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Sample records for cell groups reveal

  1. Vibrational fingerprint mapping reveals spatial distribution of functional groups of lignin in plant cell wall.

    PubMed

    Liu, Bin; Wang, Ping; Kim, Jeong Im; Zhang, Delong; Xia, Yuanqin; Chapple, Clint; Cheng, Ji-Xin

    2015-09-15

    Highly lignified vascular plant cell walls represent the majority of cellulosic biomass. Complete release of the biomass to deliver renewable energy by physical, chemical, and biological pretreatments is challenging due to the "protection" provided by polymerized lignin, and as such, additional tools to monitor lignin deposition and removal during plant growth and biomass deconstruction would be of great value. We developed a hyperspectral stimulated Raman scattering microscope with 9 cm(-1) spectral resolution and submicrometer spatial resolution. Using this platform, we mapped the aromatic ring of lignin, aldehyde, and alcohol groups in lignified plant cell walls. By multivariate curve resolution of the hyperspectral images, we uncovered a spatially distinct distribution of aldehyde and alcohol groups in the thickened secondary cell wall. These results collectively contribute to a deeper understanding of lignin chemical composition in the plant cell wall.

  2. Single-cell genomics reveals co-metabolic interactions within uncultivated Marine Group A bacteria

    NASA Astrophysics Data System (ADS)

    Hawley, A. K.; Hallam, S. J.

    2016-02-01

    Marine Group A (MGA) bacteria represent a ubiquitous and abundant candidate phylum enriched in oxygen minimum zones (OMZs) and the deep ocean. Despite MGA prevalence little is known about their ecology and biogeochemistry. Here we chart the metabolic potential of 26 MGA single-cell amplified genomes sourced from different environments spanning ecothermodynamic gradients including open ocean waters, OMZs and methanogenic environments including a terephthalate-degrading bioreactor. Metagenomic contig recruitment to SAGs combined with tetra-nucleotide frequency distribution patterns resolved nine MGA population genome bins. All population genomes exhibited genomic streamlining with open ocean MGA being the most reduced. Different strategies for carbohydrate utilization, carbon fixation energy metabolism and respiratory pathways were identified between population genome bins, including various roles in the nitrogen and sulfur cycles. MGA inhabiting OMZ oxyclines encoded genes for partial denitrification with potential to feed into anammox and nitrification as well as a polysulfide reductase with a potential role in the cryptic sulfur cycle. MGA inhabiting anoxic waters, encoded NiFe hydrogenase and nitrous oxide reductase with the potential to complete partial denitrification pathways previously linked to sulfur oxidation in SUP05 bacteria. MGA from methanogenic environments encoded genes mediating cascading syntrophic interactions with fatty acid degraders and methanogens including reverse electron transport potential. The MGA phylum appears to have evolved alternative metabolic innovations adapting specific subgroups to occupy specific niches along ecothermodynamic gradients. Additionally, expression of MGA genes from different OMZ environments supports that these subgroups manifest an increasing propensity for co-metabolic interactions under energy limiting conditions that mandates a cooperative mode of existence with important implications for C, N and S cycling in

  3. Conditional anterograde tracing reveals distinct targeting of individual serotonin cell groups (B5-B9) to the forebrain and brainstem.

    PubMed

    Muzerelle, Aude; Scotto-Lomassese, Sophie; Bernard, Jean François; Soiza-Reilly, Mariano; Gaspar, Patricia

    2016-01-01

    Serotoninergic innervation of the central nervous system is provided by hindbrain raphe nuclei (B1-B9). The extent to which each raphe subdivision has distinct topographic organization of their projections is still unclear. We provide a comprehensive description of the main targets of the rostral serotonin (5-HT) raphe subgroups (B5-B9) in the mouse brain. Adeno-associated viruses that conditionally express GFP under the control of the 5-HT transporter promoter were used to label small groups of 5-HT neurons in the dorsal (B7d), ventral (B7v), lateral (B7l), and caudal (B6) subcomponents of the dorsal raphe (DR) nucleus as well as in the rostral and caudal parts of the median raphe (MR) nucleus (B8 and B5, respectively), and in the supralemniscal (B9) cell group. We illustrate the distinctive and largely non-overlapping projection areas of these cell groups: for instance, DR (B7) projects to basal parts of the forebrain, such as the amygdala, whereas MR (B8) is the main 5-HT source to the hippocampus, septum, and mesopontine tegmental nuclei. Distinct subsets of B7 have preferential brain targets: B7v is the main source of 5-HT for the cortex and amygdala while B7d innervates the hypothalamus. We reveal for the first time the target areas of the B9 cell group, demonstrating projections to the caudate, prefrontal cortex, substantia nigra, locus coeruleus and to the raphe cell groups. The broad topographic organization of the different raphe subnuclei is likely to underlie the different functional roles in which 5-HT has been implicated in the brain. The present mapping study could serve as the basis for genetically driven specific targeting of the different subcomponents of the mouse raphe system.

  4. Single-cell RNA-seq reveals activation of unique gene groups as a consequence of stem cell-parenchymal cell fusion.

    PubMed

    Freeman, Brian T; Jung, Jangwook P; Ogle, Brenda M

    2016-03-21

    Fusion of donor mesenchymal stem cells with parenchymal cells of the recipient can occur in the brain, liver, intestine and heart following transplantation. The therapeutic benefit or detriment of resultant hybrids is unknown. Here we sought a global view of phenotypic diversification of mesenchymal stem cell-cardiomyocyte hybrids and associated time course. Using single-cell RNA-seq, we found hybrids consistently increase ribosome components and decrease genes associated with the cell cycle suggesting an increase in protein production and decrease in proliferation to accommodate the fused state. But in the case of most other gene groups, hybrids were individually distinct. In fact, though hybrids can express a transcriptome similar to individual fusion partners, approximately one-third acquired distinct expression profiles in a single day. Some hybrids underwent reprogramming, expressing pluripotency and cardiac precursor genes latent in parental cells and associated with developmental and morphogenic gene groups. Other hybrids expressed genes associated with ontologic cancer sets and two hybrids of separate experimental replicates clustered with breast cancer cells, expressing critical oncogenes and lacking tumor suppressor genes. Rapid transcriptional diversification of this type garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion.

  5. Group B, type III streptococcal cell wall: composition and structural aspects revealed through endo-N-acetylmuramidase-catalyzed hydrolysis.

    PubMed Central

    De Cueninck, B J; Shockman, G D; Swenson, R M

    1982-01-01

    Cell walls from a group B, type III streptococcus strain were prepared, purified by extraction with sodium dodecyl sulfate, and solubilized by the M-1 fraction of mutanolysin, an endo-N-acetylmuramidase obtained from Streptomyces globisporus. The lysate was resolved into three fractions by ion-exchange chromatography: a fraction containing peptidoglycan (PG) fragments, free of neutral and acidic sugars and of phosphate; a complex of PG fragments and group B-specific polysaccharide; and a complex of PG fragments and group B-specific polysaccharide and type III-specific polysaccharide. The PG-polysaccharide complexes were large and heterogeneous in molecular size. When subjected to base-catalyzed beta-elimination, both complexes were disintegrated, and polysaccharides and low-molecular-weight PG fragments could then be separated by gel filtration. The low-molecular-weight PG fragment-containing fraction contained muramic acid, glucosamine, alanine, lysine, glutamic acid, and serine in molar ratios (to lysine) of 0.92:0.98:3.01:1.00:1.00:0.05. Wall-derived, purified group polysaccharide contained rhamnose, galactose, glucosamine, and phosphorus in molar ratios (to galactose) of 5.03:1.00:1.00:1.05. It also contained an unidentified sugar. Wall-derived, purified type III polysaccharide contained galactose, glucosamine, glucose, and N-acetylneuraminic acid in molar ratios (to glucose) of 1.94:0.85:1.00:1.39. On a dry-weight basis, the whole wall lysate contained 19.8 and 20.6% of group and type polysaccharide, respectively. Neither glycerol nor ribitol was found, and all of the cell wall phosphorus was accounted for as polysaccharide, indicating the absence of a wall teichoic acid. PMID:7035367

  6. Group A streptococcus cell-associated pathogenic proteins as revealed by growth in hyaluronic acid-enriched media.

    PubMed

    Zhang, Meng; McDonald, Fiona M; Sturrock, Shane S; Charnock, Simon J; Humphery-Smith, Ian; Black, Gary W

    2007-05-01

    Group A streptococcus (GAS), also know as Streptococcus pyogenes, is a human pathogen and can cause several fatal invasive diseases such as necrotising fasciitis, the so-called flesh-eating disease, and toxic shock syndrome. The destruction of connective tissue and the hyaluronic acid (HA) therein, is a key element of GAS pathogenesis. We therefore propagated GAS in HA-enriched growth media in an attempt to create a simple biological system that could reflect some elements of GAS pathogenesis. Our results show that several recognised virulence factors were up-regulated in HA-enriched media, including the M1 protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential GAS virulence factors.

  7. The IC 342-Maffei 1 Group Revealed

    NASA Astrophysics Data System (ADS)

    McCall, M. L.; Buta, R.

    1996-12-01

    Deep wide-field CCD images of thirteen members of the IC 342-Maffei 1 Group in the Northern Milky Way have been acquired in the Johnson V and Cousins I photometric systems. The observations were obtained with the Kitt Peak Burrell-Schmidt telescope in Arizona during six nights in November 1995. Almost none of these galaxies was effectively studied in the past because of the heavy foreground extinction and significant foreground star contamination in the direction of the group. The tens of thousands of foreground stars included on the Schmidt images have been successfully subtracted using DAOPHOT, revealing the true extent and total brightness of most of the galaxies for the first time. In the absence of galactic extinction, Maffei 1, Maffei 2, and IC 342 would be among the five brightest galaxies in the northern sky, and both Maffei 1 and IC 342 would subtend angles as large as the full Moon. The results are critical for assessing the degree to which the group influenced the dynamical evolution of the Local Group. In this poster, we will present deep photographs, total magnitudes and color indices, luminosity profiles, and distance estimates for the member galaxies.

  8. Group V secretory phospholipase A2 reveals its role in house dust mite-induced allergic pulmonary inflammation by regulation of dendritic cell function

    PubMed Central

    Giannattasio, Giorgio; Fujioka, Daisuke; Xing, Wei; Katz, Howard R.; Boyce, Joshua A.; Balestrieri, Barbara

    2010-01-01

    We have previously shown that group V secretory phospholipase A2 (sPLA2) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. Here we report that group V sPLA2 (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae (Df) had markedly reduced pulmonary inflammation and goblet cell metaplasia compared to wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to Df compared to WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by Df had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of Df-challenged mice. Adoptively transferred Df-loaded Pla2g5-null BMDCs were less able than Df-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null Df-loaded BMDCs exhibited significantly reduced local inflammatory responses to Df, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA2 in APC regulates Ag processing and maturation of dendritic cells, and contributes to pulmonary inflammation and immune response against Df. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA2 is upregulated by Df and whose function is also regulated by group V sPLA2. PMID:20817863

  9. Genetic structure of Tunisian ethnic groups revealed by paternal lineages.

    PubMed

    Fadhlaoui-Zid, Karima; Martinez-Cruz, Begoña; Khodjet-el-khil, Houssein; Mendizabal, Isabel; Benammar-Elgaaied, Amel; Comas, David

    2011-10-01

    Tunisia has experienced a variety of human migrations that have modeled the myriad cultural groups inhabiting the area. Both Arabic and Berber-speaking populations live in Tunisia. Berbers are commonly considered as in situ descendants of peoples who settled roughly in Palaeolithic times, and posterior demographic events such as the arrival of the Neolithic, the Arab migrations, and the expulsion of the "Moors" from Spain, had a strong cultural influence. Nonetheless, the genetic structure and the population relationships of the ethnic groups living in Tunisia have been poorly assessed. In order to gain insight into the paternal genetic landscape and population structure, more than 40 Y-chromosome single nucleotide polymorphisms and 17 short tandem repeats were analyzed in five Tunisian ethnic groups (three Berber-speaking isolates, one Andalusian, and one Cosmopolitan Arab). The most common lineage was the North African haplogroup E-M81 (71%), being fixed in two Berber samples (Chenini-Douiret and Jradou), suggesting isolation and genetic drift. Differential levels of paternal gene flow from the Near East were detected in the Tunisian samples (J-M267 lineage over 30%); however, no major sub-Saharan African or European influence was found. This result contrasts with the high amount of sub-Saharan and Eurasian maternal lineages previously described in Tunisia. Overall, our results reveal a certain genetic inter-population diversity, especially among Berber groups, and sexual asymmetry, paternal lineages being mostly of autochthonous origin. In addition, Andalusians, who are supposed to be migrants from southern Spain, do not exhibit any substantial contribution of European lineages, suggesting a North African origin for this ethnic group. Copyright © 2011 Wiley-Liss, Inc.

  10. Dermatoglyphics from all Chinese ethnic groups reveal geographic patterning.

    PubMed

    Zhang, Hai-Guo; Chen, Yao-Fong; Ding, Ming; Jin, Li; Case, D Troy; Jiao, Yun-Ping; Wang, Xian-Ping; Bai, Chong-Xian; Jin, Gang; Yang, Jiang-Ming; Wang, Han; Yuan, Jian-Bing; Huang, Wei; Wang, Zhu-Gang; Chen, Ren-Biao

    2010-01-20

    Completion of a survey of dermatoglyphic variables for all ethnic groups in an ethnically diverse country like China is a huge research project, and an achievement that anthropological and dermatoglyphic scholars in the country could once only dream of. However, through the endeavors of scientists in China over the last 30 years, the dream has become reality. This paper reports the results of a comprehensive analysis of dermatoglyphics from all ethnic groups in China. Using cluster analysis and principal component analysis of dermatoglyphics, it has been found that Chinese populations can be generally divided into a southern group and a northern group. Furthermore, there has been considerable debate about the origins of many Chinese populations and about proper assignment of these peoples to larger ethnic groups. In this paper, we suggest that dermatoglyphic data can inform these debates by helping to classify a Chinese population as a northern or southern group, using selected reference populations and quantitative methods. This study is the first to assemble and investigate dermatoglyphics from all 56 Chinese ethnic groups. It is fortunate that data on population dermatoglyphics, a field of physical anthropology, have now been collected for all 56 Chinese ethnic groups, because intermarriage between individuals from different Chinese ethnic groups occurs more frequently in recent times, making population dermatoglyphic research an ever more challenging field of inquiry.

