). Our results, in accordance with other studies in the field showed: a) the limited levels of genetic polymorphism within the cultivars; b) the high morphological and genetic divergence existing among them as indicated by the genetic distance values calculated, which could be attributed to selection, inbreeding and bottleneck effects; and c) the lack of concordance among morphological descriptors and molecular markers. Despite these, our analysis showed that the utilization of combinations of markers is an effective method for the characterization of plant material providing also useful diagnostic tools for the identification and authentication of the selected Greek cultivars.

Fluorescent Biosensors Based on Single-Molecule Counting.

Science.gov (United States)

Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

2016-09-20

Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the

Insertion of a solo LTR retrotransposon associates with spur mutations in 'Red Delicious' apple (Malus × domestica).

Science.gov (United States)

Han, Mengxue; Sun, Qibao; Zhou, Junyong; Qiu, Huarong; Guo, Jing; Lu, Lijuan; Mu, Wenlei; Sun, Jun

2017-09-01

Insertion of a solo LTR, which possesses strong bidirectional, stem-specific promoter activities, is associated with the evolution of a dwarfing apple spur mutation. Spur mutations in apple scions revolutionized global apple production. Since long terminal repeat (LTR) retrotransposons are tightly related to natural mutations, inter-retrotransposon-amplified polymorphism technique and genome walking were used to find sequences in the apple genome based on these LTRs. In 'Red Delicious' spur mutants, a novel, 2190-bp insertion was identified as a spur-specific, solo LTR (sLTR) located at the 1038th nucleotide of another sLTR, which was 1536 bp in length. This insertion-within-an-insertion was localized within a preexisting Gypsy-50 retrotransposon at position 3,762,767 on chromosome 4. The analysis of transcriptional activity of the two sLTRs (the 2190- and 1536-bp inserts) indicated that the 2190-bp sLTR is a promoter, capable of bidirectional transcription. GUS expression in the 2190-bp-sense and 2190-bp-antisense transgenic lines was prominent in stems. In contrast, no promoter activity from either the sense or the antisense strand of the 1536-bp sLTR was detected. From ~150 kb of DNA on each side of the 2190 bp, sLTR insertion site, corresponding to 300 kb of the 'Golden Delicious' genome, 23 genes were predicted. Ten genes had predicted functions that could affect shoot development. This first report, of a sLTR insertion associated with the evolution of apple spur mutation, will facilitate apple breeding, cloning of spur-related genes, and discovery of mechanisms behind dwarf habit.

Fluorescence diffuse tomography of small animals with DsRed2 fluorescent protein

Science.gov (United States)

Turchin, I. V.; Plehanov, V. I.; Orlova, A. G.; Kamenskiy, V. A.; Kleshnin, M. S.; Shirmanova, M. V.; Shakhova, N. M.; Balalaeva, I. V.; Savitskiy, A. P.

2006-05-01

Fluorescent compounds are used as markers to diagnose oncological diseases, to study molecular processes typical for carcinogenesis, and to investigate metastasis formation and tumor regress under the influence of therapeutics. Different types of tomography, such as continuous wave (CW), frequency-domain (FD), and time-domain (TD) tomography, allow fluorescence imaging of tumors located deep in human or animal tissue. In this work, preliminary results of the frequency domain fluorescent diffuse tomography (FDT) method in application to DsRed2 protein as a fluorescent agent are presented. For the first step of our experiments, we utilized low-frequency amplitude modulation (1 kHz) of second harmonic of Nd: YAG (532 nm). The transilluminative configuration was used in the setup. The results of post mortem experiments with capsules containing DsRed2 inserted inside the esophagus of a 3-day-old hairless rat to simulate tumor are shown. An algorithm of processing fluorescent images based on calculating the zero of maximum curvature has been applied to detect fluorescent inclusion boundaries in the image. This work demonstrates the potential capability of the FDT method for imaging deep fluorescent tumors in human tissue or animal models of human cancer. Improvement of the setup can be accomplished by using high-frequency modulation (using a 110-MHz acoustooptical modulator).

Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro.

Science.gov (United States)

Cristofari, G; Gabus, C; Ficheux, D; Bona, M; Le Grice, S F; Darlix, J L

1999-12-17

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.

Regulation of rice root development by a retrotransposon acting as a microRNA sponge.

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Cho, Jungnam; Paszkowski, Jerzy

2017-08-26

It is well documented that transposable elements (TEs) can regulate the expression of neighbouring genes. However, their ability to act in trans and influence ectopic loci has been reported rarely. We searched in rice transcriptomes for tissue-specific expression of TEs and found them to be regulated developmentally. They often shared sequence homology with co-expressed genes and contained potential microRNA-binding sites, which suggested possible contributions to gene regulation. In fact, we have identified a retrotransposon that is highly transcribed in roots and whose spliced transcript constitutes a target mimic for miR171. miR171 destabilizes mRNAs encoding the root-specific family of SCARECROW-Like transcription factors. We demonstrate that retrotransposon-derived transcripts act as decoys for miR171, triggering its degradation and thus results in the root-specific accumulation of SCARECROW-Like mRNAs. Such transposon-mediated post-transcriptional control of miR171 levels is conserved in diverse rice species.

Evaluation of genetic diversity in open pollinated guava by iPBS primers

International Nuclear Information System (INIS)

Mehmood, A.; Jaskani, M.J.; Ahmad, S.; Ahmad, R.

2013-01-01

DNA markers are important tools for assessing genetic diversity and relationships among species, cultivars and breeding materials. Many horticultural species are lacking genomic information. DNA markers that do not require prior knowledge of DNA sequences are therefore appealing for horticultural research. A retrotransposon-based DNA marker system, iPBS (inter primer binding sites) developed from conserved primer binding sites within retrotransposons, was used to study the genetic variation and relationships in ornamental guava. PCR from 6 iPBS primers (dominant markers) produced a total of 113 bands (52.38-100% polymorphic) ranging from 150 bp to 3000 bp, and the mean PIC value for each primer ranging from 0.1245 to 0.3698. Molecular information generated from both iPBS was separately scored in a matrix for phylogenetic dendrogram construction. The phylogenetic dendrogram based on iPBS markers reflected morphologic classifications of the accessions that were studied. The iPBS PCR-based genome fingerprinting technology in this study is low-cost and provides another effective alternative in differentiation of accessions in guava (Psidium guajava Linn.) and related species or genera. (author)

The role of retrotransposons in gene family expansions in the human and mouse genomes

Czech Academy of Sciences Publication Activity Database

Janoušek, Václav; Laukaitis, C. M.; Yanchukov, Alexey; Karn, R. C.

2016-01-01

Roč. 8, č. 9 (2016), s. 2632-2650 ISSN 1759-6653 R&D Projects: GA MŠk EE2.3.20.0303 Institutional support: RVO:68081766 Keywords : gene families * transposable elements * retrotransposons * LINE * LTR * SINE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.979, year: 2016

Genome-wide analysis of LTR-retrotransposon diversity and its impact on the evolution of the genus Helianthus (L.).

Science.gov (United States)

Mascagni, Flavia; Giordani, Tommaso; Ceccarelli, Marilena; Cavallini, Andrea; Natali, Lucia

2017-08-18

Genome divergence by mobile elements activity and recombination is a continuous process that plays a key role in the evolution of species. Nevertheless, knowledge on retrotransposon-related variability among species belonging to the same genus is still limited. Considering the importance of the genus Helianthus, a model system for studying the ecological genetics of speciation and adaptation, we performed a comparative analysis of the repetitive genome fraction across ten species and one subspecies of sunflower, focusing on long terminal repeat retrotransposons at superfamily, lineage and sublineage levels. After determining the relative genome size of each species, genomic DNA was isolated and subjected to Illumina sequencing. Then, different assembling and clustering approaches allowed exploring the repetitive component of all genomes. On average, repetitive DNA in Helianthus species represented more than 75% of the genome, being composed mostly by long terminal repeat retrotransposons. Also, the prevalence of Gypsy over Copia superfamily was observed and, among lineages, Chromovirus was by far the most represented. Although nearly all the same sublineages are present in all species, we found considerable variability in the abundance of diverse retrotransposon lineages and sublineages, especially between annual and perennial species. This large variability should indicate that different events of amplification or loss related to these elements occurred following species separation and should have been involved in species differentiation. Our data allowed us inferring on the extent of interspecific repetitive DNA variation related to LTR-RE abundance, investigating the relationship between changes of LTR-RE abundance and the evolution of the genus, and determining the degree of coevolution of different LTR-RE lineages or sublineages between and within species. Moreover, the data suggested that LTR-RE abundance in a species was affected by the annual or perennial

A graphene oxide-based fluorescent aptasensor for the turn-on detection of epithelial tumor marker mucin 1.

Science.gov (United States)

He, Yue; Lin, Yi; Tang, Hongwu; Pang, Daiwen

2012-03-21

Mucin 1 (MUC1) which presents in epithelial malignancies, is a well-known tumor biomarker. In this paper, a highly sensitive and selective fluorescent aptasensor for Mucin 1 (MUC1) detection is constructed, utilizing graphene oxide (GO) as a quencher which can quench the fluorescence of single-stranded dye-labeled MUC1 specific aptamer. In the absence of MUC1, the adsorption of the dye-labeled aptamer on GO brings the dyes in close proximity to the GO surface resulting in high efficiency quenching of dye fluorescence. Therefore, the fluorescence of the designed aptasensor is completely quenched by GO, and the system shows very low background fluorescence. Conversely, and very importantly, upon the adding of MUC1, the quenched fluorescence is recovered significantly, and MUC1 can be detected in a wide range of 0.04-10 μM with a detection limit of 28 nM and good selectivity. Moreover, the results have also been verified for real sample application by testing 2% serum containing buffer solution spiked with a series of concentrations of MUC1. This journal is © The Royal Society of Chemistry 2012

Low levels of LTR retrotransposon deletion by ectopic recombination in the gigantic genomes of salamanders.

Science.gov (United States)

Frahry, Matthew Blake; Sun, Cheng; Chong, Rebecca A; Mueller, Rachel Lockridge

2015-02-01

Across the tree of life, species vary dramatically in nuclear genome size. Mutations that add or remove sequences from genomes-insertions or deletions, or indels-are the ultimate source of this variation. Differences in the tempo and mode of insertion and deletion across taxa have been proposed to contribute to evolutionary diversity in genome size. Among vertebrates, most of the largest genomes are found within the salamanders, an amphibian clade with genome sizes ranging from ~14 to ~120 Gb. Salamander genomes have been shown to experience slower rates of DNA loss through small (i.e., genomes. However, no studies have addressed DNA loss from salamander genomes resulting from larger deletions. Here, we focus on one type of large deletion-ectopic-recombination-mediated removal of LTR retrotransposon sequences. In ectopic recombination, double-strand breaks are repaired using a "wrong" (i.e., ectopic, or non-allelic) template sequence-typically another locus of similar sequence. When breaks occur within the LTR portions of LTR retrotransposons, ectopic-recombination-mediated repair can produce deletions that remove the internal transposon sequence and the equivalent of one of the two LTR sequences. These deletions leave a signature in the genome-a solo LTR sequence. We compared levels of solo LTRs in the genomes of four salamander species with levels present in five vertebrates with smaller genomes. Our results demonstrate that salamanders have low levels of solo LTRs, suggesting that ectopic-recombination-mediated deletion of LTR retrotransposons occurs more slowly than in other vertebrates with smaller genomes.

Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium

International Nuclear Information System (INIS)

Saloma, Caesar; Palmes-Saloma, Cynthia; Kondoh, Hisato

1998-01-01

Normally transparent biological structures in a turbid medium are imaged using a laser confocal microscope and multiwavelength site-specific fluorescence labelling. The spatial filtering capability of the detector pinhole in the confocal microscope limits the number of scattered fluorescent photons that reach the photodetector. Simultaneous application of different fluorescent markers on the same sample site minimizes photobleaching by reducing the excitation time for each marker. A high-contrast grey-level image is also produced by summing confocal images of the same site taken at different fluorescence wavelengths. Monte Carlo simulations are performed to obtain the quantitative behaviour of confocal fluorescence imaging in turbid media. Confocal images of the following samples were also obtained: (i) 15 μm diameter fluorescent spheres placed 1.16 mm deep beneath an aqueous suspension of 0.0823 μm diameter polystyrene latex spheres, and (ii) hindbrain of a whole-mount mouse embryo (age 10 days) that was stained to fluoresce at 515 nm and 580 nm peak wavelengths. Expression of RNA transcripts of a gene within the embryo hindbrain was detected by a fluorescence-based whole-mount in situ hybridization procedure that we recently tested. (author)

Fluorescence lifetime based bioassays

Science.gov (United States)

Meyer-Almes, Franz-Josef

2017-12-01

Fluorescence lifetime (FLT) is a robust intrinsic property and material constant of fluorescent matter. Measuring this important physical indicator has evolved from a laboratory curiosity to a powerful and established technique for a variety of applications in drug discovery, medical diagnostics and basic biological research. This distinct trend was mainly driven by improved and meanwhile affordable laser and detection instrumentation on the one hand, and the development of suitable FLT probes and biological assays on the other. In this process two essential working approaches emerged. The first one is primarily focused on high throughput applications employing biochemical in vitro assays with no requirement for high spatial resolution. The second even more dynamic trend is the significant expansion of assay methods combining highly time and spatially resolved fluorescence data by fluorescence lifetime imaging. The latter approach is currently pursued to enable not only the investigation of immortal tumor cell lines, but also specific tissues or even organs in living animals. This review tries to give an actual overview about the current status of FLT based bioassays and the wide range of application opportunities in biomedical and life science areas. In addition, future trends of FLT technologies will be discussed.

Identification and chromosomal distribution of copia-like retrotransposon sequences in the coffee (Coffea L. genome

Directory of Open Access Journals (Sweden)

Juan-Carlos Herrera

2013-12-01

Full Text Available The presence of copia-like transposable elements in seven coffee (Coffea sp. species, including the cultivated Coffea arabica, was investigated. The highly conserved domains of the reverse transcriptase (RT present in the copia retrotransposons were amplified by PCR using degenerated primers. Fragments of roughly 300 bp were obtained and the nucleotide sequence was determined for 36 clones, 19 of which showed good quality. The deduced amino acid sequences were compared by multiple alignment analysis. The data suggested two distinct coffee RT groups, designated as CRTG1 and CRTG2. The sequence identities among the groups ranged from 52 to 60% for CRTG1 and 74 to 85% for CRTG2. The multiple alignment analysis revealed that some of the clones in CRTG1 were closely related to the representative elements present in other plant species such as Brassica napus, Populus ciliata and Picea abis. Furthermore, the chromosomal localization of the RT domains in C. arabica and their putative ancestors was investigated by fluorescence in situ hybridization (FISH analysis. FISH signals were observed throughout the chromosomes following a similar dispersed pattern with some localized regions exhibiting higher concentrations of those elements, providing new evidence of their relative conservation and stability in the coffee genome

Elimination of ghost markers during dual sensor-based infrared tracking of multiple individual reflective markers

International Nuclear Information System (INIS)

Stroian, G.; Falco, T.; Seuntjens, J.P.

2004-01-01

The accuracy of dose delivery in radiotherapy is affected by the uncertainty in tumor localization. Motion of internal anatomy due to physiological processes such as respiration may lead to significant displacements which compromise tumor coverage and generate irradiation of healthy tissue. Real-time tracking with infrared-based systems is often used for tracking thoracic motion in radiation therapy. We studied the origin of ghost markers ('crosstalk') which may appear during dual sensor-based infrared tracking of independent reflective markers. Ghost markers occur when two or more reflective markers are coplanar with each other and with the sensors of the two camera-based infrared tracking system. Analysis shows that sensors are not points but they have a finite extent and this extent determines for each marker a 'ghost volume'. If one reflective marker enters the ghost volume of another marker, ghost markers will be reported by the tracking system; if the reflective markers belong to a surface their 'ghost volume' is reduced to a 'ghost surface' (ghost zone). Appearance of ghost markers is predicted for markers taped on the torso of an anthropomorphic phantom. This study illustrates the dependence of the shape, extent, and location of the ghost zones on the shape of the anthropomorphic phantom, the angle of view of the tracking system, and the distance between the tracking system and the anthropomorphic phantom. It is concluded that the appearance of ghost markers can be avoided by positioning the markers outside the ghost zones of the other markers. However, if this is not possible and the initial marker configuration is ghost marker-free, ghost markers can be eliminated during real-time tracking by virtue of the fact that they appear in the coordinate data sequence only temporarily

Informative genomic microsatellite markers for efficient genotyping applications in sugarcane.

Science.gov (United States)

Parida, Swarup K; Kalia, Sanjay K; Kaul, Sunita; Dalal, Vivek; Hemaprabha, G; Selvi, Athiappan; Pandit, Awadhesh; Singh, Archana; Gaikwad, Kishor; Sharma, Tilak R; Srivastava, Prem Shankar; Singh, Nagendra K; Mohapatra, Trilochan

2009-01-01

Genomic microsatellite markers are capable of revealing high degree of polymorphism. Sugarcane (Saccharum sp.), having a complex polyploid genome requires more number of such informative markers for various applications in genetics and breeding. With the objective of generating a large set of microsatellite markers designated as Sugarcane Enriched Genomic MicroSatellite (SEGMS), 6,318 clones from genomic libraries of two hybrid sugarcane cultivars enriched with 18 different microsatellite repeat-motifs were sequenced to generate 4.16 Mb high-quality sequences. Microsatellites were identified in 1,261 of the 5,742 non-redundant clones that accounted for 22% enrichment of the libraries. Retro-transposon association was observed for 23.1% of the identified microsatellites. The utility of the microsatellite containing genomic sequences were demonstrated by higher primer designing potential (90%) and PCR amplification efficiency (87.4%). A total of 1,315 markers including 567 class I microsatellite markers were designed and placed in the public domain for unrestricted use. The level of polymorphism detected by these markers among sugarcane species, genera, and varieties was 88.6%, while cross-transferability rate was 93.2% within Saccharum complex and 25% to cereals. Cloning and sequencing of size variant amplicons revealed that the variation in the number of repeat-units was the main source of SEGMS fragment length polymorphism. High level of polymorphism and wide range of genetic diversity (0.16-0.82 with an average of 0.44) assayed with the SEGMS markers suggested their usefulness in various genotyping applications in sugarcane.

Identify super quality markers from prototype-based pharmacokinetic markers of Tangzhiqing tablet (TZQ) based on in vitro dissolution/ permeation and in vivo absorption correlations.

Science.gov (United States)

Li, Ziqiang; Liu, Jia; Li, Yazhuo; Du, Xi; Li, Yanfen; Wang, Ruihua; Lv, Chunxiao; He, Xin; Wang, Baohe; Huang, Yuhong; Zhang, Deqin

2018-06-01

A quality marker (Q-marker) is defined as an inherent chemical compound that is used for the quality control of a drug. Its biological activities are closely related to safety and therapeutic effects. Generally, a multiple-component herbal medicine may have many Q-markers. We therefore proposed a concept of "super Q-marker" satisfying both the criterion of Q-markers and PK-markers to be used in more effective quality control of herbal medicine. The first aim was to find suitable prototype-based PK-markers from Tangzhiqing tablets (TZQ), a Chinese patent medicine. Then super Q-markers were expected to be identified from the prototype-based PK-markers based on an in vitro-in vivo correlation study. Potentially eligible prototype-based PK-markers were identified in a single- and multiple-dose pharmacokinetic study on TZQ in 30 healthy volunteers. The in vitro dissolution and permeation profiles of the prototype-based PK-markers of TZQ were evaluated by the physiologically-based drug dissolution/absorption simulating system (DDASS). An in vitro-in vivo correlation analysis was conducted between the dissolution/permeation behaviors in DDASS and the actual absorption profiles in human to test the transferability and traceability of the promising super Q-markers for TZQ. In human, plasma paeoniflorin and nuciferine as prototype-based PK-markers exhibited the appropriate pharmacokinetic properties, including dose-dependent systemic exposure (AUC, C max ) and a proper elimination half-life (1∼3h). In DDASS, it was predicted that paeoniflorin and nuciferine are highly permeable but the absorption rates are primarily limited by the dissolution rates. Moreover, the established in vitro-in vivo correlations of paeoniflorin and nuciferine were in support of the super Q-markers features. Paeoniflorin and nuciferine are identified as the super Q-markers from the prototype-based PK-markers of TZQ based on findings from a combination of in vitro, in vivo, and in vitro-in vivo

Preparation and application of new fluorescein-labeled fumonisins B1 in fluorescence polarization analysis technique

Science.gov (United States)

Objective: To prepare a new fluorescent tracer against common mycotoxins such as fumonisin B1 in order to replace 6-(4,6-Dichlorotriazinyl) aminofluorescein (6-DTAF), an expensive marker, and to develop a technique for quick detection of fumonisin B1 based on the principle of fluorescence polarizati...

[A cell-based detection of ciguatoxin using sodium fluorescence probe].

Science.gov (United States)

Yuan, Jian-hui; Yang, Hui; Tang, Huan-wen; Huang, Wei; Xu, Xin-yun; Liu, Jian-jun; Ke, Yue-bin; Cheng, Jin-quan; Zhuang, Zhi-xiong

2011-04-01

To establish a cell-based detection method of ciguatoxin using fluorescence assay. Mouse neuroblastoma N-2A cells were exposed to ouabain and veratridine and different concentrations of standard ciguatoxin samples (P-CTX-1) to establish the curvilinear relationship between the toxin dosage and fluorescence intensity using the sodium fluorescence probe CoroNaTM Green. The toxicity curvilinear relationship was also generated between the toxin dosage and cell survival using CCK-8 method. Based on these standard curves, the presence of ciguatoxin was detected in 33 samples of deep-sea coral fish. A correlation was found between the detection results of cell-based fluorescence assay and cytotoxicity assay, whose detection limit reached 103 g/ml and 1012 g/ml, respectively. The cell-based fluorescent assay sensitivity showed a higher sensitivity than cytotoxicity assay with a 2-4 h reduction of the detection time. The cell-based fluorescent assay can quickly and sensitively detect ciguatoxin and may serve as a good option for preliminary screening of the toxin.

Iterative h-minima-based marker-controlled watershed for cell nucleus segmentation.

Science.gov (United States)

Koyuncu, Can Fahrettin; Akhan, Ece; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

2016-04-01

Automated microscopy imaging systems facilitate high-throughput screening in molecular cellular biology research. The first step of these systems is cell nucleus segmentation, which has a great impact on the success of the overall system. The marker-controlled watershed is a technique commonly used by the previous studies for nucleus segmentation. These studies define their markers finding regional minima on the intensity/gradient and/or distance transform maps. They typically use the h-minima transform beforehand to suppress noise on these maps. The selection of the h value is critical; unnecessarily small values do not sufficiently suppress the noise, resulting in false and oversegmented markers, and unnecessarily large ones suppress too many pixels, causing missing and undersegmented markers. Because cell nuclei show different characteristics within an image, the same h value may not work to define correct markers for all the nuclei. To address this issue, in this work, we propose a new watershed algorithm that iteratively identifies its markers, considering a set of different h values. In each iteration, the proposed algorithm defines a set of candidates using a particular h value and selects the markers from those candidates provided that they fulfill the size requirement. Working with widefield fluorescence microscopy images, our experiments reveal that the use of multiple h values in our iterative algorithm leads to better segmentation results, compared to its counterparts. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Detection of intaoral lesions using a fluorescence camera

Science.gov (United States)

Thoms, Michael

2006-02-01

Optical methods for the detection of carious lesions, calculus and plaque have the advantage of being minimally invasive. The use of endogeneous fluorescence markers like porphyrins could simplify the application of fluorescence techniques in the dental practice. It is known that porphyrins are produced by some of the bacterial species that are present in the oral cavity. Since porphyrins have an excitation band at about 400nm they have the potential to be used as fluorescent markers of locations in the oral cavity where the production of bacteria is out of the limits of healthy regions. Further, modern and efficient GaN-based semiconductor diodes emit light in this spectral range and thus make the implementation of fluorescence sensors with excitation at this wavelength easy. Carious lesions, calculus and plaque have been measured using a self build fluorescence camera using GaN-diodes for illumination at 405nm. Further, emission spectra under this excitation were recorded. For the latter purpose freshly extracted teeth were used. It has been found that already in the case of an initial carious lesion red porphyrin-fluorescence is emitted whereas it is absent in healthy enamel. In already brown coloured carious lesions the emission bands of porphyrin are present but the observed overall fluorescence intensity is lower, probably due to the absorption of the fluorescence by the carious defect itself. In dental calculus, dental plaque and subgingival concrements porphyrin originated luminescence was found as well. Since in these cases the emission spectra differ slightly it can be concluded that they originate from different types of porphyrins and thus also from different bacteria. These results show that this fluorescence technique can be a promising method to diagnose carious lesions, calculus and plaque.

Multi-site and multi-depth in vivo cancer localization enhancement after auto-fluorescence removal

International Nuclear Information System (INIS)

Montcuquet, A.S.; Herve, L.; Navarro, F.; Dinten, J.M.; Mars, J.I.

2011-01-01

Fluorescence imaging in diffusive media locates tumors tagged by injected fluorescent markers in NIR wavelengths. For deep embedded markers, natural auto-fluorescence of tissues comes to be a limiting factor to tumor detection and accurate FDOT reconstructions. A spectroscopic approach coupled with Non-negative Matrix Factorization source separation method is explored to discriminate fluorescence sources according to their fluorescence spectra and remove unwanted auto-fluorescence. We successfully removed auto-fluorescence from acquisitions on living mice with a single subcutaneous tumor or two capillary tubes inserted at different depths. (authors)

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

OpenAIRE

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-01-01

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based mi...

340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays

DEFF Research Database (Denmark)

Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter

2016-01-01

We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng....../L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flash lamp and the LED based system and discussed temporal, spatial, and spectral features of the LED...... excitation for time-resolved fluorimetry. Optimization of the suggested key parameters of the LED promises significant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems....

340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays.

Science.gov (United States)

Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Petersen, Paul Michael; Pedersen, Christian

2016-09-19

We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng/L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flash lamp and the LED based system and discussed temporal, spatial, and spectral features of the LED excitation for time-resolved fluorimetry. Optimization of the suggested key parameters of the LED promises significant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems.

DNA-Based Self-Assembly of Fluorescent Nanodiamonds.

Science.gov (United States)

Zhang, Tao; Neumann, Andre; Lindlau, Jessica; Wu, Yuzhou; Pramanik, Goutam; Naydenov, Boris; Jelezko, Fedor; Schüder, Florian; Huber, Sebastian; Huber, Marinus; Stehr, Florian; Högele, Alexander; Weil, Tanja; Liedl, Tim

2015-08-12

As a step toward deterministic and scalable assembly of ordered spin arrays we here demonstrate a bottom-up approach to position fluorescent nanodiamonds (NDs) with nanometer precision on DNA origami structures. We have realized a reliable and broadly applicable surface modification strategy that results in DNA-functionalized and perfectly dispersed NDs that were then self-assembled in predefined geometries. With optical studies we show that the fluorescence properties of the nitrogen-vacancy color centers in NDs are preserved during surface modification and DNA assembly. As this method allows the nanoscale arrangement of fluorescent NDs together with other optically active components in complex geometries, applications based on self-assembled spin lattices or plasmon-enhanced spin sensors as well as improved fluorescent labeling for bioimaging could be envisioned.

Comprehensive cytological characterization of the Gossypium hirsutum genome based on the development of a set of chromosome cytological markers

Institute of Scientific and Technical Information of China (English)

Wenbo; Shan; Yanqin; Jiang; Jinlei; Han; Kai; Wang

2016-01-01

Cotton is the world’s most important natural fiber crop. It is also a model system for studying polyploidization, genomic organization, and genome-size variation. Integrating the cytological characterization of cotton with its genetic map will be essential for understanding its genome structure and evolution, as well as for performing further genetic-map based mapping and cloning. In this study, we isolated a complete set of bacterial artificial chromosome clones anchored to each of the 52 chromosome arms of the tetraploid cotton Gossypium hirsutum. Combining these with telomere and centromere markers, we constructed a standard karyotype for the G. hirsutum inbred line TM-1. We dissected the chromosome arm localizations of the 45 S and 5S r DNA and suggest a centromere repositioning event in the homoeologous chromosomes AT09 and DT09. By integrating a systematic karyotype analysis with the genetic linkage map, we observed different genome sizes and chromosomal structures between the subgenomes of the tetraploid cotton and those of its diploid ancestors. Using evidence of conserved coding sequences, we suggest that the different evolutionary paths of non-coding retrotransposons account for most of the variation in size between the subgenomes of tetraploid cotton and its diploid ancestors. These results provide insights into the cotton genome and will facilitate further genome studies in G. hirsutum.

Comprehensive cytological characterization of the Gossypium hirsutum genome based on the development of a set of chromosome cytological markers

Directory of Open Access Journals (Sweden)

Wenbo Shan

2016-08-01

Full Text Available Cotton is the world's most important natural fiber crop. It is also a model system for studying polyploidization, genomic organization, and genome-size variation. Integrating the cytological characterization of cotton with its genetic map will be essential for understanding its genome structure and evolution, as well as for performing further genetic-map based mapping and cloning. In this study, we isolated a complete set of bacterial artificial chromosome clones anchored to each of the 52 chromosome arms of the tetraploid cotton Gossypium hirsutum. Combining these with telomere and centromere markers, we constructed a standard karyotype for the G. hirsutum inbred line TM-1. We dissected the chromosome arm localizations of the 45S and 5S rDNA and suggest a centromere repositioning event in the homoeologous chromosomes AT09 and DT09. By integrating a systematic karyotype analysis with the genetic linkage map, we observed different genome sizes and chromosomal structures between the subgenomes of the tetraploid cotton and those of its diploid ancestors. Using evidence of conserved coding sequences, we suggest that the different evolutionary paths of non-coding retrotransposons account for most of the variation in size between the subgenomes of tetraploid cotton and its diploid ancestors. These results provide insights into the cotton genome and will facilitate further genome studies in G. hirsutum.

Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.

Science.gov (United States)

Sunami, Takeshi; Caschera, Filippo; Morita, Yuuki; Toyota, Taro; Nishimura, Kazuya; Matsuura, Tomoaki; Suzuki, Hiroaki; Hanczyc, Martin M; Yomo, Tetsuya

2010-10-05

We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.

Biomass-based magnetic fluorescent nanoparticles: One-step scalable synthesis, application as drug carriers and mechanism study.

Science.gov (United States)

Li, Lei; Wang, Feijun; Shao, Ziqiang

2018-03-15

A biomass-based magnetic fluorescent nanoparticle (MFNPs) was successively in situ synthesized via a one-step high-gravity approach, which constructed by a magnetic core of Fe 3 O 4 nanoparticles, the fluorescent marker of carbon dots (CDs), and shells of chitosan (CS). The obtained MFNPs had a 10 nm average diameter and narrow particle size distribution, low cytotoxicity, superior fluorescent emission and superparamagnetic properties. The encapsulating and release 5-fluorouracil experiments confirmed that the introduction of CS/CDs effectively improved the drug loading capacity. Mechanism and kinetic studies proved that: (i) the monolayer adsorption was the main sorption mode under the studied conditions; (ii) the whole adsorption process was controlled by intra-liquid diffusion mass transfer and governed by chemisorption; and (iii) the release process was controlled by Fickian diffusion. These results demonstrated this method to one-step continuously produce MFNPs and the construction of non-toxic nanostructure possessed great superiority in currently Nano-delivery systems, which would show high application value in targeted drug delivery, magnetic fluid hyperthermia treatment, magnetic resonance imaging (MRI), in vitro testing and relative research. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation and characterization of reverse transcriptase fragments of LTR retrotransposons from the genome of Chenopodium quinoa (Amaranthaceae).

Science.gov (United States)

Kolano, Bozena; Bednara, Edyta; Weiss-Schneeweiss, Hanna

2013-10-01

High heterogeneity was observed among conserved domains of reverse transcriptase ( rt ) isolated from quinoa. Only one Ty1- copia rt was highly amplified. Reverse transcriptase sequences were located predominantly in pericentromeric region of quinoa chromosomes. The heterogeneity, genomic abundance, and chromosomal distribution of reverse transcriptase (rt)-coding fragments of Ty1-copia and Ty3-gypsy long terminal repeat retrotransposons were analyzed in the Chenopodium quinoa genome. Conserved domains of the rt gene were amplified and characterized using degenerate oligonucleotide primer pairs. Sequence analyses indicated that half of Ty1-copia rt (51 %) and 39 % of Ty3-gypsy rt fragments contained intact reading frames. High heterogeneity among rt sequences was observed for both Ty1-copia and Ty3-gypsy rt amplicons, with Ty1-copia more heterogeneous than Ty3-gypsy. Most of the isolated rt fragments were present in quinoa genome in low copy numbers, with only one highly amplified Ty1-copia rt sequence family. The gypsy-like RNase H fragments co-amplified with Ty1-copia-degenerate primers were shown to be highly amplified in the quinoa genome indicating either higher abundance of some gypsy families of which rt domains could not be amplified, or independent evolution of this gypsy-region in quinoa. Both Ty1-copia and Ty3-gypsy retrotransposons were preferentially located in pericentromeric heterochromatin of quinoa chromosomes. Phylogenetic analyses of newly amplified rt fragments together with well-characterized retrotransposon families from other organisms allowed identification of major lineages of retroelements in the genome of quinoa and provided preliminary insight into their evolutionary dynamics.

Assisted Interpretation of Laser-Induced Fluorescence Spectra of Egg-Based Binding Media Using Total Emission Fluorescence Spectroscopy

International Nuclear Information System (INIS)

Anglos, D.; Nevin, A.

2006-01-01

Laser-induced fluorescence (LIF) spectroscopy can provide nondestructive, qualitative analysis of protein-based binding media found in artworks. Fluorescence emissions from proteins in egg yolk and egg white are due to auto fluorescent aromatic amino acids as well as other native and age-related fluorophores, but the potential of fluorescence spectroscopy for the differentiation between binding media is dependent on the choice of a suitable excitation wavelength and limited by problems in interpretation. However, a better understanding of emission spectra associated with LIF can be achieved following comparisons with total emission fluorescence spectra where a series of consecutive emission spectra are recorded over a specific range. Results using nanosecond UV laser sources for LIF of egg-based binding media are presented which are rationalised following comparisons with total emission spectra. Specifically, fluorescence is assigned to tryptophan and oxidation products of amino acids; in the case of egg yolk, fatty-acid polymerisation and age-related degradation products account for the formation of fluorophores.

A green fluorescent protein with photoswitchable emission from the deep sea.

Directory of Open Access Journals (Sweden)

Alexander Vogt

Full Text Available A colorful variety of fluorescent proteins (FPs from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that approximately 15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37 degrees C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.

Coevolution between a family of parasite virulence effectors and a class of LINE-1 retrotransposons.

Directory of Open Access Journals (Sweden)

Soledad Sacristán

2009-10-01

Full Text Available Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVR(k1 powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVR(k1 gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1 the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2 the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome.

Diaspora, a large family of Ty3-gypsy retrotransposons in Glycine max, is an envelope-less member of an endogenous plant retrovirus lineage.

Science.gov (United States)

Yano, Sho T; Panbehi, Bahman; Das, Arpita; Laten, Howard M

2005-05-05

The chromosomes of higher plants are littered with retrotransposons that, in many cases, constitute as much as 80% of plant genomes. Long terminal repeat retrotransposons have been especially successful colonizers of the chromosomes of higher plants and examinations of their function, evolution, and dispersal are essential to understanding the evolution of eukaryotic genomes. In soybean, several families of retrotransposons have been identified, including at least two that, by virtue of the presence of an envelope-like gene, may constitute endogenous retroviruses. However, most elements are highly degenerate and are often sequestered in regions of the genome that sequencing projects initially shun. In addition, finding potentially functional copies from genomic DNA is rare. This study provides a mechanism to surmount these issues to generate a consensus sequence that can then be functionally and phylogenetically evaluated. Diaspora is a multicopy member of the Ty3-gypsy-like family of LTR retrotransposons and comprises at least 0.5% of the soybean genome. Although the Diaspora family is highly degenerate, and with the exception of this report, is not represented in the Genbank nr database, a full-length consensus sequence was generated from short overlapping sequences using a combination of experimental and in silico methods. Diaspora is 11,737 bp in length and contains a single 1892-codon ORF that encodes a gag-pol polyprotein. Phylogenetic analysis indicates that it is closely related to Athila and Calypso retroelements from Arabidopsis and soybean, respectively. These in turn form the framework of an endogenous retrovirus lineage whose members possess an envelope-like gene. Diaspora appears to lack any trace of this coding region. A combination of empirical sequencing and retrieval of unannotated Genome Survey Sequence database entries was successfully used to construct a full-length representative of the Diaspora family in Glycine max. Diaspora is presently the

Comparative studies of the endonucleases from two related Xenopus laevis retrotransposons, Tx1L and Tx2L: target site specificity and evolutionary implications.

