Digital Commons Network^™

Comparative Genomics Of Proteins Involved In Rna Nucleocytoplasmic Export, Mariana Serpeloni, Newton M. Vidal, Samuel Goldenberg, Andréa R. Ávila, Federico G. Hoffmann

School of Biological Sciences: Faculty Publications

Background: The establishment of the nuclear membrane resulted in the physical separation of transcription and translation, and presented early eukaryotes with a formidable challenge: how to shuttle RNA from the nucleus to the locus of protein synthesis. In prokaryotes, mRNA is translated as it is being synthesized, whereas in eukaryotes mRNA is synthesized and processed in the nucleus, and it is then exported to the cytoplasm. In metazoa and fungi, the different RNA species are exported from the nucleus by specialized pathways. For example, tRNA is exported by exportin-t in a RanGTP-dependent fashion. By contrast, mRNAs are associated to …

Suitemsa: Visual Tools For Multiple Sequence Alignment Comparison And Molecular Sequence Simulation, Catherine L. Anderson, Cory L. Strope, Etsuko N. Moriyama

School of Biological Sciences: Faculty Publications

Background: Multiple sequence alignment (MSA) plays a central role in nearly all bioinformatics and molecular evolutionary applications. MSA reconstruction is thus one of the most heavily scrutinized bioinformatics fields. Evaluating the quality of MSA reconstruction is often hindered by the lack of good reference MSAs. The use of sequence evolution simulation can provide such reference MSAs. Furthermore, none of the MSA viewing/editing programs currently available allows the user to make direct comparisons between two or more MSAs. Considering the importance of MSA quality in a wide range of research, it is desirable if MSA assessment can be performed more …

Variations On A Theme: Diversification Of Cuticular Hydrocarbons In A Clade Of Cactophilic Drosophila, Cássia C. De Oliveira, Maura H. Manfrin, Fábio De M Sene, Larry L. Jackson, William J. Etges

School of Biological Sciences: Faculty Publications

Background: We characterized variation and chemical composition of epicuticular hydrocarbons (CHCs) in the seven species of the Drosophila buzzatii cluster with gas chromatography/mass spectrometry. Despite the critical role of CHCs in providing resistance to desiccation and involvement in communication, such as courtship behavior, mating, and aggregation, few studies have investigated how CHC profiles evolve within and between species in a phylogenetic context. We analyzed quantitative differences in CHC profiles in populations of the D. buzzatii species cluster in order to assess the concordance of CHC differentiation with species divergence.

Results: Thirty-six CHC components were scored in single fly …

Molecular Evolution Of Urea Amidolyase And Urea Carboxylase In Fungi, Pooja K. Strope, Kenneth W. Nickerson, Steven D. Harris, Etsuko N. Moriyama

School of Biological Sciences: Faculty Publications

Background: Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea …

The Effects Of Belowground Resources On Aboveground Allometric Growth In Bornean Tree Species, Katherine D. Heineman, Ethan Jensen, Autumn Shapland, Brett Bogenrief, Sylvester Tan, Richard Rebarber, Sabrina E. Russo

School of Biological Sciences: Faculty Publications

Tree height and crown allometries reflect adaptations for resource acquisition and structural stability, as well as plastic responses to a heterogeneous environment. While both light and soil resources limit growth and influence competitive responses in tropical forests, the effects of belowground resources on allometries are less understood, especially within the understory. To characterize outcomes of tree competition along an edaphic resource gradient, we quantified variation in height and crown allometries of six Bornean tree species from contrasting regeneration niches (lightdemanding vs. shade-tolerant) on two soil habitats (clay-fine loam and sandy loam) within a 52-ha forest dynamics plot. Using empiricallyfit allometric …

Increased Transsulfuration Mediates Longevity And Dietary Restriction In Drosophila, Hadise Kabil, Omer Kabil, Ruma Banerjee, Lawrence G. Harshman, Scott D. Pletcher