  11. Dermatoglyphics from All Chinese Ethnic Groups Reveal Geographic Patterning

    PubMed Central

    Jin, Li; Case, D. Troy; Jiao, Yun-Ping; Wang, Xian-Ping; Bai, Chong-Xian; Jin, Gang; Yang, Jiang-Ming; Wang, Han; Yuan, Jian-Bing; Huang, Wei; Wang, Zhu-Gang; Chen, Ren-Biao

    2010-01-01

    Completion of a survey of dermatoglyphic variables for all ethnic groups in an ethnically diverse country like China is a huge research project, and an achievement that anthropological and dermatoglyphic scholars in the country could once only dream of. However, through the endeavors of scientists in China over the last 30 years, the dream has become reality. This paper reports the results of a comprehensive analysis of dermatoglyphics from all ethnic groups in China. Using cluster analysis and principal component analysis of dermatoglyphics, it has been found that Chinese populations can be generally divided into a southern group and a northern group. Furthermore, there has been considerable debate about the origins of many Chinese populations and about proper assignment of these peoples to larger ethnic groups. In this paper, we suggest that dermatoglyphic data can inform these debates by helping to classify a Chinese population as a northern or southern group, using selected reference populations and quantitative methods. This study is the first to assemble and investigate dermatoglyphics from all 56 Chinese ethnic groups. It is fortunate that data on population dermatoglyphics, a field of physical anthropology, have now been collected for all 56 Chinese ethnic groups, because intermarriage between individuals from different Chinese ethnic groups occurs more frequently in recent times, making population dermatoglyphic research an ever more challenging field of inquiry. PMID:20098698

  12. Plastome data reveal multiple geographic origins of Quercus Group Ilex

    PubMed Central

    Grimm, Guido W.; Papini, Alessio; Vessella, Federico; Cardoni, Simone; Tordoni, Enrico; Piredda, Roberta; Franc, Alain; Denk, Thomas

    2016-01-01

    Nucleotide sequences from the plastome are currently the main source for assessing taxonomic and phylogenetic relationships in flowering plants and their historical biogeography at all hierarchical levels. One major exception is the large and economically important genus Quercus (oaks). Whereas differentiation patterns of the nuclear genome are in agreement with morphology and the fossil record, diversity patterns in the plastome are at odds with established taxonomic and phylogenetic relationships. However, the extent and evolutionary implications of this incongruence has yet to be fully uncovered. The DNA sequence divergence of four Euro-Mediterranean Group Ilex oak species (Quercus ilex L., Q. coccifera L., Q. aucheri Jaub. & Spach., Q. alnifolia Poech.) was explored at three chloroplast markers (rbcL, trnK/matK, trnH-psbA). Phylogenetic relationships were reconstructed including worldwide members of additional 55 species representing all Quercus subgeneric groups. Family and order sequence data were harvested from gene banks to better frame the observed divergence in larger taxonomic contexts. We found a strong geographic sorting in the focal group and the genus in general that is entirely decoupled from species boundaries. High plastid divergence in members of Quercus Group Ilex, including haplotypes shared with related, but long isolated oak lineages, point towards multiple geographic origins of this group of oaks. The results suggest that incomplete lineage sorting and repeated phases of asymmetrical introgression among ancestral lineages of Group Ilex and two other main Groups of Eurasian oaks (Cyclobalanopsis and Cerris) caused this complex pattern. Comparison with the current phylogenetic synthesis also suggests an initial high- versus mid-latitude biogeographic split within Quercus. High plastome plasticity of Group Ilex reflects geographic area disruptions, possibly linked with high tectonic activity of past and modern distribution ranges, that did not

  13. Plastome data reveal multiple geographic origins of Quercus Group Ilex.

    PubMed

    Simeone, Marco Cosimo; Grimm, Guido W; Papini, Alessio; Vessella, Federico; Cardoni, Simone; Tordoni, Enrico; Piredda, Roberta; Franc, Alain; Denk, Thomas

    2016-01-01

    Nucleotide sequences from the plastome are currently the main source for assessing taxonomic and phylogenetic relationships in flowering plants and their historical biogeography at all hierarchical levels. One major exception is the large and economically important genus Quercus (oaks). Whereas differentiation patterns of the nuclear genome are in agreement with morphology and the fossil record, diversity patterns in the plastome are at odds with established taxonomic and phylogenetic relationships. However, the extent and evolutionary implications of this incongruence has yet to be fully uncovered. The DNA sequence divergence of four Euro-Mediterranean Group Ilex oak species (Quercus ilex L., Q. coccifera L., Q. aucheri Jaub. & Spach., Q. alnifolia Poech.) was explored at three chloroplast markers (rbcL, trnK/matK, trnH-psbA). Phylogenetic relationships were reconstructed including worldwide members of additional 55 species representing all Quercus subgeneric groups. Family and order sequence data were harvested from gene banks to better frame the observed divergence in larger taxonomic contexts. We found a strong geographic sorting in the focal group and the genus in general that is entirely decoupled from species boundaries. High plastid divergence in members of Quercus Group Ilex, including haplotypes shared with related, but long isolated oak lineages, point towards multiple geographic origins of this group of oaks. The results suggest that incomplete lineage sorting and repeated phases of asymmetrical introgression among ancestral lineages of Group Ilex and two other main Groups of Eurasian oaks (Cyclobalanopsis and Cerris) caused this complex pattern. Comparison with the current phylogenetic synthesis also suggests an initial high- versus mid-latitude biogeographic split within Quercus. High plastome plasticity of Group Ilex reflects geographic area disruptions, possibly linked with high tectonic activity of past and modern distribution ranges, that did not

  14. ALBEDOS OF SMALL HILDA GROUP ASTEROIDS AS REVEALED BY SPITZER

    SciTech Connect

    Ryan, Erin Lee; Woodward, Charles E. E-mail: chelsea@astro.umn.edu

    2011-06-15

    We present thermal 24 {mu}m observations from the Spitzer Space Telescope of 62 Hilda asteroid group members with diameters ranging from 3 to 12 km. Measurements of the thermal emission, when combined with reported absolute magnitudes, allow us to constrain the albedo and diameter of each object. From our Spitzer sample, we find the mean geometric albedo, p{sub V} = 0.07 {+-} 0.05, for small (D < 10 km) Hilda group asteroids. This Spitzer-derived value of p{sub V} is greater than and spans a larger range in albedo space than the mean albedo of large (D {approx}> 10 km) Hilda group asteroids which is p{sub V} = 0.04 {+-} 0.01. Though this difference may be attributed to space weathering, the small Hilda group population reportedly displays greater taxonomic range from C-, D-, and X-type whose albedo distributions are commensurate with the range of determined albedos. We discuss the derived Hilda size-frequency distribution, color-color space, and geometric albedo for our survey sample in the context of the expected migration induced 'seeding' of the Hilda asteroid group with outer solar system proto-planetesimals as outlined in the 'Nice' formalism.

  15. A Large Study on Immunological Response to a Whole-Cell Killed Oral Cholera Vaccine Reveals That There Are Significant Geographical Differences in Response and That O Blood Group Individuals Do Not Elicit a Higher Response▿ †

    PubMed Central

    Ramamurthy, T.; Wagener, Diane; Chowdhury, Goutam; Majumder, Partha P.

    2010-01-01

    The ABO blood group system has been implicated in susceptibility to cholera or in explaining variability in the immune response to a cholera vaccine. O blood group individuals were found to be more susceptible to cholera and elicited lower vibriocidal antibody response to cholera toxin B subunit-killed oral vaccine. Based on the observations that O blood group individuals were more susceptible to cholera and that high mortality was associated with cholera, an evolutionary explanation was provided for the extremely low prevalence of the O blood group in the Gangetic Delta (West Bengal, India, and Bangladesh). However, conflicting results were reported from a later study conducted in Indonesia using a live attenuated oral cholera vaccine; O blood group individuals showed a higher vibriocidal antibody response. In a study conducted in a region of India where cholera is endemic (Kolkata, West Bengal) that comprised 992 individuals vaccinated by a killed whole-cell oral cholera vaccine, we found no statistically significant difference between O and non-O individuals either in the frequency distributions of the fold increase or in the postvaccination increase in geometric mean titer compared to the baseline. Further, in contrast to the earlier observation that the O allele frequency is extremely low in the Gangetic Delta, we have noted that the O allele frequency exceeds 0.5 in the vast majority of ethnic groups of this region. In addition, we have found large differences in response to the vaccine among residents of an area where cholera is not endemic compared to an area where cholera is endemic to The percentages of vaccinees who seroconverted in an area where cholera is not endemic (Son La province of Vietnam) was >90% compared to ∼50% in Kolkata, India, an area where cholera is endemic. PMID:20554804

  16. Low Mass Members in Nearby Young Moving Groups Revealed

    NASA Astrophysics Data System (ADS)

    Schlieder, Joshua; Simon, Michal; Rice, Emily; Lepine, Sebastien

    2010-08-01

    We are now ready to expand our program that identifies highly probable low-mass members of the nearby young moving groups (NYMGs) to stars of mass ~ 0.1 Msun. This is important 1) To provide high priority targets for exoplanet searches by direct imaging, 2) To complete the census of the membership in the NYMGs, and 3) To provide a well-characterized sample of nearby young stars for detailed study of their physical properties and multiplicity (the median distances of the (beta) Pic and AB Dor groups are ~ 35 pc with ages ~ 12 and 50 Myr respectively). Our proven technique starts with a proper motion selection algorithm, proceeds to vet the sample for indicators of youth, and requires as its last step the measurement of candidate member radial velocities (RVs). So far, we have obtained all RV measurements with the high resolution IR spectrometer at the NASA-IRTF and have reached the limits of its applicability. To identify probable new members in the south, and also those of the lowest mass, we need the sensitivity of PHOENIX at Gemini-S and NIRSPEC at Keck-II.

  17. Neural activity reveals perceptual grouping in working memory.

    PubMed

    Rabbitt, Laura R; Roberts, Daniel M; McDonald, Craig G; Peterson, Matthew S

    2017-03-01

    There is extensive evidence that the contralateral delay activity (CDA), a scalp recorded event-related brain potential, provides a reliable index of the number of objects held in visual working memory. Here we present evidence that the CDA not only indexes visual object working memory, but also the number of locations held in spatial working memory. In addition, we demonstrate that the CDA can be predictably modulated by the type of encoding strategy employed. When individual locations were held in working memory, the pattern of CDA modulation mimicked previous findings for visual object working memory. Specifically, CDA amplitude increased monotonically until working memory capacity was reached. However, when participants were instructed to group individual locations to form a constellation, the CDA was prolonged and reached an asymptote at two locations. This result provides neural evidence for the formation of a unitary representation of multiple spatial locations.

  18. A fifth major genetic group among honeybees revealed in Syria

    PubMed Central

    2013-01-01

    Background Apiculture has been practiced in North Africa and the Middle-East from antiquity. Several thousand years of selective breeding have left a mosaic of Apis mellifera subspecies in the Middle-East, many uniquely adapted and survived to local environmental conditions. In this study we explore the genetic diversity of A. mellifera from Syria (n = 1258), Lebanon (n = 169) and Iraq (n = 35) based on 14 short tandem repeat (STR) loci in the context of reference populations from throughout the Old World (n = 732). Results Our data suggest that the Syrian honeybee Apis mellifera syriaca occurs in both Syrian and Lebanese territories, with no significant genetic variability between respective populations from Syria and Lebanon. All studied populations clustered within a new fifth independent nuclear cluster, congruent with an mtDNA Z haplotype identified in a previous study. Syrian honeybee populations are not associated with Oriental lineage O, except for sporadic introgression into some populations close to the Turkish and Iraqi borders. Southern Syrian and Lebanese populations demonstrated high levels of genetic diversity compared to the northern populations. Conclusion This study revealed the effects of foreign queen importations on Syrian bee populations, especially for the region of Tartus, where extensive introgression of A. m. anatolica and/or A. m. caucasica alleles were identified. The policy of creating genetic conservation centers for the Syrian subspecies should take into consideration the influence of the oriental lineage O from the northern Syrian border and the large population of genetically divergent indigenous honeybees located in southern Syria. PMID:24314104

  19. Single-Cell RNA-Seq Reveals Hypothalamic Cell Diversity.

    PubMed

    Chen, Renchao; Wu, Xiaoji; Jiang, Lan; Zhang, Yi

    2017-03-28

    The hypothalamus is one of the most complex brain structures involved in homeostatic regulation. Defining cell composition and identifying cell-type-specific transcriptional features of the hypothalamus is essential for understanding its functions and related disorders. Here, we report single-cell RNA sequencing results of adult mouse hypothalamus, which defines 11 non-neuronal and 34 neuronal cell clusters with distinct transcriptional signatures. Analyses of cell-type-specific transcriptomes reveal gene expression dynamics underlying oligodendrocyte differentiation and tanycyte subtypes. Additionally, data analysis provides a comprehensive view of neuropeptide expression across hypothalamic neuronal subtypes and uncover Crabp1(+) and Pax6(+) neuronal populations in specific hypothalamic sub-regions. Furthermore, we found food deprivation exhibited differential transcriptional effects among the different neuronal subtypes, suggesting functional specification of various neuronal subtypes. Thus, the work provides a comprehensive transcriptional perspective of adult hypothalamus, which serves as a valuable resource for dissecting cell-type-specific functions of this complex brain region.