Science.gov (United States)

Christensen, S; Pont-Kingdon, G; Carroll, D

2000-01-01

In the genome of the South African frog, Xenopus laevis, there are two complex families of transposable elements, Tx1 and Tx2, that have identical overall structures, but distinct sequences. In each family there are approximately 1500 copies of an apparent DNA-based element (Tx1D and Tx2D). Roughly 10% of these elements in each family are interrupted by a non-LTR retrotransposon (Tx1L and Tx2L). Each retrotransposon is flanked by a 23-bp target duplication of a specific D element sequence. In earlier work, we showed that the endonuclease domain (Tx1L EN) located in the second open reading frame (ORF2) of Tx1L encodes a protein that makes a single-strand cut precisely at the expected site within its target sequence, supporting the idea that Tx1L is a site-specific retrotransposon. In this study, we express the endonuclease domain of Tx2L (Tx2L EN) and compare the target preferences of the two enzymes. Each endonuclease shows some preference for its cognate target, on the order of 5-fold over the non-cognate target. The observed discrimination is not sufficient, however, to explain the observation that no cross-occupancy is observed - that is, L elements of one family have never been found within D elements of the other family. Possible sources of additional specificity are discussed. We also compare two hypotheses regarding the genome duplication event that led to the contemporary pseudotetraploid character of Xenopus laevis in light of the Tx1L and Tx2L data.

Simultaneous determination of glycols based on fluorescence anisotropy

International Nuclear Information System (INIS)

Garcia Sanchez, F.; Navas Diaz, A.; Lopez Guerrero, M.M.

2007-01-01

Simultaneous determination of non-fluorescent glycols in mixtures without separation or chemical transformation steps is described. Two methods based in the measure of fluorescence anisotropy of a probe such as fluorescein dissolved in the analyte or analyte mixtures are described. In the first method, the anisotropy spectra of pure and mixtures of analytes are used to quantitative determination (if the fluorophor concentration is in a range where fluorescence intensity is proportional to concentration). In the second method, a calibration curve anisotropy-concentration based on the application of the Perrin equation is established. The methods presented here are capable of directly resolving binary mixtures of non-fluorescent glycols on the basis of differences on the fluorescence anisotropy of a fluorescence tracer. Best analytical performances were obtained by application of the method based on Perrin equation. This method is simple, rapid and allows the determination of mixtures of glycols with reasonable accuracy and precision. Detection limits are limited by the quantum yield and anisotropy values of the tracer in the solvents. Recovery values are related to the differences in anisotropy values of the tracer in the pure solvents. Mixtures of glycerine/ethylene glycol (GL/EG), ethylene glycol/1,2-propane diol (EG/1,2-PPD) and polyethylene glycol 400/1,2-propane diol (PEG 400/1,2-PPD) were analysed and recovery values are within 95-120% in the Perrin method. Relative standard deviation are in the range 1.3-2.9% and detection limits in the range 3.9-8.9%

Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis.

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Nikhil R Gandasi

Full Text Available Fluorescent proteins (FPs have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.

LTR retrotransposons in fungi.

Directory of Open Access Journals (Sweden)

Anna Muszewska

Full Text Available Transposable elements with long terminal direct repeats (LTR TEs are one of the best studied groups of mobile elements. They are ubiquitous elements present in almost all eukaryotic genomes. Their number and state of conservation can be a highlight of genome dynamics. We searched all published fungal genomes for LTR-containing retrotransposons, including both complete, functional elements and remnant copies. We identified a total of over 66,000 elements, all of which belong to the Ty1/Copia or Ty3/Gypsy superfamilies. Most of the detected Gypsy elements represent Chromoviridae, i.e. they carry a chromodomain in the pol ORF. We analyzed our data from a genome-ecology perspective, looking at the abundance of various types of LTR TEs in individual genomes and at the highest-copy element from each genome. The TE content is very variable among the analyzed genomes. Some genomes are very scarce in LTR TEs (8000 elements. The data shows that transposon expansions in fungi usually involve an increase both in the copy number of individual elements and in the number of element types. The majority of the highest-copy TEs from all genomes are Ty3/Gypsy transposons. Phylogenetic analysis of these elements suggests that TE expansions have appeared independently of each other, in distant genomes and at different taxonomical levels. We also analyzed the evolutionary relationships between protein domains encoded by the transposon pol ORF and we found that the protease is the fastest evolving domain whereas reverse transcriptase and RNase H evolve much slower and in correlation with each other.

CA 19-9 Pancreatic Tumor Marker Fluorescence Immunosensing Detection via Immobilized Carbon Quantum Dots Conjugated Gold Nanocomposite.

Science.gov (United States)

Alarfaj, Nawal Ahmad; El-Tohamy, Maha Farouk; Oraby, Hesham Farouk

2018-04-11

The clinical detection of carbohydrate antigen 19-9 (CA 19-9), a tumor marker in biological samples, improves and facilitates the rapid screening and diagnosis of pancreatic cancer. A simple, low cost, fast, and green synthesis method to prepare a viable carbon quantum dots/gold (CQDs/Au) nanocomposite fluorescence immunosensing solution for the detection of CA 19-9 was reported. The present method is conducted by preparing glucose-derived CQDs using a microwave-assisted method. CQDs were employed as reducing and stabilizing agents for the preparation of a CQDs/Au nanocomposite. The immobilized anti-CA 19-9-labeled horseradish peroxidase enzyme (Ab-HRP) was anchored to the surface of a CQDs/Au nanocomposite by a peptide interaction between the carboxylic and amine active groups. The CA 19-9 antigen was trapped by another monoclonal antibody that was coated on the surface of microtiter wells. The formed sandwich capping antibody-antigen-antibody enzyme complex had tunable fluorescence properties that were detected under excitation and emission wavelengths of 420 and 530 nm. The increase in fluorescence intensities of the immunoassay sensing solution was proportional to the CA 19-9 antigen concentration in the linear range of 0.01-350 U mL -1 and had a lower detection limit of 0.007 U mL -1 . The proposed CQDs/Au nanocomposite immunoassay method provides a promising tool for detecting CA 19-9 in human serum.

Flavin mononucleotide (FMN)-based fluorescent protein (FbFP) as reporter for gene expression in the anaerobe Bacteroides fragilis.

Science.gov (United States)

Lobo, Leandro A; Smith, Charles J; Rocha, Edson R

2011-04-01

In this study, we show the expression of flavin mononucleotide-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. Bacteroides fragilis 638R strain carrying osu∷bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared with nonfluorescent cells under glucose-repressed conditions. Bacteria carrying ahpC∷bs2 or dps∷bs2 constructs were fluorescent following induction by oxygen compared with nonfluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2, was detected in both anaerobic and aerobic cultures, confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and in the absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC∷bs2 or dps∷bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Monitoring the Behavior of Emerging Contaminants in Wastewater-Impacted Rivers Based on the Use of Fluorescence Excitation Emission Matrixes (EEM).

Science.gov (United States)

Sgroi, Massimiliano; Roccaro, Paolo; Korshin, Gregory V; Vagliasindi, Federico G A

2017-04-18

This study investigated the applicability of fluorescence indexes based on the interpretation of excitation emission matrices (EEMs) by PARAFAC analysis and by selecting fluorescence intensities at a priori defined excitation/emission pairs as surrogates for monitoring the behavior of emerging organic compounds (EOCs) in two catchment basins impacted by wastewater discharges. Relevant EOC and EEM data were obtained for a 90 km stretch of the Simeto River, the main river in Sicily, and the smaller San Leonardo River, which was investigated for a 17 km stretch. The use of fluorescence indexes developed by these two different approaches resulted in similar observations. Changes of the fluorescence indexes that correspond to a group of humic-like fluorescing species were determined to be highly correlated with the concentrations of recalcitrant contaminants such as sucralose, sulfamethoxazole and carbamazepine, which are typical wastewater markers in river water. Changes of the fluorescence indexes related to tyrosine-like substances were well correlated with the concentrations of ibuprofen and caffeine, anthropogenic indicators of untreated wastewater discharges. Chemical oxygen demand and dissolved organic carbon concentrations were correlated with humic-like fluorescence indexes. The observed correlations were site-specific and characterized by different regression parameters for every collection event. Caffeine and carbamazepine showed correlations with florescence indexes in the San Leonardo River and in the alluvial plain stretch of the Simeto River, whereas sucralose, sulfamethoxazole and ibuprofen have always been well correlated in all the investigated river stretches. However, when data of different collection events from river stretches where correlations were observed were combined, good linear correlations were obtained for data sets generated via the normalization of the measured concentrations by the average value for the corresponding collection event

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole-labeled biotin.

Science.gov (United States)

Zhu, Xianwei; Shinohara, Hiroaki; Miyatake, Ryuta; Hohsaka, Takahiro

2016-10-01

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole-labeled biotin. BODIPY-FL-aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position-specifically incorporated into Trp120 position of streptavidin by four-base codon method. On the other hand, carbazole-labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole-labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole-labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole-labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

Science.gov (United States)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

Preparation and characterization of alginate based-fluorescent magnetic nanoparticles for fluorescence/magnetic resonance multimodal imaging applications

Science.gov (United States)

Kwon, Yong-Su; Choi, Kee-Bong; Lim, Hyungjun; Lee, Sunghwi; Lee, Jae-Jong

2018-06-01

Simple and versatile methodologies have been reported that customize the surface of superparamagnetic iron oxide (SPIO) nanoparticles and impart additional fluorescence capabilities to these contrast agents. Herein, we present the rational design, synthesis, characterization, and biological applications of a new magnetic-based fluorescent probe. The dual modality imaging protocol was developed by labeling fluorophore with alginate natural polymers that have excellent biocompatibility and biodegradability, and using gelification method to form nanocomposites containing SPIO. The formation of alginate-based fluorescent magnetic (AFM) nanoparticles was observed in spherical and elliptical forms with a diameter of less than 500 nm by a transmission electron microscope (TEM). The fluorescent wavelength band in the range of 560 nm was also confirmed in the UV–visible spectrophotometer. In this study, we demonstrate that the multi-tasking design of AFM nanoparticles provides an ideal platform for building balanced dual-image probes of magnetic resonance imaging and optical imaging.

Diagnostic imaging of cervical intraepithelial neoplasia based on hematoxylin and eosin fluorescence.

Science.gov (United States)

Castellanos, Mario R; Szerszen, Anita; Gundry, Stephen; Pirog, Edyta C; Maiman, Mitchell; Rajupet, Sritha; Gomez, John Paul; Davidov, Adi; Debata, Priya Ranjan; Banerjee, Probal; Fata, Jimmie E

2015-07-25

Pathological classification of cervical intraepithelial neoplasia (CIN) is problematic as it relies on subjective criteria. We developed an imaging method that uses spectroscopy to assess the fluorescent intensity of cervical biopsies derived directly from hematoxylin and eosin (H&E) stained tissues. Archived H&E slides were identified containing normal cervical tissue, CIN I, and CIN III cases, from a Community Hospital and an Academic Medical Center. Cases were obtained by consensus review of at least 2 senior pathologists. Images from H&E slides were captured first with bright field illumination and then with fluorescent illumination. We used a Zeiss Axio Observer Z1 microscope and an AxioVision 4.6.3-AP1 camera at excitation wavelength of 450-490 nm with emission captured at 515-565 nm. The 32-bit grayscale fluorescence images were used for image analysis. We reviewed 108 slides: 46 normal, 33 CIN I and 29 CIN III. Fluorescent intensity increased progressively in normal epithelial tissue as cells matured and advanced from the basal to superficial regions of the epithelium. In CIN I cases this change was less prominent as compared to normal. In high grade CIN lesions, there was a slight or no increase in fluorescent intensity. All groups examined were statistically different. Presently, there are no markers to help in classification of CIN I-III lesions. Our imaging method may complement standard H&E pathological review and provide objective criteria to support the CIN diagnosis.

Environmentally Sensitive Fluorescent Sensors Based on Synthetic Peptides

Directory of Open Access Journals (Sweden)

Laurence Choulier

2010-03-01

Full Text Available Biosensors allow the direct detection of molecular analytes, by associating a biological receptor with a transducer able to convert the analyte-receptor recognition event into a measurable signal. We review recent work aimed at developing synthetic fluorescent molecular sensors for a variety of analytes, based on peptidic receptors labeled with environmentally sensitive fluorophores. Fluorescent indicators based on synthetic peptides are highly interesting alternatives to protein-based sensors, since they can be synthesized chemically, are stable, and can be easily modified in a site-specific manner for fluorophore coupling and for immobilization on solid supports.

Smart phone based bacterial detection using bio functionalized fluorescent nanoparticles

International Nuclear Information System (INIS)

Rajendran, Vinoth Kumar; Bakthavathsalam, Padmavathy; Ali, Baquir Mohammed Jaffar

2014-01-01

We are describing immunochromatographic test strips with smart phone-based fluorescence readout. They are intended for use in the detection of the foodborne bacterial pathogens Salmonella spp. and Escherichia coli O157. Silica nanoparticles (SiNPs) were doped with FITC and Ru(bpy), conjugated to the respective antibodies, and then used in a conventional lateral flow immunoassay (LFIA). Fluorescence was recorded by inserting the nitrocellulose strip into a smart phone-based fluorimeter consisting of a light weight (40 g) optical module containing an LED light source, a fluorescence filter set and a lens attached to the integrated camera of the cell phone in order to acquire high-resolution fluorescence images. The images were analysed by exploiting the quick image processing application of the cell phone and enable the detection of pathogens within few minutes. This LFIA is capable of detecting pathogens in concentrations as low as 10 5 cfu mL −1 directly from test samples without pre-enrichment. The detection is one order of magnitude better compared to gold nanoparticle-based LFIAs under similar condition. The successful combination of fluorescent nanoparticle-based pathogen detection by LFIAs with a smart phone-based detection platform has resulted in a portable device with improved diagnosis features and having potential application in diagnostics and environmental monitoring. (author)

Recurrent emergence of structural variants of LTR retrotransposon CsRn1 evolving novel expression strategy and their selective expansion in a carcinogenic liver fluke, Clonorchis sinensis.

Science.gov (United States)

Kim, Seon-Hee; Kong, Yoon; Bae, Young-An

2017-06-01

Autonomous retrotransposons, in which replication and transcription are coupled, encode the essential gag and pol genes as a fusion or separate overlapping form(s) that are expressed in single transcripts regulated by a common upstream promoter. The element-specific expression strategies have driven development of relevant translational recoding mechanisms including ribosomal frameshifting to satisfy the protein stoichiometry critical for the assembly of infectious virus-like particles. Retrotransposons with different recoding strategies exhibit a mosaic distribution pattern across the diverse families of reverse transcribing elements, even though their respective distributions are substantially skewed towards certain family groups. However, only a few investigations to date have focused on the emergence of retrotransposons evolving novel expression strategy and causal genetic drivers of the structural variants. In this study, the bulk of genomic and transcribed sequences of a Ty3/gypsy-like CsRn1 retrotransposon in Clonorchis sinensis were analyzed for the comprehensive examination of its expression strategy. Our results demonstrated that structural variants with single open reading frame (ORF) have recurrently emerged from precedential CsRn1 copies encoding overlapping gag-pol ORFs by a single-nucleotide insertion in an upstream region of gag stop codon. In the parasite genome, some of the newly evolved variants appeared to undergo proliferative burst as active master lineages together with their ancestral copies. The genetic event was similarly observed in Opisthorchis viverrini, the closest neighbor of C. sinensis, whereas the resulting structural variants might have failed to overcome purifying selection and comprised minor remnant copies in the Opisthorchis genome. Copyright © 2017 Elsevier B.V. All rights reserved.

Short interspersed CAN SINE elements as prognostic markers in canine mammary neoplasia.

Science.gov (United States)

Gelaleti, Gabriela B; Granzotto, Adriana; Leonel, Camila; Jardim, Bruna V; Moschetta, Marina G; Carareto, Claudia M A; Zuccari, Debora Ap P C

2014-01-01

The genome of mammals is characterized by a large number of non-LTR retrotransposons, and among them, the CAN SINEs are characteristics of the canine species. Small amounts of DNA freely circulate in normal blood serum and high amounts are found in human patients with cancer, characterizing it as a candidate tumor-biomarker. The aim of this study was to estimate, through its absolute expression, the number of copies of CAN SINE sequences present in free circulating DNA of female dogs with mammary cancer, in order to correlate with the clinical and pathological characteristics and the follow-up period. The copy number of CAN SINE sequences was estimated by qPCR in 28 female dogs with mammary neoplasia. The univariate analysis showed an increased number of copies in female dogs with mammary tumor in female dogs >10 years old (p=0.02) and tumor time >18 months (pSINE fragments can be good markers for the detection of tumor DNA in blood and may characterize it as a marker of poor prognosis, being related to female dogs with shorter survival times. This estimate can be used as a prognostic marker in non-invasive breast cancer research and is useful in predicting tumor progression and patient monitoring.

Intermolecular G-quadruplex structure-based fluorescent DNA detection system.

Science.gov (United States)

Zhou, Hui; Wu, Zai-Sheng; Shen, Guo-Li; Yu, Ru-Qin

2013-03-15

Adopting multi-donors to pair with one acceptor could improve the performance of fluorogenic detection probes. However, common dyes (e.g., fluorescein) in close proximity to each other would self-quench the fluorescence, and the fluorescence is difficult to restore. In this contribution, we constructed a novel "multi-donors-to-one acceptor" fluorescent DNA detection system by means of the intermolecular G-quadruplex (IGQ) structure-based fluorescence signal enhancement combined with the hairpin oligonucleotide. The novel IGQ-hairpin system was characterized using the p53 gene as the model target DNA. The proposed system showed an improved assay performance due to the introduction of IGQ-structure into fluorescent signaling probes, which could inhibit the background fluorescence and increase fluorescence restoration amplitude of fluoresceins upon target DNA hybridization. The proof-of-concept scheme is expected to provide new insight into the potential of G-quadruplex structure and promote the application of fluorescent oligonucleotide probes in fundamental research, diagnosis, and treatment of genetic diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

Screening of Oryza sativa L. for HptGene and Evaluation of Hpt Positive Samples Using Houba Retransposon-Based IRAP Markers

Directory of Open Access Journals (Sweden)

Gözde YÜZBAŞIOĞLU

2017-02-01

Full Text Available Increasing world population needs to enhance agricultural production because of food starvation. Genetically modified organism(GMOis a way to solve this problem. During gene transfers, DNA is inserted into a plant’s genome in a random way. Thisproducesspontaneous genetic changeswith movement of transposable elements, and even increasesvariations. Houbawas described as one of the active retrotransposonsin rice. The aim of this study was to screen rice samples collected from Turkey, and analyse Houba retrotransposon movements with IRAP technique intransgenic ones and their controls. For this purpose, 71 different rice seeds obtained from different regions of Turkey were used for GMO analysis.All samples were screened by real time PCR to test cauliflower mosaic virus (CaMV 35S promoter (P-35S regions, T-NOS (nopaline synthase terminator regions, figwort mosaic virus (FMV regions, bar, patand Cry1ab/ac,and hpt(hygromycin resistance genes. Hptgene was identified in 6samples as a result of real time PCR analysis. These 6transgenic samples with their controls were used for IRAP-PCRanalysisand 0-56% polymorphism ratios were observed in analysed samples. This study is one of the first detailed experimental data of transgenic Oryza sativaL. samples in terms of retrotransposon-based variation.

A fluorescence switch based on a controllable photochromic naphthopyran group

International Nuclear Information System (INIS)

Chen Lizhen; Wang Guang; Zhao Xiancai

2011-01-01

A fluorescence switch based on photoisomerization of naphthopyran (NP) has been designed by employing 2-(pyridin-2-yl)-benzimidazole (BPI) and the naphthopyran containing two pyran rings (NP) as fluorescent dye and photochromic compound, respectively. The fluorescence switch of benzimidazole derivative can be modulated either by controlling the irradiation time of UV light or by adjusting the amount ratio of fluorescent benzimidazole derivative to photochromic naphthopyran in both solution and polymethyl methacrylate (PMMA) film. The experimental results indicated that the decrease of fluorescence intensity of benzimidazole derivative is attributed to the interaction of benzimidazole with naphthopyran. - Highlights: → Naphthopyran was first used to fabricate fluorescence switch with benzimidazole derivative. → Fluorescence intensity can be modulated by controlling the UV irradiation time. → Fluorescence intensity can be adjusted by changing the ratio of benzimidazole derivative to naphthopyran. → Decrease of fluorescence intensity is attributed to the interaction of benzimidazole derivative and naphthopyran.

Ghost marker detection and elimination in marker-based optical tracking systems for real-time tracking in stereotactic body radiotherapy

International Nuclear Information System (INIS)

Yan, Guanghua; Li, Jonathan; Huang, Yin; Mittauer, Kathryn; Lu, Bo; Liu, Chihray

2014-01-01

Purpose: To propose a simple model to explain the origin of ghost markers in marker-based optical tracking systems (OTS) and to develop retrospective strategies to detect and eliminate ghost markers. Methods: In marker-based OTS, ghost markers are virtual markers created due to the cross-talk between the two camera sensors, which can lead to system execution failure or inaccuracy in patient tracking. As a result, the users have to limit the number of markers and avoid certain marker configurations to reduce the chances of ghost markers. In this work, the authors propose retrospective strategies to detect and eliminate ghost markers. The two camera sensors were treated as mathematical points in space. The authors identified the coplanar within limit (CWL) condition as the necessary condition for ghost marker occurrence. A simple ghost marker detection method was proposed based on the model. Ghost marker elimination was achieved through pattern matching: a ghost marker-free reference set was matched with the optical marker set observed by the OTS; unmatched optical markers were eliminated as either ghost markers or misplaced markers. The pattern matching problem was formulated as a constraint satisfaction problem (using pairwise distances as constraints) and solved with an iterative backtracking algorithm. Wildcard markers were introduced to address missing or misplaced markers. An experiment was designed to measure the sensor positions and the limit for the CWL condition. The ghost marker detection and elimination algorithms were verified with samples collected from a five-marker jig and a nine-marker anthropomorphic phantom, rotated with the treatment couch from −60° to +60°. The accuracy of the pattern matching algorithm was further validated with marker patterns from 40 patients who underwent stereotactic body radiotherapy (SBRT). For this purpose, a synthetic optical marker pattern was created for each patient by introducing ghost markers, marker position

Ghost marker detection and elimination in marker-based optical tracking systems for real-time tracking in stereotactic body radiotherapy

Energy Technology Data Exchange (ETDEWEB)

Yan, Guanghua, E-mail: yan@ufl.edu; Li, Jonathan; Huang, Yin; Mittauer, Kathryn; Lu, Bo; Liu, Chihray [Department of Radiation Oncology, University of Florida, Gainesville, Florida 32610 (United States)

2014-10-15

Purpose: To propose a simple model to explain the origin of ghost markers in marker-based optical tracking systems (OTS) and to develop retrospective strategies to detect and eliminate ghost markers. Methods: In marker-based OTS, ghost markers are virtual markers created due to the cross-talk between the two camera sensors, which can lead to system execution failure or inaccuracy in patient tracking. As a result, the users have to limit the number of markers and avoid certain marker configurations to reduce the chances of ghost markers. In this work, the authors propose retrospective strategies to detect and eliminate ghost markers. The two camera sensors were treated as mathematical points in space. The authors identified the coplanar within limit (CWL) condition as the necessary condition for ghost marker occurrence. A simple ghost marker detection method was proposed based on the model. Ghost marker elimination was achieved through pattern matching: a ghost marker-free reference set was matched with the optical marker set observed by the OTS; unmatched optical markers were eliminated as either ghost markers or misplaced markers. The pattern matching problem was formulated as a constraint satisfaction problem (using pairwise distances as constraints) and solved with an iterative backtracking algorithm. Wildcard markers were introduced to address missing or misplaced markers. An experiment was designed to measure the sensor positions and the limit for the CWL condition. The ghost marker detection and elimination algorithms were verified with samples collected from a five-marker jig and a nine-marker anthropomorphic phantom, rotated with the treatment couch from −60° to +60°. The accuracy of the pattern matching algorithm was further validated with marker patterns from 40 patients who underwent stereotactic body radiotherapy (SBRT). For this purpose, a synthetic optical marker pattern was created for each patient by introducing ghost markers, marker position

DNA methylation inhibits expression and transposition of the Neurospora Tad retrotransposon.

Science.gov (United States)

Zhou, Y; Cambareri, E B; Kinsey, J A

2001-06-01

Tad is a LINE-like retrotransposon of the filamentous fungus Neurospora crassa. We have analyzed both expression and transposition of this element using strains with a single copy of Tad located in the 5' noncoding sequences of the am (glutamate dehydrogenase) gene. Tad in this position has been shown to carry a de novo cytosine methylation signal which causes reversible methylation of both Tad and am upstream sequences. Here we find that methylation of the Tad sequences inhibits both Tad expression and transposition. This inhibition can be relieved by the use of 5-azacytidine, a drug which reduces cytosine methylation, or by placing the Tad/am sequences in a dim-2 genetic background.

Highly Sensitive Ratiometric Fluorescent Sensor for Trinitrotoluene Based on the Inner Filter Effect between Gold Nanoparticles and Fluorescent Nanoparticles.

Science.gov (United States)

Lu, Hongzhi; Quan, Shuai; Xu, Shoufang

2017-11-08

In this work, we developed a simple and sensitive ratiometric fluorescent assay for sensing trinitrotoluene (TNT) based on the inner filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent nanoparticles (RFNs), which was designed by hybridizing green emissive carbon dots (CDs) and red emissive quantum dots (QDs) into a silica sphere as a fluorophore pair. AuNPs in their dispersion state can be a powerful absorber to quench CDs, while the aggregated AuNPs can quench QDs in the IFE-based fluorescent assays as a result of complementary overlap between the absorption spectrum of AuNPs and emission spectrum of RFNs. As a result of the fact that TNT can induce the aggregation of AuNPs, with the addition of TNT, the fluorescent of QDs can be quenched, while the fluorescent of CDs would be recovered. Then, ratiometric fluorescent detection of TNT is feasible. The present IFE-based ratiometric fluorescent sensor can detect TNT ranging from 0.1 to 270 nM, with a detection limit of 0.029 nM. In addition, the developed method was successfully applied to investigate TNT in water and soil samples with satisfactory recoveries ranging from 95 to 103%, with precision below 4.5%. The simple sensing approach proposed here could improve the sensitivity of colorimetric analysis by changing the ultraviolet analysis to ratiometric fluorescent analysis and promote the development of a dual-mode detection system.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

Science.gov (United States)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Synthesis, quantitative structure-property relationship study of novel fluorescence active 2-pyrazolines and application

Science.gov (United States)

Girgis, Adel S.; Basta, Altaf H.; El-Saied, Houssni; Mohamed, Mohamed A.; Bedair, Ahmad H.; Salim, Ahmad S.

2018-03-01

A variety of fluorescence-active fluorinated pyrazolines 13-33 was synthesized in good yields through cyclocondensation reaction of propenones 1-9 with aryl hydrazines 10-12. Some of the synthesized compounds provided promising fluorescence properties with quantum yield (Φ) higher than that of quinine sulfate (standard reference). Quantitative structure-property relationship studies were undertaken supporting the exhibited fluorescence properties and estimating the parameters governing properties. Five synthesized fluorescence-active pyrazolines (13, 15, 18, 19 and 23) with variable Φ were selected for treating two types of paper sheets (Fabriano and Bible paper). These investigated fluorescence compounds, especially compounds 19 and 23, provide improvements in strength properties of paper sheets. Based on the observed performance they can be used as markers in security documents.

Retrotransposon long interspersed nucleotide element-1 (LINE-1) is activated during salamander limb regeneration

Science.gov (United States)

Zhu, Wei; Kuo, Dwight; Nathanson, Jason; Satoh, Akira; Pao, Gerald M.; Yeo, Gene W.; Bryant, Susan V.; Voss, S. Randal; Gardiner, David M.; Hunter, Tony

2012-01-01

Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE-1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl limb regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of limb regeneration. PMID:22913491

Catheter-based time-gated near-infrared fluorescence/OCT imaging system

Science.gov (United States)

Lu, Yuankang; Abran, Maxime; Cloutier, Guy; Lesage, Frédéric

2018-02-01

We developed a new dual-modality intravascular imaging system based on fast time-gated fluorescence intensity imaging and spectral domain optical coherence tomography (SD-OCT) for the purpose of interventional detection of atherosclerosis. A pulsed supercontinuum laser was used for fluorescence and OCT imaging. A double-clad fiber (DCF)- based side-firing catheter was designed and fabricated to have a 23 μm spot size at a 2.2 mm working distance for OCT imaging. Its single-mode core is used for OCT, while its inner cladding transports fluorescence excitation light and collects fluorescent photons. The combination of OCT and fluorescence imaging was achieved by using a DCF coupler. For fluorescence detection, we used a time-gated technique with a novel single-photon avalanche diode (SPAD) working in an ultra-fast gating mode. A custom-made delay chip was integrated in the system to adjust the delay between the excitation laser pulse and the SPAD gate-ON window. This technique allowed to detect fluorescent photons of interest while rejecting most of the background photons, thus leading to a significantly improved signal to noise ratio (SNR). Experiments were carried out in turbid media mimicking tissue with an indocyanine green (ICG) inclusion (1 mM and 100 μM) to compare the time-gated technique and the conventional continuous detection technique. The gating technique increased twofold depth sensitivity, and tenfold SNR at large distances. The dual-modality imaging capacity of our system was also validated with a silicone-based tissue-mimicking phantom.

Fluorescence-based bioassays for the detection and evaluation of food materials.

Science.gov (United States)

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-10-13

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

Directory of Open Access Journals (Sweden)

Kentaro Nishi

2015-10-01

Full Text Available We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Cucurbiturils: molecular nanocapsules for time-resolved fluorescence-based assays.

Science.gov (United States)

Marquez, Cesar; Huang, Fang; Nau, Werner M

2004-03-01

A new fluorescent host-guest system based on the inclusion of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) into the cavity of the molecular container compound cucurbit[7]uril (CB7) has been designed which possesses an exceedingly long-lived emission (690 ns in aerated water). The large binding constant of (4 +/- 1) x 10(5) M(-1) along with the resistance of the CB7.DBO complex toward external fluorescence quenchers allow the use of CB7 as an enhancer in time-resolved fluorescence-based assays, e.g., to screen enzyme activity or inhibition by using DBO-labeled peptides as substrates. The response of CB7.DBO to different environmental conditions and possible quenchers are described.

Universal Multifunctional Nanoplatform Based on Target-Induced in Situ Promoting Au Seeds Growth to Quench Fluorescence of Upconversion Nanoparticles.

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Wu, Qiongqiong; Chen, Hongyu; Fang, Aijin; Wu, Xinyang; Liu, Meiling; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo

2017-12-22

Construction of a new multifunctional chemo/biosensing platform for small biomolecules and tumor markers is of great importance in analytical chemistry. Herein, a novel universal multifunctional nanoplatform for biomolecules and enzyme activity detection was proposed based on fluorescence resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) and target-inducing enlarged gold nanoparticles (AuNPs). The reductive molecule such as H 2 O 2 can act as the reductant to reduce HAuCl 4 , which will make the Au seeds grow. The enlarged AuNPs can effectively quench the fluorescence of UCNPs owing to the good spectral overlap between the absorption band of the AuNPs and the emission band of the UCNPs. Utilizing the FRET between the UCNPs and enlarged AuNPs, good linear relationship between the fluorescence of UCNPs and the concentration of H 2 O 2 can be found. Based on this strategy, H 2 O 2 related molecules such as l-lactate, glucose, and uric acid can also be quantified. On the basis of UCNPs and PVP/HAuCl 4 , a general strategy for other reductants such as ascorbic acid (AA), dopamine (DA), or enzyme activity can be established. Therefore, the universal multifunctional nanoplatform based on UCNPs and the target-inducing in situ enlarged Au NPs will show its potential as a simple method for the detection of some life related reductive molecules, enzyme substrates, as well as enzyme activity.

Evaluation of a fluorescence-based method for antibabesial drug screening.

Science.gov (United States)

Guswanto, Azirwan; Sivakumar, Thillaiampalam; Rizk, Mohamed Abdo; Elsayed, Shimaa Abd Elsalam; Youssef, Mohamed Ahmed; ElSaid, ElSaid El Shirbini; Yokoyama, Naoaki; Igarashi, Ikuo

2014-08-01

In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r(2)) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC(50)s determined by the fluorescence-based method (408 nM and 8.13 μM, respectively) and microscopy (400.3 nM and 9.4 μM, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 μM) was similar to the recently described microscopy-based value (21.7 μM) for B. bovis. Additionally, the Z' factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Identification of an internal ribosome entry segment in the 5' region of the mouse VL30 retrotransposon and its use in the development of retroviral vectors.

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López-Lastra, M; Ulrici, S; Gabus, C; Darlix, J L

1999-10-01

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5' region of VL30m could replace the 5' leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5' region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5' region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5' region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors.

Wireless implantable electronic platform for chronic fluorescent-based biosensors.

Science.gov (United States)

Valdastri, Pietro; Susilo, Ekawahyu; Förster, Thilo; Strohhöfer, Christof; Menciassi, Arianna; Dario, Paolo

2011-06-01

The development of a long-term wireless implantable biosensor based on fluorescence intensity measurement poses a number of technical challenges, ranging from biocompatibility to sensor stability over time. One of these challenges is the design of a power efficient and miniaturized electronics, enabling the biosensor to move from bench testing to long term validation, up to its final application in human beings. In this spirit, we present a wireless programmable electronic platform for implantable chronic monitoring of fluorescent-based autonomous biosensors. This system is able to achieve extremely low power operation with bidirectional telemetry, based on the IEEE802.15.4-2003 protocol, thus enabling over three-year battery lifetime and wireless networking of multiple sensors. During the performance of single fluorescent-based sensor measurements, the circuit drives a laser diode, for sensor excitation, and acquires the amplified signals from four different photodetectors. In vitro functionality was preliminarily tested for both glucose and calcium monitoring, simply by changing the analyte-binding protein of the biosensor. Electronics performance was assessed in terms of timing, power consumption, tissue exposure to electromagnetic fields, and in vivo wireless connectivity. The final goal of the presented platform is to be integrated in a complete system for blood glucose level monitoring that may be implanted for at least one year under the skin of diabetic patients. Results reported in this paper may be applied to a wide variety of biosensors based on fluorescence intensity measurement.

A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

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Alexander Gust

2014-09-01

Full Text Available Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

Angular shaping of fluorescence from synthetic opal-based photonic crystal.

Science.gov (United States)

Boiko, Vitalii; Dovbeshko, Galyna; Dolgov, Leonid; Kiisk, Valter; Sildos, Ilmo; Loot, Ardi; Gorelik, Vladimir

2015-01-01

Spectral, angular, and temporal distributions of fluorescence as well as specular reflection were investigated for silica-based artificial opals. Periodic arrangement of nanosized silica globules in the opal causes a specific dip in the defect-related fluorescence spectra and a peak in the reflectance spectrum. The spectral position of the dip coincides with the photonic stop band. The latter is dependent on the size of silica globules and the angle of observation. The spectral shape and intensity of defect-related fluorescence can be controlled by variation of detection angle. Fluorescence intensity increases up to two times at the edges of the spectral dip. Partial photobleaching of fluorescence was observed. Photonic origin of the observed effects is discussed.

Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery

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Benkman Craig W

2010-03-01

Full Text Available Abstract Background Massively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines (Pinus spp. have hindered the development of genomic resources, despite the ecological and economical importance of the group. While most genomic studies have focused on a single species (P. taeda, genomic level resources for other pines are insufficiently developed to facilitate ecological genomic research. Lodgepole pine (P. contorta is an ecologically important foundation species of montane forest ecosystems and exhibits substantial adaptive variation across its range in western North America. Here we describe a sequencing study of expressed genes from P. contorta, including their assembly and annotation, and their potential for molecular marker development to support population and association genetic studies. Results We obtained 586,732 sequencing reads from a 454 GS XLR70 Titanium pyrosequencer (mean length: 306 base pairs. A combination of reference-based and de novo assemblies yielded 63,657 contigs, with 239,793 reads remaining as singletons. Based on sequence similarity with known proteins, these sequences represent approximately 17,000 unique genes, many of which are well covered by contig sequences. This sequence collection also included a surprisingly large number of retrotransposon sequences, suggesting that they are highly transcriptionally active in the tissues we sampled. We located and characterized thousands of simple sequence repeats and single nucleotide polymorphisms as potential molecular markers in our assembled and annotated sequences. High quality PCR primers were designed for a substantial number of the SSR loci

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

Science.gov (United States)

1994-01-01

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate. PMID:7519623

SYTO probes: markers of apoptotic cell demise.

Science.gov (United States)

Wlodkowic, Donald; Skommer, Joanna

2007-10-01

As mechanistic studies on tumor cell death advance towards their ultimate translational goal, there is a need for specific, rapid, and high-throughput analytical tools to detect diverse cell demise modes. Patented DNA-binding SYTO probes, for example, are gaining increasing interest as easy-to-use markers of caspase-dependent apoptotic cell death. They are proving convenient for tracking apoptosis in diverse hematopoietic cell lines and primary tumor samples, and, due to their spectral characteristics, appear to be useful for the development of multiparameter flow cytometry assays. Herein, several protocols for multiparametric assessment of apoptotic events using SYTO probes are provided. There are protocols describing the use of green fluorescent SYTO 16 and red fluorescent SYTO 17 dyes in combination with plasma membrane permeability markers. Another protocol highlights the multiparametric use of SYTO 16 dye in conjunction with the mitochondrial membrane potential sensitive probe, tetramethylrhodamine methyl ester (TMRM), and the plasma membrane permeability marker, 7-aminoactinomycin D (7-AAD).