School of Biological Sciences: Faculty Publications

The mechanisms through which dietary restriction enhances health and longevity in diverse species are unclear. The transsulfuration pathway (TSP) is a highly conserved mechanism for metabolizing the sulfur-containing amino acids, methionine and cysteine. Here we show that Drosophila cystathionine β-synthase (dCBS), which catalyzes the rate-determining step in the TSP, is a positive regulator of lifespan in Drosophila and that the pathway is required for the effects of diet restriction on animal physiology and lifespan. dCBS activity was up-regulated in flies exposed to reduced nutrient conditions, and ubiquitous or neuron-specific transgenic overexpression of dCBS enhanced longevity in fully fed animals. Inhibition …

Morphologic Clues To The Origins Of Iron Oxide–Cemented Spheroids, Boxworks, And Pipelike Concretions, Navajo Sandstone Of South-Central Utah, U.S.A., David B. Loope, Richard M. Kettler, Karrie A. Weber

School of Biological Sciences: Faculty Publications

Concretions cemented by iron oxide are abundant and diverse in the Jurassic Navajo Sandstone of southern Utah. Some of these structures are considered terrestrial analogs for concretions imaged on Mars. The Navajo concretions can be spheroidal, pipelike, or tabular with multicompartmented boxworks. These iron oxide concretions typically display a rinded structure: dense sandstone rinds cemented by iron oxide surround pale, iron-poor sandstone cores. Within a single structure, the iron-rich rinds may be single or multiple. Pseudomorphs of siderite are present in local residual, iron-rich cores of boxworks. Workers in the late nineteenth through mid-twentieth centuries, many of whom found evidence …

Phylogenetic Diversity Of Fe(Iii)-Reducing Microorganisms In Rice Paddy Soil: Enrichment Cultures With Different Short-Chain Fatty Acids As Electron Donors, Huijuan Li, Jingjing Peng, Karrie A. Weber, Yongguan Zhu

School of Biological Sciences: Faculty Publications

Purpose — Microbial ferric iron reduction is an important biogeochemical process in nonsulfidogenic anoxic environments, yet the structure of microbial communities involved is poorly understood because of the lack of a functional gene marker. Here, with ferrihydrite as the iron source, we characterized the potential Fe(III)-reducing bacteria from the paddy soil in the presence of different short-chain fatty acids, formate, acetate, propionate, pyruvate, succinate, and citrate.

Materials and methods — Enrichment culture was used to characterize the potential Fe(III)-reducing bacteria in the present study. Clone library and terminal restriction fragment length polymorphism (T-RFLP) analyses of bacterial 16S rRNA gene sequence …

Survival Rates Of Planidial Larvae Of The Parasitoid Fly Ormia Ochracea (Diptera: Tachinidae), Oliver M. Beckers, Cassandra M. Martin, William E. Wagner Jr.

School of Biological Sciences: Faculty Publications

The tachinid fly, Ormia ochracea Bigot 1889, is a major parasitoid of field crickets in North America. The fly ranges from Florida to California, and it has also been introduced to Hawaii. Across this range, O. ochracea uses at least 6 different Gryllus species as hosts for its larvae (Cade, 1975; Walker, 1986; Walker and Wineriter, 1991; Zuk et al., 1993; Wagner, 1996; Hedrick and Kortet, 2006). O. ochracea finds it hosts by eavesdropping on male mating songs (Cade, 1975). The fly deposits up to 3 planidial larvae (Adamo et al., 1995a) directly on the male and these …

Male Field Crickets Infested By Parasitoid Flies Express Phenotypes That May Benefit The Parasitoids, Oliver M. Beckers, William E. Wagner

School of Biological Sciences: Faculty Publications

Parasites can cause changes in the phenotypes of their hosts that may benefit the parasite, the host, or both. To understand the evolutionary dynamics of host–parasite interactions it is necessary to first examine the effect of parasitic infestation on the host phenotype and whether the host or parasite benefits from these changes. The fly Ormia ochracea parasitizes the variable field cricket, Gryllus lineaticeps, and it uses male song to locate hosts for its lethal larvae. Adult flies preferentially orient to male songs with faster and longer chirps. We tested the effect of larval infestation on two types of host traits. …