  20. Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners

    PubMed Central

    Strübbe, Gero; Popp, Christian; Schmidt, Alexander; Pauli, Andrea; Ringrose, Leonie; Beisel, Christian; Paro, Renato

    2011-01-01

    The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function. PMID:21415365

  1. Leydig Cell Hyperplasia Revealed by Gynecomastia

    PubMed Central

    Tazi, Mohamed Fadl; Mellas, Soufiane; El Fassi, Mohamed Jamal; Farih, Moulay Hassan

    2008-01-01

    Leydig cell tumors are rare and represent 1% to 3% of all tumors of the testis. Leydig cell tumors affect males at any age, but there are 2 peak periods of incidence: between 5 and 10 years and between 25 and 35 years. Their main clinical presentation is a testicular mass associated with endocrinal manifestations that are variable according to age and appearance of the tumor. Our patient, a 17-year-old adolescent, presented with an isolated and painless hypertrophy of the right mammary gland. Clinical examination found gynecomastia and no testicular mass. Hormonal levels and tumor markers were normal. Testicular sonography showed an ovular and homogeneous right intratesticular mass 6 mm in diameter. We treated the patient with an inguinal right orchidectomy. The anatomopathological study found a nodule of Leydig cell hyperplasia. The patient recovered without recurrence at 8-month follow-up. The patient opted for mammoplasty 2 months after his orchidectomy rather than wait for the spontaneous gradual regression of his gynecomastia, which requires at least 1 year. Leydig cell hyperplasia manifests in the adult by signs of hypogonadism, most frequently gynecomastia. Although many teams prefer total orchidectomy because of the diagnostic difficulty associated with malignant forms, simple subcapsular orchidectomy should become the first-line treatment, provided it be subsequently followed by close surveillance, as it preserves maximum fertility, and these tumors usually resolve favorably. PMID:18660859

  2. Leydig cell hyperplasia revealed by gynecomastia.

    PubMed

    Tazi, Mohamed Fadl; Mellas, Soufiane; El Fassi, Mohamed Jamal; Farih, Moulay Hassan

    2008-01-01

    Leydig cell tumors are rare and represent 1% to 3% of all tumors of the testis. Leydig cell tumors affect males at any age, but there are 2 peak periods of incidence: between 5 and 10 years and between 25 and 35 years. Their main clinical presentation is a testicular mass associated with endocrinal manifestations that are variable according to age and appearance of the tumor. Our patient, a 17-year-old adolescent, presented with an isolated and painless hypertrophy of the right mammary gland. Clinical examination found gynecomastia and no testicular mass. Hormonal levels and tumor markers were normal. Testicular sonography showed an ovular and homogeneous right intratesticular mass 6 mm in diameter. We treated the patient with an inguinal right orchidectomy. The anatomopathological study found a nodule of Leydig cell hyperplasia. The patient recovered without recurrence at 8-month follow-up. The patient opted for mammoplasty 2 months after his orchidectomy rather than wait for the spontaneous gradual regression of his gynecomastia, which requires at least 1 year. Leydig cell hyperplasia manifests in the adult by signs of hypogonadism, most frequently gynecomastia. Although many teams prefer total orchidectomy because of the diagnostic difficulty associated with malignant forms, simple subcapsular orchidectomy should become the first-line treatment, provided it be subsequently followed by close surveillance, as it preserves maximum fertility, and these tumors usually resolve favorably.

  3. Batteries and fuel cells working group report

    SciTech Connect

    Eberhardt, J. . Office of Advanced Transportation Materials); Landgrebe, A. . Electric and Hybrid Propulsion Systems); Lemons, R.; Wilson, M. ); MacAurther, D. (CH

    1991-01-01

    Electrochemical energy systems are dominated by interfacial phenomena. Catalysis, corrosion, electrical and ionic contact, and wetting behavior are critical to the performance of fuel cells and batteries. Accordingly, development of processing techniques to control these surface properties is important to successful commercialization of advanced batteries and fuel cells. Many of the surface processing issues are specific to a particular electrochemical system. Therefore, the working group focused on systems that are of specific interest to DOE/Conservation and Renewable Energy. These systems addressed were: Polymer Electrolyte Membrane (PEM) Fuel Cells, Direct Methanol Oxidation (DMO) Fuel Cells, and Lithium/Polymer Batteries. The approach used by the working group for each of these systems was to follow the current path through the system and to identify the principal interfaces. The function of each interface was specified together with its desired properties. The degree to which surface properties limit performance in present systems was rated. Finally, the surface processing needs associated with the performance limiting interfaces were identified. This report summarizes this information.

  4. Batteries and fuel cells working group report

    SciTech Connect

    Eberhardt, J.; Landgrebe, A.; Lemons, R.; Wilson, M.; MacAurther, D.; Savenell, R.; Swathirajan, S.; Wilson, D.

    1991-12-31

    Electrochemical energy systems are dominated by interfacial phenomena. Catalysis, corrosion, electrical and ionic contact, and wetting behavior are critical to the performance of fuel cells and batteries. Accordingly, development of processing techniques to control these surface properties is important to successful commercialization of advanced batteries and fuel cells. Many of the surface processing issues are specific to a particular electrochemical system. Therefore, the working group focused on systems that are of specific interest to DOE/Conservation and Renewable Energy. These systems addressed were: Polymer Electrolyte Membrane (PEM) Fuel Cells, Direct Methanol Oxidation (DMO) Fuel Cells, and Lithium/Polymer Batteries. The approach used by the working group for each of these systems was to follow the current path through the system and to identify the principal interfaces. The function of each interface was specified together with its desired properties. The degree to which surface properties limit performance in present systems was rated. Finally, the surface processing needs associated with the performance limiting interfaces were identified. This report summarizes this information.

  5. Computer simulations of cellular group selection reveal mechanism for sustaining cooperation.

    PubMed

    Markvoort, Albert J; Sinai, Sam; Nowak, Martin A

    2014-09-21

    We present a computer simulation of group selection that is inspired by proto-cell division. Two types of replicating molecules, cooperators and defectors, reside inside membrane bound compartments. Cooperators pay a cost for other replicators in the cell to receive a benefit. Defectors pay no cost and distribute no benefits. The total population size fluctuates as a consequence of births and deaths of individual replicators. Replication requires activated substrates that are generated at a constant rate. Our model includes mutation between cooperators and defectors and selection on two levels: within proto-cells and between proto-cells. We find surprising similarities and differences between models with and without cell death. In both cases, a necessary requirement for group selection to favor some level of cooperation is the continuous formation of a minimum fraction of pure cooperator groups. Subsequently these groups become undermined by defectors, because of mutation and selection within cells. Cell division mechanisms which generate pure cooperator groups more efficiently are stronger promoters of cooperation. For example, division of a proto-cell into many daughter cells is more powerful in enhancing cooperation than division into two daughter cells. Our model differs from previous studies of group selection in that we explore a variety of different features and relax several restrictive assumptions that would be needed for analytic calculations.

  6. Arc spot grouping: An entanglement of arc spot cells

    SciTech Connect

    Kajita, Shin; Hwangbo, Dogyun; Ohno, Noriyasu; Tsventoukh, Mikhail M.; Barengolts, Sergey A.

    2014-12-21

    In recent experiments, clear transitions in velocity and trail width of an arc spot initiated on nanostructured tungsten were observed on the boundary of the thick and thin nanostructured layer regions. The velocity of arc spot was significantly decreased on the thick nanostructured region. It was suggested that the grouping decreased the velocity of arc spot. In this study, we try to explain the phenomena using a simple random walk model that has properties of directionality and self-avoidance. And grouping feature was added by installing an attractive force between spot cells with dealing with multi-spots. It was revealed that an entanglement of arc spot cells decreased the spot velocity, and spot cells tend to stamp at the same location many times.

  7. [Eosinophilic pneumonia revealing B-cell non-Hodgkin lymphoma].

    PubMed

    Fikal, Siham; Sajiai, Hafsa; Serhane, Hind; Aitbatahar, Salma; Amro, Lamyae

    2016-01-01

    The diagnosis of eosinophilic pneumonia is rare and malignant etiology remains exceptional. Eosinophilic pneumonia etiology varies and is mainly dominated by allergic and drug causes. We report the case of a 61-year-old patient with B-cell non-Hodgkin lymphoma revealed by eosinophilic pneumonia. The diagnosis of eosinophilic pneumonia was confirmed by eosinophil count of 56% in bronchoalveolar lavage. Immunohistochemical examination of bone marrow biopsy revealed malignant Small B cells non-Hodgkin lymphoma.

  8. Phylogenetic analysis of vertebrate CXC chemokines reveals novel lineage specific groups in teleost fish.

    PubMed

    Chen, Jun; Xu, Qiaoqing; Wang, Tiehui; Collet, Bertrand; Corripio-Miyar, Yolanda; Bird, Steve; Xie, Ping; Nie, Pin; Secombes, Christopher J; Zou, Jun

    2013-10-01

    In this study, we have identified 421 molecules across the vertebrate spectrum and propose a unified nomenclature for CXC chemokines in fish, amphibians and reptiles based on phylogenetic analysis. Expanding on earlier studies in teleost fish, lineage specific CXC chemokines that have no apparent homologues in mammals were confirmed. Furthermore, in addition to the two subgroups of the CXCL8 homologues known in teleost fish, a third group was identified (termed CXCL8_L3), as was a further subgroup of the fish CXC genes related to CXCL11. Expression of the CXC chemokines found in rainbow trout, Oncorhynchus mykiss, was studied in response to stimulation with inflammatory and antiviral cytokines, and bacterial. Tissue distribution analysis revealed distinct expression profiles for these trout CXC chemokines. Lastly three of the trout chemokines, including two novel fish specific CXC chemokines containing three pairs of cysteines, were produced as recombinant proteins and their effect on trout leucocyte migration studied. These molecules increased the relative expression of CD4 and MCSFR in migrated cells in an in vitro chemotaxis assay.

  9. Single-cell chromatin accessibility reveals principles of regulatory variation.

    PubMed

    Buenrostro, Jason D; Wu, Beijing; Litzenburger, Ulrike M; Ruff, Dave; Gonzales, Michael L; Snyder, Michael P; Chang, Howard Y; Greenleaf, William J

    2015-07-23

    Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'.

  10. Automated live cell imaging systems reveal dynamic cell behavior.

    PubMed

    Chirieleison, Steven M; Bissell, Taylor A; Scelfo, Christopher C; Anderson, Jordan E; Li, Yong; Koebler, Doug J; Deasy, Bridget M

    2011-07-01

    Automated time-lapsed microscopy provides unique research opportunities to visualize cells and subcellular components in experiments with time-dependent parameters. As accessibility to these systems is increasing, we review here their use in cell science with a focus on stem cell research. Although the use of time-lapsed imaging to answer biological questions dates back nearly 150 years, only recently have the use of an environmentally controlled chamber and robotic stage controllers allowed for high-throughput continuous imaging over long periods at the cell and subcellular levels. Numerous automated imaging systems are now available from both companies that specialize in live cell imaging and from major microscope manufacturers. We discuss the key components of robots used for time-lapsed live microscopic imaging, and the unique data that can be obtained from image analysis. We show how automated features enhance experimentation by providing examples of uniquely quantified proliferation and migration live cell imaging data. In addition to providing an efficient system that drastically reduces man-hours and consumes fewer laboratory resources, this technology greatly enhances cell science by providing a unique dataset of temporal changes in cell activity. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  11. Fluorescent aminoglycosides reveal intracellular trafficking routes in mechanosensory hair cells

    PubMed Central

    Hailey, Dale W.; Esterberg, Robert; Linbo, Tor H.; Rubel, Edwin W.; Raible, David W.

    2016-01-01

    Aminoglycosides (AGs) are broad-spectrum antibiotics that are associated with kidney damage, balance disorders, and permanent hearing loss. This damage occurs primarily by killing of proximal tubule kidney cells and mechanosensory hair cells, though the mechanisms underlying cell death are not clear. Imaging molecules of interest in living cells can elucidate how molecules enter cells, traverse intracellular compartments, and interact with sites of activity. Here, we have imaged fluorescently labeled AGs in live zebrafish mechanosensory hair cells. We determined that AGs enter hair cells via both nonendocytic and endocytic pathways. Both routes deliver AGs from the extracellular space to lysosomes, and structural differences between AGs alter the efficiency of this delivery. AGs with slower delivery to lysosomes were immediately toxic to hair cells, and impeding lysosome delivery increased AG-induced death. Therefore, pro-death cascades induced at early time points of AG exposure do not appear to derive from the lysosome. Our findings help clarify how AGs induce hair cell death and reveal properties that predict toxicity. Establishing signatures for AG toxicity may enable more efficient evaluation of AG treatment paradigms and structural modifications to reduce hair cell damage. Further, this work demonstrates how following fluorescently labeled drugs at high resolution in living cells can reveal important details about how drugs of interest behave. PMID:27991862

  12. Macrophage characteristics of stem cells revealed by transcriptome profiling

    SciTech Connect

    Charriere, Guillaume M.; Cousin, Beatrice; Arnaud, Emmanuelle; Saillan-Barreau, Corinne; Andre, Mireille; Massoudi, Ali; Dani, Christian; Penicaud, Luc; Casteilla, Louis . E-mail: casteil@toulouse.inserm.fr

    2006-10-15

    We previously showed that the phenotypes of adipocyte progenitors and macrophages were close. Using functional analyses and microarray technology, we first tested whether this intriguing relationship was specific to adipocyte progenitors or could be shared with other progenitors. Measurements of phagocytic activity and gene profiling analysis of different progenitor cells revealed that the latter hypothesis should be retained. These results encouraged us to pursue and to confirm our analysis with a gold-standard stem cell population, embryonic stem cells or ESC. The transcriptomic profiles of ESC and macrophages were clustered together, unlike differentiated ESC. In addition, undifferentiated ESC displayed higher phagocytic activity than other progenitors, and they could phagocytoze apoptotic bodies. These data suggest that progenitors and stem cells share some characteristics of macrophages. This opens new perspectives on understanding stem cell phenotype and functionalities such as a putative role of stem cells in tissue remodeling by discarding dead cells but also their immunomodulation or fusion properties.