Synthesis of dye/fluorescent functionalized dendrons based on cyclotriphosphazene

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Aurélien Hameau

2011-11-01

Full Text Available Functionalized phenols based on tyramine were synthesized in order to be selectively grafted on to hexachlorocyclotriphosphazene, affording a variety of functionalized dendrons of type AB5. The B functions comprised fluorescent groups (dansyl or dyes (dabsyl, whereas the A function was provided by either an aldehyde or an amine. The characterization of these dendrons is reported. An unexpected behaviour of a fluorescent and water-soluble dendron based on dansyl groups in mixtures of dioxane/water was observed.

Fluorescence ELISA for sensitive detection of ochratoxin A based on glucose oxidase-mediated fluorescence quenching of CdTe QDs

International Nuclear Information System (INIS)

Liang, Yi; Huang, Xiaolin; Yu, Ruijin; Zhou, Yaofeng; Xiong, Yonghua

2016-01-01

The present study described a novel fluorescence enzyme-linked immunosorbent assay (ELISA) used to detect ochratoxin A (OTA) by using the glucose oxidase (GOx)-mediated fluorescence quenching of mercaptopropionic acid-capped CdTe quantum dots (MPA-QDs), in which GOx was used as an alternative to horseradish peroxidase (HRP) for the oxidization of glucose into hydrogen peroxide (H_2O_2) and gluconic acid. The MPA-QDs were used as a fluorescent signal output, whose fluorescence variation was extremely sensitive to the presence of H_2O_2 or hydrogen ions in the solution. Under the optimized conditions, the proposed fluorescence ELISA demonstrated a good linear detection of OTA in corn extract from 2.4 pg mL"−"1 to 625 pg mL"−"1 with a limit of detection of 2.2 pg mL"−"1, which was approximately 15-fold lower than that of conventional HRP-based ELISA. Our developed fluorescence immunoassay was also similar to HRP-based ELISA in terms of selectivity, accuracy, and reproducibility. In summary, this study was the first to use the GOx-mediated fluorescence quenching of QDs in immunoassay to detect OTA, offering a new possibility for the analysis of other mycotoxins and biomolecules. - Highlights: • A novel fluorescence ELISA was first developed for the detection of OTA by using GOx-mediated fluorescence quenching of QDs. • The pH- and H_2O_2-sensitive MPA-capped CdTe QDs were used as a fluorescent signal output to improve the detection sensitivity. • This novel method open up a different vision to detect other mycotoxins and biomolecules.

Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

Directory of Open Access Journals (Sweden)

Mann David GJ

2012-05-01

Full Text Available Abstract Background The expression of fluorescent protein (FP genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

Fluorescent nanocellulosic hydrogels based on graphene quantum dots for sensing laccase

International Nuclear Information System (INIS)

Ruiz-Palomero, Celia; Benítez-Martínez, Sandra; Soriano, M. Laura; Valcárcel, Miguel

2017-01-01

A novel low-cost fluorimetric platform based on sulfur, nitrogen-codoped graphene quantum dots immersed into nanocellulosic hydrogels is designed and applied in detecting the laccase enzyme. Although most of methods for detecting laccase are based on their catalytic activity, which is strongly dependent on environmental parameters, we report a sensitive and selective method based on the fluorescence response of hydrogels containing graphene quantum dots (GQDs) acting as luminophore towards laccase. The easily-prepared gel matrix not only improves the fluorescence signal of GQDs by avoiding their self-quenching but also stabilizes their fluorescence signal and improves their sensitivity towards laccase. Noncovalent interactions between the sensor and the analyte are believed to be causing this significant quenching without peak-shifts of GQD fluorescence via energy transfer. The selective extraction of laccase was proved in different shampoos as complex matrices achieving a detection limit of 0.048 U mL −1 and recoveries of 86.2–94.1%. As the unusual properties of nanocellulose and GQDs, the fluorescent sensor is simple, eco-friendly and cost-efficient. This straightforward strategy is able to detect and stabilize laccase, being an added-value for storage and recycling enzymes. - Highlights: • Fluorescent hydrogels were constructed by combining nanocellulose and graphene quantum dots. • The resulting hydrogels exhibited fluorescence quenching in presence of laccase. • Equilibrium in the optical signal of S,N-graphene quantum dots in presence of laccase was achieved faster within hydrogels. • The proposed method to determine laccase using fluorescent hydrogels was successfully applied in shampoo.

Enhanced-locality fiber-optic two-photon-fluorescence live-brain interrogation

Energy Technology Data Exchange (ETDEWEB)

Fedotov, I. V.; Doronina-Amitonova, L. V. [International Laser Center, Physics Department, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 1430125 (Russian Federation); Kurchatov Institute National Research Center, Moscow (Russian Federation); Sidorov-Biryukov, D. A.; Fedotov, A. B. [International Laser Center, Physics Department, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 1430125 (Russian Federation); Anokhin, K. V. [Kurchatov Institute National Research Center, Moscow (Russian Federation); P.K. Anokhin Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow (Russian Federation); Kilin, S. Ya. [B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk (Belarus); Sakoda, K. [National Institute for Materials Science, 1-1 Namiki, Tsukuba 305-0044 (Japan); Zheltikov, A. M. [International Laser Center, Physics Department, M.V. Lomonosov Moscow State University, Moscow 119992 (Russian Federation); Russian Quantum Center, ul. Novaya 100, Skolkovo, Moscow Region 1430125 (Russian Federation); Department of Physics and Astronomy, Texas A and M University, College Station, Texas 77843 (United States); Center of Photochemistry, Russian Academy of Sciences, ul. Novatorov 7a, Moscow 117421 (Russian Federation)

2014-02-24

Two-photon excitation is shown to substantially enhance the locality of fiber-based optical interrogation of strongly scattering biotissues. In our experiments, a high-numerical-aperture, large-core-are fiber probe is used to deliver the 200-fs output of a 100-MHz mode-locked ytterbium fiber laser to samples of live mouse brain, induce two-photon fluorescence of nitrogen–vacancy centers in diamond markers in brain sample. Fiber probes with a high numerical aperture and a large core area are shown to enable locality enhancement in fiber-laser–fiber-probe two-photon brain excitation and interrogation without sacrificing the efficiency of fluorescence response collection.

Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads

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Dae Hong Jeong

2012-03-01

Full Text Available Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.

Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions

Science.gov (United States)

Sharifzadeh, Mohsen; Bernstein, Paul S.; Gellermann, Werner

2006-10-01

We have developed a CCD-camera-based nonmydriatic instrument that detects fluorescence from retinal lipofuscin chromophores ("autofluorescence") as a means to indirectly quantify and spatially image the distribution of macular pigment (MP). The lipofuscin fluorescence intensity is reduced at all retinal locations containing MP, since MP has a competing absorption in the blue-green wavelength region. Projecting a large diameter, 488 nm excitation spot onto the retina, centered on the fovea, but extending into the macular periphery, and comparing lipofuscin fluorescence intensities outside and inside the foveal area, it is possible to spatially map out the distribution of MP. Spectrally selective detection of the lipofuscin fluorescence reveals an important wavelength dependence of the obtainable image contrast and deduced MP optical density levels, showing that it is important to block out interfering fluorescence contributions in the detection setup originating from ocular media such as the lens. Measuring 70 healthy human volunteer subjects with no ocular pathologies, we find widely varying spatial extent of MP, distinctly differing distribution patterns of MP, and strongly differing absolute MP levels among individuals. Our population study suggests that MP imaging based on lipofuscin fluorescence is useful as a relatively simple, objective, and quantitative noninvasive optical technique suitable to rapidly screen MP levels and distributions in healthy humans with undilated pupils.

Plastic lab-on-a-chip for fluorescence excitation with integrated organic semiconductor lasers.

Science.gov (United States)

Vannahme, Christoph; Klinkhammer, Sönke; Lemmer, Uli; Mappes, Timo

2011-04-25

Laser light excitation of fluorescent markers offers highly sensitive and specific analysis for bio-medical or chemical analysis. To profit from these advantages for applications in the field or at the point-of-care, a plastic lab-on-a-chip with integrated organic semiconductor lasers is presented here. First order distributed feedback lasers based on the organic semiconductor tris(8-hydroxyquinoline) aluminum (Alq3) doped with the laser dye 4-dicyanomethylene-2-methyl-6-(p-dimethylaminostyril)-4H-pyrane (DCM), deep ultraviolet induced waveguides, and a nanostructured microfluidic channel are integrated into a poly(methyl methacrylate) (PMMA) substrate. A simple and parallel fabrication process is used comprising thermal imprint, DUV exposure, evaporation of the laser material, and sealing by thermal bonding. The excitation of two fluorescent marker model systems including labeled antibodies with light emitted by integrated lasers is demonstrated.

The lipid dependence of melittin action investigated by dual-color fluorescence burst analysis

NARCIS (Netherlands)

Bogaart, Geert van den; Mika, Jacek T.; Krasnikov, Viktor; Poolman, Bert

Dual-color fluorescence-burst analysis was used to study melittin-induced leakage of macromolecules from liposomes of various lipid compositions. To perform dual-color fluorescence-burst analysis, fluorescently labeled size-marker molecules were encapsulated into liposomes, labeled with a second

Mechanical Damage Detection of Indonesia Local Citrus Based on Fluorescence Imaging

Science.gov (United States)

Siregar, T. H.; Ahmad, U.; Sutrisno; Maddu, A.

2018-05-01

Citrus experienced physical damage in peel will produce essential oils that contain polymethoxylated flavone. Polymethoxylated flavone is fluorescence substance; thus can be detected by fluorescence imaging. This study aims to study the fluorescence spectra characteristic and to determine the damage region in citrus peel based on fluorescence image. Pulung citrus from Batu district, East Java, as a famous citrus production area in Indonesia, was used in the experiment. It was observed that the image processing could detect the mechanical damage region. Fluorescence imaging can be used to classify the citrus into two categories, sound and defect citruses.

Fluorescence ELISA for sensitive detection of ochratoxin A based on glucose oxidase-mediated fluorescence quenching of CdTe QDs

Energy Technology Data Exchange (ETDEWEB)

Liang, Yi [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047 (China); Huang, Xiaolin [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Yu, Ruijin [College of Science, Northwest A& F University, Yangling, Shaanxi 712100 (China); Zhou, Yaofeng [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047 (China)

2016-09-14

The present study described a novel fluorescence enzyme-linked immunosorbent assay (ELISA) used to detect ochratoxin A (OTA) by using the glucose oxidase (GOx)-mediated fluorescence quenching of mercaptopropionic acid-capped CdTe quantum dots (MPA-QDs), in which GOx was used as an alternative to horseradish peroxidase (HRP) for the oxidization of glucose into hydrogen peroxide (H{sub 2}O{sub 2}) and gluconic acid. The MPA-QDs were used as a fluorescent signal output, whose fluorescence variation was extremely sensitive to the presence of H{sub 2}O{sub 2} or hydrogen ions in the solution. Under the optimized conditions, the proposed fluorescence ELISA demonstrated a good linear detection of OTA in corn extract from 2.4 pg mL{sup −1} to 625 pg mL{sup −1} with a limit of detection of 2.2 pg mL{sup −1}, which was approximately 15-fold lower than that of conventional HRP-based ELISA. Our developed fluorescence immunoassay was also similar to HRP-based ELISA in terms of selectivity, accuracy, and reproducibility. In summary, this study was the first to use the GOx-mediated fluorescence quenching of QDs in immunoassay to detect OTA, offering a new possibility for the analysis of other mycotoxins and biomolecules. - Highlights: • A novel fluorescence ELISA was first developed for the detection of OTA by using GOx-mediated fluorescence quenching of QDs. • The pH- and H{sub 2}O{sub 2}-sensitive MPA-capped CdTe QDs were used as a fluorescent signal output to improve the detection sensitivity. • This novel method open up a different vision to detect other mycotoxins and biomolecules.

An eco-friendly molecularly imprinted fluorescence composite material based on carbon dots for fluorescent detection of 4-nitrophenol

International Nuclear Information System (INIS)

Hao, Tongfan; Wei, Xiao; Nie, Yijing; Zhou, Zhiping; Xu, Yeqing; Yan, Yongsheng

2016-01-01

We on report an eco-friendly molecularly imprinted material based on carbon dots (C-dots) via a facile and efficient sol–gel polymerization for selective fluorescence detection of 4-nitrophenol (4-NP). The amino-modified C-dots were firstly synthesized by a hydrothermal process using citric acid as the carbon source and poly(ethyleneimine) as the surface modifier, and then after a sol–gel molecular imprinting process, the molecularly imprinted fluorescence material was obtained. The material (MIP-C-dots) showed strong fluorescence from C-dots and high selectivity due to the presence of a molecular imprint. After the detection conditions were optimized, the relative fluorescence intensity (F_0/F) of MIP-C-dots presented a good linearity with 4-NP concentrations in the linear range of 0.2 − 50 μmol L"-"1 with a detection limit (3σ/k) of 0.06 μmol L"-"1. In addition, the correlation coefficient was 0.9978 and the imprinting factor was 2.76. The method was applicable to the determination of trace 4-NP in Yangtze River water samples and good recoveries from 92.6–107.3 % were obtained. The present study provides a general strategy to fabricate materials based on C-dots with good fluorescence property for selective fluorescence detection of organic pollutants. (author)

Fluorescence quenching of Rhodamine B base by two amines

Science.gov (United States)

Bakkialakshmi, S.; Selvarani, P.; Chenthamarai, S.

2013-03-01

Fluorescence quenching of Rhodamine B base (RhB) in DMF solution has been studied at different concentrations of the amine Triethyl amine (TEA) and n-butyl amine (NBA) at room temperature. It has been observed that the fluorescence intensity of RhB decrease with increase in the concentration of the TEA and NBA. It has been observed that the quenching due to amines proceeds via dynamic quenching process. The rate constants for the quenching process have been calculated using Stern-Volmer equation. Time resolved fluorescence study and 1H NMR spectral study have also been carried out and discussed.

Genomic change, retrotransposon mobilization and extensive cytosine methylation alteration in Brassica napus introgressions from two intertribal hybridizations.

Science.gov (United States)

Zhang, Xueli; Ge, Xianhong; Shao, Yujiao; Sun, Genlou; Li, Zaiyun

2013-01-01

Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4-39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.

Tomato seeds maturity detection system based on chlorophyll fluorescence

Science.gov (United States)

Li, Cuiling; Wang, Xiu; Meng, Zhijun

2016-10-01

Chlorophyll fluorescence intensity can be used as seed maturity and quality evaluation indicator. Chlorophyll fluorescence intensity of seed coats is tested to judge the level of chlorophyll content in seeds, and further to judge the maturity and quality of seeds. This research developed a detection system of tomato seeds maturity based on chlorophyll fluorescence spectrum technology, the system included an excitation light source unit, a fluorescent signal acquisition unit and a data processing unit. The excitation light source unit consisted of two high power LEDs, two radiators and two constant current power supplies, and it was designed to excite chlorophyll fluorescence of tomato seeds. The fluorescent signal acquisition unit was made up of a fluorescence spectrometer, an optical fiber, an optical fiber scaffolds and a narrowband filter. The data processing unit mainly included a computer. Tomato fruits of green ripe stage, discoloration stage, firm ripe stage and full ripe stage were harvested, and their seeds were collected directly. In this research, the developed tomato seeds maturity testing system was used to collect fluorescence spectrums of tomato seeds of different maturities. Principal component analysis (PCA) method was utilized to reduce the dimension of spectral data and extract principal components, and PCA was combined with linear discriminant analysis (LDA) to establish discriminant model of tomato seeds maturity, the discriminant accuracy was greater than 90%. Research results show that using chlorophyll fluorescence spectrum technology is feasible for seeds maturity detection, and the developed tomato seeds maturity testing system has high detection accuracy.

Example-Based Super-Resolution Fluorescence Microscopy.

Science.gov (United States)

Jia, Shu; Han, Boran; Kutz, J Nathan

2018-04-23

Capturing biological dynamics with high spatiotemporal resolution demands the advancement in imaging technologies. Super-resolution fluorescence microscopy offers spatial resolution surpassing the diffraction limit to resolve near-molecular-level details. While various strategies have been reported to improve the temporal resolution of super-resolution imaging, all super-resolution techniques are still fundamentally limited by the trade-off associated with the longer image acquisition time that is needed to achieve higher spatial information. Here, we demonstrated an example-based, computational method that aims to obtain super-resolution images using conventional imaging without increasing the imaging time. With a low-resolution image input, the method provides an estimate of its super-resolution image based on an example database that contains super- and low-resolution image pairs of biological structures of interest. The computational imaging of cellular microtubules agrees approximately with the experimental super-resolution STORM results. This new approach may offer potential improvements in temporal resolution for experimental super-resolution fluorescence microscopy and provide a new path for large-data aided biomedical imaging.

An extremely sensitive monoboronic acid based fluorescent sensor for glucose

International Nuclear Information System (INIS)

Sun Xiangying; Liu Bin; Jiang Yunbao

2004-01-01

An extremely sensitive monoboronic acid based fluorescent sensor for glucose was developed. This was carried out by assembling a fluorescent monoboronic acid, 3-aminophenylboronic acid (PBA) indirectly onto gold surface via its electrostatic interaction with cysteine (Cys) that was directly assembled on the gold surface. The formation of self-assembled bilayers (SAB) was confirmed and primarily characterized by cyclic voltammetry and X-ray photoelectron spectra (XPS). The SAB containing PBA was found fluorescent and its fluorescence showed an extremely high sensitivity to the presence of glucose and other monosaccharides such as galactose and fructose with quenching constants at 10 8 M -1 order of magnitude compared to those at 10 2 M -1 in bulk solutions. The quenching constants were found to vary in the order of D-glucose>D-galactose>D-fructose>D-mannose that is different from that in bulk solution which shows the highest binding affinity toward D-fructose and very low sensitivity toward glucose. The reported monoboronic acid based SAB fluorescent sensor showed the highest sensitivity towards glucose with the capacity of detecting saccharides of concentration down to nanomolar level. It was also demonstrated that the fluorescence from PBA/Cys/Au can be easily recovered after each measurement event and therefore also represents a new reusable method for immobilizing reagent in fabricating chemosensors

Phenylboronic acid functionalized reduced graphene oxide based fluorescence nano sensor for glucose sensing

Energy Technology Data Exchange (ETDEWEB)

Basiruddin, SK; Swain, Sarat K., E-mail: swainsk2@yahoo.co.in

2016-01-01

Reduced graphene has emerged as promising tools for detection based application of biomolecules as it has high surface area with strong fluorescence quenching property. We have used the concept of fluorescent quenching property of reduced graphene oxide to the fluorescent probes which are close vicinity of its surface. In present work, we have synthesized fluorescent based nano-sensor consist of phenylboronic acid functionalized reduced graphene oxide (rGO–PBA) and di-ol modified fluorescent probe for detection of biologically important glucose molecules. This fluorescent graphene based nano-probe has been characterized by high resolution transmission electron microscope (HRTEM), Atomic force microscope (AFM), UV–visible, Photo-luminescence (PL) and Fourier transformed infrared (FT-IR) spectroscopy. Finally, using this PBA functionalized reduced GO based nano-sensor, we were able to detect glucose molecule in the range of 2 mg/mL to 75 mg/mL in aqueous solution of pH 7.4. - Highlights: • Easy and simple synthesis of PBA functionalized reduced GO based nano probe. • PBA functionalized reduced GO graphene based nano-probes are characterized. • PBA functionalized reduced GO nano probe is used to detect glucose molecules. • It is very cost-effective and enzyme-free detection of glucose in solution.

5-ALA/PpIX fluorescence detection of gastrointestinal neoplasia

Science.gov (United States)

Borisova, Ekaterina G.; Vladimirov, Borislav; Terziev, Ivan; Ivanova, Radina; Avramov, Latchezar

2009-07-01

In the recent study delta-ALA/PpIX is used as fluorescent marker for dysplasia and tumor detection in esophagus, stomach and colon. ALA is administered per os six to eight (depending on the lesion location) hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built for the LED to use the light guide of standard video-endoscopic system. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to re-absorption of oxy-hemoglobin in this spectral area. Endogenous and exogenous fluorescence spectra are used to develop simple but effective algorithm, based on dimensionless ratio of the signals at 560 and 635 nm, for differentiation of normal/abnormal gastrointestinal tissues. Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

Advances in Spiropyrans/Spirooxazines and Applications Based on Fluorescence Resonance Energy Transfer (FRET with Fluorescent Materials

Directory of Open Access Journals (Sweden)

Hongyan Xia

2017-12-01

Full Text Available Studies on the following were reviewed: (1 the structure of spiropyrans and spirooxazines (two kinds of spiro compounds under external stimuli and (2 the construction and applications of composite systems based on fluorescence resonance energy transfer (FRET with fluorescent materials. When treated with different stimuli (light, acids and bases, solvents, metal ions, temperature, redox potential, and so on, spiropyrans/spirooxazines undergo transformations between the ring-closed form (SP, the ring-opened merocyanine (MC form, and the protonated ring-opened form (MCH. This is due to the breakage of the spiro C–O bond and the protonation of MC, along with a color change. Various novel, multifunctional materials based on photochromic spiropyrans and spirooxazines have been successfully developed because of the vastly differently physiochemical properties posssed by the SP, MC and MCH forms. Among the three different structural forms, the MC form has been studied most extensively. The MC form not only gives complexes with various inorganic particles, biological molecules, and organic chemicals but also acts as the energy acceptor (of energy from fluorescent molecules during energy transfer processes that take place under proper conditions. Furthermore, spiropyran and spirooxazine compounds exhibit reversible physicochemical property changes under proper stimuli; this provides more advantages compared with other photochromic compounds. Additionally, the molecular structures of spiropyrans and spirooxazines can be easily modified and extended, so better compounds can be obtained to expand the scope of already known applications. Described in detail are: (1 the structural properties of spiropyrans and spirooxazines and related photochromic mechanisms; (2 composite systems based on spiropyrans and spirooxazines, and (3 fluorescent materials which have potential applications in sensing, probing, and a variety of optical elements.

A Graphene Oxide-Based Fluorescent Aptasensor for the Turn-on Detection of CCRF-CEM.

Science.gov (United States)

Tan, Jie; Lai, Zongqiang; Zhong, Liping; Zhang, Zhenghua; Zheng, Rong; Su, Jing; Huang, Yong; Huang, Panpan; Song, Hui; Yang, Nuo; Zhou, Sufang; Zhao, Yongxiang

2018-04-01

A convenient, low-cost, and highly sensitive fluorescent aptasensor for detection of leukemia has been developed based on graphene oxide-aptamer complex (GO-apt). Graphene oxide (GO) can absorb carboxyfluorescein-labeled Sgc8 aptamer (FAM-apt) by π-π stacking and quench the fluorescence through fluorescence resonance energy transfer (FRET). In the absence of Sgc8 target cell CCRF-CEM, the fluorescence is almost all quenched. Conversely, when the CCRF-CEM cells are added, the quenched fluorescence can be recovered rapidly and significantly. Therefore, based on the change of fluorescence signals, we can detect the number of CCRF-CEM cells in a wide range from 1 × 10 2 to 1 × 10 7  cells/mL with a limit of detection (LOD) of 10 cells/mL. Therefore, this strategy of graphene oxide-based fluorescent aptasensor may be promising for the detection of cancer.

An evaluation of a SVA retrotransposon in the FUS promoter as a transcriptional regulator and its association to ALS.

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Abigail L Savage

Full Text Available Genetic mutations of FUS have been linked to many diseases including Amyotrophic Lateral Sclerosis (ALS and Frontotemporal Lobar Degeneration. A primate specific and polymorphic retrotransposon of the SINE-VNTR-Alu (SVA family is present upstream of the FUS gene. Here we have demonstrated that this retrotransposon can act as a classical transcriptional regulatory domain in the context of a reporter gene construct both in vitro in the human SK-N-AS neuroblastoma cell line and in vivo in a chick embryo model. We have also demonstrated that the SVA is composed of multiple distinct regulatory domains, one of which is a variable number tandem repeat (VNTR. The ability of the SVA and its component parts to direct reporter gene expression supported a hypothesis that this region could direct differential FUS expression in vivo. The SVA may therefore contribute to the modulation of FUS expression exhibited in and associated with neurological disorders including ALS where FUS regulation may be an important parameter in progression of the disease. As VNTRs are often clinical associates for disease progression we determined the extent of polymorphism within the SVA. In total 2 variants of the SVA were identified based within a central VNTR. Preliminary analysis addressed the association of these SVA variants within a small sporadic ALS cohort but did not reach statistical significance, although we did not include other parameters such as SNPs within the SVA or an environmental factor in this analysis. The latter may be particularly important as the transcriptional and epigenetic properties of the SVA are likely to be directed by the environment of the cell.

The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae.

Science.gov (United States)

Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Pham, Thu Ha; Phan, Tuan-Nghia; Tran, Van-Tuan

2016-12-01

Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

Distribution of mating-type alleles and M13 PCR markers in the black leaf spot fungus Mycosphaerella fijiensis of bananas in Brazil.

Science.gov (United States)

Queiroz, C B; Miranda, E C; Hanada, R E; Sousa, N R; Gasparotto, L; Soares, M A; Silva, G F

2013-02-08

The fungus Mycosphaerella fijiensis is the causative agent of black sigatoka, which is one of the most destructive diseases of banana plants. Infection with this pathogen results in underdeveloped fruit, with no commercial value. We analyzed the distribution of the M. fijiensis mating-type system and its genetic variability using M13 phage DNA markers. We found a 1:1 distribution of mating-type alleles, indicating MAT1-1 and MAT1-2 idiomorphs. A polymorphism analysis using three different primers for M13 markers showed that only the M13 minisatellite primers generated polymorphic products. We then utilized this polymorphism to characterize 40 isolates from various Brazilian states. The largest genetic distances were found between isolates from the same location and between isolates from different parts of the country. Therefore, there was no correlation between the genetic similarity and the geographic origin of the isolates. The M13 marker was used to generate genetic fingerprints for five isolates; these fingerprints were compared with the band profiles obtained from inter-simple sequence repeat (UBC861) and inter-retrotransposon amplified polymorphism analyses. We found that the M13 marker was more effective than the other two markers for differentiating these isolates.

Early detection of tumor masses by in vivo hematoporphyrin-mediated fluorescence imaging

International Nuclear Information System (INIS)

Autiero, Maddalena; Celentano, Luigi; Cozzolino, Rosanna; Laccetti, Paolo; Marotta, Marcello; Mettivier, Giovanni; Cristina Montesi, Maria; Quarto, Maria; Riccio, Patrizia; Roberti, Giuseppe; Russo, Paolo

2007-01-01

We investigated the capability of fluorescence reflectance imaging (FRI) for the early detection of surface tumors in mice. We used a hematoporphyrin (HP) compound (HP dichlorohydrate) as a red fluorescent marker and a low noise, high sensitivity, digital CCD camera for fluorescence imaging. In this preliminary study, highly malignant anaplastic human thyroid carcinoma cells were implanted subcutaneously in one mouse and their growth was monitored daily for 5 days by FRI. The selective HP uptake by the tumor tissues was successfully observed: we observed the fluorescence of tumor only 3 days after cancer cells injection, i.e. when the tumor mass was neither visible (to the naked eye) or palpable. These measurements indicate that FRI is a suitable technique to detect minute subcutaneous tumor masses. This FRI system will be coupled to a radionuclide imaging system based on a CdTe detector for in vivo multimodal imaging in mice

Fluorescence quantum yields of natural organic matter and organic compounds: Implications for the fluorescence-based interpretation of organic matter composition

DEFF Research Database (Denmark)

Wünsch, Urban; Murphy, Kathleen R.; Stedmon, Colin

2015-01-01

to more than 200 modeled spectra (PARAFAC components) in the OpenFluor database. Apparent matches, based on spectral similarity, were subsequently evaluated using molar fluorescence and absorbance. Five organic compounds were potential matches with PARAFAC components from 16 studies; however, the ability......Absorbance and fluorescence spectroscopy are economical tools for tracing the supply, turnover and fate of dissolved organic matter (DOM). The colored and fluorescent fractions of DOM (CDOM and FDOM, respectively) are linked by the apparent fluorescence quantum yield (AQY) of DOM, which reflects...... the likelihood that chromophores emit fluorescence after absorbing light. Compared to the number of studies investigating CDOM and FDOM, few studies have systematically investigated AQY spectra for DOM, and linked them to fluorescence quantum yields (Φ) of organic compounds. To offer a standardized approach...

Fluorescence Behavior and Dural Infiltration of Meningioma Analyzed by 5-Aminolevulinic Acid-Based Fluorescence: Operating Microscope Versus Mini-Spectrometer.

Science.gov (United States)

Knipps, Johannes; Beseoglu, Kerim; Kamp, Marcel; Fischer, Igor; Felsberg, Joerg; Neumann, Lisa M; Steiger, Hans-Jakob; Cornelius, Jan F

2017-12-01

To compare fluorescence intensity of tumor specimens, as measured by a fluorescence-guided surgery microscope and a spectrometer, to evaluate tumor infiltration of dura mater around meningiomas with help of these 2 different 5-aminolevulinic acid (5-ALA)-based fluorescence tools, and to correlate fluorescence intensity with histopathologic data. In a clinical series, meningiomas were resected by 5-ALA fluorescence-guided surgery. Fluorescence intensity was semiquantitatively rated by the surgeon at predefined points. Biopsies were harvested and fluorescence intensity measured by a spectrometer and histopathologically analyzed. Sampling was realized at the level of the dura in a centrifugal direction. A total of 104 biopsies (n = 13 tumors) were analyzed. Specificity and sensitivity of the microscope were 0.96 and 0.53 and of the spectrometer 0.95 and 0.93, respectively. Fluorescence intensity as measured by the spectrometer was correlated to histologically confirmed tumor burden. In a centrifugal direction, tumor burden and fluorescence intensity continuously decreased (along the dural tail). Below a threshold value of 639 arbitrary units no tumor was histologically detectable. At the level of the dura the spectrometer was highly sensitive for detection of meningioma cells. The surgical microscope showed false negative results and missed residual tumor cells in more than one half of the cases. The complementary use of both fluorescence tools may improve resection quality. Copyright © 2017 Elsevier Inc. All rights reserved.

MASiVEdb: the Sirevirus Plant Retrotransposon Database

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Bousios Alexandros

2012-04-01

Full Text Available Abstract Background Sireviruses are an ancient genus of the Copia superfamily of LTR retrotransposons, and the only one that has exclusively proliferated within plant genomes. Based on experimental data and phylogenetic analyses, Sireviruses have successfully infiltrated many branches of the plant kingdom, extensively colonizing the genomes of grass species. Notably, it was recently shown that they have been a major force in the make-up and evolution of the maize genome, where they currently occupy ~21% of the nuclear content and ~90% of the Copia population. It is highly likely, therefore, that their life dynamics have been fundamental in the genome composition and organization of a plethora of plant hosts. To assist studies into their impact on plant genome evolution and also facilitate accurate identification and annotation of transposable elements in sequencing projects, we developed MASiVEdb (Mapping and Analysis of SireVirus Elements Database, a collective and systematic resource of Sireviruses in plants. Description Taking advantage of the increasing availability of plant genomic sequences, and using an updated version of MASiVE, an algorithm specifically designed to identify Sireviruses based on their highly conserved genome structure, we populated MASiVEdb (http://bat.infspire.org/databases/masivedb/ with data on 16,243 intact Sireviruses (total length >158Mb discovered in 11 fully-sequenced plant genomes. MASiVEdb is unlike any other transposable element database, providing a multitude of highly curated and detailed information on a specific genus across its hosts, such as complete set of coordinates, insertion age, and an analytical breakdown of the structure and gene complement of each element. All data are readily available through basic and advanced query interfaces, batch retrieval, and downloadable files. A purpose-built system is also offered for detecting and visualizing similarity between user sequences and Sireviruses, as

Genetic diversity analysis of sugarcane germplasm based on fluorescence-labeled simple sequence repeat markers and a capillary electrophoresis-based genotyping platform

Science.gov (United States)

Genetic diversity analysis, which refers to the elaboration of total extent of genetic characteristics in the genetic makeup of a certain species, constitutes a classical strategy for the study of diversity, population genetic structure, and breeding practices. In this study, fluorescence-labeled se...

A fluorescence-based rapid screening assay for cytotoxic compounds

International Nuclear Information System (INIS)

Montoya, Jessica; Varela-Ramirez, Armando; Estrada, Abril; Martinez, Luis E.; Garza, Kristine; Aguilera, Renato J.

2004-01-01

A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death

Preparation of bioconjugates of CdTe nanocrystals for cancer marker detection

International Nuclear Information System (INIS)

Hu Fengqin; Ran Yuliang; Zhou Zhuan; Gao Mingyuan

2006-01-01

Highly fluorescent CdTe quantum dots (Q-dots) stabilized by 3-mercaptopropionic acid (MPA) were prepared by an aqueous solution approach and used as fluorescent labels in detecting a cancer marker, carcinoembryonic antigen (CEA), expressed on human colon carcinoma cell line LS 180. Nonspecific adsorptions of CdTe Q-dots on carcinoma cells were observed and effectively eliminated by replacing MPA with a thiolated PEG (poly(ethylene glycol), Mn = 750) synthesized according to literature. It was unexpectedly found out that the PEG-coated CdTe Q-dots exhibited very strong and specific affinity to anti-CEA monoclonal antibody rch 24 (rch 24 mAb). The resultant CdTe-(rch 24 mAb) conjugates were successfully used in detections of CEA expressed on the surface of cell line LS 180. Further experiments demonstrated that the fluorescent CdTe Q-dots exhibited much better photostability and a brighter fluorescence than FITC, which consequently led to a higher efficiency in the cancer marker detection

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

Directory of Open Access Journals (Sweden)

Erin Wilson

2018-05-01

Full Text Available A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP. The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD600 and plate counting (colony-forming units (CFUs. While both OD600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.

Genomic change, retrotransposon mobilization and extensive cytosine methylation alteration in Brassica napus introgressions from two intertribal hybridizations.

Directory of Open Access Journals (Sweden)

Xueli Zhang

Full Text Available Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP, sequence-specific amplification polymorphism (SSAP and methylation-sensitive amplified polymorphism (MSAP were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4-39.8% and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

Science.gov (United States)

Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

2012-07-01

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.

Mixed Marker-Based/Marker-Less Visual Odometry System for Mobile Robots

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Fabrizio Lamberti

2013-05-01

Full Text Available Abstract When moving in generic indoor environments, robotic platforms generally rely solely on information provided by onboard sensors to determine their position and orientation. However, the lack of absolute references often leads to the introduction of severe drifts in estimates computed, making autonomous operations really hard to accomplish. This paper proposes a solution to alleviate the impact of the above issues by combining two vision-based pose estimation techniques working on relative and absolute coordinate systems, respectively. In particular, the unknown ground features in the images that are captured by the vertical camera of a mobile platform are processed by a vision-based odometry algorithm, which is capable of estimating the relative frame-to-frame movements. Then, errors accumulated in the above step are corrected using artificial markers displaced at known positions in the environment. The markers are framed from time to time, which allows the robot to maintain the drifts bounded by additionally providing it with the navigation commands needed for autonomous flight. Accuracy and robustness of the designed technique are demonstrated using an off-the-shelf quadrotor via extensive experimental tests.

Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B. bubalis chromosome 20

Directory of Open Access Journals (Sweden)

Schäffer Alejandro A

2008-11-01

Full Text Available Abstract Background Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification dissociation curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and dissociation curve analysis for use as a novel scoring method. Results As proof of principle for this dissociation curve method, we generated new maps of river buffalo (Bubalus bubalis chromosome 20 by both dissociation curve analysis and conventional marker scoring. We also obtained sequence data to augment dissociation curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46% were suitable as cross-species markers for dissociation curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly. Conclusion Dissociation curve

Preliminary evidence for associations between molecular markers and quantitative traits in a set of bread wheat (Triticum aestivum L.) cultivars and breeding lines.

Science.gov (United States)

Abdollahi Mandoulakani, Babak; Nasri, Shilan; Dashchi, Sahar; Arzhang, Sorour; Bernousi, Iraj; Abbasi Holasou, Hossein

The identification of polymorphic markers associated with various quantitative traits allows us to test their performance for the exploitation of the extensive quantitative variation maintained in gene banks. In the current study, a set of 97 wheat germplasm accessions including 48 cultivars and 49 breeding lines were evaluated for 18 agronomic traits. The accessions were also genotyped with 23 ISSR, nine IRAP and 20 REMAP markers, generating a total of 658 clear and scorable bands, 86% of which were polymorphic. Both neighbor-joining dendrogram and Bayesian analysis of clustering of individuals revealed that the accessions could be divided into four genetically distinct groups, indicating the presence of a population structure in current wheat germplasm. Associations between molecular markers and 18 agronomic traits were analyzed using the mixed linear model (MLM) approach. A total of 94 loci were found to be significantly associated with agronomic traits (P≤0.01). The highest number of bands significantly associated with the 18 traits varied from 11 for number of spikelets spike -1 (NSS) to two for grain yield in row (GRY). Loci ISSR16-9 and REMAP13-10 were associated with three different traits. The results of the current study provide useful information about the performance of retrotransposon-based and ISSR molecular markers that could be helpful in selecting potentially elite gene bank samples for wheat-breeding programs. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

Science.gov (United States)

Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

2017-01-01

In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

Marker-based or model-based RSA for evaluation of hip resurfacing arthroplasty? A clinical validation and 5-year follow-up.