A Cytoplasm-Specific Activity Encoded By The Trithorax-Like Atx1 Gene, Ivan Ndamukong, Hanna Lapko, Ronald L. Cerny, Zoya Avramova

School of Biological Sciences: Faculty Publications

Eukaryotes produce multiple products from a single gene locus by alternative splicing, translation or promoter usage as mechanisms expanding the complexity of their proteome. Trithorax proteins, including the Arabidopsis Trithorax-like protein ATX1, are histone modifiers regulating gene activity. Here, we report that a novel member of the Trithorax family has a role unrelated to chromatin. It is encoded from an internal promoter in the ATX1 locus as an isoform containing only the SET domain (soloSET). It is located exclusively in the cytoplasm and its substrate is the elongation factor 1A (EF1A). Loss of SET, but not of the histone modifying …

Dtorsin, The Drosophila Ortholog Of The Early-Onset Dystonia Tor1a (Dyt1), Plays A Novel Role In Dopamine Metabolism, Noriko Wakabayashi-Ito, Olugbenga M. Doherty, Hideaki Moriyama, Xandra O. Breakefield, James F. Gusella, Janis M. O’Donnell, Naoto Ito

School of Biological Sciences: Faculty Publications

Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned. TOR1A (formally DYT1), the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the prepupal stage and the few surviving …

Effects Of Postmortem Interval And Preservation Method On Rna Isolated From Field-Preserved Avian Tissues Efectos Del Intervalo Post-Mortem Y Del Método De Preservación En El Arn Aislado De Tejidos De Aves Preservados En El Campo, Zachary A. Cheviron, Matthew D. Carling, Robb T. Brumfield

School of Biological Sciences: Faculty Publications

Studies of gene expression and transcriptome profiling often require the isolation of high-quality mRNA. Here, we examined how preservation method and the lag between collection and processing (postmortem interval; PMI) affect RNA quality and yield in field-collected samples. We subjected tissue samples of 11 House Sparrows (Passer domesticus) to two preservation treatments (flash freezing in liquid nitrogen versus treatment with Ambion’s RNAlater prior to freezing) and eight PMI treatments (intervals ranging from 0 to 360 min). Mean RNA yields were significantly higher for flash-frozen tissues. Total RNA yield, as measured by a UV-vis spectrophotometer (Nanodrop), was not negatively associated with …

Vapc6, A Ribonucleolytic Toxin Regulates Thermophilicity In The Crenarchaeote Sulfolobus Solfataricus, Yukari Maezato, Amanda Daugherty, Karl Dana, Edith Soo, Charlotte Cooper, Sabrina Tachdjian, Robert M. Kelly, Paul H. Blum

School of Biological Sciences: Faculty Publications

The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxin– antitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 …

Evidence For Air Movement Signals In The Agonistic Behaviour Of A Nocturnal Arachnid (Order Amblypygi), Roger D. Santer, Eileen A. Hebets

School of Biological Sciences: Faculty Publications

Many arthropods possess filiform hair sensilla (termed trichobothria in arachnids), which are extremely sensitive detectors of medium particle displacement. Electrophysiological evidence in some taxa suggests that these sensilla can detect air particle displacements resulting from intraspecific communication signals. However, it has not yet been shown for any species that the air particle displacements detected by the filiform hairs are themselves perceived as a ‘signal’ (i.e. that individuals make behavioural decisions based upon the responses of these organs to the displays of conspecifics). We investigate the agonistic behaviour of the whip spider Phrynus marginemaculatus and the role of its trichobothria in …

Composite Effects Of Polymorphisms Near Multiple Regulatory Elements Create A Major-Effect Qtl, Ryan D. Bickel, Artyom Kopp, Sergey V. Nuzhdin