  13. High-frequency microrheology reveals cytoskeleton dynamics in living cells

    NASA Astrophysics Data System (ADS)

    Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

    2017-08-01

    Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

  14. Morphogenesis and Cell Fate Determination within the Adaxial Cell Equivalence Group of the Zebrafish Myotome

    PubMed Central

    Nguyen-Chi, Mai E.; Bryson-Richardson, Robert; Sonntag, Carmen; Hall, Thomas E.; Gibson, Abigail; Sztal, Tamar; Chua, Wendy; Schilling, Thomas F.; Currie, Peter D.

    2012-01-01

    One of the central questions of developmental biology is how cells of equivalent potential—an equivalence group—come to adopt specific cellular fates. In this study we have used a combination of live imaging, single cell lineage analyses, and perturbation of specific signaling pathways to dissect the specification of the adaxial cells of the zebrafish embryo. We show that the adaxial cells are myogenic precursors that form a cell fate equivalence group of approximately 20 cells that consequently give rise to two distinct sub-types of muscle fibers: the superficial slow muscle fibers (SSFs) and muscle pioneer cells (MPs), distinguished by specific gene expression and cell behaviors. Using a combination of live imaging, retrospective and indicative fate mapping, and genetic studies, we show that MP and SSF precursors segregate at the beginning of segmentation and that they arise from distinct regions along the anterior-posterior (AP) and dorsal-ventral (DV) axes of the adaxial cell compartment. FGF signaling restricts MP cell fate in the anterior-most adaxial cells in each somite, while BMP signaling restricts this fate to the middle of the DV axis. Thus our results reveal that the synergistic actions of HH, FGF, and BMP signaling independently create a three-dimensional (3D) signaling milieu that coordinates cell fate within the adaxial cell equivalence group. PMID:23133395

  15. Music-supported motor training after stroke reveals no superiority of synchronization in group therapy

    PubMed Central

    Van Vugt, Floris T.; Ritter, Juliane; Rollnik, Jens D.; Altenmüller, Eckart

    2014-01-01

    Background: Music-supported therapy has been shown to be an effective tool for rehabilitation of motor deficits after stroke. A unique feature of music performance is that it is inherently social: music can be played together in synchrony. Aim: The present study explored the potential of synchronized music playing during therapy, asking whether synchronized playing could improve fine motor rehabilitation and mood. Method: Twenty-eight patients in neurological early rehabilitation after stroke with no substantial previous musical training were included. Patients learned to play simple finger exercises and familiar children's songs on the piano for 10 sessions of half an hour. Patients first received three individual therapy sessions and then continued in pairs. The patient pairs were divided into two groups. Patients in one group played synchronously (together group) whereas the patients in the other group played one after the other (in-turn group). To assess fine motor skill recovery the patients performed standard clinical tests such as the nine-hole-pegboard test (9HPT) and index finger-tapping speed and regularity, and metronome-paced finger tapping. Patients' mood was established using the Profile of Mood States (POMS). Results: Both groups showed improvements in fine motor control. In metronome-paced finger tapping, patients in both groups improved significantly. Mood tests revealed reductions in depression and fatigue in both groups. During therapy, patients in the in-turn group rated their partner as more sympathetic than the together-group in a visual-analog scale. Conclusions: Our results suggest that music-supported stroke rehabilitation can improve fine motor control and mood not only individually but also in patient pairs. Patients who were playing in turn rather than simultaneously tended to reveal greater improvement in fine motor skill. We speculate that patients in the former group may benefit from the opportunity to learn from observation. PMID

  16. EEG reveals an early influence of social conformity on visual processing in group pressure situations.

    PubMed

    Trautmann-Lengsfeld, Sina Alexa; Herrmann, Christoph Siegfried

    2013-01-01

    Humans are social beings and often have to perceive and perform within groups. In conflict situations, this puts them under pressure to either adhere to the group opinion or to risk controversy with the group. Psychological experiments have demonstrated that study participants adapt to erroneous group opinions in visual perception tasks, which they can easily solve correctly when performing on their own. Until this point, however, it is unclear whether this phenomenon of social conformity influences early stages of perception that might not even reach awareness or later stages of conscious decision-making. Using electroencephalography, this study has revealed that social conformity to the wrong group opinion resulted in a decrease of the posterior-lateral P1 in line with a decrease of the later centro-parietal P3. These results suggest that group pressure situations impact early unconscious visual perceptual processing, which results in a later diminished stimulus discrimination and an adaptation even to the wrong group opinion. These findings might have important implications for understanding social behavior in group settings and are discussed within the framework of social influence on eyewitness testimony.

  17. Core/shell-type nanorods of Tb3+-doped LaPO4, modified with amine groups, revealing reduced cytotoxicity

    NASA Astrophysics Data System (ADS)

    Runowski, Marcin; Dąbrowska, Krystyna; Grzyb, Tomasz; Miernikiewicz, Paulina; Lis, Stefan

    2013-11-01

    A simple co-precipitation reaction between Ln3+ cations (Ln = lanthanide) and phosphate ions in the presence of polyethylene glycol (PEG), including post-treatment under hydrothermal conditions, leads to the formation of Tb3+-doped LaPO4 crystalline nanorods. The nanoparticles obtained can be successfully coated with amorphous and porous silica, forming core/shell-type nanorods. Both products reveal intensive green luminescence under UV lamp irradiation. The surface of the core/shell-type product can also be modified with -NH2 groups via silylation procedure, using 3-aminopropyltriethoxysilane as a modifier. Powder X-ray diffraction, transmission electron microscopy, and scanning electron microscopy confirm the desired structure and needle-like shape of the products synthesized. Fourier transform infrared spectroscopy and specific surface area measurements by Brunauer-Emmett-Teller method reveal a successful surface modification with amine groups of the core/shell-type nanoparticles prepared. The nanomaterials synthesized exhibit green luminescence characteristic of Tb3+ ions, as solid powders and aqueous colloids, examined by spectrofluorometry. The in vitro cytotoxicity studies reveal different degree toxicity of the products. LaPO4:Tb3+@SiO2@NH2 exhibits the smallest toxicity against B16F0 mouse melanoma cancer cells and human skin microvascular endothelial cell lines, in contrast to the most toxic LaPO4:Tb3+@SiO2.

  18. Spontaneous Movements of a Computer Mouse Reveal Egoism and In-group Favoritism.

    PubMed

    Maliszewski, Norbert; Wojciechowski, Łukasz; Suszek, Hubert

    2017-01-01

    The purpose of the project was to assess whether the first spontaneous movements of a computer mouse, when making an assessment on a scale presented on the screen, may express a respondent's implicit attitudes. In Study 1, the altruistic behaviors of 66 students were assessed. The students were led to believe that the task they were performing was also being performed by another person and they were asked to distribute earnings between themselves and the partner. The participants performed the tasks under conditions with and without distractors. With the distractors, in the first few seconds spontaneous mouse movements on the scale expressed a selfish distribution of money, while later the movements gravitated toward more altruism. In Study 2, 77 Polish students evaluated a painting by a Polish/Jewish painter on a scale. They evaluated it under conditions of full or distracted cognitive abilities. Spontaneous movements of the mouse on the scale were analyzed. In addition, implicit attitudes toward both Poles and Jews were measured with the Implicit Association Test (IAT). A significant association between implicit attitudes (IAT) and spontaneous evaluation of images using a computer mouse was observed in the group with the distractor. The participants with strong implicit in-group favoritism of Poles revealed stronger preference for the Polish painter's work in the first few seconds of mouse movement. Taken together, these results suggest that spontaneous mouse movements may reveal egoism (in-group favoritism), i.e., processes that were not observed in the participants' final decisions (clicking on the scale).

  19. Raman spectrum reveals Mesenchymal stem cells inhibiting HL60 cells growth

    NASA Astrophysics Data System (ADS)

    Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; Lu, Xiaoxu; Tian, Jindong; Fan, Jinping; Zhong, Liyun

    2017-04-01

    Though some research results reveals that Mesenchymal stem cells (MSCs) have the ability of inhibiting tumor cells proliferation, it remains controversial about the precise interaction mechanism during MSCs and tumor cells co-culture. In this study, combing Raman spectroscopic data and principle component analysis (PCA), the biochemical changes of MSCs or Human promyelocytic leukemia (HL60) cells during their co-culture were presented. The obtained results showed that some main Raman peaks of HL60 assigned to nucleic acids or proteins were greatly higher in intensity in the late stage of co-culture than those in the early stage of co-culture while they were still lower relative to the control group, implicating that the effect of MSCs inhibiting HL60 proliferation appeared in the early stage but gradually lost the inhibiting ability in the late stage of co-culture. Moreover, some other peaks of HL60 assigned to proteins were decreased in intensity in the early stage of co-culture relative to the control group but rebounded to the level similar to the control group in the late stage, showing that the content and structure changes of these proteins might be generated in the early stage but returned to the original state in the late stage of co-culture. As a result, in the early stage of MSCs-HL60 co-culture, along with the level of Akt phosphorylation of HL60 was lowered relative to its control group, the proliferation rate of HL60 cells was decreased. And in the late stage of co-culture, along with the level of Akt phosphorylation was rebounded, the reverse transfer of Raman peaks within 875-880 cm- 1 appeared, thus MSCs lost the ability to inhibit HL60 growth and HL60 proliferation was increased. In addition, it was observed that the peak at 811 cm- 1, which is a marker of RNA, was higher in intensity in the late stage than that in the control group, indicating that MSCs might be differentiated into myofibroblast-like MSCs. In addition, PCA results also exhibited

  20. Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

    PubMed Central

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit; Saari, Heikki; Ibañez, Elisa Lazaro; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2015-01-01

    Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level. PMID:26649679

  1. Analysis of virus genomes from glacial environments reveals novel virus groups with unusual host interactions

    PubMed Central

    Bellas, Christopher M.; Anesio, Alexandre M.; Barker, Gary

    2015-01-01

    Microbial communities in glacial ecosystems are diverse, active, and subjected to strong viral pressures and infection rates. In this study we analyse putative virus genomes assembled from three dsDNA viromes from cryoconite hole ecosystems of Svalbard and the Greenland Ice Sheet to assess the potential hosts and functional role viruses play in these habitats. We assembled 208 million reads from the virus-size fraction and developed a procedure to select genuine virus scaffolds from cellular contamination. Our curated virus library contained 546 scaffolds up to 230 Kb in length, 54 of which were circular virus consensus genomes. Analysis of virus marker genes revealed a wide range of viruses had been assembled, including bacteriophages, cyanophages, nucleocytoplasmic large DNA viruses and a virophage, with putative hosts identified as Cyanobacteria, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes, eukaryotic algae and amoebae. Whole genome comparisons revealed the majority of circular genome scaffolds (CGS) formed 12 novel groups, two of which contained multiple phage members with plasmid-like properties, including a group of phage-plasmids possessing plasmid-like partition genes and toxin-antitoxin addiction modules to ensure their replication and a satellite phage-plasmid group. Surprisingly we also assembled a phage that not only encoded plasmid partition genes, but a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas adaptive bacterial immune system. One of the spacers was an exact match for another phage in our virome, indicating that in a novel use of the system, the lysogen was potentially capable of conferring immunity on its bacterial host against other phage. Together these results suggest that highly novel and diverse groups of viruses are present in glacial environments, some of which utilize very unusual life strategies and genes to control their replication and maintain a long-term relationship with their hosts

  2. Analysis of virus genomes from glacial environments reveals novel virus groups with unusual host interactions.

    PubMed

    Bellas, Christopher M; Anesio, Alexandre M; Barker, Gary

    2015-01-01

    Microbial communities in glacial ecosystems are diverse, active, and subjected to strong viral pressures and infection rates. In this study we analyse putative virus genomes assembled from three dsDNA viromes from cryoconite hole ecosystems of Svalbard and the Greenland Ice Sheet to assess the potential hosts and functional role viruses play in these habitats. We assembled 208 million reads from the virus-size fraction and developed a procedure to select genuine virus scaffolds from cellular contamination. Our curated virus library contained 546 scaffolds up to 230 Kb in length, 54 of which were circular virus consensus genomes. Analysis of virus marker genes revealed a wide range of viruses had been assembled, including bacteriophages, cyanophages, nucleocytoplasmic large DNA viruses and a virophage, with putative hosts identified as Cyanobacteria, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes, eukaryotic algae and amoebae. Whole genome comparisons revealed the majority of circular genome scaffolds (CGS) formed 12 novel groups, two of which contained multiple phage members with plasmid-like properties, including a group of phage-plasmids possessing plasmid-like partition genes and toxin-antitoxin addiction modules to ensure their replication and a satellite phage-plasmid group. Surprisingly we also assembled a phage that not only encoded plasmid partition genes, but a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas adaptive bacterial immune system. One of the spacers was an exact match for another phage in our virome, indicating that in a novel use of the system, the lysogen was potentially capable of conferring immunity on its bacterial host against other phage. Together these results suggest that highly novel and diverse groups of viruses are present in glacial environments, some of which utilize very unusual life strategies and genes to control their replication and maintain a long-term relationship with their hosts.

  3. Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.

    PubMed

    Colarusso, Pina; Spring, Kenneth R

    2002-02-01

    High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane.

  4. Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.

    PubMed Central

    Colarusso, Pina; Spring, Kenneth R

    2002-01-01

    High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane. PMID:11806917

  5. Revealing Transcriptome Landscape of Mouse Spermatogonial Cells by Tiling Microarray

    PubMed Central

    Lee, Tin-Lap.; Rennert, Owen M.; Chan, Wai-Yee.

    2014-01-01

    Summary Spermatogenesis is a highly regulated developmental process by which spermatogonia develop into mature spermatozoa. This process involves many testis- or male germ cell-specific events through tightly regulated gene expression programs. In the past decade the advent of microarray technologies has allowed functional genomic studies of male germ cell development, resulting in the identification of genes governing various processes. A major limitation with conventional gene expression microarray is that there is a bias from gene probe design. The gene probes for expression microarrays are usually represented by a small number probes located at the 3’ end of a transcirpt. Tiling microarrays eliminate such issue by interrogating the genome in an unbiased fashion through probes tiled for the entire genome. These arrays provide a higher genomic resolution and allow identification of novel transcripts. To reveal the complexity of the genomic landscape of developing male germ cells, we applied tiling microarray to evaluate the transcriptome in spermatogonial cells. Over 50% of the mouse and rat genome are expressed during testicular development. More than 47% of transcripts are uncharacterized. The results suggested the transcription machinery in spermaotogonial cells are more complex than previously envisioned. PMID:22144238

  6. Cell cycle synchronization reveals greater G2/M-phase accumulation of lung epithelial cells exposed to titanium dioxide nanoparticles.