Science.gov (United States)

Lorenzen, Nina Dyrberg; Stilling, Maiken; Jakobsen, Stig Storgaard; Gustafson, Klas; Søballe, Kjeld; Baad-Hansen, Thomas

2013-11-01

The stability of implants is vital to ensure a long-term survival. RSA determines micro-motions of implants as a predictor of early implant failure. RSA can be performed as a marker- or model-based analysis. So far, CAD and RE model-based RSA have not been validated for use in hip resurfacing arthroplasty (HRA). A phantom study determined the precision of marker-based and CAD and RE model-based RSA on a HRA implant. In a clinical study, 19 patients were followed with stereoradiographs until 5 years after surgery. Analysis of double-examination migration results determined the clinical precision of marker-based and CAD model-based RSA, and at the 5-year follow-up, results of the total translation (TT) and the total rotation (TR) for marker- and CAD model-based RSA were compared. The phantom study showed that comparison of the precision (SDdiff) in marker-based RSA analysis was more precise than model-based RSA analysis in TT (p CAD RSA analysis (p = 0.002), but showed no difference between the marker- and CAD model-based RSA analysis regarding the TR (p = 0.91). Comparing the mean signed values regarding the TT and the TR at the 5-year follow-up in 13 patients, the TT was lower (p = 0.03) and the TR higher (p = 0.04) in the marker-based RSA compared to CAD model-based RSA. The precision of marker-based RSA was significantly better than model-based RSA. However, problems with occluded markers lead to exclusion of many patients which was not a problem with model-based RSA. HRA were stable at the 5-year follow-up. The detection limit was 0.2 mm TT and 1° TR for marker-based and 0.5 mm TT and 1° TR for CAD model-based RSA for HRA.

Intense fluorescence of Au ₂₀

Energy Technology Data Exchange (ETDEWEB)

Yu, Chongqi; Harbich, Wolfgang; Sementa, Luca; Ghiringhelli, Luca; Apra, Edoardo; Stener, Mauro; Fortunelli, Alessandro; Brune, Harald

2017-08-21

Ligand-protected Au clusters are non-bleaching fluorescence markers in bio- and medical applications. We show that their fluorescence is an intrinsic property of the Au cluster itself. We find a very intense and sharp fluorescence peak located at λ =739.2 nm (1.68 eV) for Au20 clusters in a Ne matrix held at 6 K. The fluorescence reflects the HOMO-LUMO diabatic bandgap of the cluster. The cluster shows a very rich absorption fine structure reminiscent of well defined molecule-like quantum levels. These levels are resolved since Au20 has only one stable isomer (tetrahedral), therefore our sample is mono-disperse in cluster size and conformation. Density-functional theory (DFT) and time-dependent DFT calculations clarify the nature of optical absorptionand predict both main absorption peaks and intrinsic fluorescence in good agreement with experiment.

Identification of an Internal Ribosome Entry Segment in the 5′ Region of the Mouse VL30 Retrotransposon and Its Use in the Development of Retroviral Vectors

Science.gov (United States)

López-Lastra, Marcelo; Ulrici, Sandrine; Gabus, Caroline; Darlix, Jean-Luc

1999-01-01

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5′ region of VL30m could replace the 5′ leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5′ region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5′ region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5′ region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors. PMID:10482590

Induced marker gene mutations in soybean

International Nuclear Information System (INIS)

Sawada, S.; Palmer, R.G.

1989-01-01

Full text: Non-fluorescent root mutants in soybean are useful as markers in genetic studies. 13 such mutants were detected among more than 150 000 seedlings derived from soybean lines treated with 6 mutagens. One of them, derived from variety 'Williams' treated with 20 kR gamma rays, did not correspond to the already known spontaneous non-fluorescent mutants. It was assigned the identification no. T285 and the gene symbol fr5. The other mutants corresponded with known loci fr1, fr2 or fr4. (author)

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

Science.gov (United States)

Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

2017-09-01

Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

A chromenoquinoline-based fluorescent off-on thiol probe for bioimaging.

Science.gov (United States)

Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Varma, Sreejith Jayasree; Talukdar, Pinaki

2012-03-11

A new chromenoquinoline-based fluorescent off-on thiol probe 2 is reported. In aqueous buffer solutions at physiological pH, the probe exhibited 223-fold enhancement in fluorescence intensity by a Michael addition of cysteine to the maleimide appended to a chromenoquinoline. Cell permeability and live cell imaging of thiols are also demonstrated. This journal is © The Royal Society of Chemistry 2012

An aptamer-based fluorescence bio-sensor for chiral recognition of arginine enantiomers.

Science.gov (United States)

Yuan, Haiyan; Huang, Yunmei; Yang, Jidong; Guo, Yuan; Zeng, Xiaoqing; Zhou, Shang; Cheng, Jiawei; Zhang, Yuhui

2018-07-05

In this study, a novel aptamer - based fluorescence bio-sensor (aptamer-AuNps) was developed for chiral recognition of arginine (Arg) enantiomers based on aptamer and gold nanoparticles (AuNps). Carboxyfluorescein (FAM) labeled aptamers (Apt) were absorbed on AuNps and their fluorescence intensity could be significantly quenched by AuNps based on fluorescence resonance energy transfer (FRET). Once d-Arg or l-Arg were added into the above solution, the aptamer specifically bind to Arg enantiomers and released from AuNps, so the fluorescence intensity of d-Arg system and l-Arg system were all enhanced. The affinity of Apt to l-Arg is tighter to d-Arg, so the enhanced fluorescence signals of l-Arg system was stronger than d-Arg system. What's more, the enhanced fluorescence were directly proportional to the concentration of d-Arg and l-Arg ranging from 0-300 nM and 0-400 nM with related coefficients of 0.9939 and 0.9952, respectively. Furthermore, the method was successfully applied to detection l-Arg in human urine samples with satisfactory results. Eventually, a simple "OR" logic gate with d-Arg &l-Arg as inputs and AuNps aggregation state as outputs was fabricated, which can help us understand the chiral recognition process deeply. Copyright © 2018 Elsevier B.V. All rights reserved.

Image navigation as a means to expand the boundaries of fluorescence-guided surgery.

Science.gov (United States)

Brouwer, Oscar R; Buckle, Tessa; Bunschoten, Anton; Kuil, Joeri; Vahrmeijer, Alexander L; Wendler, Thomas; Valdés-Olmos, Renato A; van der Poel, Henk G; van Leeuwen, Fijs W B

2012-05-21

Hybrid tracers that are both radioactive and fluorescent help extend the use of fluorescence-guided surgery to deeper structures. Such hybrid tracers facilitate preoperative surgical planning using (3D) scintigraphic images and enable synchronous intraoperative radio- and fluorescence guidance. Nevertheless, we previously found that improved orientation during laparoscopic surgery remains desirable. Here we illustrate how intraoperative navigation based on optical tracking of a fluorescence endoscope may help further improve the accuracy of hybrid surgical guidance. After feeding SPECT/CT images with an optical fiducial as a reference target to the navigation system, optical tracking could be used to position the tip of the fluorescence endoscope relative to the preoperative 3D imaging data. This hybrid navigation approach allowed us to accurately identify marker seeds in a phantom setup. The multispectral nature of the fluorescence endoscope enabled stepwise visualization of the two clinically approved fluorescent dyes, fluorescein and indocyanine green. In addition, the approach was used to navigate toward the prostate in a patient undergoing robot-assisted prostatectomy. Navigation of the tracked fluorescence endoscope toward the target identified on SPECT/CT resulted in real-time gradual visualization of the fluorescent signal in the prostate, thus providing an intraoperative confirmation of the navigation accuracy.

Plasmonic-based instrument response function for time-resolved fluorescence: toward proper lifetime analysis

Energy Technology Data Exchange (ETDEWEB)

Szlazak, Radoslaw; Tutaj, Krzysztof; Grudzinski, Wojciech; Gruszecki, Wieslaw I.; Luchowski, Rafal, E-mail: rafal.luchowski@umcs.pl [Maria Curie-Sklodowska University, Department of Biophysics, Institute of Physics (Poland)

2013-06-15

In this report, we investigated the so-called plasmonic platforms prepared to target ultra-short fluorescence and accurate instrumental response function in a time-domain spectroscopy and microscopy. The interaction of metallic nanoparticles with nearby fluorophores results in the increase of the dye fluorescence quantum yield, photostability and decrease of the lifetime parameter. The mentioned properties of platforms were applied to achieve a picosecond fluorescence lifetime (21 ps) of erythrosin B, used later as a better choice for deconvolution of fluorescence decays measured with 'color' sensitive photo-detectors. The ultra-short fluorescence standard based on combination of thin layers of silver film, silver colloidal nanoparticles (about 60 nm in diameter), and top layer of erythrosin B embedded in 0.2 % poly(vinyl) alcohol. The response functions were monitored on two photo-detectors; microchannel plate photomultiplier and single photon avalanche photodiode as a Rayleigh scattering and ultra-short fluorescence. We demonstrated that use of the plasmonic base fluorescence standard as an instrumental response function results in the absence of systematic error in lifetime measurements and analysis.

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

Science.gov (United States)

Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

2016-04-01

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer

International Nuclear Information System (INIS)

Kokko, Tiina; Kokko, Leena; Soukka, Tero; Loevgren, Timo

2007-01-01

A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin-quencher is bound to Eu-streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher-biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2-2 nmol L -1 . In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay

A fluorescence detection of D-penicillamine based on Cu(2+)-induced fluorescence quenching system of protein-stabilized gold nanoclusters.

Science.gov (United States)

Wang, Peng; Li, Bang Lin; Li, Nian Bing; Luo, Hong Qun

2015-01-25

In this contribution, a luminescent gold nanoclusters which were synthesized by bovine serum albumin as novel fluorescent probes were successfully utilized for the determination of D-penicillamine for the first time. Cupric ion was employed to quench the strong fluorescence of the gold nanoclusters, whereas the addition of D-penicillamine caused obvious restoration of fluorescence intensity of the Cu(2+)-gold nanoclusters system. Under optimum conditions, the increment in fluorescence intensity of Cu(2+)-gold nanoclusters system caused by D-penicillamine was linearly proportional to the concentration of D-penicillamine in the range of 2.0×10(-5)-2.39×10(-4) M. The detection limit for D-penicillamine was 5.4×10(-6) M. With the off-on fluorescence signal at 650 nm approaching the near-infrared region, the present sensor for D-penicillamine detection had high sensitivity and low spectral interference. Furthermore, the novel gold nanoclusters-based fluorescent sensor has been applied to the determination of D-penicillamine in real biological samples with satisfactory results. Copyright © 2014 Elsevier B.V. All rights reserved.

Fluorescent properties of novel dendrimer dyes based on thiazole orange

International Nuclear Information System (INIS)

Fei Xuening; Gu Yingchun; Lan Yunquan; Shi Bin

2011-01-01

In this paper, polyamidoamine (PAMAM) dendrimers with active amino group of some generations (G=0.5-2) were prepared from commercial aminoacetaldehyde diethyl acetal by the divergent method. After that, thiazole orange (TO) with -COOH was incorporated with dendrimers of G=1 and 2 to afford novel dendrimer-TO dyes. The fluorescent properties studies showed that the fluorescent intensity of the same concentration of dendrimer-TO (G=2) was higher than that of the dendrimer-TO (G=1), and both of them were much stronger than free TO with -COOH. There was a fluorescent enhancement of the dendrimer dyes compared with free dye. The dendrimer dyes were of well-defined chemical structure,with little aggregation and self-quenching as well as good fluorescence properties of good stability, high intensity and sensitivity, which could be used in labeling cancer cells and further in diagnosis and detection of early-stage tumors. - Highlights: → A kind of dendrimer probe based on TO was designed and synthesized. → Dendrimers showed an obvious fluorescence enhancement compared to free dye. → Dendrimers labeled with BSA also showed fluorescence enhancement. → Dendrimers may be used in diagnosis and detection of early-stage tumors.

Simulation and application of micro X-ray fluorescence based on an ellipsoidal capillary

Energy Technology Data Exchange (ETDEWEB)

Yang, Jing; Li, Yude; Wang, Xingyi; Zhang, Xiaoyun; Lin, Xiaoyan, E-mail: yangjing_928@126.com

2017-06-15

Highlights: • A micro X-ray fluorescence setup based on an ellipsoidal capillary was presented. • The optimal parameters of ellipsoidal capillary were designed. • The 2D mapping image of biological sample was obtained. - Abstract: A micro X-ray fluorescence setup was presented, based on an ellipsoidal capillary and a traditional laboratorial X-ray source. Using Ray-tracing principle, we have simulated the transmission path of X-ray beam in the ellipsoidal capillary and designed the optimal parameters of the ellipsoidal capillary for the micro X-ray fluorescence setup. We demonstrate that ellipsoidal capillary is well suited as condenser for the micro X-ray fluorescence based on traditional laboratorial X-ray source. Furthermore, we obtain the 2D mapping image of the leaf blade sample by using the ellipsoidal capillary we designed.

Depth profiling of marker layers using x-ray waveguide structures

International Nuclear Information System (INIS)

Gupta, Ajay; Rajput, Parasmani; Saraiya, Amit; Reddy, V. R.; Gupta, Mukul; Bernstorff, Sigrid; Amenitsch, H.

2005-01-01

It is demonstrated that x-ray waveguide structures can be used for depth profiling of a marker layer inside the guiding layer with an accuracy of better than 0.2 nm. A combination of x-ray fluorescence and x-ray reflectivity measurements can provide detailed information about the structure of the guiding layer. The position and thickness of the marker layer affect different aspects of the angle-dependent x-ray fluorescence pattern, thus making it possible to determine the structure of the marker layer in an unambiguous manner. As an example, effects of swift heavy ion irradiation on a Si/M/Si trilayer (M=Fe, W), forming the cavity of the waveguide structure, have been studied. It is found that in accordance with the prediction of thermal spike model, Fe is much more sensitive to swift heavy ion induced modifications as compared to W, even in thin film form. However, a clear evidence of movement of the Fe marker layer towards the surface is observed after irradiation, which cannot be understood in terms of the thermal spike model alone

Depth profiling of marker layers using x-ray waveguide structures

Science.gov (United States)

Gupta, Ajay; Rajput, Parasmani; Saraiya, Amit; Reddy, V. R.; Gupta, Mukul; Bernstorff, Sigrid; Amenitsch, H.

2005-08-01

It is demonstrated that x-ray waveguide structures can be used for depth profiling of a marker layer inside the guiding layer with an accuracy of better than 0.2 nm. A combination of x-ray fluorescence and x-ray reflectivity measurements can provide detailed information about the structure of the guiding layer. The position and thickness of the marker layer affect different aspects of the angle-dependent x-ray fluorescence pattern, thus making it possible to determine the structure of the marker layer in an unambiguous manner. As an example, effects of swift heavy ion irradiation on a Si/M/Si trilayer ( M=Fe , W), forming the cavity of the waveguide structure, have been studied. It is found that in accordance with the prediction of thermal spike model, Fe is much more sensitive to swift heavy ion induced modifications as compared to W, even in thin film form. However, a clear evidence of movement of the Fe marker layer towards the surface is observed after irradiation, which cannot be understood in terms of the thermal spike model alone.

Pancreatic tumor detection using hypericin-based fluorescence spectroscopy and cytology

Science.gov (United States)

Lavu, Harish; Geary, Kevin; Fetterman, Harold R.; Saxton, Romaine E.

2005-04-01

Hypericin is a novel, highly fluorescent photosensitizer that exhibits selective tumor cell uptake properties and is particularly resistant to photobleaching. In this study, we have characterized hypericin uptake in human pancreatic tumor cells with relation to incubation time, cell number, and drug concentration. Ex vivo hypericin based fluorescence spectroscopy was performed to detect the presence of MIA PaCa-2 pancreatic tumor cells in the peritoneal cavity of BALB/c nude mice, as well as to quantify gross tumor burden. Hypericin based cytology of peritoneal lavage samples, using both one and two photon laser confocal microscopy, demonstrated more than a two-fold increase in fluorescence emission of pancreatic tumor cells as compared to control samples. In vitro treatment of pancreatic cancer cells with hypericin based photodynamic therapy showed tumor cell cytotoxicity in a drug dose, incident laser power, and time dependent manner. For these experiments, a continuous wavelength solid-state laser source (532 nm) was operated at power levels in the range of 100-400 mW. Potential applications of hypericin in tumor diagnosis, staging, and therapy will be presented.

Molecularly imprinted fluorescent probe based on FRET for selective and sensitive detection of doxorubicin

Energy Technology Data Exchange (ETDEWEB)

Xu, Zhifeng, E-mail: 897061147@qq.com [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China); Deng, Peihong; Li, Junhua [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China); Xu, Li [Department of Applied Chemistry, College of Materials and Energy, South China Agricultural University, Guangzhou 510642 (China); Tang, Siping [College of Chemistry and Materials Science, Hengyang Normal University, Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008 (China)

2017-04-15

Highlights: • FRET-based molecularly imprinted probe for detection of doxorubicin was prepared. • The detection limit of the probe was 13.8 nM for doxorubicin. • The FRET-based probe had a higher selectivity for the template than ordinary MIMs. - Abstract: In this work, a new type of fluorescent probe for detection of doxorubicin has been constructed by the combined use of fluorescence resonance energy transfer (FRET) technology and molecular imprinting technique (MIT). Using doxorubicin as the template, the molecularly imprinted polymer thin layer was fabricated on the surfaces of carbon dot (CD) modified silica by sol-gel polymerization. The excitation energy of the fluorescent donor (CDs) could be transferred to the fluorescent acceptor (doxorubicin). The FRET based fluorescent probe demonstrated high sensitivity and selectivity for doxorubicin. The detection limit was 13.8 nM. The fluorescent probe was successfully applied for detecting doxorubicin in doxorubicin-spiked plasmas with a recovery of 96.8–103.8%, a relative standard deviation (RSD) of 1.3–2.8%. The strategy for construction of FRET-based molecularly imprinted materials developed in this work is very promising for analytical applications.

Parallel scan hyperspectral fluorescence imaging system and biomedical application for microarrays

International Nuclear Information System (INIS)

Liu Zhiyi; Ma Suihua; Liu Le; Guo Jihua; He Yonghong; Ji Yanhong

2011-01-01

Microarray research offers great potential for analysis of gene expression profile and leads to greatly improved experimental throughput. A number of instruments have been reported for microarray detection, such as chemiluminescence, surface plasmon resonance, and fluorescence markers. Fluorescence imaging is popular for the readout of microarrays. In this paper we develop a quasi-confocal, multichannel parallel scan hyperspectral fluorescence imaging system for microarray research. Hyperspectral imaging records the entire emission spectrum for every voxel within the imaged area in contrast to recording only fluorescence intensities of filter-based scanners. Coupled with data analysis, the recorded spectral information allows for quantitative identification of the contributions of multiple, spectrally overlapping fluorescent dyes and elimination of unwanted artifacts. The mechanism of quasi-confocal imaging provides a high signal-to-noise ratio, and parallel scan makes this approach a high throughput technique for microarray analysis. This system is improved with a specifically designed spectrometer which can offer a spectral resolution of 0.2 nm, and operates with spatial resolutions ranging from 2 to 30 μm . Finally, the application of the system is demonstrated by reading out microarrays for identification of bacteria.

Scanning electron microscopy of individual nanoparticle bio-markers in liquid

Energy Technology Data Exchange (ETDEWEB)

Liv, Nalan, E-mail: n.liv@tudelft.nl; Lazić, Ivan; Kruit, Pieter; Hoogenboom, Jacob P.

2014-08-01

We investigated SEM imaging of nanoparticle biomarkers suspended below a thin membrane, with the ultimate goal of integrating functional fluorescence and structural SEM measurements of samples kept at ambient or hydrated conditions. In particular, we investigated how resolving power in liquid SEM is affected by the interaction of the electron beam with the membrane. Simulations with the Geant4-based Monte Carlo scheme developed by Kieft and Bosch (2008) [1] are compared to experimental results with suspended nanoparticles. For 20 nm and 50 nm thin membranes, we found a beam broadening of 1.5 nm and 3 nm, respectively, with an excellent agreement between simulations and experiments. 15 nm Au nanoparticles and bio-functionalized core-shell quantum dots can be individually resolved in denser clusters. We demonstrated the imaging of single EGF-conjugated quantum dots docked at filopodia during cellular uptake with both fluorescence microscopy and SEM simultaneously. These results open novel opportunities for correlating live fluorescence microscopy with structural electron microscopy. - Highlights: • We investigate the achievable resolution in liquid scanning electron microscopy (SEM). • We demonstrate liquid SEM imaging of individual fluorescent nanoparticle bio-markers • We show imaging of cellular QDot uptake with simultaneous fluorescence microscopy and SEM. • The positions of individual QDots can be resolved with details on cellular structure.

Scanning electron microscopy of individual nanoparticle bio-markers in liquid

International Nuclear Information System (INIS)

Liv, Nalan; Lazić, Ivan; Kruit, Pieter; Hoogenboom, Jacob P.

2014-01-01

We investigated SEM imaging of nanoparticle biomarkers suspended below a thin membrane, with the ultimate goal of integrating functional fluorescence and structural SEM measurements of samples kept at ambient or hydrated conditions. In particular, we investigated how resolving power in liquid SEM is affected by the interaction of the electron beam with the membrane. Simulations with the Geant4-based Monte Carlo scheme developed by Kieft and Bosch (2008) [1] are compared to experimental results with suspended nanoparticles. For 20 nm and 50 nm thin membranes, we found a beam broadening of 1.5 nm and 3 nm, respectively, with an excellent agreement between simulations and experiments. 15 nm Au nanoparticles and bio-functionalized core-shell quantum dots can be individually resolved in denser clusters. We demonstrated the imaging of single EGF-conjugated quantum dots docked at filopodia during cellular uptake with both fluorescence microscopy and SEM simultaneously. These results open novel opportunities for correlating live fluorescence microscopy with structural electron microscopy. - Highlights: • We investigate the achievable resolution in liquid scanning electron microscopy (SEM). • We demonstrate liquid SEM imaging of individual fluorescent nanoparticle bio-markers • We show imaging of cellular QDot uptake with simultaneous fluorescence microscopy and SEM. • The positions of individual QDots can be resolved with details on cellular structure

Simultaneous quantitative detection of multiple tumor markers with a rapid and sensitive multicolor quantum dots based immunochromatographic test strip.

Science.gov (United States)

Wang, Chunying; Hou, Fei; Ma, Yicai

2015-06-15

A novel multicolor quantum dots (QDs) based immunochromatographic test strip (ICTS) was developed for simultaneous quantitative detection of multiple tumor markers, by utilizing alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) as models. The immunosensor could realize simultaneous quantitative detection of tumor markers with only one test line and one control line on the nitrocellulose membrane (NC membrane) due to the introduction of multicolor QDs. In this method, a mixture of mouse anti-AFP McAb and mouse anti-CEA McAb was coated on NC membrane as test line and goat anti-mouse IgG antibody was coated as control line. Anti-AFP McAb-QDs546 conjugates and anti-CEA McAb-QDs620 conjugates were mixed and applied to the conjugate pad. Simultaneous quantitative detection of multiple tumor markers was achieved by detecting the fluorescence intensity of captured QDs labels on test line and control line using a test strip reader. Under the optimum conditions, AFP and CEA could be detected as low as 3 ng/mL and 2 ng/mL in 15 min with a sample volume of 80 μL, and no obvious cross-reactivity was observed. The immunosensor was validated with 130 clinical samples and in which it exhibited high sensitivity (93% for AFP and 87% for CEA) and specificity (94% for AFP and 97% for CEA). The immunosensor also demonstrated high recoveries (87.5-113% for AFP and 90-97.3% for CEA) and low relative standard deviations (RSDs) (2.8-6.2% for AFP and 4.9-9.6% for CEA) when testing spiked human serum. This novel multicolor QDs based ICTS provides an easy and rapid, simultaneous quantitative detecting strategy for point-of-care testing of tumor markers. Copyright © 2014 Elsevier B.V. All rights reserved.

Strategies of molecular imprinting-based fluorescence sensors for chemical and biological analysis.

Science.gov (United States)

Yang, Qian; Li, Jinhua; Wang, Xiaoyan; Peng, Hailong; Xiong, Hua; Chen, Lingxin

2018-07-30

One pressing concern today is to construct sensors that can withstand various disturbances for highly selective and sensitive detecting trace analytes in complicated samples. Molecularly imprinted polymers (MIPs) with tailor-made binding sites are preferred to be recognition elements in sensors for effective targets detection, and fluorescence measurement assists in highly sensitive detection and user-friendly control. Accordingly, molecular imprinting-based fluorescence sensors (MI-FL sensors) have attracted great research interest in many fields such as chemical and biological analysis. Herein, we comprehensively review the recent advances in MI-FL sensors construction and applications, giving insights on sensing principles and signal transduction mechanisms, focusing on general construction strategies for intrinsically fluorescent or nonfluorescent analytes and improvement strategies in sensing performance, particularly in sensitivity. Construction strategies are well overviewed, mainly including the traditional indirect methods of competitive binding against pre-bound fluorescent indicators, employment of fluorescent functional monomers and embedding of fluorescence substances, and novel rational designs of hierarchical architecture (core-shell/hollow and mesoporous structures), post-imprinting modification, and ratiometric fluorescence detection. Furthermore, MI-FL sensor based microdevices are discussed, involving micromotors, test strips and microfluidics, which are more portable for rapid point-of-care detection and in-field diagnosing. Finally, the current challenges and future perspectives of MI-FL sensors are proposed. Copyright © 2018 Elsevier B.V. All rights reserved.

Biomedical wellness monitoring system based upon molecular markers

Science.gov (United States)

Ingram, Whitney

2012-06-01

We wish to assist caretakers with a sensor monitoring systems for tracking the physiological changes of homealone patients. One goal is seeking biomarkers and modern imaging sensors like stochastic optical reconstruction microscopy (STORM), which has achieved visible imaging at the nano-scale range. Imaging techniques like STORM can be combined with a fluorescent functional marker in a system to capture the early transformation signs from wellness to illness. By exploiting both microscopic knowledge of genetic pre-disposition and the macroscopic influence of epigenetic factors we hope to target these changes remotely. We adopt dual spectral infrared imaging for blind source separation (BSS) to detect angiogenesis changes and use laser speckle imaging for hypertension blood flow monitoring. Our design hypothesis for the monitoring system is guided by the user-friendly, veteran-preferred "4-Non" principles (noninvasive, non-contact, non-tethered, non-stop-to-measure) and by the NIH's "4Ps" initiatives (predictive, personalized, preemptive, and participatory). We augment the potential storage system with the recent know-how of video Compressive Sampling (CSp) from surveillance cameras. In CSp only major changes are saved, which reduces the manpower cost of caretakers and medical analysts. This CSp algorithm is based on smart associative memory (AM) matrix storage: change features and detailed scenes are written by the outer-product and read by the inner product without the usual Harsh index for image searching. From this approach, we attempt to design an effective household monitoring approach to save healthcare costs and maintain the quality of life of seniors.

Graphene oxide based photoinduced charge transfer label-free near-infrared fluorescent biosensor for dopamine.

Science.gov (United States)

Chen, Jin-Long; Yan, Xiu-Ping; Meng, Kang; Wang, Shu-Feng

2011-11-15

While the super fluorescence quenching capacity of graphene and graphene oxide (GO) has been extensively employed to develop fluorescent sensors, their own unique fluorescence and its potential for chemo-/biosensing have seldom been explored. Here we report a GO-based photoinduced charge transfer (PCT) label-free near-infrared (near-IR) fluorescent biosensor for dopamine (DA). The multiple noncovalent interactions between GO and DA and the ultrafast decay at the picosecond range of the near-IR fluorescence of GO resulted in effective self-assembly of DA molecules on the surface of GO, and significant fluorescence quenching, allowing development of a PCT-based biosensor with direct readout of the near-IR fluorescence of GO for selective and sensitive detection of DA. The developed method gave a detection limit of 94 nM and a relative standard deviation of 2.0% for 11 replicate detections of 2.0 μM DA and was successfully applied to the determination of DA in biological fluids with quantitative recovery (98-115%).

Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

Energy Technology Data Exchange (ETDEWEB)

Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz; Lukasik, Beata; Garner, Logan E.; Chworos, Arkadiusz

2017-05-01

Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining was determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.

Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

Science.gov (United States)

Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

2015-01-01

This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

Marker chromosome 21 identified by microdissection and FISH

Energy Technology Data Exchange (ETDEWEB)

Sun, Y.; Palmer, C.G. [Indiana Univ. School of Medicine, Indianapolis, IN (United States); Rubinstein, J. [Univ. Affiliated Cincinnati Center for Developmental Disorders, OH (United States)] [and others

1995-03-27

A child without Down`s syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with {alpha}-satellite DNA probes for acrocentric chromosomes and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21. 14 refs., 3 figs.

Fluorescent nanohybrids based on asymmetrical cyanine dyes decorated carbon nanotubes

OpenAIRE

Çavuşlar, Özge; Cavuslar, Ozge

2015-01-01

In this thesis, we focused on imparting new optical properties to carbon nanotubes (CNTs) to allow their optical detection and visualization in biomedical applications. We investigated the interactions of CNTs and DNA wrapped CNTs with asymmetrical cyanine dye molecules to study the applicability of resulting hybrid materials to fluorescent based systems. When CNTs interacted with asymmetrical cyanine dyes, they constructed a light absorbing nanoarray. However, the fluorescence emission of th...

Multistage morphological segmentation of bright-field and fluorescent microscopy images

Science.gov (United States)

Korzyńska, A.; Iwanowski, M.

2012-06-01

This paper describes the multistage morphological segmentation method (MSMA) for microscopic cell images. The proposed method enables us to study the cell behaviour by using a sequence of two types of microscopic images: bright field images and/or fluorescent images. The proposed method is based on two types of information: the cell texture coming from the bright field images and intensity of light emission, done by fluorescent markers. The method is dedicated to the image sequences segmentation and it is based on mathematical morphology methods supported by other image processing techniques. The method allows for detecting cells in image independently from a degree of their flattening and from presenting structures which produce the texture. It makes use of some synergic information from the fluorescent light emission image as the support information. The MSMA method has been applied to images acquired during the experiments on neural stem cells as well as to artificial images. In order to validate the method, two types of errors have been considered: the error of cell area detection and the error of cell position using artificial images as the "gold standard".

Ratiometric fluorescent nanosensor based on carbon dots for the detection of mercury ion

Science.gov (United States)

Ma, Yusha; Mei, Jing; Bai, Jianliang; Chen, Xu; Ren, Lili

2018-05-01

A novel ratiometric fluorescent nanosensor based on carbon dots has been synthesized via bonding rhodamine B hydrazide to the carbon dots surface by an amide reaction. The ratiometric fluorescent nanosensor showed only a single blue fluorescence emission around 450 nm. While, as mercury ion was added, due to the open-ring of rhodamine moiety bonded on the CDs surface, the orange emission of the open-ring rhodamine would increase obviously according to the concentration of mercury ion, resulting in the distinguishable dual emissions at 450 nm and 575 nm under a single 360 excitation wavelength. Meanwhile, the ratiometric fluorescent nanosensor based on carbon dots we prepared is more sensitive to qualitative and semi-quantitative detection of mercury ion in the range of 0–100 μM, because fluorescence changes gradually from blue to orange emission under 365 nm lamp with the increasing of mercury ion in the tested solution.

A Novel Marker Based Method to Teeth Alignment in MRI

Science.gov (United States)

Luukinen, Jean-Marc; Aalto, Daniel; Malinen, Jarmo; Niikuni, Naoko; Saunavaara, Jani; Jääsaari, Päivi; Ojalammi, Antti; Parkkola, Riitta; Soukka, Tero; Happonen, Risto-Pekka

2018-04-01

Magnetic resonance imaging (MRI) can precisely capture the anatomy of the vocal tract. However, the crowns of teeth are not visible in standard MRI scans. In this study, a marker-based teeth alignment method is presented and evaluated. Ten patients undergoing orthognathic surgery were enrolled. Supraglottal airways were imaged preoperatively using structural MRI. MRI visible markers were developed, and they were attached to maxillary teeth and corresponding locations on the dental casts. Repeated measurements of intermarker distances in MRI and in a replica model was compared using linear regression analysis. Dental cast MRI and corresponding caliper measurements did not differ significantly. In contrast, the marker locations in vivo differed somewhat from the dental cast measurements likely due to marker placement inaccuracies. The markers were clearly visible in MRI and allowed for dental models to be aligned to head and neck MRI scans.

Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly.

Science.gov (United States)

Patra, Digambara

2010-01-15

A synchronous fluorescence probe based biosensor for estimation of albumin with high sensitivity and selectivity was developed. Unlike conventional fluorescence emission or excitation spectral measurements, synchronous fluorescence measurement offered exclusively a new synchronous fluorescence peak in the shorter wavelength range upon binding of chrysene with protein making it an easy identification tool for albumin determination. The cooperative binding of a fluorescence probe, chrysene, in a supramolecular host-protein assembly during various albumin assessments was investigated. The presence of supramolecular host molecules such as beta-cyclodextrin, curucurbit[6]uril or curucurbit[7]uril have little influence on sensitivity or limit of detection during albumin determination but reduced dramatically interference from various coexisting metal ion quenchers/enhancers. Using the present method the limit of detection for BSA and gamma-Globulin was found to be 0.005 microM which is more sensitive than reported values. Copyright 2009 Elsevier B.V. All rights reserved.

Highly stable lipid-encapsulation of fluorescent nanodiamonds for bioimaging applications.

Science.gov (United States)

Sotoma, Shingo; Hsieh, Feng-Jen; Chen, Yen-Wei; Tsai, Pei-Chang; Chang, Huan-Cheng

2018-01-23

Highly stable lipid-encapsulated fluorescent nanodiamonds (FNDs) are produced by photo-crosslinking of diacetylene-containing lipids physically attached to the FND surface. Not only is this coating method simple and fast, but also it gives the FND-lipid hybrids favorable properties for bioapplications. The hybrids are useful as fluorescent biolabels as well as fiducial markers for correlative light and electron microscopy.

Selective detection of heavy metal ions by calixarene-based fluorescent molecular sensors

Science.gov (United States)

Zhang, Haitao; Faye, Djibril; Zhang, Han; Lefevre, Jean-Pierre; Delaire, J. A.; Leray, Isabelle

2012-06-01

The synthesis, spectroscopic characterization and complexing properties of calixarene-based fluorescent sensors are reported. The calixarene bearing four dansyl fluorophores (Calix-DANS4) exhibits a very high affinity for the detection of lead. A fluorimetric micro-device based on the use of a Y-shape microchannel was developed and allows lead detection with a 5 ppb detection limit. For mercury detection, a fluorescent molecular sensor containing a calixarene anchored with four 8-quinolinoloxy groups (Calix-Q) has been synthesized. The absorption and fluorescence spectra of this sensor are sensitive to the presence of metal cations. An efficient fluorescence quenching is observed upon mercury complexation because of a photoinduced electron transfer from the fluorophore to the bound mercury. Calix-Q shows a high selectivity towards Hg2+ over interfering cations (Na+, K+, Ca2+, Cu2+, Zn2+, Cd2+ and Pb2+) and a 70 ppb sensitivity.

From quality markers to data mining and intelligence assessment: A smart quality-evaluation strategy for traditional Chinese medicine based on quality markers.

Science.gov (United States)

Bai, Gang; Zhang, Tiejun; Hou, Yuanyuan; Ding, Guoyu; Jiang, Min; Luo, Guoan

2018-01-31

The quality of traditional Chinese medicine (TCM) forms the foundation of its clinical efficacy. The standardization of TCM is the most important task of TCM modernization. In recent years, there has been great progress in the quality control of TCM. However, there are still many issues related to the current quality standards, and it is difficult to objectively evaluate and effectively control the quality of TCM. To face these challenge, we summarized the current quality marker (Q-marker) research based on its characteristics and benefits, and proposed a reasonable and intelligentized quality evaluation strategy for the development and application of Q-markers. Ultra-performance liquid chromatography-quadrupole/time-of-flight with partial least squares-discriminant analysis was suggested to screen the chemical markers from Chinese medicinal materials (CMM), and a bioactive-guided evaluation method was used to select the Q-markers. Near-infrared spectroscopy (NIRS), based on the distinctive wavenumber zones or points from the Q-markers, was developed for its determination. Then, artificial intelligence algorithms were used to clarify the complex relationship between the Q-markers and their integral functions. Internet and mobile communication technology helped us to perform remote analysis and determine the information feedback of test samples. The quality control research, evaluation, standard establishment and quality control of TCM must be based on the systematic analysis of Q-markers to study and describe the material basis of TCM efficacy, define the chemical markers in the plant body, and understand the process of herb drug acquisition, change and transmission laws affecting metabolism and exposure. Based on the advantages of chemometrics, new sensor technologies, including infrared spectroscopy, hyperspectral imaging, chemical imaging, electronic nose and electronic tongue, have become increasingly important in the quality evaluation of CMM. Inspired by the

Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

Science.gov (United States)

Montoya, Leticia A; Pluth, Michael D

2014-06-17

Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

Construction of a multiple fluorescence labeling system for use in co-invasion studies of Listeria monocytogenes

DEFF Research Database (Denmark)

Andersen, Jens Bo; Roldgaard, Bent; Lindner, A. B.

2006-01-01

strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations. The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay......, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible. Conclusion The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between...... deviations in the observed capacity for infection when animal models are used. One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains...

Comparison between low-cost marker-less and high-end marker-based motion capture systems for the computer-aided assessment of working ergonomics.