School of Biological Sciences: Faculty Publications

Many agriculturally, evolutionarily, and medically important characters vary in a quantitative fashion. Unfortunately, the genes and sequence variants accounting for this variation remain largely unknown due to a variety of biological and technical challenges. Drosophila melanogaster contains high levels of sequence variation and low linkage disequilibrium, allowing us to dissect the effects of many causative variants within a single locus. Here, we take advantage of these features to identify and characterize the sequence polymorphisms that comprise major effect QTL alleles segregating at the bric-a-brac locus. We show that natural bric-a-brac alleles with large effects on cuticular pigmentation reflect a cumulative …

Epigenetic Regulatory Mechanisms In Plants, Zoya Avramova

School of Biological Sciences: Faculty Publications

Genomes are defined by their primary sequence, which provides the genetic blueprint of a species. Eukaryotic DNA functions within the context of chromatin, which provides additional layers of gene regulation referred to as “epigenetic.” The commonly found definition of epigenetics is that of a “study of heritable changes in genome function that occur without a change in DNA sequence.” However, evidence that neuronal gene-expression states are also regulated by epigenetic mechanisms, despite evidence that neuronal cells do not divide, has opened space for a broader unifying definition that keeps “the sense of prevailing usage but avoids constraints imposed by stringently …

The Arabidopsis Trithorax-Like Factor Atx1 Functions In Dehydration Stress Responses Via Aba-Dependent And Aba-Independent Pathways, Yong Ding, Zoya Avramova, Michael Fromm

School of Biological Sciences: Faculty Publications

Emerging evidence suggests that the molecular mechanisms driving the responses of plants to environmental stresses are associated with specific chromatin modifications. Here, we demonstrate that the Arabidopsis trithorax-like factor ATX1, which trimethylates histone H3 at lysine 4 (H3K4me3), is involved in dehydration stress signaling in both abscisic acid (ABA)-dependent and ABA-independent pathways. The loss of function of ATX1 results in decreased germination rates, larger stomatal apertures, more rapid transpiration and decreased tolerance to dehydration stress in atx1 plants. This deficiency is caused in part by reduced ABA biosynthesis in atx1 plants resulting from decreased transcript levels from NCED3, which …

Geographic Distribution: Osteopilus Septentrionalis (Cuban Treefrog). Usa: Florida: Bradford Co., Linda S. Stevenson, Louis A. Somma

Papers in Herpetology

OSTEOPILUS SEPTENTRIONALIS (Cuban Treefrog). USA: FLORIDA: B IlAI)flOIlD Co.: Starke. 703 South Orange Street (29.940 15"N, 82. 11532S'W; WGS84) . 12 December 2009. Linda S. Sleven son. Verified by Melvin P. Gra.mke. Florida Museum of Natural History. University of Florida. UF 162973. First county record (Meshaka et al. 2004. The Exotic Amphibians and Reptiles of Florida. Krieger Publ. Co., Malabar, Florida; Somma 2009. In Nonindigenous Aq uatic Species. USGS: hI rp: II nas.e r.usgs.gov I q ue ries! FactSheet. asp?specieslD=57). One adult collected from inside a mailbox on wall of a residence. Individ ual O. septentrionalis have been sighted at …

Complex Courtship Displays Facilitate Male Reproductive Success And Plasticity In Signaling Across Variable Environments, Dustin Wilgers, Eileen Hebets

Eileen Hebets Publications

Effective signal transmission is essential for communication. In environments where signal transmission is highly variable, signalers may utilize complex signals, which incorporate multiple components and modalities, to maintain effective communication. Male Rabidosa rabida wolf spiders produce complex courtship signals, consisting of both visual and seismic components. We test the hypothesis that the complex signaling of R. rabida contributes to male reproductive success in variable signaling environments. We first examine the condition-dependence of foreleg ornamentation (a presumed visual signal) and seismic signal components and find that both may provide potentially redundant information on foraging history. Next, we assessed reproductive success across …

Chemical Communication In A Multimodal Context, Eileen A. Hebets, Aaron S. Rundus