    PubMed

    Medina-Reyes, Estefany I; Bucio-López, Laura; Freyre-Fonseca, Verónica; Sánchez-Pérez, Yesennia; García-Cuéllar, Claudia M; Morales-Bárcenas, Rocío; Pedraza-Chaverri, José; Chirino, Yolanda I

    2015-03-01

    Titanium dioxide has been classified in the 2B group as a possible human carcinogen by the International Agency for Research on Cancer, and amid concerns of its exposure, cell cycle alterations are an important one. However, several studies show inconclusive effects, mainly because it is difficult to compare cell cycle effects caused by TiO2 nanoparticle (NP) exposure between different shapes and sizes of NP, cell culture types, and time of exposure. In addition, cell cycle is frequently analyzed without cell cycle synchronization, which may also mask some effects. We hypothesized that synchronization after TiO2 NP exposure could reveal dissimilar cell cycle progression when compared with unsynchronized cell population. To test our hypothesis, we exposed lung epithelial cells to 1 and 10 μg/cm(2) TiO2 NPs for 7 days and one population was synchronized by serum starvation and inhibition of ribonucleotide reductase using hydroxyurea. Another cell population was exposed to TiO2 NPs under the same experimental conditions, but after treatments, cell cycle was analyzed without synchronization. Our results showed that TiO2 NP-exposed cells without synchronization had no changes in cell cycle distribution; however, cell population synchronized after 1 and 10 μg/cm(2) TiO2 NP treatment showed a 1.5-fold and 1.66-fold increase, respectively, in proliferation. Synchronized cells also reveal a faster capability of TiO2 NP-exposed cells to increase cell population in the G2/M phase in the following 9 h after synchronization. We conclude that synchronization discloses a greater percentage of cells in the G2/M phase and higher proliferation than TiO2 NP-synchronized cells.

  7. Spontaneous Movements of a Computer Mouse Reveal Egoism and In-group Favoritism

    PubMed Central

    Maliszewski, Norbert; Wojciechowski, Łukasz; Suszek, Hubert

    2017-01-01

    The purpose of the project was to assess whether the first spontaneous movements of a computer mouse, when making an assessment on a scale presented on the screen, may express a respondent’s implicit attitudes. In Study 1, the altruistic behaviors of 66 students were assessed. The students were led to believe that the task they were performing was also being performed by another person and they were asked to distribute earnings between themselves and the partner. The participants performed the tasks under conditions with and without distractors. With the distractors, in the first few seconds spontaneous mouse movements on the scale expressed a selfish distribution of money, while later the movements gravitated toward more altruism. In Study 2, 77 Polish students evaluated a painting by a Polish/Jewish painter on a scale. They evaluated it under conditions of full or distracted cognitive abilities. Spontaneous movements of the mouse on the scale were analyzed. In addition, implicit attitudes toward both Poles and Jews were measured with the Implicit Association Test (IAT). A significant association between implicit attitudes (IAT) and spontaneous evaluation of images using a computer mouse was observed in the group with the distractor. The participants with strong implicit in-group favoritism of Poles revealed stronger preference for the Polish painter’s work in the first few seconds of mouse movement. Taken together, these results suggest that spontaneous mouse movements may reveal egoism (in-group favoritism), i.e., processes that were not observed in the participants’ final decisions (clicking on the scale). PMID:28163689

  8. Visual target modulation of functional connectivity networks revealed by self-organizing group ICA.

    PubMed

    van de Ven, Vincent; Bledowski, Christoph; Prvulovic, David; Goebel, Rainer; Formisano, Elia; Di Salle, Francesco; Linden, David E J; Esposito, Fabrizio

    2008-12-01

    We applied a data-driven analysis based on self-organizing group independent component analysis (sogICA) to fMRI data from a three-stimulus visual oddball task. SogICA is particularly suited to the investigation of the underlying functional connectivity and does not rely on a predefined model of the experiment, which overcomes some of the limitations of hypothesis-driven analysis. Unlike most previous applications of ICA in functional imaging, our approach allows the analysis of the data at the group level, which is of particular interest in high order cognitive studies. SogICA is based on the hierarchical clustering of spatially similar independent components, derived from single subject decompositions. We identified four main clusters of components, centered on the posterior cingulate, bilateral insula, bilateral prefrontal cortex, and right posterior parietal and prefrontal cortex, consistently across all participants. Post hoc comparison of time courses revealed that insula, prefrontal cortex and right fronto-parietal components showed higher activity for targets than for distractors. Activation for distractors was higher in the posterior cingulate cortex, where deactivation was observed for targets. While our results conform to previous neuroimaging studies, they also complement conventional results by showing functional connectivity networks with unique contributions to the task that were consistent across subjects. SogICA can thus be used to probe functional networks of active cognitive tasks at the group-level and can provide additional insights to generate new hypotheses for further study. Copyright 2007 Wiley-Liss, Inc.

  9. Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

    PubMed

    Pei, Weike; Feyerabend, Thorsten B; Rössler, Jens; Wang, Xi; Postrach, Daniel; Busch, Katrin; Rode, Immanuel; Klapproth, Kay; Dietlein, Nikolaus; Quedenau, Claudia; Chen, Wei; Sauer, Sascha; Wolf, Stephan; Höfer, Thomas; Rodewald, Hans-Reimer

    2017-08-24

    Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

  10. Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

    PubMed

    von Burg, Nicole; Chappaz, Stéphane; Baerenwaldt, Anne; Horvath, Edit; Bose Dasgupta, Somdeb; Ashok, Devika; Pieters, Jean; Tacchini-Cottier, Fabienne; Rolink, Antonius; Acha-Orbea, Hans; Finke, Daniela

    2014-09-02

    Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1β stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

  11. Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

    PubMed

    Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

    2017-05-31

    Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

  12. Tree ferns: monophyletic groups and their relationships as revealed by four protein-coding plastid loci.

    PubMed

    Korall, Petra; Pryer, Kathleen M; Metzgar, Jordan S; Schneider, Harald; Conant, David S

    2006-06-01

    Tree ferns are a well-established clade within leptosporangiate ferns. Most of the 600 species (in seven families and 13 genera) are arborescent, but considerable morphological variability exists, spanning the giant scaly tree ferns (Cyatheaceae), the low, erect plants (Plagiogyriaceae), and the diminutive endemics of the Guayana Highlands (Hymenophyllopsidaceae). In this study, we investigate phylogenetic relationships within tree ferns based on analyses of four protein-coding, plastid loci (atpA, atpB, rbcL, and rps4). Our results reveal four well-supported clades, with genera of Dicksoniaceae (sensu ) interspersed among them: (A) (Loxomataceae, (Culcita, Plagiogyriaceae)), (B) (Calochlaena, (Dicksonia, Lophosoriaceae)), (C) Cibotium, and (D) Cyatheaceae, with Hymenophyllopsidaceae nested within. How these four groups are related to one other, to Thyrsopteris, or to Metaxyaceae is weakly supported. Our results show that Dicksoniaceae and Cyatheaceae, as currently recognised, are not monophyletic and new circumscriptions for these families are needed.

  13. Natural grouping of neural responses reveals spatially segregated clusters in prearcuate cortex.

    PubMed

    Kiani, Roozbeh; Cueva, Christopher J; Reppas, John B; Peixoto, Diogo; Ryu, Stephen I; Newsome, William T

    2015-03-18

    A fundamental challenge in studying the frontal lobe is to parcellate this cortex into "natural" functional modules despite the absence of topographic maps, which are so helpful in primary sensory areas. Here we show that unsupervised clustering algorithms, applied to 96-channel array recordings from prearcuate gyrus, reveal spatially segregated subnetworks that remain stable across behavioral contexts. Looking for natural groupings of neurons based on response similarities, we discovered that the recorded area includes at least two spatially segregated subnetworks that differentially represent behavioral choice and reaction time. Importantly, these subnetworks are detectable during different behavioral states and, surprisingly, are defined better by "common noise" than task-evoked responses. Our parcellation process works well on "spontaneous" neural activity, and thus bears strong resemblance to the identification of "resting-state" networks in fMRI data sets. Our results demonstrate a powerful new tool for identifying cortical subnetworks by objective classification of simultaneously recorded electrophysiological activity.

  14. Event-related potentials can reveal differences between two decision-making groups.

    PubMed

    Cutmore, T R; Muckert, T D

    1998-02-01

    Previous research has shown that a complex decision is dependent on an underlying utility metric that is used by decision making processes to accumulate preference for one alternative. This study postulated that a state of indecision may arise if this underlying metric is poorly organized. The underlying metric was examined with a paired comparison task while measuring event-related potentials (ERP) for subjects classified as 'career decided' and 'career undecided'. Stimuli for comparison were presented either sequentially or simultaneously. The simultaneous condition produced results consistent with the hypothesis that undecided subjects have a poorly organized value metric as revealed in both the behavioral data and the P3 component. A relationship between P3 amplitude and word distance on the underlying metric was found only for the decided group. This was interpreted in terms of the previously documented relationship between P3 and the constructs of decision confidence and task difficulty.

  15. Norovirus Capture with Histo-Blood Group Antigens Reveals Novel Virus-Ligand Interactions

    PubMed Central

    Harrington, Patrick R.; Vinjé, Jan; Moe, Christine L.; Baric, Ralph S.

    2004-01-01

    Noroviruses are genetically diverse, uncultivable, positive-sense RNA viruses and are the most common cause of epidemic acute gastroenteritis in humans in the United States. Recent studies of norovirus attachment in vitro by using recombinant virus-like particles (VLPs) suggest that various norovirus strains exhibit different patterns of attachment to ABH histo-blood group antigens, which are carbohydrate epitopes present in high concentrations on mucosal cell surfaces of the gut. However, attachment of live norovirus strains to histo-blood group antigens has not been investigated to date. Utilizing a newly designed magnetic bead-virus capture method, we characterized histo-blood group antigen attachment properties of various norovirus strains obtained from clinical stool specimens to compare the attachment properties of wild-type virus and VLPs and to further map norovirus attachment. Consistent with previous reports using VLPs, various strains of noroviruses exhibited different patterns of attachment to histo- blood group antigens. Norwalk virus bound specifically to H type 1, H type 3, and Leb. Two genogroup II noroviruses, one representing the Toronto genotype and the other from a novel genotype, bound specifically to Leb. A Desert Shield-like strain did not attach to H types 1, 2, or 3, H type 1 and 3 precursors, Lea, or Leb. Surprisingly, wild-type Snow Mountain virus (SMV) attached specifically to H type 3, which contradicted previous findings with SMV VLPs. On further investigation, we found that stool components promote this attachment, providing the first known observation that one or more components of human feces could promote and enhance norovirus attachment to histo-blood group antigens. PMID:14990722

  16. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state

    PubMed Central

    Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A.; Goren, Alon; Weitz, David A.; Bernstein, Bradley E.

    2015-01-01

    Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells, and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell is sparse, comprising on the order of 1000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of sub-populations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone. PMID:26458175

  17. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state.

    PubMed

    Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A; Goren, Alon; Weitz, David A; Bernstein, Bradley E

    2015-11-01

    Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.

  18. Complex evolutionary history of the Aeromonas veronii group revealed by host interaction and DNA sequence data.

    PubMed

    Silver, Adam C; Williams, David; Faucher, Joshua; Horneman, Amy J; Gogarten, J Peter; Graf, Joerg

    2011-02-16

    Aeromonas veronii biovar sobria, Aeromonas veronii biovar veronii, and Aeromonas allosaccharophila are a closely related group of organisms, the Aeromonas veronii Group, that inhabit a wide range of host animals as a symbiont or pathogen. In this study, the ability of various strains to colonize the medicinal leech as a model for beneficial symbiosis and to kill wax worm larvae as a model for virulence was determined. Isolates cultured from the leech out-competed other strains in the leech model, while most strains were virulent in the wax worms. Three housekeeping genes, recA, dnaJ and gyrB, the gene encoding chitinase, chiA, and four loci associated with the type three secretion system, ascV, ascFG, aexT, and aexU were sequenced. The phylogenetic reconstruction failed to produce one consensus tree that was compatible with most of the individual genes. The Approximately Unbiased test and the Genetic Algorithm for Recombination Detection both provided further support for differing evolutionary histories among this group of genes. Two contrasting tests detected recombination within aexU, ascFG, ascV, dnaJ, and gyrB but not in aexT or chiA. Quartet decomposition analysis indicated a complex recent evolutionary history for these strains with a high frequency of horizontal gene transfer between several but not among all strains. In this study we demonstrate that at least for some strains, horizontal gene transfer occurs at a sufficient frequency to blur the signal from vertically inherited genes, despite strains being adapted to distinct niches. Simply increasing the number of genes included in the analysis is unlikely to overcome this challenge in organisms that occupy multiple niches and can exchange DNA between strains specialized to different niches. Instead, the detection of genes critical in the adaptation to specific niches may help to reveal the physiological specialization of these strains.