Science.gov (United States)

Patrizi, Alfredo; Pennestrì, Ettore; Valentini, Pier Paolo

2016-01-01

The paper deals with the comparison between a high-end marker-based acquisition system and a low-cost marker-less methodology for the assessment of the human posture during working tasks. The low-cost methodology is based on the use of a single Microsoft Kinect V1 device. The high-end acquisition system is the BTS SMART that requires the use of reflective markers to be placed on the subject's body. Three practical working activities involving object lifting and displacement have been investigated. The operational risk has been evaluated according to the lifting equation proposed by the American National Institute for Occupational Safety and Health. The results of the study show that the risk multipliers computed from the two acquisition methodologies are very close for all the analysed activities. In agreement to this outcome, the marker-less methodology based on the Microsoft Kinect V1 device seems very promising to promote the dissemination of computer-aided assessment of ergonomics while maintaining good accuracy and affordable costs. PRACTITIONER’S SUMMARY: The study is motivated by the increasing interest for on-site working ergonomics assessment. We compared a low-cost marker-less methodology with a high-end marker-based system. We tested them on three different working tasks, assessing the working risk of lifting loads. The two methodologies showed comparable precision in all the investigations.

Pea Marker Database (PMD) - A new online database combining known pea (Pisum sativum L.) gene-based markers.

Science.gov (United States)

Kulaeva, Olga A; Zhernakov, Aleksandr I; Afonin, Alexey M; Boikov, Sergei S; Sulima, Anton S; Tikhonovich, Igor A; Zhukov, Vladimir A

2017-01-01

Pea (Pisum sativum L.) is the oldest model object of plant genetics and one of the most agriculturally important legumes in the world. Since the pea genome has not been sequenced yet, identification of genes responsible for mutant phenotypes or desirable agricultural traits is usually performed via genetic mapping followed by candidate gene search. Such mapping is best carried out using gene-based molecular markers, as it opens the possibility for exploiting genome synteny between pea and its close relative Medicago truncatula Gaertn., possessing sequenced and annotated genome. In the last 5 years, a large number of pea gene-based molecular markers have been designed and mapped owing to the rapid evolution of "next-generation sequencing" technologies. However, the access to the complete set of markers designed worldwide is limited because the data are not uniformed and therefore hard to use. The Pea Marker Database was designed to combine the information about pea markers in a form of user-friendly and practical online tool. Version 1 (PMD1) comprises information about 2484 genic markers, including their locations in linkage groups, the sequences of corresponding pea transcripts and the names of related genes in M. truncatula. Version 2 (PMD2) is an updated version comprising 15944 pea markers in the same format with several advanced features. To test the performance of the PMD, fine mapping of pea symbiotic genes Sym13 and Sym27 in linkage groups VII and V, respectively, was carried out. The results of mapping allowed us to propose the Sen1 gene (a homologue of SEN1 gene of Lotus japonicus (Regel) K. Larsen) as the best candidate gene for Sym13, and to narrow the list of possible candidate genes for Sym27 to ten, thus proving PMD to be useful for pea gene mapping and cloning. All information contained in PMD1 and PMD2 is available at www.peamarker.arriam.ru.

Fluorescence based molecular in vivo imaging

International Nuclear Information System (INIS)

Ebert, Bernd

2008-01-01

Molecular imaging represents a modern research area that allows the in vivo study of molecular biological process kinetics using appropriate probes and visualization methods. This methodology may be defined- apart from the contrast media injection - as non-abrasive. In order to reach an in vivo molecular process imaging as accurate as possible the effects of the used probes on the biological should not be too large. The contrast media as important part of the molecular imaging can significantly contribute to the understanding of molecular processes and to the development of tailored diagnostics and therapy. Since more than 15 years PTB is developing optic imaging systems that may be used for fluorescence based visualization of tissue phantoms, small animal models and the localization of tumors and their predecessors, and for the early recognition of inflammatory processes in clinical trials. Cellular changes occur during many diseases, thus the molecular imaging might be of importance for the early diagnosis of chronic inflammatory diseases. Fluorescent dyes can be used as unspecific or also as specific contrast media, which allow enhanced detection sensitivity

Diagnostic markers of urothelial cancer based on DNA methylation analysis

International Nuclear Information System (INIS)

Chihara, Yoshitomo; Hirao, Yoshihiko; Kanai, Yae; Fujimoto, Hiroyuki; Sugano, Kokichi; Kawashima, Kiyotaka; Liang, Gangning; Jones, Peter A; Fujimoto, Kiyohide; Kuniyasu, Hiroki

2013-01-01

Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation. In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers. Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity. Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect

Localization Based on Magnetic Markers for an All-Wheel Steering Vehicle

Directory of Open Access Journals (Sweden)

Yeun Sub Byun

2016-11-01

Full Text Available Real-time continuous localization is a key technology in the development of intelligent transportation systems. In these systems, it is very important to have accurate information about the position and heading angle of the vehicle at all times. The most widely implemented methods for positioning are the global positioning system (GPS, vision-based system, and magnetic marker system. Among these methods, the magnetic marker system is less vulnerable to indoor and outdoor environment conditions; moreover, it requires minimal maintenance expenses. In this paper, we present a position estimation scheme based on magnetic markers and odometry sensors for an all-wheel-steering vehicle. The heading angle of the vehicle is determined by using the position coordinates of the last two detected magnetic markers and odometer data. The instant position and heading angle of the vehicle are integrated with an extended Kalman filter to estimate the continuous position. GPS data with the real-time kinematics mode was obtained to evaluate the performance of the proposed position estimation system. The test results show that the performance of the proposed localization algorithm is accurate (mean error: 3 cm; max error: 9 cm and reliable under unexpected missing markers or incorrect markers.

Retention of external and internal markers by southern pine beetles (Coleoptera: Scolytidae) during gallery

Science.gov (United States)

Douglas J. Rhodes; Jane Leslie Hayes; Chris Steiner

1998-01-01

If retained, markers used in mark-release-recapture studies of bark beetle dispersal could provide valuable tools in the determination of post-dispersal fate. Retention of the internal marker rubidium (Rb) and of the external marker fluorescent powder during egg gallery construction, oviposition, and feeding were quantified at intervals from 0 to 96 hours by allowing...

A Fluorescent Tile DNA Diagnocode System for In Situ Rapid and Selective Diagnosis of Cytosolic RNA Cancer Markers

Science.gov (United States)

Park, Kyung Soo; Shin, Seung Won; Jang, Min Su; Shin, Woojung; Yang, Kisuk; Min, Junhong; Cho, Seung-Woo; Oh, Byung-Keun; Bae, Jong Wook; Jung, Sunghwan; Choi, Jeong-Woo; Um, Soong Ho

2015-01-01

Accurate cancer diagnosis often requires extraction and purification of genetic materials from cells, and sophisticated instrumentations that follow. Otherwise in order to directly treat the diagnostic materials to cells, multiple steps to optimize dose concentration and treatment time are necessary due to diversity in cellular behaviors. These processes may offer high precision but hinder fast analysis of cancer, especially in clinical situations that need rapid detection and characterization of cancer. Here we present a novel fluorescent tile DNA nanostructure delivered to cancer cytosol by employing nanoparticle technology. Its structural anisotropicity offers easy manipulation for multifunctionalities, enabling the novel DNA nanostructure to detect intracellular cancer RNA markers with high specificity within 30 minutes post treatment, while the nanoparticle property bypasses the requirement of treatment optimization, effectively reducing the complexity of applying the system for cancer diagnosis. Altogether, the system offers a precise and rapid detection of cancer, suggesting the future use in the clinical fields. PMID:26678430

Ensemble empirical mode decomposition based fluorescence spectral noise reduction for low concentration PAHs

Science.gov (United States)

Wang, Shu-tao; Yang, Xue-ying; Kong, De-ming; Wang, Yu-tian

2017-11-01

A new noise reduction method based on ensemble empirical mode decomposition (EEMD) is proposed to improve the detection effect for fluorescence spectra. Polycyclic aromatic hydrocarbons (PAHs) pollutants, as a kind of important current environmental pollution source, are highly oncogenic. Using the fluorescence spectroscopy method, the PAHs pollutants can be detected. However, instrument will produce noise in the experiment. Weak fluorescent signals can be affected by noise, so we propose a way to denoise and improve the detection effect. Firstly, we use fluorescence spectrometer to detect PAHs to obtain fluorescence spectra. Subsequently, noises are reduced by EEMD algorithm. Finally, the experiment results show the proposed method is feasible.

Highly selective rhodamine-based fluorescence turn-on chemosensor for Al3+ ion

Science.gov (United States)

Manjunath, Rangasamy; Kannan, Palaninathan

2018-05-01

A new rhodamine-based colorimetric and fluorescent turn-on chemosensor (L) has been designed and synthesized for selective and sensitive detection of Al3+ ion. The sensing behavior toward metal ion was investigated by UV/Vis and fluorescence spectroscopy. Upon addition of Al3+ ion to solution of L provided a visual color change as well as significantly fluorescent enhancement, while other metal ions including Na+, Mg2+, K+, Mn2+, Fe3+, Ni2+, Cu2+, Zn2+, Pb2+, Cd2+ and Hg2+ ions fails to generate a distinct color and spectral changes, the distinct color change and rapid switch-on fluorescence also provide naked eye detection for Al3+ ion. The mechanism involved equilibrium between non-fluorescent spirocyclic form and highly fluorescent ring open form process was utilized and 1:2 stoichiometry for L-Al3+ complex formed with an association constant of 1.42 × 103 M-1. Moreover, chemosensor L was applied for living cell imaging and confirmed that can be used as a fluorescent probe for monitoring Al3+ ion in living cells.

Photocured thiol-ene based optical fluorescence sensor for determination of gold(III)

Energy Technology Data Exchange (ETDEWEB)

Çubuk, Soner, E-mail: sonercubuk@marmara.edu.tr; Kahraman, Memet Vezir; Yetimoğlu, Ece Kök; Kenan, Sibel

2014-02-17

Graphical abstract: -- Highlights: •Photopolymerized fluorescence sensor for Au(III) analysis has been developed. •Preparation of polymeric sensor is simple and quick. •Fluorescence sensor used for analysis of Au(III) in real samples. -- Abstract: This study describes the preparation and the characterization of a new thiol-ene based polymeric fluorescence sensor by photo initiated polymerization of trimethylolpropane tris(3-mercaptopropionate), 2-hydroxyethylacrylate, and 2,4,6-triallyloxy-1,3,5-triazine which are used as monomers and also a photo initiator (2,2-dimethoxy-2-phenylacetophenone) for its usage as optical sensor for gold ions. The thiol-ene based polymeric membrane sensor was characterized by using attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM). The response characteristics of the sensors including dynamic range, pH effect, response time, and the effect of foreign ions were investigated. Fluorescence spectra showed that the excitation/emission maxima of the membrane were at 379/425 nm, respectively.

Photocured thiol-ene based optical fluorescence sensor for determination of gold(III)

International Nuclear Information System (INIS)

Çubuk, Soner; Kahraman, Memet Vezir; Yetimoğlu, Ece Kök; Kenan, Sibel

2014-01-01

Graphical abstract: -- Highlights: •Photopolymerized fluorescence sensor for Au(III) analysis has been developed. •Preparation of polymeric sensor is simple and quick. •Fluorescence sensor used for analysis of Au(III) in real samples. -- Abstract: This study describes the preparation and the characterization of a new thiol-ene based polymeric fluorescence sensor by photo initiated polymerization of trimethylolpropane tris(3-mercaptopropionate), 2-hydroxyethylacrylate, and 2,4,6-triallyloxy-1,3,5-triazine which are used as monomers and also a photo initiator (2,2-dimethoxy-2-phenylacetophenone) for its usage as optical sensor for gold ions. The thiol-ene based polymeric membrane sensor was characterized by using attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM). The response characteristics of the sensors including dynamic range, pH effect, response time, and the effect of foreign ions were investigated. Fluorescence spectra showed that the excitation/emission maxima of the membrane were at 379/425 nm, respectively

Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

International Nuclear Information System (INIS)

Nienhaus, Karin; Nienhaus, G Ulrich

2016-01-01

Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments. (topical review)

Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

Science.gov (United States)

Nienhaus, Karin; Nienhaus, G. Ulrich

2016-11-01

Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review

International Nuclear Information System (INIS)

Sednev, Maksim V; Belov, Vladimir N; Hell, Stefan W

2015-01-01

The review deals with commercially available organic dyes possessing large Stokes shifts and their applications as fluorescent labels in optical microscopy based on stimulated emission depletion (STED). STED microscopy breaks Abbe’s diffraction barrier and provides optical resolution beyond the diffraction limit. STED microscopy is non-invasive and requires photostable fluorescent markers attached to biomolecules or other objects of interest. Up to now, in most biology-related STED experiments, bright and photoresistant dyes with small Stokes shifts of 20–40 nm were used. The rapid progress in STED microscopy showed that organic fluorophores possessing large Stokes shifts are indispensable in multi-color super-resolution techniques. The ultimate result of the imaging relies on the optimal combination of a dye, the bio-conjugation procedure and the performance of the optical microscope. Modern bioconjugation methods, basics of STED microscopy, as well as structures and spectral properties of the presently available fluorescent markers are reviewed and discussed. In particular, the spectral properties of the commercial dyes are tabulated and correlated with the available depletion wavelengths found in STED microscopes produced by LEICA Microsytems, Abberior Instruments and Picoquant GmbH. (topical review)

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.

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Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. This chapter focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here, we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the chimeric ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. © 2017 Elsevier Inc. All rights reserved.

Dynamic fluorescence imaging with molecular agents for cancer detection

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Kwon, Sun Kuk

Non-invasive dynamic optical imaging of small animals requires the development of a novel fluorescence imaging modality. Herein, fluorescence imaging is demonstrated with sub-second camera integration times using agents specifically targeted to disease markers, enabling rapid detection of cancerous regions. The continuous-wave fluorescence imaging acquires data with an intensified or an electron-multiplying charge-coupled device. The work presented in this dissertation (i) assessed dose-dependent uptake using dynamic fluorescence imaging and pharmacokinetic (PK) models, (ii) evaluated disease marker availability in two different xenograft tumors, (iii) compared the impact of autofluorescence in fluorescence imaging of near-infrared (NIR) vs. red light excitable fluorescent contrast agents, (iv) demonstrated dual-wavelength fluorescence imaging of angiogenic vessels and lymphatics associated with a xenograft tumor model, and (v) examined dynamic multi-wavelength, whole-body fluorescence imaging with two different fluorescent contrast agents. PK analysis showed that the uptake of Cy5.5-c(KRGDf) in xenograft tumor regions linearly increased with doses of Cy5.5-c(KRGDf) up to 1.5 nmol/mouse. Above 1.5 nmol/mouse, the uptake did not increase with doses, suggesting receptor saturation. Target to background ratio (TBR) and PK analysis for two different tumor cell lines showed that while Kaposi's sarcoma (KS1767) exhibited early and rapid uptake of Cy5.5-c(KRGDf), human melanoma tumors (M21) had non-significant TBR differences and early uptake rates similar to the contralateral normal tissue regions. The differences may be due to different compartment location of the target. A comparison of fluorescence imaging with NIR vs. red light excitable fluorescent dyes demonstrates that NIR dyes are associated with less background signal, enabling rapid tumor detection. In contrast, animals injected with red light excitable fluorescent dyes showed high autofluorescence. Dual

Label-free and enzyme-free detection of transcription factors with graphene oxide fluorescence switch-based multifunctional G-quadruplex-hairpin probe.

Science.gov (United States)

Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

2016-01-15

Transcription factors (TFs) play pivotal roles in the regulation of a variety of essential cellular processes and some of them have been recognized as potential diagnostic markers and therapeutic targets of some diseases. Sensitive and accurate detection of TFs is of great importance to better understanding their roles in gene regulation and evaluation of disease state. Here, we developed a simple, label-free and enzyme-free new fluorescent strategy for the detection of TFs by graphene oxide (GO) fluorescence switch-based multifunctional G-quadruplex-hairpin probe (MGHP). The MGHP possessed of three functions simultaneously, adsorbing onto GO with the loop part, binding to target with the stem part and serving as signal carrier with the terminal G-quadruplex. First, the MGHP was adsorbed quickly to GO. Next, the TF bound to the stem part of MGHP to form a huge target-MGHP complex, which led to desorption of the complex from GO. Finally, NMM was inserted into G-quadruplex in the complex to yield an enhanced fluorescence response. The GO used here, as a fluorescence switch, could quickly and efficiently quench the fluorescence of NMM inserted into the MGHP absorbed on the GO, guaranteeing a high signal-to-noise ratio. Sensitive detection of purified NF-κB p50 and HeLa cell nuclear extracts were achieved with detection limits of 0.2nM and 7.8ng/µL, respectively. Moreover, this proposed strategy could be used to screen inhibitors of NF-κB p50 activity. The strategy proposed here might offer a new potential approach for reliable quantification of TFs in clinical diagnostics and treatment research of some diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Simulation and application of micro X-ray fluorescence based on an ellipsoidal capillary

Science.gov (United States)

Yang, Jing; Li, Yude; Wang, Xingyi; Zhang, Xiaoyun; Lin, Xiaoyan

2017-06-01

A micro X-ray fluorescence setup was presented, based on an ellipsoidal capillary and a traditional laboratorial X-ray source. Using Ray-tracing principle, we have simulated the transmission path of X-ray beam in the ellipsoidal capillary and designed the optimal parameters of the ellipsoidal capillary for the micro X-ray fluorescence setup. We demonstrate that ellipsoidal capillary is well suited as condenser for the micro X-ray fluorescence based on traditional laboratorial X-ray source. Furthermore, we obtain the 2D mapping image of the leaf blade sample by using the ellipsoidal capillary we designed.

Fluorescence from the maillard reaction and its potential applications in food science.

Science.gov (United States)

Matiacevich, Silvia B; Santagapita, Patricio R; Buera, M Pilar

2005-01-01

The chemistry of the Maillard reaction involves a complex set of steps, and its interpretation represents a challenge in basic and applied aspects of Food Science. Fluorescent compounds have been recognized as important early markers of the reaction in food products since 1942. However, the recent advances in the characterization of fluorophores' development were observed in biological and biomedical areas. The in vivo non-enzymatic glycosylation of proteins produces biological effects, promoting health deterioration. The characteristic fluorescence of advanced glycosylation end products (AGEs) is similar to that of Maillard food products and represents an indicator of the level of AGE-modified proteins, but the structure of the fluorescent groups is, typically, unknown. Application of fluorescence measurement is considered a potential tool for addressing key problems of food deterioration as an early marker or index of the damage of biomolecules. Fluorophores may be precursors of the brown pigments and/or end products. A general scheme of the Maillard reaction is proposed in this article, incorporating the pool concept. A correct interpretation of the effect of environmental and compositional conditions and their influences on the reaction kinetics may help to define the meaning of fluorescence development for each particular system.

Development of DArT-based PCR markers for selecting drought-tolerant spring barley.

Science.gov (United States)

Fiust, Anna; Rapacz, Marcin; Wójcik-Jagła, Magdalena; Tyrka, Mirosław

2015-08-01

The tolerance of spring barley (Hordeum vulgare L.) cultivars to spring drought is an important agronomic trait affecting crop yield and quality in Poland. Therefore, breeders require new molecular markers to select plants with lower spring drought susceptibility. With the advent of genomic selection technology, simple molecular tools may still be applicable to screen material for markers of the most important traits and in-depth genome scanning. In previous studies, diversity arrays technology (DArT)-based genetic maps were constructed for F2 populations of Polish fodder and malt barley elite breeding lines, and 15 and 18 quantitative trait loci (QTLs) related to spring drought tolerance were identified, respectively. In this paper, we show the results of a conversion of 30 DArT markers corresponding to 11 QTLs into simple sequence repeat (SSR) and sequence tagged site (STS) markers. Twenty-two polymorphic markers were obtained, including 13 DArT-based SSRs. Additionally, 31 SSR markers, located in close proximity to the DArT markers, were selected from the GrainGenes database and tested. Further analyses of 24 advanced breeding lines with different drought tolerances confirmed that five out of the 30 converted markers, as well as three out of the 31 additional SSR markers, were effective in marker-assisted selection for drought tolerance. The possible function of clones related to these markers in drought tolerance is discussed.

A new Schiff base based on vanillin and naphthalimide as a fluorescent probe for Ag+ in aqueous solution

Science.gov (United States)

Zhou, Yanmei; Zhou, Hua; Ma, Tongsen; Zhang, Junli; Niu, Jingyang

2012-03-01

A new Schiff base based on vanillin and naphthalimide was designed and synthesized as fluorescent probe. The probe showed high selectivity for Ag+ over other metal ions such as Pb2+, Na+, K+, Cd2+, Ba2+, Cr3+, Zn2+, Cu2+, Ni2+, Ca2+, Al3+ and Mg2+ in aqueous solution. A new fluorescence emission was observed at 682 nm in the presence of Ag+ ion. The fluorescence intensity quenched with increasing the concentration of Ag+ at 682 nm. The method of job's plot confirmed the 1:2 complex between Ag+ and probe, and the mechanism was proposed.

Molecular markers. Amplified fragment length polymorphism

Directory of Open Access Journals (Sweden)

Pržulj Novo

2005-01-01

Full Text Available Amplified Fragment Length Polymorphism molecular markers (AFLPs has been developed combining procedures of RFLPs and RAPDs molekular markers, i.e. the first step is restriction digestion of the genomic DNA that is followed by selective amplification of the restricted fragments. The advantage of the AFLP technique is that it allows rapid generation of a large number of reproducible markers. The reproducibility of AFLPs markers is assured by the use of restriction site-specific adapters and adapter-specific primers for PCR reaction. Only fragments containing the restriction site sequence plus the additional nucleotides will be amplified and the more selected nucleotides added on the primer sequence the fewer the number of fragments amplified by PCR. The amplified products are normally separated on a sequencing gel and visualized after exposure to X-ray film or by using fluorescent labeled primers. AFLP shave proven to be extremely proficient in revealing diversity at below the species level. A disadvantage of AFLP technique is that AFLPs are essentially a dominant marker system and not able to identify heterozygotes.

A Fluorescent Thermometer Based on a Pyrene-Labeled Thermoresponsive Polymer

Directory of Open Access Journals (Sweden)

Ulrich S. Schubert

2010-08-01

Full Text Available Thermoresponsive polymers that undergo a solubility transition by variation of the temperature are important materials for the development of ‘smart’ materials. In this contribution we exploit the solubility phase transition of poly(methoxy diethylene glycol methacrylate, which is accompanied by a transition from hydrophilic to hydrophobic, for the development of a fluorescent thermometer. To translate the polymer phase transition into a fluorescent response, the polymer was functionalized with pyrene resulting in a change of the emission based on the microenvironment. This approach led to a soluble polymeric fluorescent thermometer with a temperature range from 11 °C to 21 °C. The polymer phase transition that occurs during sensing is studied in detail by dynamic light scattering.

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

Science.gov (United States)

Zhang, Ming; Chakraborty, Subhasish K.; Sampath, Padma; Rojas, Juan J.; Hou, Weizhou; Saurabh, Saumya; Thorne, Steve H.; Bruchez, Marcel P.; Waggoner, Alan S.

2015-01-01

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene. PMID:26348895

A Dansyl-Rhodamine Based Fluorescent Probe for Detection of Hg2+ and Cu2.

Science.gov (United States)

Yuan, Shizhuang; Su, Wei; Wang, Enju

2017-09-01

A novel fluorescent probe based on dansyl-appended rhodamine B was developed. The probe can selectively recognize and sense Hg2+ and Cu2+ from other common metal ions by showing unique fluorescence and absorption characteristics. In MeCN/HEPES buffer solution, the probe gives a ratiometric fluorescent response to Hg2+, which was ascribed to the fluorescence resonance energy transfer from dansyl moiety to the ring-opened rhodamine B moiety, while the presence of Cu2+ causes fluorescence quenching. Beside the fluorescence change, the presence of Cu2+ and Hg2+ can induce intensive absorption at about 555 nm, which resulted in a color change from colorless to pink.

NABIC marker database: A molecular markers information network of agricultural crops.

Science.gov (United States)

Kim, Chang-Kug; Seol, Young-Joo; Lee, Dong-Jun; Jeong, In-Seon; Yoon, Ung-Han; Lee, Gang-Seob; Hahn, Jang-Ho; Park, Dong-Suk

2013-01-01

In 2013, National Agricultural Biotechnology Information Center (NABIC) reconstructs a molecular marker database for useful genetic resources. The web-based marker database consists of three major functional categories: map viewer, RSN marker and gene annotation. It provides 7250 marker locations, 3301 RSN marker property, 3280 molecular marker annotation information in agricultural plants. The individual molecular marker provides information such as marker name, expressed sequence tag number, gene definition and general marker information. This updated marker-based database provides useful information through a user-friendly web interface that assisted in tracing any new structures of the chromosomes and gene positional functions using specific molecular markers. The database is available for free at http://nabic.rda.go.kr/gere/rice/molecularMarkers/

Extracting Fluorescent Reporter Time Courses of Cell Lineages from High-Throughput Microscopy at Low Temporal Resolution

Science.gov (United States)

Downey, Mike J.; Jeziorska, Danuta M.; Ott, Sascha; Tamai, T. Katherine; Koentges, Georgy; Vance, Keith W.; Bretschneider, Till

2011-01-01

The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell

Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution.

Directory of Open Access Journals (Sweden)

Mike J Downey

Full Text Available The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted

Multicolor Fluorescence Writing Based on Host-Guest Interactions and Force-Induced Fluorescence-Color Memory.

Science.gov (United States)

Matsunaga, Yuki; Yang, Jye-Shane

2015-06-26

A new strategy is reported for multicolor fluorescence writing on thin solid films with mechanical forces. This concept is illustrated by the use of a green-fluorescent pentiptycene derivative 1, which forms variably colored fluorescent exciplexes: a change from yellow to red was observed with anilines, and fluorescence quenching (a change to black) occurred in the presence of benzoquinone. Mechanical forces, such as grinding and shearing, induced a crystalline-to-amorphous phase transition in both the pristine and guest-adsorbed solids that led to a change in the fluorescence color (mechanofluorochromism) and a memory of the resulting color. Fluorescence drawings of five or more colors were created on glass or paper and could be readily erased by exposure to air and dichloromethane fumes. The structural and mechanistic aspects of the observations are also discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Marker-Based Multi-Sensor Fusion Indoor Localization System for Micro Air Vehicles.

Science.gov (United States)

Xing, Boyang; Zhu, Quanmin; Pan, Feng; Feng, Xiaoxue

2018-05-25

A novel multi-sensor fusion indoor localization algorithm based on ArUco marker is designed in this paper. The proposed ArUco mapping algorithm can build and correct the map of markers online with Grubbs criterion and K-mean clustering, which avoids the map distortion due to lack of correction. Based on the conception of multi-sensor information fusion, the federated Kalman filter is utilized to synthesize the multi-source information from markers, optical flow, ultrasonic and the inertial sensor, which can obtain a continuous localization result and effectively reduce the position drift due to the long-term loss of markers in pure marker localization. The proposed algorithm can be easily implemented in a hardware of one Raspberry Pi Zero and two STM32 micro controllers produced by STMicroelectronics (Geneva, Switzerland). Thus, a small-size and low-cost marker-based localization system is presented. The experimental results show that the speed estimation result of the proposed system is better than Px4flow, and it has the centimeter accuracy of mapping and positioning. The presented system not only gives satisfying localization precision, but also has the potential to expand other sensors (such as visual odometry, ultra wideband (UWB) beacon and lidar) to further improve the localization performance. The proposed system can be reliably employed in Micro Aerial Vehicle (MAV) visual localization and robotics control.

Marker-Based Multi-Sensor Fusion Indoor Localization System for Micro Air Vehicles

Directory of Open Access Journals (Sweden)

Boyang Xing

2018-05-01

Full Text Available A novel multi-sensor fusion indoor localization algorithm based on ArUco marker is designed in this paper. The proposed ArUco mapping algorithm can build and correct the map of markers online with Grubbs criterion and K-mean clustering, which avoids the map distortion due to lack of correction. Based on the conception of multi-sensor information fusion, the federated Kalman filter is utilized to synthesize the multi-source information from markers, optical flow, ultrasonic and the inertial sensor, which can obtain a continuous localization result and effectively reduce the position drift due to the long-term loss of markers in pure marker localization. The proposed algorithm can be easily implemented in a hardware of one Raspberry Pi Zero and two STM32 micro controllers produced by STMicroelectronics (Geneva, Switzerland. Thus, a small-size and low-cost marker-based localization system is presented. The experimental results show that the speed estimation result of the proposed system is better than Px4flow, and it has the centimeter accuracy of mapping and positioning. The presented system not only gives satisfying localization precision, but also has the potential to expand other sensors (such as visual odometry, ultra wideband (UWB beacon and lidar to further improve the localization performance. The proposed system can be reliably employed in Micro Aerial Vehicle (MAV visual localization and robotics control.

The Effect of Pulsed Streamer-like Discharge in Liquid on Transcriptional Activation of Retrotransposon Genes of a Red Alga, Porphyra Yezoensis

OpenAIRE

Ohno, T.; Li, Z.; Lin, X.F.; Zhang, W.B.; Takano, H.; Takio, S.; Namihira, T.; Akiyama, H.; オオノ, ツヨシ; ナミヒラ, タカオ; アキヤマ, ヒデノリ; 大野, 剛史; 浪平, 隆男; 秋山, 秀典

2007-01-01

Retrotransposons are mobile genetic elements thataccomplished transposition via an RNA intermediate.These elements can be transcriptionally activated by stressfactors, such as UV light, ozone, pathogens, woundingand drought. A red alga, porphyra yezoensis has recentlybeen recognized as a model plant for fundamental andapplied study in marine biological science. In this paper,pulsed streamer-like discharge in liquid was used as a newstress condition, and the transcription level of a copia-like...

A coumarin based Schiff base probe for selective fluorescence detection of Al3 + and its application in live cell imaging

Science.gov (United States)

Sen, Bhaskar; Sheet, Sanjoy Kumar; Thounaojam, Romita; Jamatia, Ramen; Pal, Amarta Kumar; Aguan, Kripamoy; Khatua, Snehadrinarayan

2017-02-01

A new coumarin based Schiff base compound, CSB-1 has been synthesized to detect metal ion based on the chelation enhanced fluorescence (CHEF). The cation binding properties of CSB-1 was thoroughly examined in UV-vis and fluorescence spectroscopy. In fluorescence spectroscopy the compound showed high selectivity toward Al3 + ion and the Al3 + can be quantified in mixed aqueous buffer solution (MeOH: 0.01 M HEPES Buffer; 9:1; v/v) at pH 7.4 as well as in BSA media. The fluorescence intensity of CSB-1 was enhanced by 24 fold after addition of only five equivalents of Al3 +. The fluorescence titration of CSB-1 with Al3 + in mixed aqueous buffer afforded a binding constant, Ka = (1.06 ± 0.2) × 104 M- 1. The colour change from light yellow to colourless and the appearance of blue fluorescence, which can be observed by the naked eye, provides a real-time method for Al3 + sensing. Further the live cell imaging study indicated that the detection of intracellular Al3 + ions are also readily possible in living cell.

Experimental evaluation of fluorescent (alizarin red S and calcein) and clip-tag markers for stock assessment of ark shell, Anadara broughtonii

Science.gov (United States)

Zhou, Shanshan; Zhang, Xiumei; Li, Wentao; Li, Long; Cai, Xingyuan

2017-03-01

Release programs to enhance stocks of ark shell ( Anadara broughtonii) have been undertaken in a number of Asian countries, but their effectiveness has rarely been investigated owing to a lack of marking methods. The quality and longevity of fluorescent markers, alizarin red S (ARS) and calcein (CAL) (200 and 300 mg/L), as well as clip tags, were tested on juvenile A. broughtonii. No significant differences in survival or shell growth were observed in juveniles stained with either of the two fluorochromes after a 160-day culture period, but the retention rate was 100% after 1 year. Fluorescent marks (≥grade 3) were observable microscopically in juveniles stained with the two fluorochromes, and some fluorescent marks (≥grade 4) were visible with the naked eye after 1 year. ARS-marked shells were brighter than those marked with CAL, and shells marked with 300 mg/L of the fluorochromes were easier to detect than those marked with 200 mg/L. Clip tags were incorporated into the shell as the bivalve grew, and the retention rate was 64.25% after 160 days. Significant differences in survival (at 30 days), shell length (at 60, 90, 120, and 160 days), and wet weight (at 90, 120, and 160 days) were observed between the clip-tagged and control groups (all P< 0.05), indicating that the tags may have passive effects on the ark shell. The results suggest that both ARS and CAL are suitable to mark A. broughtonii for large-scale restocking programs, and that optimal marking quality was achieved with 300 mg/L ARS. Lighter and smaller clip tags need to be developed to reduce injury and increase survival rate of clams.

Chlorophyll Fluorescence and Reflectance-Based Non-Invasive Quantification of Blast, Bacterial Blight and Drought Stresses in Rice

Czech Academy of Sciences Publication Activity Database

Šebela, David; Quiňones, C.; Cruz, C.; Ona, I.; Olejníčková, Julie; Jagadish, K. S. V.

2018-01-01

Roč. 59, č. 1 (2018), s. 30-43 ISSN 0032-0781 R&D Projects: GA MŠk(CZ) LO1415 EU Projects: European Commission(XE) 284443 - EPPN Institutional support: RVO:86652079 Keywords : marker-assisted selection * oryza-sativa l. * water -stress * magnaporthe-grisea * disease resistance * photosynthetic efficiency * spectral reflectance * carotenoid content * eucalyptus leaves * diurnal changes * Bacterial blight * Chlorophyll fluorescence * Drought stress * Reflectance * Rice bast Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 4.760, year: 2016

Structural properties of oligonucleotide monolayers on gold surfaces probed by fluorescence investigations.

Science.gov (United States)

Rant, Ulrich; Arinaga, Kenji; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard; Tornow, Marc

2004-11-09

We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.

Segregation analysis of microsatellite (SSR) markers in sugarcane polyploids.

Science.gov (United States)

Lu, X; Zhou, H; Pan, Y-B; Chen, C Y; Zhu, J R; Chen, P H; Li, Y-R; Cai, Q; Chen, R K

2015-12-28

No information is available on segregation analysis of DNA markers involving both pollen and self-progeny. Therefore, we used capillary electrophoresis- and fluorescence-based DNA fingerprinting together with single pollen collection and polymerase chain reaction (PCR) to investigate simple sequence repeat (SSR) marker segregation among 964 single pollens and 288 self-progenies (S1) of sugarcane cultivar LCP 85-384. Twenty SSR DNA fragments (alleles) were amplified by five polymorphic SSR markers. Only one non-parental SSR allele was observed in 2392 PCRs. SSR allele inheritance was in accordance with Mendelian laws of segregation and independent assortment. Highly significant correlation coefficients were found between frequencies of observed and expected genotypes in pollen and S1 populations. Within the S1 population, the most frequent genotype of each SSR marker was the parental genotype of the same marker. The number of genotypes was higher in pollen than S1 population. PIC values of the five SSR markers were greater in pollen than S1 populations. Eleven of 20 SSR alleles (55%) were segregated in accordance with Mendelian segregation ratios expected from pollen and S1 populations of a 2n = 10x polyploid. Six of 20 SSR alleles were segregated in a 3:1 (presence:absence) ratio and were simplex markers. Four and one alleles were segregated in 77:4 and 143:1 ratios and considered duplex and triplex markers, respectively. Segregation ratios of remaining alleles were unexplainable. The results provide information about selection of crossing parents, estimation of seedling population optimal size, and promotion of efficient selection, which may be valuable for sugarcane breeders.

Novel chalcone-based fluorescent human histamine H3 receptor ligands as pharmacological tools

Directory of Open Access Journals (Sweden)

Holger eStark

2012-03-01

Full Text Available Novel fluorescent chalcone-based ligands at human histamine H3 receptors (hH3R have been designed, synthesized and characterized. Compounds described are non-imidazole analogues of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH3R in the same concentration range like that of the reference antagonist ciproxifan (hH3R pKi value of 7.2. Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be taken to visualize hH3R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH3R visualization in different tissues.

Identification and chromosome mapping of repetitive elements in the Astyanax scabripinnis (Teleostei: Characidae) species complex.

Science.gov (United States)

Barbosa, Patrícia; de Oliveira, Luiz Antonio; Pucci, Marcela Baer; Santos, Mateus Henrique; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo; Nogaroto, Viviane; de Almeida, Mara Cristina; Artoni, Roberto Ferreira

2015-02-01

Most part of the eukaryotic genome is composed of repeated sequences or multiple copies of DNA, which were considered as "junk DNA", and may be associated to the heterochromatin. In this study, three populations of Astyanax aff. scabripinnis from Brazilian rivers of Guaratinguetá and Pindamonhangaba (São Paulo) and a population from Maringá (Paraná) were analyzed concerning the localization of the nucleolar organizer regions (Ag-NORs), the As51 satellite DNA, the 18S ribosomal DNA (rDNA), and the 5S rDNA. Repeated sequences were also isolated and identified by the Cot - 1 method, which indicated similarity (90%) with the LINE UnaL2 retrotransposon. The fluorescence in situ hybridization (FISH) showed the retrotransposon dispersed and more concentrated markers in centromeric and telomeric chromosomal regions. These sequences were co-localized and interspaced with 18S and 5S rDNA and As51, confirmed by fiber-FISH essay. The B chromosome found in these populations pointed to a conspicuous hybridization with LINE probe, which is also co-located in As51 sequences. The NORs were active at unique sites of a homologous pair in the three populations. There were no evidences that transposable elements and repetitive DNA had influence in the transcriptional regulation of ribosomal genes in our analyses.