Eileen Hebets Publications

All animals are equipped with multiple sensory systems (e.g., visual, chemical, acoustic, tactile, electrical, thermal), and signals perceived via these sensory systems facilitate communication. Such communication often involves displays that incorporate more than one signal from more than one sensory modality, resulting in multimodal signaling. The number of empirical and theoretical studies addressing issues of multimodal signaling is ever-increasing and this chapter highlights why crustaceans, as a taxonomic group, are ideal for advancing such studies. Early classifications of multimodal signaling sought to categorize signal components as either redundant or nonredundant, while more recent classifications lay out specific hypotheses relating to …

The Degree Of Response To Increased Predation Risk Corresponds To Male Secondary Sexual Traits, Kasey Fowler-Finn, Eileen Hebets

Eileen Hebets Publications

Secondary sexual traits are expected to reflect a balance between sexual selection and natural selection. We test the hypothesis that plasticity in sexual advertisement behaviors can influence this trade-off, allowing showier traits than expected for a given level of predation risk. Specifically, we tested whether the degree of behavioral plasticity exhibited in response to chemical cues of a co-occurring predatory wolf spider corresponds to courtship rate and the degree of ornamentation in male wolf spiders. Both ornamented (brush-legged) males and non-ornamented males decreased locomotion, decreased their likelihood to court, and increased their time to initiate courtship in response to predator …

Copper Tolerance Of Saccharomyces Cerevisiae Nonsense-Mediated Mrna Decay Mutants, Rafael Deliz-Aguirre, Audrey L. Atkin, Bessie W. Kebaara

School of Biological Sciences: Faculty Publications

The eukaryotic nonsense-mediated mRNA (NMD) is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons, and importantly some natural mRNAs as well. Natural mRNAs with atypically long 3′-untranslated regions (UTRs) are degraded by NMD in Saccharomyces cerevisiae. A number of S. cerevisiae mRNAs undergo alternative 3′-end processing producing mRNA isoforms that differ in their 3′-UTR lengths. Some of these alternatively 3′-end processed mRNA isoforms have atypically long 3′-UTRs and would be likely targets for NMD-mediated degradation. Here, we investigated the role NMD plays in the regulation of expression of CTR2, …

Sex Chromosome-Specific Regulation In The Drosophila Male Germline But Little Evidence For Chromosomal Dosage Compensation Or Meiotic Inactivation, Colin D. Meiklejohn, Emily L. Landeen, Jodi M. Cook, Sarah B. Kingan, Daven C. Presgraves

School of Biological Sciences: Faculty Publications

The evolution of heteromorphic sex chromosomes (e.g., XY in males or ZW in females) has repeatedly elicited the evolution of two kinds of chromosome-specific regulation: dosage compensation—the equalization of X chromosome gene expression in males and females— and meiotic sex chromosome inactivation (MSCI)—the transcriptional silencing and heterochromatinization of the X during meiosis in the male (or Z in the female) germline. How the X chromosome is regulated in the Drosophila melanogaster male germline is unclear. Here we report three new findings concerning gene expression from the X in Drosophila testes. First, X chromosome-wide dosage compensation appears to be absent from …

Normalization And Centering Of Array-Based Heterologous Genome Hybridization Based On Divergent Control Probes, Brian J. Darby, Kenneth L. Jones, David Wheeler, Michael A. Herman

School of Biological Sciences: Faculty Publications

Background: Hybridization of heterologous (non-specific) nucleic acids onto arrays designed for model-organisms has been proposed as a viable genomic resource for estimating sequence variation and gene expression in nonmodel organisms. However, conventional methods of normalization that assume equivalent distributions (such as quantile normalization) are inappropriate when applied to non-specific (heterologous) hybridization. We propose an algorithm for normalizing and centering intensity data from heterologous hybridization that makes no prior assumptions of distribution, reduces the false appearance of homology, and provides a way for researchers to confirm whether heterologous hybridization is suitable.