  19. Complex Evolutionary History of the Aeromonas veronii Group Revealed by Host Interaction and DNA Sequence Data

    PubMed Central

    Faucher, Joshua; Horneman, Amy J.; Gogarten, J. Peter; Graf, Joerg

    2011-01-01

    Aeromonas veronii biovar sobria, Aeromonas veronii biovar veronii, and Aeromonas allosaccharophila are a closely related group of organisms, the Aeromonas veronii Group, that inhabit a wide range of host animals as a symbiont or pathogen. In this study, the ability of various strains to colonize the medicinal leech as a model for beneficial symbiosis and to kill wax worm larvae as a model for virulence was determined. Isolates cultured from the leech out-competed other strains in the leech model, while most strains were virulent in the wax worms. Three housekeeping genes, recA, dnaJ and gyrB, the gene encoding chitinase, chiA, and four loci associated with the type three secretion system, ascV, ascFG, aexT, and aexU were sequenced. The phylogenetic reconstruction failed to produce one consensus tree that was compatible with most of the individual genes. The Approximately Unbiased test and the Genetic Algorithm for Recombination Detection both provided further support for differing evolutionary histories among this group of genes. Two contrasting tests detected recombination within aexU, ascFG, ascV, dnaJ, and gyrB but not in aexT or chiA. Quartet decomposition analysis indicated a complex recent evolutionary history for these strains with a high frequency of horizontal gene transfer between several but not among all strains. In this study we demonstrate that at least for some strains, horizontal gene transfer occurs at a sufficient frequency to blur the signal from vertically inherited genes, despite strains being adapted to distinct niches. Simply increasing the number of genes included in the analysis is unlikely to overcome this challenge in organisms that occupy multiple niches and can exchange DNA between strains specialized to different niches. Instead, the detection of genes critical in the adaptation to specific niches may help to reveal the physiological specialization of these strains. PMID:21359176

  20. Biochemical and molecular tools reveal two diverse Xanthomonas groups in bananas.

    PubMed

    Adriko, J; Aritua, V; Mortensen, C N; Tushemereirwe, W K; Mulondo, A L; Kubiriba, J; Lund, O S

    2016-02-01

    Xanthomonas campestris pv. musacearum (Xcm) causing the banana Xanthomonas wilt (BXW) disease has been the main xanthomonad associated with bananas in East and Central Africa based on phenotypic and biochemical characteristics. However, biochemical methods cannot effectively distinguish between pathogenic and non-pathogenic xanthomonads. In this study, gram-negative and yellow-pigmented mucoid bacteria were isolated from BXW symptomatic and symptomless bananas collected from different parts of Uganda. Biolog, Xcm-specific (GspDm), Xanthomonas vasicola species-specific (NZ085) and Xanthomonas genus-specific (X1623) primers in PCR, and sequencing of ITS region were used to identify and characterize the isolates. Biolog tests revealed several isolates as xanthomonads. The GspDm and NZ085 primers accurately identified three isolates from diseased bananas as Xcm and these were pathogenic when re-inoculated into bananas. DNA from more isolates than those amplified by GspDm and NZ085 primers were amplified by the X1623 primers implying they are xanthomonads, these were however non-pathogenic on bananas. In the 16-23 ITS sequence based phylogeny, the pathogenic bacteria clustered together with the Xcm reference strain, while the non-pathogenic xanthomonads isolated from both BXW symptomatic and symptomless bananas clustered with group I xanthomonads. The findings reveal dynamic Xanthomonas populations in bananas, which can easily be misrepresented by only using phenotyping and biochemical tests. A combination of tools provides the most accurate identity and characterization of these plant associated bacteria. The interactions between the pathogenic and non-pathogenic xanthomonads in bananas may pave way to understanding effect of microbial interactions on BXW disease development and offer clues to biocontrol of Xcm. Copyright © 2016. Published by Elsevier GmbH.

  1. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells.

    PubMed

    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-03-29

    The 'neural plate border' of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure.

  2. Group C adenovirus DNA sequences in human lymphoid cells

    SciTech Connect

    Horvath, J.; Palkonyay, L.; Weber, J.

    1986-07-01

    Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 (Ad1), Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infectious in childhood result in the persistence of the viral genome in circulating lymphocytes.

  3. Mpath maps multi-branching single-cell trajectories revealing progenitor cell progression during development

    PubMed Central

    Chen, Jinmiao; Schlitzer, Andreas; Chakarov, Svetoslav; Ginhoux, Florent; Poidinger, Michael

    2016-01-01

    Single-cell RNA-sequencing offers unprecedented resolution of the continuum of state transition during cell differentiation and development. However, tools for constructing multi-branching cell lineages from single-cell data are limited. Here we present Mpath, an algorithm that derives multi-branching developmental trajectories using neighborhood-based cell state transitions. Applied to mouse conventional dendritic cell (cDC) progenitors, Mpath constructs multi-branching trajectories spanning from macrophage/DC progenitors through common DC progenitor to pre-dendritic cells (preDC). The Mpath-generated trajectories detect a branching event at the preDC stage revealing preDC subsets that are exclusively committed to cDC1 or cDC2 lineages. Reordering cells along cDC development reveals sequential waves of gene regulation and temporal coupling between cell cycle and cDC differentiation. Applied to human myoblasts, Mpath recapitulates the time course of myoblast differentiation and isolates a branch of non-muscle cells involved in the differentiation. Our study shows that Mpath is a useful tool for constructing cell lineages from single-cell data. PMID:27356503

  4. Revealing the hidden networks of interaction in mobile animal groups allows prediction of complex behavioral contagion

    PubMed Central

    Rosenthal, Sara Brin; Twomey, Colin R.; Hartnett, Andrew T.; Wu, Hai Shan; Couzin, Iain D.

    2015-01-01

    Coordination among social animals requires rapid and efficient transfer of information among individuals, which may depend crucially on the underlying structure of the communication network. Establishing the decision-making circuits and networks that give rise to individual behavior has been a central goal of neuroscience. However, the analogous problem of determining the structure of the communication network among organisms that gives rise to coordinated collective behavior, such as is exhibited by schooling fish and flocking birds, has remained almost entirely neglected. Here, we study collective evasion maneuvers, manifested through rapid waves, or cascades, of behavioral change (a ubiquitous behavior among taxa) in schooling fish (Notemigonus crysoleucas). We automatically track the positions and body postures, calculate visual fields of all individuals in schools of ∼150 fish, and determine the functional mapping between socially generated sensory input and motor response during collective evasion. We find that individuals use simple, robust measures to assess behavioral changes in neighbors, and that the resulting networks by which behavior propagates throughout groups are complex, being weighted, directed, and heterogeneous. By studying these interaction networks, we reveal the (complex, fractional) nature of social contagion and establish that individuals with relatively few, but strongly connected, neighbors are both most socially influential and most susceptible to social influence. Furthermore, we demonstrate that we can predict complex cascades of behavioral change at their moment of initiation, before they actually occur. Consequently, despite the intrinsic stochasticity of individual behavior, establishing the hidden communication networks in large self-organized groups facilitates a quantitative understanding of behavioral contagion. PMID:25825752

  5. Revealing the hidden networks of interaction in mobile animal groups allows prediction of complex behavioral contagion.

    PubMed

    Rosenthal, Sara Brin; Twomey, Colin R; Hartnett, Andrew T; Wu, Hai Shan; Couzin, Iain D

    2015-04-14

    Coordination among social animals requires rapid and efficient transfer of information among individuals, which may depend crucially on the underlying structure of the communication network. Establishing the decision-making circuits and networks that give rise to individual behavior has been a central goal of neuroscience. However, the analogous problem of determining the structure of the communication network among organisms that gives rise to coordinated collective behavior, such as is exhibited by schooling fish and flocking birds, has remained almost entirely neglected. Here, we study collective evasion maneuvers, manifested through rapid waves, or cascades, of behavioral change (a ubiquitous behavior among taxa) in schooling fish (Notemigonus crysoleucas). We automatically track the positions and body postures, calculate visual fields of all individuals in schools of ∼150 fish, and determine the functional mapping between socially generated sensory input and motor response during collective evasion. We find that individuals use simple, robust measures to assess behavioral changes in neighbors, and that the resulting networks by which behavior propagates throughout groups are complex, being weighted, directed, and heterogeneous. By studying these interaction networks, we reveal the (complex, fractional) nature of social contagion and establish that individuals with relatively few, but strongly connected, neighbors are both most socially influential and most susceptible to social influence. Furthermore, we demonstrate that we can predict complex cascades of behavioral change at their moment of initiation, before they actually occur. Consequently, despite the intrinsic stochasticity of individual behavior, establishing the hidden communication networks in large self-organized groups facilitates a quantitative understanding of behavioral contagion.

  6. Group choreography: mechanisms orchestrating the collective movement of border cells.

    PubMed

    Montell, Denise J; Yoon, Wan Hee; Starz-Gaiano, Michelle

    2012-10-01

    Cell movements are essential for animal development and homeostasis but also contribute to disease. Moving cells typically extend protrusions towards a chemoattractant, adhere to the substrate, contract and detach at the rear. It is less clear how cells that migrate in interconnected groups in vivo coordinate their behaviour and navigate through natural environments. The border cells of the Drosophila melanogaster ovary have emerged as an excellent model for the study of collective cell movement, aided by innovative genetic, live imaging, and photomanipulation techniques. Here we provide an overview of the molecular choreography of border cells and its more general implications.

  7. Natural grouping of neural responses reveals spatially segregated clusters in prearcuate cortex

    PubMed Central

    Kiani, Roozbeh; Cueva, Christopher J.; Reppas, John B.; Peixoto, Diogo; Ryu, Stephen I.; Newsome, William T.

    2015-01-01

    Summary A fundamental challenge in studying the frontal lobe is to parcellate this cortex into ‘natural’ functional modules despite the absence of topographic maps, which are so helpful in primary sensory areas. Here we show that unsupervised clustering algorithms, applied to 96-channel array recordings from prearcuate gyrus, reveal spatially segregated sub-networks that remain stable across behavioral contexts. Looking for natural groupings of neurons based on response similarities, we discovered that the recorded area includes at least two spatially segregated sub-networks that differentially represent behavioral choice and reaction time. Importantly, these sub-networks are detectable during different behavioral states, and surprisingly, are defined better by ‘common noise’ than task-evoked responses. Our parcellation process works well on ‘spontaneous’ neural activity, and thus bears strong resemblance to the identification of ‘resting state’ networks in fMRI datasets. Our results demonstrate a powerful new tool for identifying cortical sub-networks by objective classification of simultaneously recorded electrophysiological activity. PMID:25728571

  8. Phylogeny of a genomically diverse group of elymus (poaceae) allopolyploids reveals multiple levels of reticulation.

    PubMed

    Mason-Gamer, Roberta J

    2013-01-01

    The grass tribe Triticeae (=Hordeeae) comprises only about 300 species, but it is well known for the economically important crop plants wheat, barley, and rye. The group is also recognized as a fascinating example of evolutionary complexity, with a history shaped by numerous events of auto- and allopolyploidy and apparent introgression involving diploids and polyploids. The genus Elymus comprises a heterogeneous collection of allopolyploid genome combinations, all of which include at least one set of homoeologs, designated St, derived from Pseudoroegneria. The current analysis includes a geographically and genomically diverse collection of 21 tetraploid Elymus species, and a single hexaploid species. Diploid and polyploid relationships were estimated using four molecular data sets, including one that combines two regions of the chloroplast genome, and three from unlinked nuclear genes: phosphoenolpyruvate carboxylase, β-amylase, and granule-bound starch synthase I. Four gene trees were generated using maximum likelihood, and the phylogenetic placement of the polyploid sequences reveals extensive reticulation beyond allopolyploidy alone. The trees were interpreted with reference to numerous phenomena known to complicate allopolyploid phylogenies, and introgression was identified as a major factor in their history. The work illustrates the interpretation of complicated phylogenetic results through the sequential consideration of numerous possible explanations, and the results highlight the value of careful inspection of multiple independent molecular phylogenetic estimates, with particular focus on the differences among them.

  9. Phylogeny of a Genomically Diverse Group of Elymus (Poaceae) Allopolyploids Reveals Multiple Levels of Reticulation

    PubMed Central

    Mason-Gamer, Roberta J.

    2013-01-01

    The grass tribe Triticeae (=Hordeeae) comprises only about 300 species, but it is well known for the economically important crop plants wheat, barley, and rye. The group is also recognized as a fascinating example of evolutionary complexity, with a history shaped by numerous events of auto- and allopolyploidy and apparent introgression involving diploids and polyploids. The genus Elymus comprises a heterogeneous collection of allopolyploid genome combinations, all of which include at least one set of homoeologs, designated St, derived from Pseudoroegneria. The current analysis includes a geographically and genomically diverse collection of 21 tetraploid Elymus species, and a single hexaploid species. Diploid and polyploid relationships were estimated using four molecular data sets, including one that combines two regions of the chloroplast genome, and three from unlinked nuclear genes: phosphoenolpyruvate carboxylase, β-amylase, and granule-bound starch synthase I. Four gene trees were generated using maximum likelihood, and the phylogenetic placement of the polyploid sequences reveals extensive reticulation beyond allopolyploidy alone. The trees were interpreted with reference to numerous phenomena known to complicate allopolyploid phylogenies, and introgression was identified as a major factor in their history. The work illustrates the interpretation of complicated phylogenetic results through the sequential consideration of numerous possible explanations, and the results highlight the value of careful inspection of multiple independent molecular phylogenetic estimates, with particular focus on the differences among them. PMID:24302986

  10. In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures.

    PubMed

    Ng, Jia-Hui; Kumar, Vibhor; Muratani, Masafumi; Kraus, Petra; Yeo, Jia-Chi; Yaw, Lai-Ping; Xue, Kun; Lufkin, Thomas; Prabhakar, Shyam; Ng, Huck-Hui

    2013-02-11

    The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale chromatin immunoprecipitation sequencing (ChIP-seq) method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac, and H2BK20ac). Comparison of active chromatin state between somatic, embryonic stem, and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. We found the nuclear receptor Nr5a2 motif to be enriched at a subset of germ cell cis-regulatory regions, and our results implicate Nr5a2 in germ cell biology. Interestingly, in germ cells, the H3K27me3 histone modification occurs more frequently at regions that are enriched for retrotransposons and MHC genes, indicating that these loci are specifically silenced in germ cells. Together, our study provides genome-wide histone modification maps of in vivo germ cells and reveals the molecular chromatin signatures of germ cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Association of type- and group-specific antigens with the cell wall of serotype III group B streptococcus.