A label-free, fluorescence based assay for microarray

Science.gov (United States)

Niu, Sanjun

DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications

Science.gov (United States)

Platt, Donald W.; Hoover, Richard B.

2009-01-01

A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments.

Fluorescent blood glucose monitor by hemin-functionalized graphene quantum dots based sensing system

Energy Technology Data Exchange (ETDEWEB)

He, Yuezhen; Wang, Xiaoxun; Sun, Jian; Jiao, Shoufeng; Chen, Hongqi; Gao, Feng; Wang, Lun, E-mail: wanglun@mail.ahnu.edu.cn

2014-01-31

Graphical abstract: -- Highlights: •Hemin is assembled onto the surfaces of graphene quantum dots (GQDs). •With the aid of hemin, H{sub 2}O{sub 2} could quench the FL signal of GQDs obviously. •Based on this effect, a fluorescent platform is proposed for the sensing of glucose. •The proposed method provides a new pathway to explore practical application of GQDs. -- Abstract: In the present work, a highly sensitive and specific fluorescent biosensor for blood glucose monitoring is developed based on hemin-functionalized graphene quantum dots (GQDs) and glucose oxidase (GOx) system. The GQDs which are simply prepared by pyrolyzing citric acid exhibit strong fluorescence and good water-solubility. Due to the noncovalent assembly between hemin and GQDs, the addition of hemin can make hydrogen peroxide (H{sub 2}O{sub 2}) to destroy the passivated surface of GQDs, leading to significant fluorescence quenching of GQDs. Based on this effect, a novel fluorescent platform is proposed for the sensing of glucose. Under the optimized conditions, the linear range of glucose is from 9 to 300 μM, and the limit of detection is 0.1 μM. As unique properties of GQDs, the proposed biosensor is green, simple, cost-efficient, and it is successfully applied to the determination of glucose in human serum. In addition, the proposed method provides a new pathway to further design the biosensors based on the assembly of GQDs with hemin for detection of biomolecules.

Transformation of Sclerotinia Sclerotiorum with the Green Fluorescent Protein Gene and Fluorescence of Hyphae in Four Inoculated Hosts

Science.gov (United States)

Sclerotinia sclerotiorum is an important pathogen of a wide variety of crops. To obtain a genetic marker to observe and study the interaction of the pathogen with its hosts, isolates ND30 and ND21 were transformed using pCT74 and gGFP constructs both containing genes for the green fluorescent protei...

A low-cost 2D fluorescence detection system for mm sized beads on-chip

NARCIS (Netherlands)

Segerink, Loes Irene; Koster, Maarten J.; Sprenkels, A.J.; van den Berg, Albert

2012-01-01

In this paper we describe a compact fluorescence detection system for on-chip analysis of beads, comprising a low-cost optical HD-DVD pickup. The complete system consists of a fluorescence detection unit, a control unit and a microfluidic chip containing microchannels and optical markers. With these

Piezoresistor-equipped fluorescence-based cantilever probe for near-field scanning.

Science.gov (United States)

Kan, Tetsuo; Matsumoto, Kiyoshi; Shimoyama, Isao

2007-08-01

Scanning near-field optical microscopes (SNOMs) with fluorescence-based probes are promising tools for evaluating the optical characteristics of nanoaperture devices used for biological investigations, and this article reports on the development of a microfabricated fluorescence-based SNOM probe with a piezoresistor. The piezoresistor was built into a two-legged root of a 160-microm-long cantilever. To improve the displacement sensitivity of the cantilever, the piezoresistor's doped area was shallowly formed on the cantilever surface. A fluorescent bead, 500 nm in diameter, was attached to the bottom of the cantilever end as a light-intensity-sensitive material in the visible-light range. The surface of the scanned sample was simply detected by the probe's end being displaced by contact with the sample. Measuring displacements piezoresistively is advantageous because it eliminates the noise arising from the use of the optical-lever method and is free of any disturbance in the absorption or the emission spectrum of the fluorescent material at the probe tip. The displacement sensitivity was estimated to be 6.1 x 10(-6) nm(-1), and the minimum measurable displacement was small enough for near-field measurement. This probe enabled clear scanning images of the light field near a 300 x 300 nm(2) aperture to be obtained in the near-field region where the tip-sample distance is much shorter than the light wavelength. This scanning result indicates that the piezoresistive way of tip-sample distance regulation is effective for characterizing nanoaperture optical devices.

A new pyrazoline-based fluorescent sensor for Al3+ in aqueous solution.

Science.gov (United States)

Hu, Shengli; Song, Jingjing; Wu, Gongying; Cheng, Cuixia; Gao, Qing

2015-02-05

A new pyrazoline-based fluorescent sensor was synthesized and the structure was confirmed by single crystal X-ray diffraction. The sensor responds to Al(3+) with high selectivity among a series of cations in aqueous methanol. This sensor forms a 1:1 complex with Al(3+) and displays fluorescent quenching. Copyright © 2014 Elsevier B.V. All rights reserved.

'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

International Nuclear Information System (INIS)

Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ulleras, Erik; Dastych, JarosIaw

2005-01-01

The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals

A novel fluorescence probe based on triphenylamine Schiff base for bioimaging and responding to pH and Fe3+

International Nuclear Information System (INIS)

Wang, Lei; Yang, Xiaodong; Chen, Xiuli; Zhou, Yuping; Lu, Xiaodan; Yan, Chenggong; Xu, Yikai; Liu, Ruiyuan; Qu, Jinqing

2017-01-01

A novel fluorescence probe 1 based on triphenylamine was synthesized and characterized by NMR, IR, high resolution mass spectrometry and elemental analysis. Its fluorescence was quenched when pH below 2. There was a linear relationship between the fluorescence intensity and pH value ranged from 2 to 7. And its fluorescence emission was reversibility in acidic and alkaline solution. Furthermore, it exhibited remarkable selectivity and high sensitivity to Fe 3+ and was able to detect Fe 3+ in aqueous solution with low detection limit of 0.511 μM. Job plot showed that the binding stoichiometry of 1 with Fe 3+ was 1:1. Further observations of 1 H NMR titration suggested that coordination interaction between Fe 3+ and nitrogen atom on C =N bond promoted the intramolecular charge transfer (ICT) or energy transfer process causing fluorescence quenching. Additionally, 1 was also able to be applied for detecting Fe 3+ in living cell and bioimaging. - Graphical abstract: Triphenylamine based fluorescence probe can detect pH and Fe 3+ simultaneously in aqueous solution and be applied for detecting Fe 3+ in living cell and bioimaging. - Highlights: • The fluorescence probe is sensitive to pH in strong acid conditions. • The fluorescence chemosensor can detect pH and Fe 3+ simultaneously. • The recognition is able to carry out in aqueous solution. • The probe can also be applied for detecting Fe 3+ in living cell and bioimaging. • The sensor is synthesized easily with one step.

Increasing of sensitivity of fluorescent immunoassay analysis of alpha-fetoprotein by means of plasmonical silver nanoparticles

International Nuclear Information System (INIS)

Vashchenko, S.V.; Min'ko, A.A.; Romanenko, A.A.; Gaponenko, S.V.; Kulakovich, O.S.

2014-01-01

A test system is proposed based on metal enhanced fluorescence to analyze low concentrations of alpha-fetoprotein (AFP), a tumor marker. Antigen-antibody reaction was performed on polystyrene plates coated with silver nanoparticles to increase sensitivity of fluorescent immunoassay and signal-to-noise ratio as compared to silver-free system. As compared to widely used ELISA technique and other immunoassay techniques the proposed approach is characterized by smaller probe volume, fast analysis and simplicity. The proposed test system uses layer-by-layer assembly approach, LED excitation and nanowatt photodetection set-up. The proposed test system offers AFP detection at concentrations used in clinical practice. Fluorescence enhancement for labeled AFP antibodies on a silver substrate was found to depend on antibodies concentration and was up to 6 times. (authors)

Three-dimensional distributions of sewage markers in Tokyo Bay water-fluorescent whitening agents (FWAs)

International Nuclear Information System (INIS)

Managaki, Satoshi; Takada, Hideshige; Kim, Dong-Myung; Horiguchi, Toshihiro; Shiraishi, Hiroaki

2006-01-01

Three-dimensional distributions of fluorescent whitening agents (FWAs: more specifically, DSBP and DAS1), which are sewage-derived water-soluble markers, were observed in Tokyo Bay water through multi-layer sampling of water at 20 locations. In summer, FWAs predominated in the surface layers, with trace but significant concentration of FWAs in bottom water due to stratification of seawater. In winter, on the other hand, FWAs were extensively mixed into the bottom layers because of the vertical mixing of seawater. In the surface layer, FWA concentrations and the DSBP/DAS1 ratio (the concentration ratio of DSBP to DAS1) were lower in summer than in winter, suggesting more efficient photodegradation of FWAs in euphotic zones during the summer due to stronger solar radiation. Horizontally, FWAs were widely distributed over the surface layer of Tokyo Bay. Surface water with DSBP concentrations above 50 ng/L, corresponding to <200 times dilution of sewage effluent, was found to have spread up to 10 km from the coastline. In addition, an offshore decline in FWA concentrations was observed, showing a half-distance of 10-20 km. The decrease was caused by dilution by seawater of fresh water containing FWAs. The eastern part of the bay was different with respect to surface layers, with higher concentrations seen in northeastern parts. Furthermore, dispersion of combined sewer overflow (CSO)-derived water mass was observed in Tokyo Bay after heavy rain

Hyper-spectral modulation fluorescent imaging using double acousto-optical tunable filter based on TeO2-crystals

International Nuclear Information System (INIS)

Zaytsev, Kirill I; Perchik, Alexey V; Chernomyrdin, Nikita V; Yurchenko, Stanislav O; Kudrin, Konstantin G; Reshetov, Igor V

2015-01-01

We have proposed a method for hyper-spectral fluorescent imaging based on acousto-optical filtering. The object of interest was pumped using ultraviolet radiation of mercury lamp equipped with monochromatic excitation filter with the window of transparency centered at 365 nm. Double TeO 2 -based acousto-optical filter, tunable in range from 430 to 780 nm and having 2 nm bandwidth of spectral transparency, was used in order to detect quasimonochromatic images of object fluorescence. Modulating of ultraviolet pump intensity was used in order to reduce an impact of non-fluorescent background on the sample fluorescent imaging. The technique for signal-to-noise ratio improvement, based on fluorescence intensity estimation via digital processing of modulated video sequence of fluorescent object, was introduced. We have implemented the proposed technique for the test sample studying and we have discussed its possible applications

Kazusa Marker DataBase: a database for genomics, genetics, and molecular breeding in plants

Science.gov (United States)

Shirasawa, Kenta; Isobe, Sachiko; Tabata, Satoshi; Hirakawa, Hideki

2014-01-01

In order to provide useful genomic information for agronomical plants, we have established a database, the Kazusa Marker DataBase (http://marker.kazusa.or.jp). This database includes information on DNA markers, e.g., SSR and SNP markers, genetic linkage maps, and physical maps, that were developed at the Kazusa DNA Research Institute. Keyword searches for the markers, sequence data used for marker development, and experimental conditions are also available through this database. Currently, 10 plant species have been targeted: tomato (Solanum lycopersicum), pepper (Capsicum annuum), strawberry (Fragaria × ananassa), radish (Raphanus sativus), Lotus japonicus, soybean (Glycine max), peanut (Arachis hypogaea), red clover (Trifolium pratense), white clover (Trifolium repens), and eucalyptus (Eucalyptus camaldulensis). In addition, the number of plant species registered in this database will be increased as our research progresses. The Kazusa Marker DataBase will be a useful tool for both basic and applied sciences, such as genomics, genetics, and molecular breeding in crops. PMID:25320561

Hybrid Integrated Silicon Microfluidic Platform for Fluorescence Based Biodetection

Directory of Open Access Journals (Sweden)

André Darveau

2007-09-01

Full Text Available The desideratum to develop a fully integrated Lab-on-a-chip device capable ofrapid specimen detection for high throughput in-situ biomedical diagnoses and Point-of-Care testing applications has called for the integration of some of the novel technologiessuch as the microfluidics, microphotonics, immunoproteomics and Micro ElectroMechanical Systems (MEMS. In the present work, a silicon based microfluidic device hasbeen developed for carrying out fluorescence based immunoassay. By hybrid attachment ofthe microfluidic device with a Spectrometer-on-chip, the feasibility of synthesizing anintegrated Lab-on-a-chip type device for fluorescence based biosensing has beendemonstrated. Biodetection using the microfluidic device has been carried out usingantigen sheep IgG and Alexafluor-647 tagged antibody particles and the experimentalresults prove that silicon is a compatible material for the present application given thevarious advantages it offers such as cost-effectiveness, ease of bulk microfabrication,superior surface affinity to biomolecules, ease of disposability of the device etc., and is thussuitable for fabricating Lab-on-a-chip type devices.

Fluorescence detection of esophageal neoplasia

Science.gov (United States)

Borisova, E.; Vladimirov, B.; Avramov, L.

2008-06-01

White-light endoscopy is well-established and wide used modality. However, despite the many technological advances that have been occurred, conventional endoscopy is suboptimal and usually detects advanced stage lesions. The limitations of standard endoscopy initiate development of spectroscopic techniques, additional to standard endoscopic equipment. One of the most sensitive approaches is fluorescence spectroscopy of gastrointestinal mucosa for neoplasia detection. In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus. The 5-ALA is administered per os six hours before measurements at dose 20 mg/kg weight. Excitation source has max of emission at 405 nm and light is delivered by the standard light guide of the endoscopic equipment. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to reabsorption of blood. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of 5-ALA/PpIX only in abnormal sites Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

Validation of tumor protein marker quantification by two independent automated immunofluorescence image analysis platforms

Science.gov (United States)

Peck, Amy R; Girondo, Melanie A; Liu, Chengbao; Kovatich, Albert J; Hooke, Jeffrey A; Shriver, Craig D; Hu, Hai; Mitchell, Edith P; Freydin, Boris; Hyslop, Terry; Chervoneva, Inna; Rui, Hallgeir

2016-01-01

Protein marker levels in formalin-fixed, paraffin-embedded tissue sections traditionally have been assayed by chromogenic immunohistochemistry and evaluated visually by pathologists. Pathologist scoring of chromogen staining intensity is subjective and generates low-resolution ordinal or nominal data rather than continuous data. Emerging digital pathology platforms now allow quantification of chromogen or fluorescence signals by computer-assisted image analysis, providing continuous immunohistochemistry values. Fluorescence immunohistochemistry offers greater dynamic signal range than chromogen immunohistochemistry, and combined with image analysis holds the promise of enhanced sensitivity and analytic resolution, and consequently more robust quantification. However, commercial fluorescence scanners and image analysis software differ in features and capabilities, and claims of objective quantitative immunohistochemistry are difficult to validate as pathologist scoring is subjective and there is no accepted gold standard. Here we provide the first side-by-side validation of two technologically distinct commercial fluorescence immunohistochemistry analysis platforms. We document highly consistent results by (1) concordance analysis of fluorescence immunohistochemistry values and (2) agreement in outcome predictions both for objective, data-driven cutpoint dichotomization with Kaplan–Meier analyses or employment of continuous marker values to compute receiver-operating curves. The two platforms examined rely on distinct fluorescence immunohistochemistry imaging hardware, microscopy vs line scanning, and functionally distinct image analysis software. Fluorescence immunohistochemistry values for nuclear-localized and tyrosine-phosphorylated Stat5a/b computed by each platform on a cohort of 323 breast cancer cases revealed high concordance after linear calibration, a finding confirmed on an independent 382 case cohort, with concordance correlation coefficients >0

Absorption and fluorescence studies of curcumin bound to liposomes and lymphocytes: effect of γ- irradiation

International Nuclear Information System (INIS)

Kunwar, Amit; Barik, A.; Indira Priyadarsini, K.; Pandey, R.

2006-01-01

Absorption and fluorescence spectral changes in curcumin were employed to follow its binding to liposomes and lymphocytes. The association constants indicated high affinity of curcumin to liposomes. Tumor lymphocytes show mere intense fluorescence of curcumin over the normal lymphocytes. The loss of curcumin in cells after γ-irradiation could be followed by reduction in curcumin fluorescence. The studies indicate that such fluorescence changes can be used as markers to understand the preferential loading of curcumin to cells. (author)

Absorption and fluorescence studies of curcumin bound to liposomes and lymphocytes: effect of {gamma}- irradiation

Energy Technology Data Exchange (ETDEWEB)

Kunwar, Amit; Barik, A; Indira Priyadarsini, K [Radiation and Photochemistry Division, Bhabha Atomic Research Centre, Mumbai (India); Pandey, R [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai (India)

2006-01-15

Absorption and fluorescence spectral changes in curcumin were employed to follow its binding to liposomes and lymphocytes. The association constants indicated high affinity of curcumin to liposomes. Tumor lymphocytes show mere intense fluorescence of curcumin over the normal lymphocytes. The loss of curcumin in cells after {gamma}-irradiation could be followed by reduction in curcumin fluorescence. The studies indicate that such fluorescence changes can be used as markers to understand the preferential loading of curcumin to cells. (author)

Static Hyperspectral Fluorescence Imaging of Viscous Materials Based on a Linear Variable Filter Spectrometer

Directory of Open Access Journals (Sweden)

Alexander W. Koch

2013-09-01

Full Text Available This paper presents a low-cost hyperspectral measurement setup in a new application based on fluorescence detection in the visible (Vis wavelength range. The aim of the setup is to take hyperspectral fluorescence images of viscous materials. Based on these images, fluorescent and non-fluorescent impurities in the viscous materials can be detected. For the illumination of the measurement object, a narrow-band high-power light-emitting diode (LED with a center wavelength of 370 nm was used. The low-cost acquisition unit for the imaging consists of a linear variable filter (LVF and a complementary metal oxide semiconductor (CMOS 2D sensor array. The translucent wavelength range of the LVF is from 400 nm to 700 nm. For the confirmation of the concept, static measurements of fluorescent viscous materials with a non-fluorescent impurity have been performed and analyzed. With the presented setup, measurement surfaces in the micrometer range can be provided. The measureable minimum particle size of the impurities is in the nanometer range. The recording rate for the measurements depends on the exposure time of the used CMOS 2D sensor array and has been found to be in the microsecond range.

Fluorescent probe based on heteroatom containing styrylcyanine: pH-sensitive properties and bioimaging in vivo

International Nuclear Information System (INIS)

Yang, Xiaodong; Gao, Ya; Huang, Zhibing; Chen, Xiaohui; Ke, Zhiyong; Zhao, Peiliang; Yan, Yichen; Liu, Ruiyuan; Qu, Jinqing

2015-01-01

A novel fluorescent probe based on heteroatom containing styrylcyanine is synthesized. The fluorescence of probe is bright green in basic and neutral media but dark orange in strong acidic environments, which could be reversibly switched. Such behavior enables it to work as a fluorescent pH sensor in the solution state and a chemosensor for detecting acidic and basic volatile organic compounds. Analyses by NMR spectroscopy confirm that the protonation or deprotonation of pyridinyl moiety is responsible for the sensing process. In addition, the fluorescent microscopic images of probe in live cells and zebrafish are achieved successfully, suggesting that the probe has good cell membrane permeability and low cytotoxicity. - Graphical abstract: A novel styrylcyanine-based fluorescent pH probe was designed and synthesized, the fluorescence of which is bright green in basic and neutral media but dark orange in strong acidic environments. Such behavior enables it to work as a fluorescent pH sensor in solution states, and a chemosensor for detecting volatile organic compounds with high acidity and basicity in solid state. In addition, it can be used for fluorescent imaging in living cell and living organism. - Highlights: • Bright green fluorescence was observed in basic and neutral media. • Dark orange fluorescence was found in strong acidic environments. • Volatile organic compounds with high acidity and basicity could be detected. • Bioimaging in living cell and living organism was achieved successfully

ALA-based fluorescent diagnosis of malignant oral lesions in the presence of bacterial porphyrin formation

Science.gov (United States)

Schleier, P.; Berndt, A.; Zinner, K.; Zenk, W.; Dietel, W.; Pfister, W.

2006-02-01

The aminolevulinic acid (5-ALA) -based fluorescence diagnosis has been found to be promising for an early detection and demarcation of superficial oral squamous cell carcinomas (OSCC). This method has previously demonstrated high sensitivity, however this clinical trial showed a specificity of approximately 62 %. This specificity was mainly restricted by tumor detection in the oral cavity in the presence of bacteria. After topical ALA application in the mouth of patients with previously diagnosed OSSC, red fluorescent areas were observed which did not correlate to confirm histological findings. Swabs and plaque samples were taken from 44 patients and cultivated microbiologically. Fluorescence was investigated (OMA-system) from 32 different bacteria strains found naturally in the oral cavity. After ALA incubation, 30 of 32 strains were found to synthesize fluorescent porphyrins, mainly Protoporphyrin IX. Also multiple fluorescent spectra were obtained having peak wavelengths of 636 nm and around 618 nm - 620 nm indicating synthesis of different porphyrins, such as the lipophylic Protoporphyrin IX (PpIX) and hydrophylic porphyrins (water soluble porphyrins, wsp). Of the 32 fluorescent bacterial strains, 18 produced wsp, often in combination with PpIX, and 5 produced solely wsp. These results clarify that ALA-based fluorescence diagnosis without consideration or suppression of bacteria fluorescence may lead to false-positive findings. It is necessary to suppress bacteria fluorescence with suitable antiseptics before starting the procedure. In this study, when specific antiseptic pre-treatment was performed bacterial associated fluorescence was significantly reduced.

Synthesis, characterization and fluorescence performance of a waterborne polyurethane-based polymeric dye

Energy Technology Data Exchange (ETDEWEB)

Xianhai, Hu, E-mail: hxyh@aiai.edu.cn [CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026 (China); School of Materials and Chemical Engineering, Building Energy Efficiency Research Institute, Anhui University of Architecture, Hefei 230022 (China); Zhang, Xingyuan, E-mail: zxym@ustc.edu.cn [CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026 (China); Liu, Jin [School of Materials and Chemical Engineering, Building Energy Efficiency Research Institute, Anhui University of Architecture, Hefei 230022 (China); Dai, Jiabing [CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026 (China)

2013-10-15

A novel anionic waterborne polyurethane-based fluorescent dye WPU-DV26 was synthesized by incorporating the molecular structure of disperse violet 26 (DV26) into the polyurethane chain. The structure of WPU-DV26 was confirmed by means of Fourier transform infrared spectroscopy and UV–vis absorption analysis. Comparing to the UV–vis spectrum of DV26, WPU-DV26 showed a hypsochromic shift from the absorption maxima of 518, 558, 609 nm to 510, 548, 586 nm, respectively. WPU-DV26 can form stable latex in water. The number average molecular weight and its distribution index, and average latex particle size for WPU-DV26 were determined to be 2.33×10{sup 4}, 1.36 and 80 nm, respectively. The improved thermal stability of WPU-DV26 can be attributed to the embedded anthraquinone unit of DV26. It was found that both the intensity and stability of the fluorescence of WPU-DV26 latex were improved significantly compared with those of DV26. -- Highlights: ► A waterborne polyurethane-based polymeric dye was synthesized. ► The fluorescence intensity of WPU-DV26 emulsion was enhanced greatly compared with that of DV26. ► The fluorescence stability of WPU-DV26 emulsion was fine not only for long term storage but also for fluorescence quencher.

Turn-on fluorescent chemosensor for Hg2+ based on multivalent rhodamine ligands

NARCIS (Netherlands)

Wang, X.; Iqbal, M.; Huskens, Jurriaan; Verboom, Willem

2012-01-01

Rhodamine-based fluorescent chemosensors 1 and 2 exhibit selective fluorescence enhancement to Fe3+ and Hg2+ over other metal ions at 580 nm in CH3CN/H2O (3/1, v/v) solution. Bis(rhodamine) chemosensor 1, under optimized conditions (CH3CN/HEPES buffer (0.02 M, pH = 7.0) (95/5, v/v)), shows a high

Micromotor-based on-off fluorescence detection of sarin and soman simulants.

Science.gov (United States)

Singh, Virendra V; Kaufmann, Kevin; Orozco, Jahir; Li, Jinxing; Galarnyk, Michael; Arya, Gaurav; Wang, Joseph

2015-06-30

Self-propelled micromotor-based fluorescent "On-Off" detection of nerve agents is described. The motion-based assay utilizes Si/Pt Janus micromotors coated with fluoresceinamine toward real-time "on-the-fly" field detection of sarin and soman simulants.

Recognizing the SINEs of Infection: Regulation of Retrotransposon Expression and Modulation of Host Cell Processes

Directory of Open Access Journals (Sweden)

William Dunker

2017-12-01

Full Text Available Short interspersed elements (SINEs are a family of retrotransposons evolutionarily derived from cellular RNA polymerase III transcripts. Over evolutionary time, SINEs have expanded throughout the human genome and today comprise ~11% of total chromosomal DNA. While generally transcriptionally silent in healthy somatic cells, SINE expression increases during a variety of types of stresses, including DNA virus infection. The relevance of SINE expression to viral infection was largely unexplored, however, recent years have seen great progress towards defining the impact of SINE expression on viral replication and host gene expression. Here we review the origin and diversity of SINE elements and their transcriptional control, with an emphasis on how their expression impacts host cell biology during viral infection.

Modulation of a fluorescence switch based on photochromic spirooxazine in composite organic nanoparticles

International Nuclear Information System (INIS)

Sheng Xiaohai; Peng Aidong; Fu Hongbing; Liu Yuanyuan; Zhao Yongsheng; Ma Ying; Yao Jiannian

2007-01-01

We describe a versatile and convenient approach to achieve fluorescence modulation by the preparation of composite nanoparticles (CNPs), based on photochromic 5-methoxy-1,3,3-trimethyl-9'-hydroxyspiroindolinenaphthoxazine (SO), fluorescent 4-(dicyanomethylene)-2-methyl-6-(p-dimethyl-aminostyryl)-4H-pyran (DCM), and emissive-assistant 1,3-bis(pyrene) propane (BPP) molecules, employing doping techniques. The mechanism of the fluorescence switch is the intermolecular energy transfer as supported by both steady-state and time-resolved spectroscopy results. The addition of BPP not only enhances the contrast of the fluorescence signal between the 'ON' and 'OFF' state, but also provides a convenient way to tune the excitation wavelength for reading the fluorescence. High-contrast ON/OFF (20:1) fluorescence switching is successfully implemented in the CNPs and also in a more practical PVA film loaded with the CNPs. This system may represent an alternative to the covalent system in potentially rewritable high-density optical data or image storage utilizing luminescence intensity readout schemes

A novel dansyl-based fluorescent probe for highly selective detection of ferric ions.

Science.gov (United States)

Yang, Min; Sun, Mingtai; Zhang, Zhongping; Wang, Suhua

2013-02-15

A novel dansyl-based fluorescent probe was synthesized and characterized. It exhibits high selectivity and sensitivity towards Fe(3+) ion. This fluorescent probe is photostable, water soluble and pH insensitive. The limit of detection is found to be 0.62 μM. These properties make it a good fluorescent probe for Fe(3+) ion detection in both chemical and biological systems. Spike recovery test confirms its practical application in tap water samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Lysergic acid amide as chemical marker for the total ergot alkaloids in rye flour - Determination by high-performance thin-layer chromatography-fluorescence detection.

Science.gov (United States)

Oellig, Claudia

2017-07-21

Ergot alkaloids are generally determined by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD) or mass selective detection, analyzing the individual compounds. However, fast and easy screening methods for the determination of the total ergot alkaloid content are more suitable, since for monitoring only the sum of the alkaloids is relevant. The herein presented screening uses lysergic acid amide (LSA) as chemical marker, formed from ergopeptine alkaloids, and ergometrine for the determination of the total ergot alkaloids in rye with high-performance thin-layer chromatography-fluorescence detection (HPTLC-FLD). An ammonium acetate buffered extraction step was followed by liquid-liquid partition for clean-up before the ergopeptine alkaloids were selectively transformed to LSA and analyzed by HPTLC-FLD on silica gel with isopropyl acetate/methanol/water/25% ammonium hydroxide solution (80:10:3.8:1.1, v/v/v/v) as the mobile phase. The enhanced native fluorescence of LSA and unaffected ergometrine was used for quantitation without any interfering matrix. Limits of detection and quantitation were 8 and 26μg LSA/kg rye, which enables the determination of the total ergot alkaloids far below the applied quality criterion limit for rye. Close to 100% recoveries for different rye flours at relevant spiking levels were obtained. Thus, reliable results were guaranteed, and the fast and efficient screening for the total ergot alkaloids in rye offers a rapid alternative to the HPLC analysis of the individual compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimized measurements of separations and angles between intra-molecular fluorescent markers

DEFF Research Database (Denmark)

Mortensen, Kim; Sung, Jongmin; Flyvbjerg, Henrik

2015-01-01

We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie...

Fluorescent visualization of macromolecules in Drosophila whole mounts.

Science.gov (United States)

Ramos, Ricardo Guelerman Pinheiro; Machado, Luciana Claudia Herculano; Moda, Livia Maria Rosatto

2010-01-01

The ability to determine the expression dynamics of individual genes "in situ" by visualizing the precise spatial and temporal distribution of their products in whole mounts by histochemical and immunocytochemical reactions has revolutionized our understanding of cellular processes. Drosophila developmental genetics was one of the fields that benefited most from these technologies, and a variety of fluorescent methods were specifically designed for investigating the localization of developmentally important proteins and cell markers during embryonic and post embryonic stages of this model organism. In this chapter we present detailed protocols for fluorescence immunocytochemistry of whole mount embryos, imaginal discs, pupal retinas, and salivary glands of Drosophila melanogaster, as well as methods for fluorescent visualization of specific subcellular structures in these tissues.

Defining a turnover index for the correlation of biomaterial degradation and cell based extracellular matrix synthesis using fluorescent tagging techniques.

Science.gov (United States)

Bardsley, Katie; Wimpenny, Ian; Wechsler, Roni; Shachaf, Yonatan; Yang, Ying; El Haj, Alicia J

2016-11-01

Non-destructive protocols which can define a biomaterial's degradation and its associated ability to support proliferation and/or promote extracellular matrix deposition will be an essential in vitro tool. In this study we investigate fluorescently tagged biomaterials, with varying rates of degradation and their ability to support cell proliferation and osteogenic differentiation. Changes in fluorescence of the biomaterials and the release of fluorescent soluble by-products were confirmed as accurate methods to quantify degradation. It was demonstrated that increasing rates of the selected biomaterials' degradation led to a decrease in cell proliferation and concurrently an increase in osteogenic matrix production. A novel turnover index (TI), which directly describes the effect of degradation of a biomaterial on cell behaviour, was calculated. Lower TIs for proliferation and high TIs for osteogenic marker production were observed on faster degrading biomaterials, indicating that these biomaterials supported an upregulation of osteogenic markers. This TI was further validated using an ex vivo chick femur model, where the faster degrading biomaterial, fibrin, led to an increased TI for mineralisation within an epiphyseal defect. This in vitro tool, TI, for monitoring the effect of biomaterial degradation on extracellular matrix production may well act as predictor of the selected biomaterials' performance during in vivo studies. This paper outlines a novel metric, Turnover Index (TI), which can be utilised in tissue-engineering for the comparison of a range of biomaterials. The metric sets out to define the relationship between the rate of degradation of biomaterials with the rate of cell proliferation and ECM synthesis, ultimately allowing us to tailor material for set clinical requirements. We have discovered some novel comparative findings that cells cultured on biomaterials with increased rates of degradation have lower rates of proliferation but alternatively

A vision-based automated guided vehicle system with marker recognition for indoor use.

Science.gov (United States)

Lee, Jeisung; Hyun, Chang-Ho; Park, Mignon

2013-08-07

We propose an intelligent vision-based Automated Guided Vehicle (AGV) system using fiduciary markers. In this paper, we explore a low-cost, efficient vehicle guiding method using a consumer grade web camera and fiduciary markers. In the proposed method, the system uses fiduciary markers with a capital letter or triangle indicating direction in it. The markers are very easy to produce, manipulate, and maintain. The marker information is used to guide a vehicle. We use hue and saturation values in the image to extract marker candidates. When the known size fiduciary marker is detected by using a bird's eye view and Hough transform, the positional relation between the marker and the vehicle can be calculated. To recognize the character in the marker, a distance transform is used. The probability of feature matching was calculated by using a distance transform, and a feature having high probability is selected as a captured marker. Four directional signals and 10 alphabet features are defined and used as markers. A 98.87% recognition rate was achieved in the testing phase. The experimental results with the fiduciary marker show that the proposed method is a solution for an indoor AGV system.

Introducing Ratiometric Fluorescence to MnO2 Nanosheet-Based Biosensing: A Simple, Label-Free Ratiometric Fluorescent Sensor Programmed by Cascade Logic Circuit for Ultrasensitive GSH Detection.

Science.gov (United States)

Fan, Daoqing; Shang, Changshuai; Gu, Wenling; Wang, Erkang; Dong, Shaojun

2017-08-09

Glutathione (GSH) plays crucial roles in various biological functions, the level alterations of which have been linked to varieties of diseases. Herein, we for the first time expanded the application of oxidase-like property of MnO 2 nanosheet (MnO 2 NS) to fluorescent substrates of peroxidase. Different from previously reported fluorescent quenching phenomena, we found that MnO 2 NS could not only largely quench the fluorescence of highly fluorescent Scopoletin (SC) but also surprisingly enhance that of nonfluorescent Amplex Red (AR) via oxidation reaction. If MnO 2 NS is premixed with GSH, it will be reduced to Mn 2+ and lose the oxidase-like property, accompanied by subsequent increase in SC's fluorescence and decrease in AR's. On the basis of the above mechanism, we construct the first MnO 2 NS-based ratiometric fluorescent sensor for ultrasensitive and selective detection of GSH. Notably, this ratiometric sensor is programmed by the cascade logic circuit (an INHIBIT gate cascade with a 1 to 2 decoder). And a linear relationship between ratiometric fluorescent intensities of the two substrates and logarithmic values of GSH's concentrations is obtained. The detection limit of GSH is as low as 6.7 nM, which is much lower than previous ratiometric fluorescent sensors, and the lowest MnO 2 NS-based fluorescent GSH sensor reported so far. Furthermore, this sensor is simple, label-free, and low-cost; it also presents excellent applicability in human serum samples.

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

Science.gov (United States)

Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

2008-09-01

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

A novel acidic pH fluorescent probe based on a benzothiazole derivative

Science.gov (United States)

Ma, Qiujuan; Li, Xian; Feng, Suxiang; Liang, Beibei; Zhou, Tiqiang; Xu, Min; Ma, Zhuoyi

2017-04-01

A novel acidic pH fluorescent probe 1 based on a benzothiazole derivative has been designed, synthesized and developed. The linear response range covers the acidic pH range from 3.44 to 6.46, which is valuable for pH researches in acidic environment. The evaluated pKa value of the probe 1 is 4.23. The fluorescence enhancement of the studied probe 1 with an increase in hydrogen ions concentration is based on the hindering of enhanced photo-induced electron transfer (PET) process. Moreover, the pH sensor possesses a highly selective response to H+ in the presence of metal ions, anions and other bioactive small molecules which would be interfere with its fluorescent pH response. Furthermore, the probe 1 responds to acidic pH with short response time that was less than 1 min. The probe 1 has been successfully applied to confocal fluorescence imaging in live HeLa cells and can selectively stain lysosomes. All of such good properties prove it can be used to monitoring pH fluctuations in acidic environment with high sensitivity, pH dependence and short response time.

Regulation of mouse retroelement MuERV-L/MERVL expression by REX1 and epigenetic control of stem cell potency.

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Jon eSchoorlemmer

2014-02-01

Full Text Available About half of mammalian genomes is occupied by DNA sequences that originatefrom transposable elements. Retrotransposons can modulate gene expression indifferent ways and, particularly retrotransposon-derived LTRs, profoundly shapeexpression of both surrounding and distant genomic loci. This is especially important inpreimplantation development, during which extensive reprogramming of the genometakes place and cells pass through totipotent and pluripotent states. At this stage, themain mechanisms responsible for retrotransposon silencing i.e. DNA methylation, isinoperative. A particular retrotransposon called muERV-L/MERVL is expressed duringpreimplantation stages and contributes to the plasticity of mouse embryonic stem cells.This review will focus on the role of MERVL-derived sequences as controllingelements of gene expression specific for preimplantation development, two-cell stagespecific gene expression and stem cell pluripotency, the epigenetic mechanisms thatcontrol their expression, and the contributions of the pluripotency marker REX1 and therelated YY1 family of transcription factors to this regulation process.

A highly sensitive fluorescent probe based on BODIPY for Hg2+ in aqueous solution

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ZHAO Junwei

2016-12-01

Full Text Available A highly sensitive fluorescent probe based on BODIPY and hydrazine for Hg2+ was designed and synthesized.This probe could detect mercury ions in aqueous solutions within 5 min.With the increase of Hg2+ mole concentration,an obvious red shift of UV-Vis absorption wavelength was observed and the fluorescence intensity significantly enhanced.It was found that the fluorescence intensity of an aqueous solution containing 0.1 μmol/L Hg2+ is much stronger than that of blank solution,which indicats that the fluorescent probe has high sensitivity.In addition,other metal ions could not cause the change of fluorescent spectra,which means this probe has good selectivity,as well.