Results: Data are normalized by adjusting for Gibbs free energy …

Developmental Regulation Of Hemoglobin Synthesis In The Green Anole Lizard Anolis Carolinensis, Jay F. Storz, Federico G. Hoffmann, Juan C. Opazo, Thomas J. Sanger, Hideaki Moriyama

Jay F. Storz Publications

Tetrapod vertebrates possess multiple alpha- and beta-like globin genes that are ontogenetically regulated, such that functionally distinct hemoglobin (Hb) isoforms are synthesized during different stages of development. The alpha- and beta-like globin genes of amphibians, birds and mammals are differentially expressed during embryonic development and postnatal life, but little is known about the developmental regulation of globin gene expression in non-avian reptiles. Here we report an investigation into the developmental regulation of Hb synthesis in the green anole lizard Anolis carolinensis. We tested two hypotheses derived from comparative genomic studies of the globin gene clusters in tetrapod vertebrates. First, …

Evolutionary Origins Of Oxygen Sensing In Animals, Kalle T. Rytkönen, Jay F. Storz

Jay F. Storz Publications

All animal life requires molecular oxygen as the terminal electron acceptor in aerobic energy production. A lack of oxygen can reduce the rate of energy production, whereas an excess of oxygen leads to the accumulation of toxic reactive oxygen species. Hence, animals have evolved sophisticated mechanisms with which to monitor and respond to fluctuations in oxygen availability, in order to maintain cellular homeostasis. In all animal taxa examined so far, the maintenance of physiological oxygen homeostasis is mediated by the oxygen-dependent post-translational hydroxylation of a heterodimeric transcription factor, termed hypoxia-inducible factor (HIF; Kaelin & Ratcliffe, 2008). The hydroxylation reaction is …

Differential Loss And Retention Of Cytoglobin, Myoglobin, And Globin-E During The Radiation Of Vertebrates, Federico G. Hoffmann, Juan C. Opazo, Jay F. Storz

Jay F. Storz Publications

If rates of postduplication gene retention are positively correlated with levels of functional constraint, then gene duplicates that have been retained in a restricted number of taxonomic lineages would be expected to exhibit relatively low levels of sequence conservation. Paradoxical patterns are presented by gene duplicates that have been retained in a small number of taxa but which are nonetheless subject to strong purifying selection relative to paralogous members of the same multigene family. This pattern suggests that such genes may have been co-opted for novel, lineage-specific functions. One possible example involves the enigmatic globin-E gene (GbE), which …

Experimental Approaches To Evaluate The Contributions Of Candidate Protein-Coding Mutations To Phenotypic Evolution, Jay F. Storz, Anthony J. Zera

Jay F. Storz Publications

Identifying mechanisms of molecular adaptation can provide important insights into the process of phenotypic evolution, but it can be exceedingly difficult to quantify the phenotypic effects of specific mutational changes. To verify the adaptive significance of genetically based changes in protein function, it is necessary to document functional differences between the products of derived and wild-type alleles and to demonstrate that such differences impinge on higher-level physiological processes (and ultimately, fitness). In the case of metabolic enzymes, this requires documenting in vivo differences in reaction rate that give rise to differences in flux through the pathway in which the enzymes …

Glypican-3 Targeting Of Liver Cancer Cells Using Multifunctional Nanoparticles, James O. Park, Zachary Stephen, Conory Sun, Omid Veiseh, Forrest M. Kievit, Chen Fang, Matthew Leung, Hyejung Mok, Miqin Zhang

Department of Agricultural and Biological Systems Engineering: Faculty Publications

Imaging is essential in accurately detecting, staging, and treating primary liver cancer (hepatocellular carcinoma [HCC]), one of the most prevalent and lethal malignancies. We developed a novel multifunctional nanoparticle (NP) specifically targeting glypican-3 (GPC3), a proteoglycan implicated in promotion of cell growth that is overexpressed in most HCCs. Quantitative real-time polymerase chain reaction was performed to confirm the differential GPC3 expression in two human HCC cells, Hep G2 (high) and HLF (negligible). These cells were treated with biotin-conjugated GPC3 monoclonal antibody (aGPC3) and subsequently targeted using superparamagnetic iron oxide NPs conjugated to streptavidin and Alexa Fluor 647. Flow cytometry demonstrated …