    PubMed Central

    Doran, T I; Mattingly, S J

    1982-01-01

    The type-specific antigens (TSA) of group B streptococcus (GBS) represent the primary virulence factors for these organisms, yet little is known about their relationship to the cell surface of GBS. Crude cell walls of serotype III GBS strain 110 were purified by extraction with sodium dodecyl sulfate, LiCl, and urea, which removed essentially all of the protein associated with the cell wall as determined by amino acid analysis. Only those amino acids found in peptidoglycan were present, which included alanine, lysine, and glutamate (3.5:1:1 molar ratio). In contrast, these procedures resulted in the release of only 4.6% of the wall-associated TSA, indicating that protein was not the primary means by which TSA was bound to the cell surface. Mutanolysin (20 micrograms/ml) treatment of purified cell walls resulted in the release of 95% of the wall-associated TSA. The covalent association of TSA, the group B polysaccharide, and the peptidoglycan was demonstrated by the presence of N-acetylmuramic acid, rhamnose, alanine, glutamate, and lysine in mutanolysin-extracted TSA material purified by DEAE-Sephacel anion exchange and Sepharose 4B gel chromatography. Chemical analysis of purified cell walls revealed that group B antigen and peptidoglycan comprised 37.4 and 36.5%, respectively, whereas TSA accounted for 22.1 to 24.5% of the weight of the purified walls. Of the total 283.5 mg of TSA produced per 10-liter culture of GBS strain 110, 8.4% was released into the supernatant fluid. The remainder (249 mg) comprised the cell wall antigen. As described above, 4.6% of the cell wall antigen was extractable by nonenzymatic methods, which represented 3.8% of the total TSA, whereas 87.8% of the total TSA produced appeared to be covalently attached to the cell wall. PMID:7047392

  12. Modelling of Yeast Mating Reveals Robustness Strategies for Cell-Cell Interactions

    PubMed Central

    Chen, Weitao; Nie, Qing; Yi, Tau-Mu; Chou, Ching-Shan

    2016-01-01

    Mating of budding yeast cells is a model system for studying cell-cell interactions. Haploid yeast cells secrete mating pheromones that are sensed by the partner which responds by growing a mating projection toward the source. The two projections meet and fuse to form the diploid. Successful mating relies on precise coordination of dynamic extracellular signals, signaling pathways, and cell shape changes in a noisy background. It remains elusive how cells mate accurately and efficiently in a natural multi-cell environment. Here we present the first stochastic model of multiple mating cells whose morphologies are driven by pheromone gradients and intracellular signals. Our novel computational framework encompassed a moving boundary method for modeling both a-cells and α-cells and their cell shape changes, the extracellular diffusion of mating pheromones dynamically coupled with cell polarization, and both external and internal noise. Quantification of mating efficiency was developed and tested for different model parameters. Computer simulations revealed important robustness strategies for mating in the presence of noise. These strategies included the polarized secretion of pheromone, the presence of the α-factor protease Bar1, and the regulation of sensing sensitivity; all were consistent with data in the literature. In addition, we investigated mating discrimination, the ability of an a-cell to distinguish between α-cells either making or not making α-factor, and mating competition, in which multiple a-cells compete to mate with one α-cell. Our simulations were consistent with previous experimental results. Moreover, we performed a combination of simulations and experiments to estimate the diffusion rate of the pheromone a-factor. In summary, we constructed a framework for simulating yeast mating with multiple cells in a noisy environment, and used this framework to reproduce mating behaviors and to identify strategies for robust cell-cell interactions. PMID

  13. Mechanism of cell alignment in groups of Myxococcus xanthus bacteria

    NASA Astrophysics Data System (ADS)

    Balgam, Rajesh; Igoshin, Oleg

    2015-03-01

    Myxococcus xanthus is a model for studying self-organization in bacteria. These flexible cylindrical bacteria move along. In groups, M. xanthus cells align themselves into dynamic cell clusters but the mechanism underlying their formation is unknown. It has been shown that steric interactions can cause alignment in self-propelled hard rods but it is not clear how flexibility and reversals affect the alignment and cluster formation. We have investigated cell alignment process using our biophysical model of M. xanthus cell in an agent-based simulation framework under realistic cell flexibility values. We observed that flexible model cells can form aligned cell clusters when reversals are suppressed but these clusters disappeared when reversals frequency becomes similar to the observed value. However, M. xanthus cells follow slime (polysaccharide gel like material) trails left by other cells and we show that implementing this into our model rescues cell clustering for reversing cells. Our results show that slime following along with periodic cell reversals act as positive feedback to reinforce existing slime trails and recruit more cells. Furthermore, we have observed that mechanical cell alignment combined with slime following is sufficient to explain the distinct clustering patterns of reversing and non-reversing cells as observed in recent experiments. This work is supported by NSF MCB 0845919 and 1411780.

  14. Molecular genetic analysis of ABO blood group variations reveals 29 novel ABO subgroup alleles.

    PubMed

    Cai, Xiaohong; Jin, Sha; Liu, Xi; Fan, Liangfeng; Lu, Qiong; Wang, Jianlian; Shen, Wei; Gong, Songsong; Qiu, Li; Xiang, Dong

    2013-11-01

    Identifying genetic variants of the ABO gene may reveal new biologic mechanisms underlying variant phenotypes of the ABO blood group. We report the molecular genetic analysis of 322 apparently unrelated ABO subgroup individuals in an estimated 2.1 million donors. We performed phenotype investigations by serology studies, analyzed the DNA sequence of the ABO gene by direct sequencing or sequencing after cloning, and evaluated promoter activity by reporter assays. In 62 rare ABO alleles, we identified 29 novel ABO subgroup alleles in 43 apparently unrelated subgroup individuals and their four available pedigrees. Of these alleles, one was a deletion-mutation allele, four were hybrid alleles, and 24 were point-mutation alleles. Most of the point mutations were detected in Exons 6 to 7, while several others were also detected in Exons 1 to 5 or splicing regions. One ABO promoter mutation, -35 to -18 del, was found and verified to reduce promoter activity, as determined by dual luciferase assays. Two mutations, 7G>T and 52C>T, carrying the premature terminal codons E3X and R18X in the 5'-region, were found to be associated with the very weak ABO subgroups "Ael" and "Bel." Twenty-nine ABO subgroup alleles were newly linked to different kinds of ABO variations. We provide the first evidence that promoter abnormality is involved in the formation of weak ABO phenotypes. We also described the first naturally occurring ABO alleles with premature terminal codons in the 5'-region that led to Ael and Bel phenotypes. © 2013 American Association of Blood Banks.

  15. Analysis of the full genome of human group C rotaviruses reveals lineage diversification and reassortment.

    PubMed

    Medici, Maria Cristina; Tummolo, Fabio; Martella, Vito; Arcangeletti, Maria Cristina; De Conto, Flora; Chezzi, Carlo; Fehér, Enikő; Marton, Szilvia; Calderaro, Adriana; Bányai, Krisztián

    2016-08-01

    Group C rotaviruses (RVC) are enteric pathogens of humans and animals. Whole-genome sequences are available only for few RVCs, leaving gaps in our knowledge about their genetic diversity. We determined the full-length genome sequence of two human RVCs (PR2593/2004 and PR713/2012), detected in Italy from hospital-based surveillance for rotavirus infection in 2004 and 2012. In the 11 RNA genomic segments, the two Italian RVCs segregated within separate intra-genotypic lineages showed variation ranging from 1.9 % (VP6) to 15.9 % (VP3) at the nucleotide level. Comprehensive analysis of human RVC sequences available in the databases allowed us to reveal the existence of at least two major genome configurations, defined as type I and type II. Human RVCs of type I were all associated with the M3 VP3 genotype, including the Italian strain PR2593/2004. Conversely, human RVCs of type II were all associated with the M2 VP3 genotype, including the Italian strain PR713/2012. Reassortant RVC strains between these major genome configurations were identified. Although only a few full-genome sequences of human RVCs, mostly of Asian origin, are available, the analysis of human RVC sequences retrieved from the databases indicates that at least two intra-genotypic RVC lineages circulate in European countries. Gathering more sequence data is necessary to develop a standardized genotype and intra-genotypic lineage classification system useful for epidemiological investigations and avoiding confusion in the literature.

  16. Structural polymorphism analysis of Chinese Mongolian ethnic group revealed by a new STR panel: genetic relationship to other groups.

    PubMed

    Gao, Ying; Han, Jun-Tao; Shen, Chun-Mei; Wu, Hua; Yuan, Guo-Lian; Zhao, Li-Jun; Yan, Jiang-Wei; Meng, Hao-Tian; Zhang, Yu-Dang; Liu, Wen-Juan; Wang, Hong-Dan; White, Richard E; Wei, Xing

    2014-07-01

    Mongolian is the eighth largest ethnic minority on Chinese population data according to the 2010 census. In the present study, we presented the first report about the allelic frequencies and forensic statistical parameters at the 21 new STRs and analyzed linkage disequilibrium of pairwise loci in the Mongolian ethnic minority, China. Hardy-Weinberg equilibrium tests demonstrated no significant deviations except for the D1S1627 locus. The cumulative power of discrimination and power of exclusion of all the loci are 0.9999999999999999992576 and 0.9999997528, respectively. The results of analysis of molecular variance showed that significant differences between the Mongolian and the other eight populations were found at 1-9 STR loci. In population genetics, the results of principal component analysis, structure analysis, and phylogenetic reconstruction analysis indicated shorter genetic distance between the Mongolian group and the Ningxia Han. All the results suggest that the 21 new STR loci will contribute to Chinese population genetics and forensic caseworks in the Mongolian group.

  17. Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

    PubMed

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary; Zhu, Hongwen; Zhang, Jun; Liu, Jie; Liu, Hailiang; deVellis, Jean; Horvath, Steve; Sun, Yi Eve; Li, Siguang

    2015-05-21

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

  18. Cell Cycle and Cell Size Dependent Gene Expression Reveals Distinct Subpopulations at Single-Cell Level

    PubMed Central

    Dolatabadi, Soheila; Candia, Julián; Akrap, Nina; Vannas, Christoffer; Tesan Tomic, Tajana; Losert, Wolfgang; Landberg, Göran; Åman, Pierre; Ståhlberg, Anders

    2017-01-01

    Cell proliferation includes a series of events that is tightly regulated by several checkpoints and layers of control mechanisms. Most studies have been performed on large cell populations, but detailed understanding of cell dynamics and heterogeneity requires single-cell analysis. Here, we used quantitative real-time PCR, profiling the expression of 93 genes in single-cells from three different cell lines. Individual unsynchronized cells from three different cell lines were collected in different cell cycle phases (G0/G1 – S – G2/M) with variable cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression through the cell cycle, but not with cell size. By applying the random forests algorithm, a supervised machine learning approach, we show how a multi-gene signature that classifies individual cells into their correct cell cycle phase and cell size can be generated. To identify the most predictive genes we used a variable selection strategy. Detailed analysis of cell cycle predictive genes allowed us to define subpopulations with distinct gene expression profiles and to calculate a cell cycle index that illustrates the transition of cells between cell cycle phases. In conclusion, we provide useful experimental approaches and bioinformatics to identify informative and predictive genes at the single-cell level, which opens up new means to describe and understand cell proliferation and subpopulation dynamics. PMID:28179914

  19. Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

    PubMed Central

    Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

    2013-01-01

    Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

  20. Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells.

    PubMed

    Winterhoff, Boris J; Maile, Makayla; Mitra, Amit Kumar; Sebe, Attila; Bazzaro, Martina; Geller, Melissa A; Abrahante, Juan E; Klein, Molly; Hellweg, Raffaele; Mullany, Sally A; Beckman, Kenneth; Daniel, Jerry; Starr, Timothy K

    2017-03-01

    The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells. A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections. Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells. Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Emergence of spatial structure in cell groups and the evolution of cooperation.

    PubMed

    Nadell, Carey D; Foster, Kevin R; Xavier, João B

    2010-03-19

    On its own, a single cell cannot exert more than a microscopic influence on its immediate surroundings. However, via strength in numbers and the expression of cooperative phenotypes, such cells can enormously impact their environments. Simple cooperative phenotypes appear to abound in the microbial world, but explaining their evolution is challenging because they are often subject to exploitation by rapidly growing, non-cooperative cell lines. Population spatial structure may be critical for this problem because it influences the extent of interaction between cooperative and non-cooperative individuals. It is difficult for cooperative cells to succeed in competition if they become mixed with non-cooperative cells, which can exploit the public good without themselves paying a cost. However, if cooperative cells are segregated in space and preferentially interact with each other, they may prevail. Here we use a multi-agent computational model to study the origin of spatial structure within growing cell groups. Our simulations reveal that the spatial distribution of genetic lineages within these groups is linked to a small number of physical and biological parameters, including cell growth rate, nutrient availability, and nutrient diffusivity. Realistic changes in these parameters qualitatively alter the emergent structure of cell groups, and thereby determine whether cells with cooperative phenotypes can locally and globally outcompete exploitative cells. We argue that cooperative and exploitative cell lineages will spontaneously segregate in space under a wide range of conditions and, therefore, that cellular cooperation may evolve more readily than naively expected.

  2. Revealing the structural and functional diversity of plant cell walls.

    PubMed

    Knox, J Paul

    2008-06-01

    The extensive knowledge of the chemistry of isolated cell wall polymers, and that relating to the identification and partial annotation of gene families involved in their synthesis and modification, is not yet matched by a sophisticated understanding of the occurrence of the polymers within cell walls of the diverse cell types within a growing organ. Currently, the main sets of tools that are used to determine cell-type-specific configurations of cell wall polymers and aspects of cell wall microstructures are antibodies, carbohydrate-binding modules (CBMs) and microspectroscopies. As these tools are applied we see that cell wall polymers are extensively developmentally regulated and that there is a range of structurally distinct primary and secondary cell walls within organs and across species. The challenge now is to document cell wall structures in relation to diverse cell biological events and to integrate this knowledge with the emerging understanding of polymer functions.

  3. Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading

    PubMed Central

    Cai, Yunfei; Xenias, Harry; Spielman, Ingrid; Shneidman, Anna V.; David, Lawrence A.; Döbereiner, Hans-Günther; Wiggins, Chris H.; Sheetz, Michael P.