The impact of fluorescent dyes on the performances of polystyrene-based plastic scintillators

International Nuclear Information System (INIS)

Zhu, Jun; Deng, Cheng; Jiang, Huimin; Zheng, Zhanlong; Gong, Rui; Bi, Yutie; Zhang, Lin; Lin, Runxiong

2016-01-01

To investigate the influence of both the first luminescent additive and the wavelength-shifter on the performance of plastic scintillator, a series of polystyrene-based scintillator had been prepared by thermal polymerization. Three first luminescent additives (PPO, p-TP and b-PBD) and four wavelength-shifters (POPOP, Bis-MSB, Me-MSB and DPA) were added to the scintillators respectively. The comparison results showed that PPO and POPOP were the most adequate fluorescent dyes for the polystyrene-based plastic scintillator. Moreover, with the increase of the concentration of PPO and POPOP, the fluorescence intensity and light yield were increased firstly and then decreased. The plastic scintillator containing 2% PPO and 0.02% POPOP had the highest fluorescence intensity and light yield.

The impact of fluorescent dyes on the performances of polystyrene-based plastic scintillators

Energy Technology Data Exchange (ETDEWEB)

Zhu, Jun [Research Center of Laser Fusion, China Academy of Engineering Physics, Mianyang 621900 (China); Engineering Research Center of High Performance Polymer and Molding Technology, Ministry of Education, Qingdao University of Science and Technology, Qingdao 266042 (China); Joint Laboratory for Extreme Conditions Matter Properties, Southwest University of Science and Technology and Research Center of Laser Fusion, China Academy of Engineering Physics, Mianyang 621010 (China); Deng, Cheng; Jiang, Huimin [Engineering Research Center of High Performance Polymer and Molding Technology, Ministry of Education, Qingdao University of Science and Technology, Qingdao 266042 (China); Zheng, Zhanlong; Gong, Rui [Research Center of Laser Fusion, China Academy of Engineering Physics, Mianyang 621900 (China); Joint Laboratory for Extreme Conditions Matter Properties, Southwest University of Science and Technology and Research Center of Laser Fusion, China Academy of Engineering Physics, Mianyang 621010 (China); Bi, Yutie [Joint Laboratory for Extreme Conditions Matter Properties, Southwest University of Science and Technology and Research Center of Laser Fusion, China Academy of Engineering Physics, Mianyang 621010 (China); Zhang, Lin, E-mail: zhlmy@sina.com [Research Center of Laser Fusion, China Academy of Engineering Physics, Mianyang 621900 (China); Lin, Runxiong, E-mail: qdlrx@qust.edu.cn [Engineering Research Center of High Performance Polymer and Molding Technology, Ministry of Education, Qingdao University of Science and Technology, Qingdao 266042 (China)

2016-11-01

To investigate the influence of both the first luminescent additive and the wavelength-shifter on the performance of plastic scintillator, a series of polystyrene-based scintillator had been prepared by thermal polymerization. Three first luminescent additives (PPO, p-TP and b-PBD) and four wavelength-shifters (POPOP, Bis-MSB, Me-MSB and DPA) were added to the scintillators respectively. The comparison results showed that PPO and POPOP were the most adequate fluorescent dyes for the polystyrene-based plastic scintillator. Moreover, with the increase of the concentration of PPO and POPOP, the fluorescence intensity and light yield were increased firstly and then decreased. The plastic scintillator containing 2% PPO and 0.02% POPOP had the highest fluorescence intensity and light yield.

Highly Selective Fluorescent Sensing of Proteins Based on a Fluorescent Molecularly Imprinted Nanosensor

Directory of Open Access Journals (Sweden)

Shuo Wang

2013-09-01

Full Text Available A fluorescent molecularly imprinted nanosensor was obtained by grafting imprinted polymer onto the surface of multi-wall carbon nanotubes and post-imprinting treatment with fluorescein isothiocyanate (FITC. The fluorescence of lysozyme-imprinted polymer (Lys-MIP was quenched more strongly by Lys than that of nonimprinted polymer (NIP, which indicated that the Lys-MIP could recognize Lys. The resulted imprinted material has the ability to selectively sense a target protein, and an imprinting factor of 3.34 was achieved. The Lys-MIP also showed selective detection for Lys among other proteins such as cytochrome C (Cyt C, hemoglobin (HB and bovine serum albumin (BSA due to the imprinted sites in the Lys-MIP. This approach combines the high selectivity of surface molecular imprinting technology and fluorescence, and converts binding events into detectable signals by monitoring fluorescence spectra. Therefore, it will have further applications for Lys sensing.

Innovative molecular-based fluorescent nanoparticles for multicolor single particle tracking in cells

International Nuclear Information System (INIS)

Daniel, Jonathan; Blanchard-Desce, Mireille; Godin, Antoine G; Palayret, Matthieu; Lounis, Brahim; Cognet, Laurent

2016-01-01

Based on an original molecular-based design, we present bright and photostable fluorescent organic nanoparticles (FONs) showing excellent colloidal stability in various aqueous environments. Complementary near-infrared emitting and green emitting FONs were prepared using a simple, fast and robust protocol. Both types of FONs could be simultaneously imaged at the single-particle level in solution as well as in biological environments using a monochromatic excitation and a dual-color fluorescence microscope. No evidence of acute cytotoxicity was found upon incubation of live cells with mixed solutions of FONs, and both types of nanoparticles were found internalized in the cells where their motion could be simultaneously tracked at video-rate up to minutes. These fluorescent organic nanoparticles open a novel non-toxic alternative to existing nanoparticles for imaging biological structures, compatible with live-cell experiments and specially fitted for multicolor single particle tracking. (paper)

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

Science.gov (United States)

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-03-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

Selective fluorescence sensors for detection of nitroaniline and metal Ions based on ligand-based luminescent metal-organic frameworks

International Nuclear Information System (INIS)

Yu, Zongchao; Wang, Fengqin; Lin, Xiangyi; Wang, Chengmiao; Fu, Yiyuan; Wang, Xiaojun; Zhao, Yongnan; Li, Guodong

2015-01-01

Metal-organic frameworks (MOFs) are porous crystalline materials with high potential for applications in fluorescence sensors. In this work, two solvent-induced Zn(II)–based metal-organic frameworks, Zn_3L_3(DMF)_2 (1) and Zn_3L_3(DMA)_2(H_2O)_3 (2) (L=4,4′-stilbenedicarboxylic acid), were investigated as selective sensing materials for detection of nitroaromatic compounds and metal ions. The sensing experiments show that 1 and 2 both exhibit selective fluorescence quenching toward nitroaniline with a low detection limit. In addition, 1 exhibits high selectivity for detection of Fe"3"+ and Al"3"+ by significant fluorescence quenching or enhancement effect. While for 2, it only exhibits significant fluorescence quenching effect for Fe"3"+. The results indicate that 1 and 2 are both promising fluorescence sensors for detecting and recognizing nitroaniline and metal ions with high sensitivity and selectivity. - Graphical abstract: Two MOFs have been selected as the fluorescence sensing materials for selectively sensing mitroaromatic compounds and metal ions. The high selectivity makes them promising fluorescence sensors for detecting and recognizing nitroaniline and Fe"3"+ or Al"3"+.

PwRn1, a novel Ty3/gypsy-like retrotransposon of Paragonimus westermani: molecular characters and its differentially preserved mobile potential according to host chromosomal polyploidy

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Kong Yoon

2008-10-01

Full Text Available Abstract Background Retrotransposons have been known to involve in the remodeling and evolution of host genome. These reverse transcribing elements, which show a complex evolutionary pathway with diverse intermediate forms, have been comprehensively analyzed from a wide range of host genomes, while the information remains limited to only a few species in the phylum Platyhelminthes. Results A LTR retrotransposon and its homologs with a strong phylogenetic affinity toward CsRn1 of Clonorchis sinensis were isolated from a trematode parasite Paragonimus westermani via a degenerate PCR method and from an insect species Anopheles gambiae by in silico analysis of the whole mosquito genome, respectively. These elements, designated PwRn1 and AgCR-1 – AgCR-14 conserved unique features including a t-RNATrp primer binding site and the unusual CHCC signature of Gag proteins. Their flanking LTRs displayed >97% nucleotide identities and thus, these elements were likely to have expanded recently in the trematode and insect genomes. They evolved heterogeneous expression strategies: a single fused ORF, two separate ORFs with an identical reading frame and two ORFs overlapped by -1 frameshifting. Phylogenetic analyses suggested that the elements with the separate ORFs had evolved from an ancestral form(s with the overlapped ORFs. The mobile potential of PwRn1 was likely to be maintained differentially in association with the karyotype of host genomes, as was examined by the presence/absence of intergenomic polymorphism and mRNA transcripts. Conclusion Our results on the structural diversity of CsRn1-like elements can provide a molecular tool to dissect a more detailed evolutionary episode of LTR retrotransposons. The PwRn1-associated genomic polymorphism, which is substantial in diploids, will also be informative in addressing genomic diversification following inter-/intra-specific hybridization in P. westermani populations.

Complete sequence of Tvv1, a family of Ty 1 copia-like retrotransposons of Vitis vinifera L., reconstituted by chromosome walking.

Science.gov (United States)

Pelsy, F.; Merdinoglu, D.

2002-09-01

A chromosome-walking strategy was used to sequence and characterize retrotransposons in the grapevine genome. The reconstitution of a family of retroelements, named Tvv1, was achieved by six successive steps. These elements share a single, highly conserved open reading frame 4,153 nucleotides-long, putatively encoding the gag, pro, int, rt and rh proteins. Comparison of the Tvv1 open reading frame coding potential with those of drosophila copia and tobacco Tnt1, revealed that Tvv1 is closely related to Ty 1 copia-like retrotransposons. A highly variable untranslated leader region, upstream of the open reading frame, allowed us to differentiate Tvv1 variants, which represent a family of at least 28 copies, in varying sizes. This internal region is flanked by two long terminal repeats in direct orientation, sized between 149 and 157 bp. Among elements theoretically sized from 4,970 to 5,550 bp, we describe the full-length sequence of a reference element Tvv1-1, 5,343 nucleotides-long. The full-length sequence of Tvv1-1 compared to pea PDR1 shows a 53.3% identity. In addition, both elements contain long terminal repeats of nearly the same size in which the U5 region could be entirely absent. Therefore, we assume that Tvv1 and PDR1 could constitute a particular class of short LTRs retroelements.

Compatibility of pedigree-based and marker-based relationship matrices for single-step genetic evaluation

DEFF Research Database (Denmark)

Christensen, Ole Fredslund

2012-01-01

Single-step methods for genomic prediction have recently become popular because they are conceptually simple and in practice such a method can completely replace a pedigree-based method for routine genetic evaluation. An issue with single-step methods is compatibility between the marker-based rel...

Modulation of a fluorescence switch based on photochromic spirooxazine in composite organic nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Sheng Xiaohai [Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080 (China); Peng Aidong [Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080 (China); Fu Hongbing [Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080 (China); Liu Yuanyuan [Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080 (China); Zhao Yongsheng [Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080 (China); Ma Ying [Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080 (China); Yao Jiannian [Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080 (China)

2007-04-11

We describe a versatile and convenient approach to achieve fluorescence modulation by the preparation of composite nanoparticles (CNPs), based on photochromic 5-methoxy-1,3,3-trimethyl-9'-hydroxyspiroindolinenaphthoxazine (SO), fluorescent 4-(dicyanomethylene)-2-methyl-6-(p-dimethyl-aminostyryl)-4H-pyran (DCM), and emissive-assistant 1,3-bis(pyrene) propane (BPP) molecules, employing doping techniques. The mechanism of the fluorescence switch is the intermolecular energy transfer as supported by both steady-state and time-resolved spectroscopy results. The addition of BPP not only enhances the contrast of the fluorescence signal between the 'ON' and 'OFF' state, but also provides a convenient way to tune the excitation wavelength for reading the fluorescence. High-contrast ON/OFF (20:1) fluorescence switching is successfully implemented in the CNPs and also in a more practical PVA film loaded with the CNPs. This system may represent an alternative to the covalent system in potentially rewritable high-density optical data or image storage utilizing luminescence intensity readout schemes.

Dendrimer-based fluorescent indicators: in vitro and in vivo applications.

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Lorenzo Albertazzi

Full Text Available BACKGROUND: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. PRINCIPAL FINDINGS: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. CONCLUSION: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups.

Smart markers for watershed-based cell segmentation.

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Can Fahrettin Koyuncu

Full Text Available Automated cell imaging systems facilitate fast and reliable analysis of biological events at the cellular level. In these systems, the first step is usually cell segmentation that greatly affects the success of the subsequent system steps. On the other hand, similar to other image segmentation problems, cell segmentation is an ill-posed problem that typically necessitates the use of domain-specific knowledge to obtain successful segmentations even by human subjects. The approaches that can incorporate this knowledge into their segmentation algorithms have potential to greatly improve segmentation results. In this work, we propose a new approach for the effective segmentation of live cells from phase contrast microscopy. This approach introduces a new set of "smart markers" for a marker-controlled watershed algorithm, for which the identification of its markers is critical. The proposed approach relies on using domain-specific knowledge, in the form of visual characteristics of the cells, to define the markers. We evaluate our approach on a total of 1,954 cells. The experimental results demonstrate that this approach, which uses the proposed definition of smart markers, is quite effective in identifying better markers compared to its counterparts. This will, in turn, be effective in improving the segmentation performance of a marker-controlled watershed algorithm.

Smart markers for watershed-based cell segmentation.

Science.gov (United States)

Koyuncu, Can Fahrettin; Arslan, Salim; Durmaz, Irem; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

2012-01-01

Automated cell imaging systems facilitate fast and reliable analysis of biological events at the cellular level. In these systems, the first step is usually cell segmentation that greatly affects the success of the subsequent system steps. On the other hand, similar to other image segmentation problems, cell segmentation is an ill-posed problem that typically necessitates the use of domain-specific knowledge to obtain successful segmentations even by human subjects. The approaches that can incorporate this knowledge into their segmentation algorithms have potential to greatly improve segmentation results. In this work, we propose a new approach for the effective segmentation of live cells from phase contrast microscopy. This approach introduces a new set of "smart markers" for a marker-controlled watershed algorithm, for which the identification of its markers is critical. The proposed approach relies on using domain-specific knowledge, in the form of visual characteristics of the cells, to define the markers. We evaluate our approach on a total of 1,954 cells. The experimental results demonstrate that this approach, which uses the proposed definition of smart markers, is quite effective in identifying better markers compared to its counterparts. This will, in turn, be effective in improving the segmentation performance of a marker-controlled watershed algorithm.

EVALUATION OF IMMUNOHISTOCHEMISTRY (IHC MARKER HER2 IN BREAST CANCER

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Prasanna G. Shete

2016-08-01

Full Text Available The paper discusses a novel approach involving algorithm implementation and hardware Devkit processing for estimating the extent of cancer in a breast tissue sample. The process aims at providing a reliable, repeatable, and fast method that could replace the traditional method of manual examination and estimation. Immunohistochemistry (IHC and Fluorescence in situ Hybridization (FISH are the two main methods used to detect the marker status in clinical practice. FISH is though more reliable than IHC, but IHC is widely used as it is cheaper, convenient to operate and conserve, the morphology is clear. The IHC markers are Estrogen receptor (ER, Progesterone receptor (PR, Human Epidermal Growth Factor (HER2 that give clear indications of the presence of cancer cells in the tissue sample. HER2 remains the most reliable marker for the detection of breast cancer. The Human Epidermal Growth Factor Receptor (HER2 markers are discussed in the paper, as it gives clear indications of the presence of cancer cells in the tissue sample. HER2 is identified based on the color and intensity of the cell membrane staining. The color and intensity is obviously based on the thresholding for classifying the cancerous cells into severity levels in terms of score to estimate the extent of spread of cancer in breast tissue. For HER2 evaluation, the percentage of staining is calculated in terms of ratio of stain pixel count to the total pixel count. The evaluation of HER2 is obtained through simulation software (MATLAB using intensity based algorithm and same is run on embedded processor evaluation board Devkit 8500. The results are validated with doctors.

Stratification of type 2 diabetes based on routine clinical markers

DEFF Research Database (Denmark)

Safai, Narges; Ali, Ashfaq; Rossing, Peter

2018-01-01

AIMS: We hypothesized that patients with dysregulated type 2 diabetes may be stratified based on routine clinical markers. METHODS: In this retrospective cohort study, diabetes related clinical measures including age at onset, diabetes duration, HbA1c, BMI, HOMA2-β, HOMA2-IR and GAD65...... autoantibodies, were used for sub-grouping patients by K-means clustering and for adjusting. Probability of diabetes complications (95% confidence interval), were calculated using logistic regression. RESULTS: Based on baseline data from patients with type 2 diabetes (n=2,290), the cluster analysis suggested up....... CONCLUSIONS: Patients with type 2 diabetes cluster into clinically relevant sub-groups based on routine clinical markers. The prevalence of diabetes complications seems to be sub-group specific. Our data suggests the need for a tailored strategy for the treatment of type 2 diabetes....

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

Science.gov (United States)

Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

2017-08-01

Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

Microvascularity, blood flow and tissue structure at the subchondral plate using an X-ray fluorescence technique

International Nuclear Information System (INIS)

Muthuvelu, P.; Ellis, R.E.; Green, E.M.; Attenburrow, D.; Arkill, K.; Colridge, D.B.; Winlove, C.P.; Bradley, D.A.

2007-01-01

The measurement of blood flow and blood in bone and cartilaginous tissues is crucial to understanding of the development of various diseases, but it presents a formidable technical challenge. We have therefore developed a method based on the detection of metallized microspheres using X-ray fluorescence. This approach provides unrivalled sensitivity and spatial resolution and also allows us simultaneously to measure other markers of the metabolic status of the tissue. (author)

Intrinsic fluorescence of protein in turbid media using empirical relation based on Monte Carlo lookup table

Science.gov (United States)

Einstein, Gnanatheepam; Udayakumar, Kanniyappan; Aruna, Prakasarao; Ganesan, Singaravelu

2017-03-01

Fluorescence of Protein has been widely used in diagnostic oncology for characterizing cellular metabolism. However, the intensity of fluorescence emission is affected due to the absorbers and scatterers in tissue, which may lead to error in estimating exact protein content in tissue. Extraction of intrinsic fluorescence from measured fluorescence has been achieved by different methods. Among them, Monte Carlo based method yields the highest accuracy for extracting intrinsic fluorescence. In this work, we have attempted to generate a lookup table for Monte Carlo simulation of fluorescence emission by protein. Furthermore, we fitted the generated lookup table using an empirical relation. The empirical relation between measured and intrinsic fluorescence is validated using tissue phantom experiments. The proposed relation can be used for estimating intrinsic fluorescence of protein for real-time diagnostic applications and thereby improving the clinical interpretation of fluorescence spectroscopic data.

Gene-based single nucleotide polymorphism markers for genetic and association mapping in common bean.

Science.gov (United States)

Galeano, Carlos H; Cortés, Andrés J; Fernández, Andrea C; Soler, Álvaro; Franco-Herrera, Natalia; Makunde, Godwill; Vanderleyden, Jos; Blair, Matthew W

2012-06-26

In common bean, expressed sequence tags (ESTs) are an underestimated source of gene-based markers such as insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). However, due to the nature of these conserved sequences, detection of markers is difficult and portrays low levels of polymorphism. Therefore, development of intron-spanning EST-SNP markers can be a valuable resource for genetic experiments such as genetic mapping and association studies. In this study, a total of 313 new gene-based markers were developed at target genes. Intronic variation was deeply explored in order to capture more polymorphism. Introns were putatively identified after comparing the common bean ESTs with the soybean genome, and the primers were designed over intron-flanking regions. The intronic regions were evaluated for parental polymorphisms using the single strand conformational polymorphism (SSCP) technique and Sequenom MassARRAY system. A total of 53 new marker loci were placed on an integrated molecular map in the DOR364 × G19833 recombinant inbred line (RIL) population. The new linkage map was used to build a consensus map, merging the linkage maps of the BAT93 × JALO EEP558 and DOR364 × BAT477 populations. A total of 1,060 markers were mapped, with a total map length of 2,041 cM across 11 linkage groups. As a second application of the generated resource, a diversity panel with 93 genotypes was evaluated with 173 SNP markers using the MassARRAY-platform and KASPar technology. These results were coupled with previous SSR evaluations and drought tolerance assays carried out on the same individuals. This agglomerative dataset was examined, in order to discover marker-trait associations, using general linear model (GLM) and mixed linear model (MLM). Some significant associations with yield components were identified, and were consistent with previous findings. In short, this study illustrates the power of intron-based markers for linkage and association mapping in

A rhodol-based fluorescent chemosensor for hydrazine and its application in live cell bioimaging

Science.gov (United States)

Tiensomjitr, Khomsan; Noorat, Rattha; Wechakorn, Kanokorn; Prabpai, Samran; Suksen, Kanoknetr; Kanjanasirirat, Phongthon; Pewkliang, Yongyut; Borwornpinyo, Suparerk; Kongsaeree, Palangpon

2017-10-01

A rhodol cinnamate fluorescent chemosensor (RC) has been developed for selective detection of hydrazine (N2H4). In aqueous medium, the rhodol-based probe exhibited high selectivity for hydrazine among other molecules. The addition of hydrazine triggered a fluorescence emission with 48-fold enhancement based on hydrazinolysis and a subsequent ring-opening process. The chemical probe also displayed a selective colorimetric response toward N2H4 from colorless solution to pink, readily observed by the naked eye. The detection limit of RC for hydrazine was calculated to be 300 nM (9.6 ppb). RC is membrane permeable and was successfully demonstrated to detect hydrazine in live HepG2 cells by confocal fluorescence microscopy.

DNA-based genetic markers for Rapid Cycling Brassica rapa (Fast Plants type designed for the teaching laboratory.

Directory of Open Access Journals (Sweden)

Eryn E. Slankster

2012-06-01

Full Text Available We have developed DNA-based genetic markers for rapid-cycling Brassica rapa (RCBr, also known as Fast Plants. Although markers for Brassica rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP based markers and 14 variable number tandem repeat (VNTR-type markers spread over four chromosomes. The DNA sequences of these markers represent variation in a wide range of genomic features. Among the VNTR-type markers, there are examples of variation in a nongenic region, variation within an intron, and variation in the coding sequence of a gene. Among the SNP-based markers there are examples of polymorphism in intronic DNA and synonymous substitution in a coding sequence. Thus these markers can serve laboratory exercises in both transmission genetics and molecular biology.

A novel fluorescence probe based on triphenylamine Schiff base for bioimaging and responding to pH and Fe{sup 3+}

Energy Technology Data Exchange (ETDEWEB)

Wang, Lei; Yang, Xiaodong; Chen, Xiuli [School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640 (China); Zhou, Yuping [Guangdong Provincial key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Lu, Xiaodan; Yan, Chenggong; Xu, Yikai [Medical Imaging Center, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Liu, Ruiyuan, E-mail: ruiyliu@smu.edu.cn [Guangdong Provincial key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Qu, Jinqing, E-mail: cejqqu@scut.edu.cn [School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640 (China)

2017-03-01

A novel fluorescence probe 1 based on triphenylamine was synthesized and characterized by NMR, IR, high resolution mass spectrometry and elemental analysis. Its fluorescence was quenched when pH below 2. There was a linear relationship between the fluorescence intensity and pH value ranged from 2 to 7. And its fluorescence emission was reversibility in acidic and alkaline solution. Furthermore, it exhibited remarkable selectivity and high sensitivity to Fe{sup 3+} and was able to detect Fe{sup 3+} in aqueous solution with low detection limit of 0.511 μM. Job plot showed that the binding stoichiometry of 1 with Fe{sup 3+} was 1:1. Further observations of {sup 1}H NMR titration suggested that coordination interaction between Fe{sup 3+} and nitrogen atom on C =N bond promoted the intramolecular charge transfer (ICT) or energy transfer process causing fluorescence quenching. Additionally, 1 was also able to be applied for detecting Fe{sup 3+} in living cell and bioimaging. - Graphical abstract: Triphenylamine based fluorescence probe can detect pH and Fe{sup 3+} simultaneously in aqueous solution and be applied for detecting Fe{sup 3+} in living cell and bioimaging. - Highlights: • The fluorescence probe is sensitive to pH in strong acid conditions. • The fluorescence chemosensor can detect pH and Fe{sup 3+} simultaneously. • The recognition is able to carry out in aqueous solution. • The probe can also be applied for detecting Fe{sup 3+} in living cell and bioimaging. • The sensor is synthesized easily with one step.

Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis.

Science.gov (United States)

Praveen, Bavishna B; Ashok, Praveen C; Mazilu, Michael; Riches, Andrew; Herrington, Simon; Dholakia, Kishan

2012-07-01

In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30  s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells

International Nuclear Information System (INIS)

Hagen, Christoph; Werner, Stephan; Carregal-Romero, Susana; Malhas, Ashraf N.; Klupp, Barbara G.; Guttmann, Peter; Rehbein, Stefan; Henzler, Katja; Mettenleiter, Thomas C.

2014-01-01

Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness. - Highlights: • Nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated cells. • New polyelectrolyte-Qdot ® 605 coated gold beads were employed as fiducials. • Saquinavir can induce a strong apoptotic phenotype in the nucleus. • CryoXT is an auspicious imaging technique in apoptosis research

Sensitive turn-on fluorescent detection of tartrazine based on fluorescence resonance energy transfer.

Science.gov (United States)

Huang, Sheng Tian; Shi, Yan; Li, Nian Bing; Luo, Hong Qun

2012-01-18

We introduce a sensitive, rapid, label-free and general fluorescent method for the determination of tartrazine by competitive binding to reduced graphene oxide (rGO) against fluorescein, and the fluorescence recovery upon fluorescein desorption from rGO provides a quantitative readout for tartrazine, giving a detection limit of 0.53 ng mL(-1).

Fluorescence spectrometric studies on the binding of puerarin to human serum albumin using warfarin, ibuprofen and digitoxin as site markers with the aid of chemometrics

International Nuclear Information System (INIS)

Zhang Guowen; Zhao Nan; Wang Lin

2011-01-01

The interaction of puerarin with human serum albumin (HSA) in pH 7.4 Tris-HCl buffer has been investigated by fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. The results revealed the presence of static type of quenching mechanism in the binding of puerarin to HSA. The association constants (K a ) between puerarin and HSA were obtained according to Modified Stern-Volmer equation. The calculated thermodynamic parameters indicated that the binding of puerarin to HSA was driven mainly by hydrophobic interaction. The competitive experiments of site markers suggested that the binding site of puerarin to HSA was located in the region of subdomain IIA (sudlow site I). Further, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way synchronous fluorescence spectra data of the competitive interaction between puerarin and warfarin with HSA. The concentration information for the three reaction components, warfarin, puerarin and puerarin-HSA, in the system at equilibrium was obtained simultaneously. The PARAFAC analysis indicated that puerarin in the puerarin-HSA complex was displaced by warfarin, which confirmed the binding site of puerarin to HSA was located in site I. Moreover, the results of CD and FT-IR spectra demonstrated that the secondary structure of HSA was changed in the presence of puerarin. - Highlights: → Puerarin can quench the fluorescence of human serum albumin (HSA). → The HSA fluorescence is quenched by puerarin through a static quenching mechanism. → The binding of puerarin to HSA is driven mainly by hydrophobic interaction. → The parallel factor analysis confirms that puerarin is located in site I of HSA. → The binding of puerarin to HSA induces changes in the secondary structure of HSA.

A Pyridazine-Based Fluorescent Probe Targeting Aβ Plaques in Alzheimer’s Disease

Directory of Open Access Journals (Sweden)

Yong Dae Park

2018-01-01

Full Text Available Accumulation of β-amyloid (Aβ plaques comprising Aβ40 and Aβ42 in the brain is the most significant factor in the pathogenesis of Alzheimer’s disease (AD. Thus, the detection of Aβ plaques has increasingly attracted interest in the context of AD diagnosis. In the present study, a fluorescent pyridazine-based dye that can detect and image Aβ plaques was designed and synthesized, and its optical properties in the presence of Aβ aggregates were evaluated. An approximately 34-fold increase in emission intensity was exhibited by the fluorescent probe after binding with Aβ aggregates, for which it showed high affinity (KD = 0.35 µM. Moreover, the reasonable hydrophobic properties of the probe (log P = 2.94 allow it to penetrate the blood brain barrier (BBB. In addition, the pyridazine-based probe was used in the histological costaining of transgenic mouse (APP/PS1 brain sections to validate the selective binding of the probe to Aβ plaques. The results suggest that the pyridazine-based compound has the potential to serve as a fluorescent probe for the diagnosis of AD.

Toxicological evaluation of Cd-based fluorescent nanoprobes by means of in vivo studies

Science.gov (United States)

Farias, Patricia M. A.; Ma-Hock, Lan; Landsiedel, Robert; van Ravenzwaay, Bennard

2018-02-01

Cadmium still represents a stigma for many research- and/or industrial applications. Some deleterious effects are attributed to Cadmium. In the present work, highly fluorescent Cadmium sulfide quantum dots are investigated by e.g. physical-chemical characterization. Most important however is their application as fluorescent probes for bio-imaging in living cells and tissues. This work presents their toxicological evaluation by means of in vivo studies. Bio-imaging experiments are performed without any pre-treatment. The toxicological studies performed, strongly indicate that the use of Cadmium based nanoparticles as fluorescent probes may be nonhazardous and not induce side effects for cells/tissues.

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

Science.gov (United States)

Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

2017-05-15

5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

Quality evaluation of the edible blue-green alga Nostoc flagelliforme using a chlorophyll fluorescence parameter and several biochemical markers.

Science.gov (United States)

Gao, Xiang; Yang, Yiwen; Ai, Yufeng; Luo, Hongyi; Qiu, Baosheng

2014-01-15

Nostoc flagelliforme is an edible blue-green alga with herbal and dietary values. Due to the diminishing supply of natural N. flagelliforme and the large investment on the development of its cultivation technology, it is anticipated that artificially cultured N. flagelliforme will soon sustain the market supply. Once this change occurs, the storage-associated quality problem will become the focus of attention for future trade. In this paper, we used a chlorophyll fluorescence parameter, maximum quantum efficiency of Photosystem II (Fv/Fm), and several biomarkers to evaluate the quality of several N. flagelliforme samples. It was found that longer storage times resulted in darker coloured solutions (released pigments) and decreased amounts of chlorophyll a (Chl a) and water-soluble sugars (WSS). Additionally, a higher Fv/Fm value suggests better physiological recovery and quality. In actual application, determination of Fv/Fm would be the first step for evaluating the quality of N. flagelliforme, and the biochemical indexes would serve as good secondary markers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Analysis of Cell Movement by Simultaneous Quantification of Local Membrane Displacement and Fluorescent Intensities Using Quimp2

NARCIS (Netherlands)

Bosgraaf, Leonard; van Haastert, Peter J. M.; Bretschneider, Till

The use of fluorescent markers in living cells has increased dramatically in the recent years. The quantitative analysis of the images requires specific analysis software. Previously, the program Quimp was launched for quantitating fluorescent intensities at the membrane or the cortex of the cell.

Basic dose response of fluorescent screen-based portal imaging device

International Nuclear Information System (INIS)

Yeo, In Hwan; Yonannes, Yonas; Zhu, Yunping

1999-01-01

The purpose of this study is to investigate fundamental aspects of the dose response of fluorescent screen-based electronic portal imaging devices (EPIDs). We acquired scanned signal across portal planes as we varied the radiation that entered the EPID by changing the thickness and anatomy of the phantom as well as the air gap between the phantom and the EPID. In addition, we simulated the relative contribution of the scintillation light signal in the EPID system. We have shown that the dose profile across portal planes is a function of the air gap and phantom thickness. We have also found that depending on the density change within the phantom geometry, errors associated with dose response based on the EPID scan can be as high as 7%. We also found that scintillation light scattering within the EPID system is an important source of error. This study revealed and demonstrated fundamental characteristics of dose response of EPID, as relative to that of ion chambers. This study showed that EPID based on fluorescent screen cannot be an accurate dosimetry system

Fluorescence intensity and lifetime-based cyanide sensitive probes for physiological safeguard

International Nuclear Information System (INIS)

Badugu, Ramachandram; Lakowicz, Joseph R.; Geddes, Chris D.

2004-01-01

We characterize six new fluorescent probes that show both intensity and lifetime changes in the presence of free uncomplexed aqueous cyanide, allowing for fluorescence based cyanide sensing up to physiological safeguard levels, i.e. 2 to the anionic R-B - (CN) 3 form, a new cyanide binding mechanism which we have recently reported. The presence of an electron deficient quaternary heterocyclic nitrogen nucleus, and the electron rich cyanide bound form, provides for the intensity changes observed. We have determined the disassociation constants of the probes to be in the range ∼15-84 μM 3 . In addition we have synthesized control compounds which do not contain the boronic acid moiety, allowing for a rationale of the cyanide responses between the probe isomers to be made. The lifetime of the cyanide bound probes are significantly shorter than the free R-B(OH) 2 probe forms, providing for the opportunity of lifetime based cyanide sensing up to physiologically lethal levels. Finally, while fluorescent probes containing the boronic acid moiety have earned a well-deserved reputation for monosaccharide sensing, we show that strong bases such as CN - and OH - preferentially bind as compared to glucose, enabling the potential use of these probes for cyanide safeguard and determination in physiological fluids, especially given that physiologies do not experience any notable changes in pH

Internal validation of two new retrotransposons-based kits (InnoQuant® HY and InnoTyper® 21) at a forensic lab.

Science.gov (United States)

Martins, Cátia; Ferreira, Paulo Miguel; Carvalho, Raquel; Costa, Sandra Cristina; Farinha, Carlos; Azevedo, Luísa; Amorim, António; Oliveira, Manuela

2018-02-01

Obtaining a genetic profile from pieces of evidence collected at a crime scene is the primary objective of forensic laboratories. New procedures, methods, kits, software or equipment must be carefully evaluated and validated before its implementation. The constant development of new methodologies for DNA testing leads to a steady process of validation, which consists of demonstrating that the technology is robust, reproducible, and reliable throughout a defined range of conditions. The present work aims to internally validate two new retrotransposon-based kits (InnoQuant ® HY and InnoTyper ® 21), under the working conditions of the Laboratório de Polícia Científica da Polícia Judiciária (LPC-PJ). For the internal validation of InnoQuant ® HY and InnoTyper ® 21 sensitivity, repeatability, reproducibility, and mixture tests and a concordance study between these new kits and those currently in use at LPC-PJ (Quantifiler ® Duo and GlobalFiler™) were performed. The results obtained for sensitivity, repeatability, and reproducibility tests demonstrated that both InnoQuant ® HY and InnoTyper ® 21 are robust, reproducible, and reliable. The results of the concordance studies demonstrate that InnoQuant ® HY produced quantification results in nearly 29% more than Quantifiler ® Duo (indicating that this new kit is more effective in challenging samples), while the differences observed between InnoTyper ® 21 and GlobalFiler™ are not significant. Therefore, the utility of InnoTyper ® 21 has been proven, especially by the successful amplification of a greater number of complete genetic profiles (27 vs. 21). The results herein presented allowed the internal validation of both InnoQuant ® HY and InnoTyper ® 21, and their implementation in the LPC-PJ laboratory routine for the treatment of challenging samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescent Endoscopy of Tumors in Upper Part of Gastrointestinal Tract

Science.gov (United States)

Borisova, E.; Vladimirov, B.; Angelov, I.; Avramov, L.

2007-04-01

In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus and stomach. The 5-ALA is administered per os six hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built for LED to use the light guide of standard video-endoscopic system (Olimpus Corp.). Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer (USB4000, OceanOptics Inc.). Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

Incorporation of conventional genetic markers and RAPD markers into an RFLP based map in maize

International Nuclear Information System (INIS)

Coe, E.H. Jr.; McMullen, M.D.; Polacco, M.; Davis, G.L.; Chao, S.

1998-01-01

Integration of classical genetic markers, in particular mutants, onto the maize Restriction Fragment Length Polymorphism (RFLP) map will provide the tools necessary to further our understanding of plant development and of complex traits. Initially integration was accomplished by visual alignment of common markers and sometimes involved the use of information from several different molecular maps to determine the relative placement of a single mutant. The maize core marker set was designed to provide a common set of markers which could be used for integration of map data. We have completed the mapping, of 56 mutants on chromosome one relative to the core marker set. Phenotypes included whole plant, seedling, and kernel effects and represented a variety of biological processes. Since these mutants were previously located to chromosome arm, mapping required the use of only seven markers per mutant to define the correct bin location. Two mistakes in marker order relative to the classical map were identified, as well as, six groups of mutants which require allelism testing. Placement of mutants and cDNAs into bins using, the core markers provides a necessary resource for identification of gene function in maize. (author)

Synthesis and validation of novel cholesterol-based fluorescent lipids designed to observe the cellular trafficking of cationic liposomes.

Science.gov (United States)

Kim, Bieong-Kil; Seu, Young-Bae; Choi, Jong-Soo; Park, Jong-Won; Doh, Kyung-Oh

2015-09-15

Cholesterol-based fluorescent lipids with ether linker were synthesized using NBD (Chol-E-NBD) or Rhodamine B (Chol-E-Rh), and the usefulnesses as fluorescent probes for tracing cholesterol-based liposomes were validated. The fluorescent intensities of liposomes containing these modified lipids were measured and observed under a microscope. Neither compound interfered with the expression of GFP plasmid, and live cell images were obtained without interferences. Changes in the fluorescent intensity of liposomes containing Chol-E-NBD were followed by flow cytometry for up to 24h. These fluorescent lipids could be useful probes for trafficking of cationic liposome-mediated gene delivery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Exudation of fluorescent beta-carbolines from Oxalis tuberosa L roots.

Science.gov (United States)

Bais, Harsh Pal; Park, Sang-Wook; Stermitz, Frank R; Halligan, Kathleen M; Vivanco, Jorge M

2002-11-01

Root fluorescence is a phenomenon in which roots of seedlings fluoresce when irradiated with ultraviolet (UV) light. Soybean (Glycine max) and rye grass (Elymus glaucus) are the only plant species that have been reported to exhibit this occurrence in germinating seedling roots. The trait has been useful as a marker in genetic, tissue culture and diversity studies, and has facilitated selection of plants for breeding purposes. However, the biological significance of this occurrence in plants and other organisms is unknown. Here we report that the Andean tuber crop species Oxalis tuberosa, known as oca in the highlands of South America, secretes a fluorescent compound as part of its root exudates. The main fluorescent compounds were characterized as harmine (7-methoxy-1-methyl-beta-carboline) and harmaline (3, 4-dihydroharmine). We also detected endogenous root fluorescence in other plant species, including Arabidopsis thaliana and Phytolacca americana, a possible indication that this phenomenon is widespread within the plant kingdom.

Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

Science.gov (United States)

Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

2002-08-01

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

Searching for an Accurate Marker-Based Prediction of an Individual Quantitative Trait in Molecular Plant Breeding.

Science.gov (United States)

Fu, Yong-Bi; Yang, Mo-Hua; Zeng, Fangqin; Biligetu, Bill

2017-01-01

Molecular plant breeding with the aid of molecular markers has played an important role in modern plant breeding over the last two decades. Many marker-based predictions for quantitative traits have been made to enhance parental selection, but the trait prediction accuracy remains generally low, even with the aid of dense, genome-wide SNP markers. To search for more accurate trait-specific prediction with informative SNP markers, we conducted a literature review on the prediction issues in molecular plant breeding and on the applicability of an RNA-Seq technique for developing function-associated specific trait (FAST) SNP markers. To understand whether and how FAST SNP markers could enhance trait prediction, we also performed a theoretical reasoning on the effectiveness of these markers in a trait-specific prediction, and verified the reasoning through computer simulation. To the end, the search yielded an alternative to regular genomic selection with FAST SNP markers that could be explored to achieve more accurate trait-specific prediction. Continuous search for better alternatives is encouraged to enhance marker-based predictions for an individual quantitative trait in molecular plant breeding.

Intrinsic fluorescence for cervical precancer detection using polarized light based in-house fabricated portable device

Science.gov (United States)

Meena, Bharat Lal; Singh, Pankaj; Sah, Amar Nath; Pandey, Kiran; Agarwal, Asha; Pantola, Chayanika; Pradhan, Asima

2018-01-01

An in-house fabricated portable device has been tested to detect cervical precancer through the intrinsic fluorescence from human cervix of the whole uterus in a clinical setting. A previously validated technique based on simultaneously acquired polarized fluorescence and polarized elastic scattering spectra from a turbid medium is used to extract the intrinsic fluorescence. Using a diode laser at 405 nm, intrinsic fluorescence of flavin adenine dinucleotide, which is the dominant fluorophore and other contributing fluorophores in the epithelium of cervical tissue, has been extracted. Different grades of cervical precancer (cervical intraepithelial neoplasia; CIN) have been discriminated using principal component analysis-based Mahalanobis distance and linear discriminant analysis. Normal, CIN I and CIN II samples have been discriminated from one another with high sensitivity and specificity at 95% confidence level. This ex vivo study with cervix of whole uterus samples immediately after hysterectomy in a clinical environment indicates that the in-house fabricated portable device has the potential to be used as a screening tool for in vivo precancer detection using intrinsic fluorescence.

A Quantitative Fluorescence-Based Lipase Assay

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Giovanna Lomolino

2012-01-01

Full Text Available An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.

Fluorescence turn-on detection of target sequence DNA based on silicon nanodot-mediated quenching.

Science.gov (United States)

Zhang, Yanan; Ning, Xinping; Mao, Guobin; Ji, Xinghu; He, Zhike

2018-05-01

We have developed a new enzyme-free method for target sequence DNA detection based on the dynamic quenching of fluorescent silicon nanodots (SiNDs) toward Cy5-tagged DNA probe. Fascinatingly, the water-soluble SiNDs can quench the fluorescence of cyanine (Cy5) in Cy5-tagged DNA probe in homogeneous solution, and the fluorescence of Cy5-tagged DNA probe can be restored in the presence of target sequence DNA (the synthetic target miRNA-27a). Based on this phenomenon, a SiND-featured fluorescent sensor has been constructed for "turn-on" detection of the synthetic target miRNA-27a for the first time. This newly developed approach possesses the merits of low cost, simple design, and convenient operation since no enzymatic reaction, toxic reagents, or separation procedures are involved. The established method achieves a detection limit of 0.16 nM, and the relative standard deviation of this method is 9% (1 nM, n = 5). The linear range is 0.5-20 nM, and the recoveries in spiked human fluids are in the range of 90-122%. This protocol provides a new tactic in the development of the nonenzymic miRNA biosensors and opens a promising avenue for early diagnosis of miRNA-associated disease. Graphical abstract The SiND-based fluorescent sensor for detection of S-miR-27a.

An easily Prepared Fluorescent pH Probe Based on Dansyl.

Science.gov (United States)

Sha, Chunming; Chen, Yuhua; Chen, Yufen; Xu, Dongmei

2016-09-01

A novel fluorescent pH probe from dansyl chloride and thiosemicarbazide was easily prepared and fully characterized by (1)H NMR, (13)C NMR, LC-MS, Infrared spectra and elemental analysis. The probe exhibited high selectivity and sensitivity to H(+) with a pK a value of 4.98. The fluorescence intensity at 510 nm quenched 99.5 % when the pH dropped from 10.88 to 1.98. In addition, the dansyl-based probe could respond quickly and reversibly to the pH variation and various common metal ions showed negligible interference. The recognition could be ascribed to the intramolecular charge transfer caused by the protonation of the nitrogen in the dimethylamino group.

Fluorescence based fiber optic and planar waveguide biosensors. A review

International Nuclear Information System (INIS)

Benito-Peña, Elena; Valdés, Mayra Granda; Glahn-Martínez, Bettina; Moreno-Bondi, Maria C.

2016-01-01

The application of optical biosensors, specifically those that use optical fibers and planar waveguides, has escalated throughout the years in many fields, including environmental analysis, food safety and clinical diagnosis. Fluorescence is, without doubt, the most popular transducer signal used in these devices because of its higher selectivity and sensitivity, but most of all due to its wide versatility. This paper focuses on the working principles and configurations of fluorescence-based fiber optic and planar waveguide biosensors and will review biological recognition elements, sensing schemes, as well as some major and recent applications, published in the last ten years. The main goal is to provide the reader a general overview of a field that requires the joint collaboration of researchers of many different areas, including chemistry, physics, biology, engineering, and material science. - Highlights: • Principles, configurations and fluorescence techniques using fiber optic and planar waveguide biosensors are discussed. • The biorecognition elements and sensing schemes used in fiber optic and planar waveguide platforms are reviewed. • Some major and recent applications of fiber optic and planar waveguide biosensors are introduced.

Fluorescence based fiber optic and planar waveguide biosensors. A review

Energy Technology Data Exchange (ETDEWEB)

Benito-Peña, Elena [Department of Analytical Chemistry, Faculty of Chemistry, Complutense University, 28040 Madrid (Spain); Valdés, Mayra Granda [Department of Analytical Chemistry, Faculty of Chemistry, University of La Habana, 10400 La Habana (Cuba); Glahn-Martínez, Bettina [Department of Analytical Chemistry, Faculty of Chemistry, Complutense University, 28040 Madrid (Spain); Moreno-Bondi, Maria C., E-mail: mcmbondi@quim.ucm.es [Department of Analytical Chemistry, Faculty of Chemistry, Complutense University, 28040 Madrid (Spain)

2016-11-02

The application of optical biosensors, specifically those that use optical fibers and planar waveguides, has escalated throughout the years in many fields, including environmental analysis, food safety and clinical diagnosis. Fluorescence is, without doubt, the most popular transducer signal used in these devices because of its higher selectivity and sensitivity, but most of all due to its wide versatility. This paper focuses on the working principles and configurations of fluorescence-based fiber optic and planar waveguide biosensors and will review biological recognition elements, sensing schemes, as well as some major and recent applications, published in the last ten years. The main goal is to provide the reader a general overview of a field that requires the joint collaboration of researchers of many different areas, including chemistry, physics, biology, engineering, and material science. - Highlights: • Principles, configurations and fluorescence techniques using fiber optic and planar waveguide biosensors are discussed. • The biorecognition elements and sensing schemes used in fiber optic and planar waveguide platforms are reviewed. • Some major and recent applications of fiber optic and planar waveguide biosensors are introduced.

Surface recognition and fluorescence sensing of histone by dansyl-appended cyclophane-based resorcinarene trimer.

Science.gov (United States)

Hayashida, Osamu; Ogawa, Naoyuki; Uchiyama, Masaki

2007-11-07

A cyclophane-based resorcinarene trimer (3) bearing a dansyl moiety as an environmentally sensitive fluorophore was prepared by stepwise condensation of a tetraaza[6.1.6.1]paracyclophane skeleton with a dansyl moiety and three resorcinarene derivatives having heptacarboxylic acid residues in this sequence. The dansyl-appended cyclophane exhibited the following fluorescence properties regarding solvent polarity dependency and histone surface recognition: With increasing dioxane contents in dioxane/water solvents, the fluorescence intensity originating from the dansyl moiety of 3 increased along with a concomitant blue shift of the fluorescence maximum (lambdaem). The microenvironmentally sensitive fluorescence properties of dansyl fluorophore were maintained, even when the dansyl moiety was covalently attached to a cyclophane. Most interestingly, the cyclophane-based resorcinarene trimer exhibited recognition and fluorescence sensing capabilities toward histone, a small basic protein of eukaryotic chromatins. The fluorescence intensity originating from 3 increased along with a concomitant blue shift of lambdaem upon the addition of histone, reflecting the formation of 3-histone complexes. A relatively large fluorescence polarization (P) value was obtained for the 3-histone complexes (0.15), reflecting highly restricted conformations of 3, and the obtained P value was much larger than that of 3 alone in aqueous medium (0.07). The binding constant (K) of 3 with histone (unit basis) was estimated to be 2.1 x 106 M-1. On the other hand, upon the addition of acetylated histone (Ac-histone) to an aqueous solution containing 3, the extent of change in fluorescence intensity originating from the dansyl group of 3 was almost negligible, indicating that the electrostatic interactions between 3 and Ac-histone were weak. In addition, the fluorescence spectral changes were also small or negligible upon the addition of other proteins such as albumin, ovalbumin, peanut agglutinin

Biomolecule-to-fluorescent-color encoder: modulation of fluorescence emission via DNA structural changes

Science.gov (United States)

Nishimura, Takahiro; Ogura, Yusuke; Yamada, Kenji; Ohno, Yuko; Tanida, Jun

2014-01-01

A biomolecule-to-fluorescent-color (B/F) encoder for optical readout of biomolecular information is proposed. In the B/F encoder, a set of fluorescence wavelengths and their intensity levels are used for coding of a biomolecular signal. A hybridization chain reaction of hairpin DNAs labeled with fluorescent reporters was performed to generate the fluorescence color codes. The fluorescence is modulated via fluorescence resonance energy transfer, which is controlled by DNA structural changes. The results demonstrate that fluorescent color codes can be configured based on two wavelengths and five intensities using the B/F encoder, and the assigned codes can be retrieved via fluorescence measurements. PMID:25071950

A fluorescence resonance energy transfer-based method for histone methyltransferases

DEFF Research Database (Denmark)

Devkota, Kanchan; Lohse, Brian; Nyby Jakobsen, Camilla

2015-01-01

A simple dye–quencher fluorescence resonance energy transfer (FRET)-based assay for methyltransferases was developed and used to determine kinetic parameters and inhibitory activity at EHMT1 and EHMT2. Peptides mimicking the truncated histone H3 tail were functionalized in each end with a dye...

Novel Insight for Organic Matter Sourcing: Interest of Time Resolved Fluorescence to Qualify and Quantify PAH Content of Solid Matrix at High Resolution

Science.gov (United States)

Quiers, M.; Perrette, Y.; Jacq, K.; Pousset, E.; Plassart, G.

2017-12-01

OM fluorescence is today a well-developed tool used to characterize and quantify organic matter (OM), but also to evaluate and discriminate OM fate and changes related to climate and environmental modifications. While fluorescence measurements on water and soils extracts provide information about organic fluxes today, solid phase fluorescence using natural archives allows to obtain high resolution records of OM evolution during time. These evolutions can be discussed in regards of climate and environmental perturbations detected in archives using different proxies, and thus provide keys for understanding factors driving carbon fluxes mechanisms. Among fluorescent organic species, Polycyclic Aromatic Hydrocarbons (PAH) have been used as probe molecules for organic contamination tracking. Moreover, monitoring studies have shown that PAH could also be used as markers to discriminates atmospheric and erosion factors leading to PAH and organic matter fluxes to the aquifer. PAH records in soils and natural archives appear as a promising proxy to follow both past atmospheric contamination and soil erosion. But, PAH fluorescence is difficult to discriminate from bulk OM fluorescence using steady-state fluorescence (SSF) technics as their fluorescence domains recover. Time resolved emission spectroscopy (TRES) increases the information provided by SSF technic, adding a time dimension to measurements and allowing to discriminate PAH fluorescence. We report here a first application of this technic on natural archives. The challenge is to obtain TRES signature along the sample, including for low PAH concentrations. This study aims to evaluate the reliability of high resolution TRES measurement as PAH carbon fluxes sources. Method is based on LIF instrument for solid phase fluorescence measurement. An instrument coupling an excitation system constituting by 2 pulsed lasers (266 and 355 nm) and a detection system was developed. This measurement provides high resolution record of

Molecular performance of commercial MTG variety oil palm based on RAPD markers

Science.gov (United States)

Putri, L. A. P.; Setyo, I. E.; Basyuni, M.; Bayu, E. S.; Setiado, H.; Reynaldi, N. F.; Laia, H.; Puteri, S. A. K.; Arifiyanto, D.; Syahputra, I.

2018-02-01

The oil palm, an economically important tree in Indonesia, has been one of the world’s major sources of edible oil and a significant precursor of biodiesel fuel. This research is conducted by taking individual tree sample of commercial MTG variety germplasm oil palm one years old. The purpose of this research is to analyse molecular performance of some oil palm MTG variety based on RAPD markers. In this experiment, the DNA profile diversity was assessed using markers of oil palm’s random RAPD markers (OPD-20, SB-19, OPM-01 and OPO-11). A total of 15 trees commercial MTG oil palm variety were used for analysis. The results of the experiment indicated out of 4 RAPD markers (OPD-20, SB-19, OPM-01 and OPO-11) showed polymorphic of PCR product. These preliminary results demonstrated RAPD marker can be used to evaluate genetic relatedness among trees of commercial MTG variety oil palm and detecting either genetic variants or mislabelled.

An amplified graphene oxide-based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling for bioassays.

Science.gov (United States)

Hu, Kun; Liu, Jinwen; Chen, Jia; Huang, Yong; Zhao, Shulin; Tian, Jianniao; Zhang, Guohai

2013-04-15

An amplified graphene oxide (GO) based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling is developed for bioassays. The dye-labeled single-strand DNA (aptamer hairpin) was adsorbed on the surface of GO, which result in the fluorescence quenching of dye, and exhibiting minimal background fluorescence. Upon the target, primer and polymerase, the stem of the aptamer hairpin was opened, and binds with the primer to triggers the circular target strand-displacement polymerization reaction, which produces huge amounts of duplex helixes DNA and lead to strong fluorescence emission due to shielding of nucelobases within its double-helix structure. During the polymerization reaction, the primer was extended, and target was displaced. And the displaced target recognizes and hybridizes with another hairpin probe, triggering the next round of polymerization reaction, and the circle process induces fluorescence signal amplification for the detection of analyte. To test the feasibility of the aptasensor systems, interferon-gamma (IFN-γ) was employed as a model analyte. A detection limit as low as 1.5 fM is obtained based on the GO aptasensor with a linear range of three orders of magnitude. The present method was successfully applied for the detection of IFN-γ in human plasma. Copyright © 2012 Elsevier B.V. All rights reserved.

A vector carrying the GFP gene (Green fluorescent protein as a yeast marker for fermentation processes Um vetor com o gene da GFP (Green fluorescent protein para a marcação de leveduras em processos fermentativos

Directory of Open Access Journals (Sweden)

Luiz Humberto Gomes

2000-12-01

Full Text Available Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3. Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.Leveduras contaminantes podem causar grandes perdas em processos fermentativos quando infectam culturas puras e degradam a qualidade do produto final. Estas leveduras podem ser detectadas por diversos métodos mas nenhum deles oferece resultados com a exatidão e precisão necessárias, quando os contaminantes estão em baixa freqüência. Culturas puras contendo um gene marcador podem ser utilizadas para a direta identificação de células e colônias contaminantes. Detecção rápida e fácil é desejada e marcadores morfológicos podem auxiliar na visualização da cultura marcada. O gene da GFP (green fluorescent protein extraído da Aequorea victoria

A dansyl-rhodamine ratiometric fluorescent probe for Hg2+ based on FRET mechanism.

Science.gov (United States)

Xie, Puhui; Guo, Fengqi; Wang, Lingyu; Yang, Sen; Yao, Denghui; Yang, Guoyu

2015-03-01

Based on resonance energy transfer (FRET) from dansyl to rhodamine 101, a new fluorescent probe (compound 1) containing rhodamine 101 and a dansyl unit was synthesized for detecting Hg(2+) through ratiometric sensing in DMSO aqueous solutions. This probe shows a fast, reversible and selective response toward Hg(2+) in a wide pH range. Hg(2+) induced ring-opening reactions of the spirolactam rhodamine moiety of 1, leading to the formation of fluorescent derivatives that can serve as the FRET acceptors. Very large stokes shift (220 nm) was observed in this case. About 97-fold increase in fluorescence intensity ratio was observed upon its binding with Hg(2+).

Compound parabolic concentrator optical fiber tip for FRET-based fluorescent sensors

DEFF Research Database (Denmark)

Hassan, Hafeez Ul; Nielsen, Kristian; Aasmul, Soren

2015-01-01

The Compound Parabolic Concentrator (CPC) optical fiber tip shape has been proposed for intensity based fluorescent sensors working on the principle of FRET (Förster Resonance Energy Transfer). A simple numerical Zemax model has been used to optimize the CPC tip geometry for a step-index multimode...... polymer optical fiber for an excitation and emission wavelength of 550 nm and 650nm, respectively. The model suggests an increase of a factor of 1.6 to 4 in the collected fluorescent power for an ideal CPC tip, as compared to the plane-cut fiber tip for fiber lengths between 5 and 45mm...

Size effects in the quantum yield of Cd Te quantum dots for optimum fluorescence bioimaging

International Nuclear Information System (INIS)

Jacinto, C.; Rocha, U.S.; Maestro, L.M.; Garcia-Sole, J.; Jaque, D.

2011-01-01

Full text: Semiconductor nano-crystals, usually referred as Quantum Dots (QDs) are nowadays regarded as one of the building-blocks in modern photonics. They constitute bright and photostable fluorescence sources whose emission and absorption properties can be adequately tailored through their size. Recent advances on the controlled modification of their surface has made possible the development of water soluble QDs, without causing any deterioration in their fluorescence properties. This has made them excellent optical selective markers to be used in fluorescence bio-imaging experiments. The suitability of colloidal QDs for bio-imaging is pushed forward by their large two-photon absorption cross section so that their visible luminescence (associated to the recombination of electro-hole pairs) can be also efficiently excited under infrared excitation (two-photon excitation). This, in turns, allows for large penetration depths in tissues, minimization of auto-fluorescence and achievement of superior spatial imaging resolution. In addition, recent works have demonstrated the ability of QDs to act as nano-thermometers based on the thermal sensitivity of their fluorescence bands. Based on all these outstanding properties, QDs have been successfully used to mark individual receptors in cell membranes, to intracellular temperature measurements and to label living embryos at different stages. Most of the QD based bio-images reported up to now were obtained by using whether CdSe or CdTe QDs since both are currently commercial available with a high degree of quality. They show similar fluorescence properties and optical performance when used in bio-imaging. Nevertheless, CdTe-QDs have very recently attracted much attention since the hyper-thermal sensitivity of their fluorescence bands was discovered. Based on this, it has been postulated that intracellular thermal sensing with resolutions as large as 0.25 deg C can be achieved based on CdTe-QDs, three times better than

Synthesis and application of a highly selective copper ions fluorescent probe based on the coumarin group

Science.gov (United States)

He, Guangjie; Liu, Xiangli; Xu, Jinhe; Ji, Liguo; Yang, Linlin; Fan, Aiying; Wang, Songjun; Wang, Qingzhi

2018-02-01

A highly selective copper ions fluorescent probe based on the coumarin-type Schiff base derivative 1 (probe) was produced by condensation reaction between coumarin carbohydrazide and 1H-indazole-3-carbaldehyde. The UV-vis spectroscopy showed that the maximum absorption peak of compound 1 appeared at 439 nm. In the presence of Cu2 + ions, the maximum peak decreased remarkably compared with other physiological important metal ions and a new absorption peak at 500 nm appeared. The job's plot experiments showed that complexes of 1:2 binding mode were formed in CH3CN:HEPES (3:2, v/v) solution. Compound 1 exhibited a strong blue fluorescence. Upon addition of copper ions, the fluorescence gradually decreased and reached a plateau with the fluorescence quenching rate up to 98.73%. The detection limit for Cu2 + ions was estimated to 0.384 ppm. Fluorescent microscopy experiments demonstrated that probe 1 had potential to be used to investigate biological processes involving Cu2 + ions within living cells.

Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex

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Pilar Santolaria

2016-01-01

Full Text Available This study was designed to determine the ability of computer-assisted sperm morphometry analysis (CASA-Morph with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa (SX and SY, respectively. Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average (P < 0.001 although with important differences between bulls. A simultaneous evaluation of all the measured features by discriminant analysis revealed that nuclear area and average fluorescence intensity were the variables selected by stepwise discriminant function analysis as the best discriminators between SX and SY. In the second experiment, the sperm nuclear morphometric results from CASA-Morph in nonsexed (mixed SX and SY and sexed (SX semen samples from four bulls were compared. FISH allowed a successful classification of spermatozoa according to their sex chromosome content. X-sexed spermatozoa displayed a larger size and fluorescence intensity than nonsexed spermatozoa (P < 0.05. We conclude that the CASA-Morph fluorescence-based method has the potential to find differences between X- and Y-chromosome-bearing spermatozoa in bovine species although more studies are needed to increase the precision of sex determination by this technique.

[Molecular beacon based PNA-FISH method combined with fluorescence scanning for rapid detection of Listeria monocytogenes].

Science.gov (United States)

Wu, Shan; Zhang, Xiaofeng; Shuai, Jiangbing; Li, Ke; Yu, Huizhen; Jin, Chenchen

2016-07-04

To simplify the PNA-FISH (Peptide nucleic acid-fluorescence in situ hybridization) test, molecular beacon based PNA probe combined with fluorescence scanning detection technology was applied to replace the original microscope observation to detect Listeria monocytogenes The 5′ end and 3′ end of the L. monocytogenes specific PNA probes were labeled with the fluorescent group and the quenching group respectively, to form a molecular beacon based PNA probe. When PNA probe used for fluorescence scanning and N1 treatment as the control, the false positive rate was 11.4%, and the false negative rate was 0; when N2 treatment as the control, the false positive rate decreased to 4.3%, but the false negative rate rose to 18.6%. When beacon based PNA probe used for fluorescence scanning, taken N1 treatment as blank control, the false positive rate was 8.6%, and the false negative rate was 1.4%; taken N2 treatment as blank control, the false positive rate was 5.7%, and the false negative rate was 1.4%. Compared with PNA probe, molecular beacon based PNA probe can effectively reduce false positives and false negatives. The success rates of hybridization of the two PNA probes were 83.3% and 95.2% respectively; and the rates of the two beacon based PNA probes were 91.7% and 90.5% respectively, which indicated that labeling the both ends of the PNA probe dose not decrease the hybridization rate with the target bacteria. The combination of liquid phase PNA-FISH and fluorescence scanning method, can significantly improve the detection efficiency.

Cellular imaging using biocompatible dendrimer-functionalized graphene oxide-based fluorescent probe anchored with magnetic nanoparticles

International Nuclear Information System (INIS)

Wate, Prateek S; Banerjee, Shashwat S; Mascarenhas, Russel R; Zope, Khushbu R; Khandare, Jayant; Jalota-Badhwar, Archana; Misra, R Devesh K

2012-01-01

We describe a novel multicomponent graphene nanostructured system that is biocompatible, and has strong NIR optical absorbance and superparamagnetic properties. The fabrication of the multicomponent nanostructure system involves the covalent attachment of 3 components; Fe 3 O 4 (Fe) nanoparticles, PAMAM-G4-NH 2 (G4) dendrimer and Cy5 (Cy) on a graphene oxide (GO) surface to synthesize a biologically relevant multifunctional system. The resultant GO-G4-Fe-Cy nanosystem exhibits high dispersion in an aqueous medium, and is magnetically responsive and fluorescent. In vitro experiments provide a clear indication of successful uptake of the GO-G4-Fe-Cy nanosystem by MCF-7 breast cancer cells, and it is seen to behave as a bright and stable fluorescent marker. The study also reveals varied cellular distribution kinetics profile for the GO nanostructured system compared to free Cy. Furthermore, the newly developed GO nanostructured system is observed to be non-toxic to MDA-MB-231 cell growth, in striking contrast to free G4 dendrimer and GO-G4 conjugate. The GO-G4-Fe-Cy nanostructured system characterized by multifunctionality suggests the merits of graphene for cellular bioimaging and the delivery of bioactives. (paper)

Evaluation of Stem Cell Markers, CD44/CD24 in Breast Cancer Cell Lines

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Masoud Hashemi Arabi

2014-05-01

Four breast cancer cell lines, MCF-7 ، T47D ، MDA-MB231 and MDA-MB468 were purchased from National cell Bank of Iran based in Iran Pasture Institute and were cultured in high glucose DMEM supplemented with 10% FCS. Cells were stained with antiCD44-PE and antiCD24-FITC antibodies and Status of CD44 and CD24 as markers of breast cancer stem cells were evaluated using flow cytometer and fluorescent microscopy.Evaluation of CD44 and CD24 as markers of breast cancer stem cells showed that MDA-MB231 with 97±1.2% CD44+/CD24-/low cells is significantly different from the others that they were mainly CD44 and CD24 positive cells(p

On the use of EPID-based implanted marker tracking for 4D radiotherapy

International Nuclear Information System (INIS)

Keall, P.J.; Todor, A.D.; Vedam, S.S.; Bartee, C.L.; Siebers, J.V.; Kini, V.R.; Mohan, R.

2004-01-01

Four-dimensional (4D) radiotherapy delivery to dynamically moving tumors requires a real-time signal of the tumor position as a function of time so that the radiation beam can continuously track the tumor during the respiration cycle. The aim of this study was to develop and evaluate an electronic portal imaging device (EPID)-based marker-tracking system that can be used for real-time tumor targeting, or 4D radiotherapy. Three gold cylinders, 3 mm in length and 1 mm in diameter, were implanted in a dynamic lung phantom. The phantom range of motion was 4 cm with a 3-s 'breathing' period. EPID image acquisition parameters were modified, allowing image acquisition in 0.1 s. Images of the stationary and moving phantom were acquired. Software was developed to segment automatically the marker positions from the EPID images. Images acquired in 0.1 s displayed higher noise and a lower signal-noise ratio than those obtained using regular (>1 s) acquisition settings. However, the markers were still clearly visible on the 0.1-s images. The motion of the phantom blurred the images of the markers and further reduced the signal-noise ratio, though they could still be successfully segmented from the images in 10-30 ms of computation time. The positions of gold markers placed in the lung phantom were detected successfully, even for phantom velocities substantially higher than those observed for typical lung tumors. This study shows that using EPID-based marker tracking for 4D radiotherapy is feasible, however, changes in linear accelerator technology and EPID-based image acquisition as well as patient studies are required before this method can be implemented clinically

Triphenylamine based benzimidazole and benzothiazole: Synthesis and applications in fluorescent chemosensors and laser dyes

Energy Technology Data Exchange (ETDEWEB)

Yang, Yang [State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033 (China); University of Chinese Academy of Sciences, Beijing 100039 (China); Li, Bin, E-mail: libinteacher@163.com [State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033 (China); Zhang, Liming; Guan, Yunlong [State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033 (China)

2014-01-15

Triphenylamine based fluorescent dyes TPA-benzimidazole and TPA-benzothiazole have been designed and synthesized. The TPA-benzimidazole chemosensor was tested for a number of metal ions and found to exhibit binding affinity for Fe{sup 3+} and Hg{sup 2+} in acetonitrile, and the fluorescence quenching was achieved through a PET process. The appearance of an isosbestic point in absorption titrations and Job's plot analysis supported 1:1 stoichiometries for Fe{sup 3+} and Hg{sup 2+} ions. Laser experiments showed that under transversal pumping with a Q-switched Nd:YAG (355 nm) laser in toluene, TPA-benzothiazole exhibits efficient and stable amplified spontaneous emissions (ASE) at 436 nm. -- Highlights: • Triphenylamine based fluorescent dyes TPA-benzimidazole and TPA-benzothiazole have been designed and synthesized. • The TPA-benzimidazole exhibits binding affinity for Fe{sup 3+} and Hg{sup 2+} in acetonitrile and the fluorescence quenching was achieved through a PET process. • Under transversal pumping at 355 nm in toluene, TPA-benzothiazole exhibits efficient and stable amplified spontaneous emissions (ASE) in 436 nm.

Triphenylamine based benzimidazole and benzothiazole: Synthesis and applications in fluorescent chemosensors and laser dyes

International Nuclear Information System (INIS)

Yang, Yang; Li, Bin; Zhang, Liming; Guan, Yunlong

2014-01-01

Triphenylamine based fluorescent dyes TPA-benzimidazole and TPA-benzothiazole have been designed and synthesized. The TPA-benzimidazole chemosensor was tested for a number of metal ions and found to exhibit binding affinity for Fe 3+ and Hg 2+ in acetonitrile, and the fluorescence quenching was achieved through a PET process. The appearance of an isosbestic point in absorption titrations and Job's plot analysis supported 1:1 stoichiometries for Fe 3+ and Hg 2+ ions. Laser experiments showed that under transversal pumping with a Q-switched Nd:YAG (355 nm) laser in toluene, TPA-benzothiazole exhibits efficient and stable amplified spontaneous emissions (ASE) at 436 nm. -- Highlights: • Triphenylamine based fluorescent dyes TPA-benzimidazole and TPA-benzothiazole have been designed and synthesized. • The TPA-benzimidazole exhibits binding affinity for Fe 3+ and Hg 2+ in acetonitrile and the fluorescence quenching was achieved through a PET process. • Under transversal pumping at 355 nm in toluene, TPA-benzothiazole exhibits efficient and stable amplified spontaneous emissions (ASE) in 436 nm

Salicylimine-Based Colorimetric and Fluorescent Chemosensor for Selective Detection of Cyanide in Aqueous Buffer

Energy Technology Data Exchange (ETDEWEB)

Noh, Jin Young; Hwang, In Hong; Kim, Hyun; Song, Eun Joo; Kim, Kyung Beom; Kim, Cheal [Seoul National Univ., Seoul (Korea, Republic of)

2013-07-15

A simple colorimetric and fluorescent anion sensor 1 based on salicylimine showed a high selectivity and sensitivity for detection of cyanide in aqueous solution. The receptor 1 showed high selectivity toward CN{sup -} ions in a 1:1 stoichiometric manner, which induces a fast color change from colorless to orange and a dramatic enhancement in fluorescence intensity selectively for cyanide anions over other anions. Such selectivity resulted from the nucleophilic addition of CN{sup -} to the carbon atom of an electron-deficient imine group. The sensitivity of the fluorescence-based assay (0.06 μM) is below the 1.9 μM suggested by the World Health Organization (WHO) as the maximum allowable cyanide concentration in drinking water, capable of being a practical system for the monitoring of CN. concentrations in aqueous samples.

Emergence of White Organic Light-Emitting Diodes Based on Thermally Activated Delayed Fluorescence

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Peng Xiao

2018-02-01

Full Text Available Recently, thermally activated delayed fluorescence (TADF organic light-emitting diodes (OLEDs have attracted both academic and industrial interest due to their extraordinary characteristics, such as high efficiency, low driving voltage, bright luminance, lower power consumption and potentially long lifetime. In this invited review, the fundamental concepts of TADF have been firstly introduced. Then, main approaches to realize WOLEDs based on TADF have been summarized. More specifically, the recent development of WOLEDs based on all TADF emitters, WOLEDs based on TADF and conventional fluorescence emitters, hybrid WOLEDs based on blue TADF and phosphorescence emitters and WOLEDs based on TADF exciplex host and phosphorescence dopants is highlighted. In particular, design strategies, device structures, working mechanisms and electroluminescent processes of the representative WOLEDs based on TADF are reviewed. Finally, challenges and opportunities for further enhancement of the performance of WOLEDs based on TADF are presented.

Fluorescently labelled multiplex lateral flow immunoassay based on cadmium-free quantum dots.

Science.gov (United States)

Beloglazova, Natalia V; Sobolev, Aleksander M; Tessier, Mickael D; Hens, Zeger; Goryacheva, Irina Yu; De Saeger, Sarah

2017-03-01

A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures. They consisted of core/shell InP/ZnS QDs enrobed in a silica shell that allowed the water solubility (QD@SiO 2 ). Then we applied the QD@SiO 2 as novel, silica shell-encapsulated fluorescent labels in immunoassays for rapid multiplexed screening. Two mycotoxins, zearalenone and deoxynivalenol, were simultaneously detected in maize and wheat, since the two QD@SiO 2 labelled conjugates emit at two different, individually detectable wavelengths. The cutoff values for the simultaneous determination were 50 and 500μgkg -1 for zearalenone and deoxynivalenol, respectively, in both maize and wheat. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the result. Copyright © 2017 Elsevier Inc. All rights reserved.

A PDMS-based cylindrical hybrid lens for enhanced fluorescence detection in microfluidic systems.

Science.gov (United States)

Lin, Bor-Shyh; Yang, Yu-Ching; Ho, Chong-Yi; Yang, Han-Yu; Wang, Hsiang-Yu

2014-02-13

Microfluidic systems based on fluorescence detection have been developed and applied for many biological and chemical applications. Because of the tiny amount of sample in the system; the induced fluorescence can be weak. Therefore, most microfluidic systems deploy multiple optical components or sophisticated equipment to enhance the efficiency of fluorescence detection. However, these strategies encounter common issues of complex manufacturing processes and high costs. In this study; a miniature, cylindrical and hybrid lens made of polydimethylsiloxane (PDMS) to improve the fluorescence detection in microfluidic systems is proposed. The hybrid lens integrates a laser focusing lens and a fluorescence collecting lens to achieve dual functions and simplify optical setup. Moreover, PDMS has advantages of low-cost and straightforward fabrication compared with conventional optical components. The performance of the proposed lens is first examined with two fluorescent dyes and the results show that the lens provides satisfactory enhancement for fluorescence detection of Rhodamine 6G and Nile Red. The overall increments in collected fluorescence signal and detection sensitivity are more than 220% of those without lens, and the detection limits of Rhodamine 6G and Nile red are lowered to 0.01 μg/mL and 0.05 μg/mL, respectively. The hybrid lens is further applied to the detection of Nile red-labeled Chlorella vulgaris cells and it increases both signal intensity and detection sensitivity by more than 520%. The proposed hybrid lens also dramatically reduces the variation in detected signal caused by the deviation in incident angle of excitation light.

Detection of DNA hybridization based on SnO2 nanomaterial enhanced fluorescence

International Nuclear Information System (INIS)

Gu Cuiping; Huang Jiarui; Ni Ning; Li Minqiang; Liu Jinhuai

2008-01-01

In this paper, enhanced fluorescence emissions were firstly investigated based on SnO 2 nanomaterial, and its application in the detection of DNA hybridization was also demonstrated. The microarray of SnO 2 nanomaterial was fabricated by the vapour phase transport method catalyzed by patterned Au nanoparticles on a silicon substrate. A probe DNA was immobilized on the substrate with patterned SnO 2 nanomaterial, respectively, by covalent and non-covalent linking schemes. When a fluorophore labelled target DNA was hybridized with a probe DNA on the substrate, fluorescence emissions were only observed on the surface of SnO 2 nanomaterial, which indicated the property of enhancing fluorescence signals from the SnO 2 nanomaterial. By comparing the different fluorescence images from covalent and non-covalent linking schemes, the covalent method was confirmed to be more effective for immobilizing a probe DNA. With the combined use of SnO 2 nanomaterial and the covalent linking scheme, the target DNA could be detected at a very low concentration of 10 fM. And the stability of SnO 2 nanomaterial under the experimental conditions was also compared with silicon nanowires. The findings strongly suggested that SnO 2 nanomaterial could be extensively applied in detections of biological samples with enhancing fluorescence property and high stability