    2008-01-01

    Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments – spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1) During early spreading, cells form initial contacts with the surface. 2) The middle spreading phase exhibits rapidly increasing attachment area. 3) Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters – a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules that, together

  4. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  5. Analytical cell adhesion chromatography reveals impaired persistence of metastatic cell rolling adhesion to P-selectin

    PubMed Central

    Oh, Jaeho; Edwards, Erin E.; McClatchey, P. Mason; Thomas, Susan N.

    2015-01-01

    ABSTRACT Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cell–cell signaling that directs extravasation into surrounding tissues. To measure the relative efficiency of cell adhesion in shear flow for in vitro drug screening, we designed and implemented a microfluidic-based analytical cell adhesion chromatography system. The juxtaposition of instantaneous rolling velocities with elution times revealed that human metastatic cancer cells, but not human leukocytes, had a reduced capacity to sustain rolling adhesion with P-selectin. We define a new parameter, termed adhesion persistence, which is conceptually similar to migration persistence in the context of chemotaxis, but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate that might modulate the probability of extravasation. Among cell types assayed, adhesion persistence to P-selectin was specifically reduced in metastatic but not leukocyte-like cells in response to a low dose of heparin. In conclusion, we demonstrate this as an effective methodology to identify selectin adhesion antagonist doses that modulate homing cell adhesion and engraftment in a cell-subtype-selective manner. PMID:26349809

  6. Meta-analysis reveals conserved cell cycle transcriptional network across multiple human cell types.

    PubMed

    Giotti, Bruno; Joshi, Anagha; Freeman, Tom C

    2017-01-05

    Cell division is central to the physiology and pathology of all eukaryotic organisms. The molecular machinery underpinning the cell cycle has been studied extensively in a number of species and core aspects of it have been found to be highly conserved. Similarly, the transcriptional changes associated with this pathway have been studied in different organisms and different cell types. In each case hundreds of genes have been reported to be regulated, however there seems to be little consensus in the genes identified across different studies. In a recent comparison of transcriptomic studies of the cell cycle in different human cell types, only 96 cell cycle genes were reported to be the same across all studies examined. Here we perform a systematic re-examination of published human cell cycle expression data by using a network-based approach to identify groups of genes with a similar expression profile and therefore function. Two clusters in particular, containing 298 transcripts, showed patterns of expression consistent with cell cycle occurrence across the four human cell types assessed. Our analysis shows that there is a far greater conservation of cell cycle-associated gene expression across human cell types than reported previously, which can be separated into two distinct transcriptional networks associated with the G1/S-S and G2-M phases of the cell cycle. This work also highlights the benefits of performing a re-analysis on combined datasets.

  7. Sarcomere dynamics in single myocardial cells as revealed by high-resolution light diffractometry.

    PubMed

    Leung, A F

    1983-08-01

    A specially designed diffractometer with a high spatial and temporal resolution recorded the diffraction of a laser beam by single enzymatically isolated myocardial cells. The fine structures within the first-order diffraction were resolved and each structure was interpreted as the diffraction from a group of sarcomeres of nearly equal length. During activation of the cell dynamics of each discrete group of sarcomeres was uniform and independent of the other groups. However, a small nonuniform component in the sarcomere dynamics was observed and attributed to the coupling between the shortening tension and the radial stress resulting from the expansion of the myofibrillar cross-section. The time-course of the diffraction fine structures during contractile activity revealed (1) the period of the contraction-relaxation cycle, (2) the latent period, (3) the shortening and relengthening speeds and (4) the variation in the line width and intensity of the fine structure. Measurements showed that the latent period was dependent on the free Ca2+ of the cell's bathing solution while the initial shortening speed was not. The diffraction line width and intensity of the shortening cell were explained by the grating model.

  8. Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment

    PubMed Central

    van Wolfswinkel, Josien C.; Wagner, Daniel E.; Reddien, Peter W.

    2014-01-01

    Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings present a new view of planarian neoblasts, in which the population is comprised of two major and functionally distinct cellular compartments. PMID:25017721

  9. Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment.

    PubMed

    van Wolfswinkel, Josien C; Wagner, Daniel E; Reddien, Peter W

    2014-09-04

    Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage, including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings indicate that planarian neoblasts comprise two major and functionally distinct cellular compartments. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Group Work Education in Social Work: A Review of the Literature Reveals Possible Solutions

    ERIC Educational Resources Information Center

    LaRocque, Sarah E.

    2017-01-01

    This article examines the growing concerns in the literature that traditional group work education in social work is not providing the foundational knowledge, skills, evidence-based practice, professional uses of self, and adherence to practice standards necessary for effective group practice. An exploration of the best available evidence on group…

  11. Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

    PubMed Central

    Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

    2009-01-01

    OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

  12. Unified effects of aggregation reveal larger prey groups take longer to find

    PubMed Central

    Ioannou, Christos C.; Bartumeus, Frederic; Krause, Jens; Ruxton, Graeme D.

    2011-01-01

    Previous work has suggested that larger groups of prey are more conspicuous to predators. However, this ignores that prey populations are finite. As groups get larger they become fewer, hence the encounter rate between predator and prey decreases with prey aggregation. Here, we present a two-dimensional model based on visual angle to unify these encounter and conspicuousness effects of aggregation. With experimental support using three-spined sticklebacks (Gasterosteus aculeatus L.), searching for chironomid larvae, we demonstrate that the increase in visual angle with increasing group size is outweighed by its corresponding decrease as the groups become fewer and thus further away from the searching predator. The net effect is that prey are found with more difficulty when they aggregate, giving an additional anti-predatory benefit to group living rather than a cost. PMID:21325333

  13. Vancomycin tolerant, methicillin-resistant Staphylococcus aureus reveals the effects of vancomycin on cell wall thickening.

    PubMed

    Cázares-Domínguez, Vicenta; Cruz-Córdova, Ariadnna; Ochoa, Sara A; Escalona, Gerardo; Arellano-Galindo, José; Rodríguez-Leviz, Alejandra; Hernández-Castro, Rigoberto; López-Villegas, Edgar O; Xicohtencatl-Cortes, Juan

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important opportunistic pathogen that causes both healthcare- and community-acquired infections. An increase in the incidence of these infections may lead to a substantial change in the rate of vancomycin usage. Incidence of reduced susceptibility to vancomycin has been increasing worldwide for the last few years, conferring different levels of resistance to vancomycin as well as producing changes in the cell wall structure. The aim of the present study was to determine the effect of vancomycin on cell wall thickening in clinical isolates of vancomycin-tolerant (VT) MRSA obtained from pediatric patients. From a collection of 100 MRSA clinical isolates from pediatric patients, 12% (12/100) were characterized as VT-MRSA, and from them, 41.66% (5/12) exhibited the heterogeneous vancomycin-intermediate S. aureus (hVISA) phenotype. Multiplex-PCR assays revealed 66.66% (8/12), 25% (3/12), and 8.33% (1/12) of the VT-MRSA isolates were associated with agr group II, I, and III polymorphisms, respectively; the II-mec gene was amplified from 83.3% (10/12) of the isolates, and the mecIVa gene was amplified from 16.66% (2/12) of the isolates. Pulsed field electrophoresis (PFGE) fingerprint analysis showed 62% similarity among the VT-MRSA isolates. Thin transverse sections analyzed by transmission electron microscopy (TEM) revealed an average increase of 24 nm (105.55%) in the cell wall thickness of VT-MRSA compared with untreated VT-MRSA isolates. In summary, these data revealed that the thickened cell walls of VT-MRSA clinical isolates with agr type II and SCCmec group II polymorphisms are associated with an adaptive resistance to vancomycin.

  14. Vancomycin Tolerant, Methicillin-Resistant Staphylococcus aureus Reveals the Effects of Vancomycin on Cell Wall Thickening

    PubMed Central

    Cázares-Domínguez, Vicenta; Cruz-Córdova, Ariadnna; Ochoa, Sara A.; Escalona, Gerardo; Arellano-Galindo, José; Rodríguez-Leviz, Alejandra; Hernández-Castro, Rigoberto; López-Villegas, Edgar O.; Xicohtencatl-Cortes, Juan

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important opportunistic pathogen that causes both healthcare- and community-acquired infections. An increase in the incidence of these infections may lead to a substantial change in the rate of vancomycin usage. Incidence of reduced susceptibility to vancomycin has been increasing worldwide for the last few years, conferring different levels of resistance to vancomycin as well as producing changes in the cell wall structure. The aim of the present study was to determine the effect of vancomycin on cell wall thickening in clinical isolates of vancomycin-tolerant (VT) MRSA obtained from pediatric patients. From a collection of 100 MRSA clinical isolates from pediatric patients, 12% (12/100) were characterized as VT-MRSA, and from them, 41.66% (5/12) exhibited the heterogeneous vancomycin-intermediate S. aureus (hVISA) phenotype. Multiplex-PCR assays revealed 66.66% (8/12), 25% (3/12), and 8.33% (1/12) of the VT-MRSA isolates were associated with agr group II, I, and III polymorphisms, respectively; the II-mec gene was amplified from 83.3% (10/12) of the isolates, and the mecIVa gene was amplified from 16.66% (2/12) of the isolates. Pulsed field electrophoresis (PFGE) fingerprint analysis showed 62% similarity among the VT-MRSA isolates. Thin transverse sections analyzed by transmission electron microscopy (TEM) revealed an average increase of 24 nm (105.55%) in the cell wall thickness of VT-MRSA compared with untreated VT-MRSA isolates. In summary, these data revealed that the thickened cell walls of VT-MRSA clinical isolates with agr type II and SCCmec group II polymorphisms are associated with an adaptive resistance to vancomycin. PMID:25793280

  15. Metabolic Differences in Microbial Cell Populations Revealed by Nanophotonic Ionization

    SciTech Connect

    Walker, Bennett; Antonakos, Cory; Retterer, Scott T; Vertes, Akos

    2013-01-01

    ellular differences are linked to cell differentiation, the proliferation of cancer and to the development of drug resistance in microbial infections. Due to sensitivity limitations, however, large- scale metabolic analysis at the single cell level is only available for cells significantly larger in volume than Saccharomyces cerevisiae (~30 fL). Here we demonstrate that by a nanophotonic ionization platform and mass spectrometry, over one hundred up to 108 metabolites, or up to 18% of the known S. cerevisiae metabolome, can be identified in very small cell populations (n < 100). Under ideal conditions, r Relative quantitation of up to 4% of the metabolites is achieved at the single cell level.

  16. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

    PubMed Central

    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-01-01

    The ‘neural plate border’ of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure. DOI: http://dx.doi.org/10.7554/eLife.21620.001 PMID:28355135

  17. Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.

    PubMed

    Truong, David M; Hewitt, F Curtis; Hanson, Joseph H; Cui, Xiaoxia; Lambowitz, Alan M

    2015-08-01

    Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the

  18. Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation

    PubMed Central

    Truong, David M.; Hewitt, F. Curtis; Hanson, Joseph H.; Cui, Xiaoxia; Lambowitz, Alan M.

    2015-01-01

    Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed “retrohoming”. Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the

  19. Communication: Dissolution DNP reveals a long-lived deuterium spin state imbalance in methyl groups

    NASA Astrophysics Data System (ADS)

    Jhajharia, Aditya; Weber, Emmanuelle M. M.; Kempf, James G.; Abergel, Daniel; Bodenhausen, Geoffrey; Kurzbach, Dennis

    2017-01-01

    We report the generation and observation of long-lived spin states in deuterated methyl groups by dissolution DNP. These states are based on population imbalances between manifolds of spin states corresponding to irreducible representations of the C3v point group and feature strongly dampened quadrupolar relaxation. Their lifetime depends on the activation energies of methyl group rotation. With dissolution DNP, we can reduce the deuterium relaxation rate by a factor up to 20, thereby extending the experimentally available time window. The intrinsic limitation of NMR spectroscopy of quadrupolar spins by short relaxation times can thus be alleviated.

  20. Advances in cell surface glycoengineering reveal biological function.

    PubMed

    Nischan, Nicole; Kohler, Jennifer J

    2016-08-01

    Cell surface glycans are critical mediators of cell-cell, cell-ligand, and cell-pathogen interactions. By controlling the set of glycans displayed on the surface of a cell, it is possible to gain insight into the biological functions of glycans. Moreover, control of glycan expression can be used to direct cellular behavior. While genetic approaches to manipulate glycosyltransferase gene expression are available, their utility in glycan engineering has limitations due to the combinatorial nature of glycan biosynthesis and the functional redundancy of glycosyltransferase genes. Biochemical and chemical strategies offer valuable complements to these genetic approaches, notably by enabling introduction of unnatural functionalities, such as fluorophores, into cell surface glycans. Here, we describe some of the most recent developments in glycoengineering of cell surfaces, with an emphasis on strategies that employ novel chemical reagents. We highlight key examples of how these advances in cell surface glycan engineering enable study of cell surface glycans and their function. Exciting new technologies include synthetic lipid-glycans, new chemical reporters for metabolic oligosaccharide engineering to allow tandem and in vivo labeling of glycans, improved chemical and enzymatic methods for glycoproteomics, and metabolic glycosyltransferase inhibitors. Many chemical and biochemical reagents for glycan engineering are commercially available, facilitating their adoption by the biological community.

  1. Quantifying memory CD8 T cells reveals regionalization of immunosurveillance

    PubMed Central

    Steinert, Elizabeth M.; Schenkel, Jason M.; Fraser, Kathryn A.; Beura, Lalit K.; Manlove, Luke S.; Igyártó, Botond Z.; Southern, Peter J.; Masopust, David

    2015-01-01

    Summary Memory CD8 T cells protect against intracellular pathogens by scanning host cell surfaces, thus infection detection rates depend on memory cell number and distribution. Population analyses rely on isolation from whole organs and interpretation is predicated on presumptions of near complete cell recovery. Paradigmatically, memory is parsed into central, effector, and resident subsets, ostensibly defined by immunosurveillance patterns, but in practice identified by phenotypic markers. Because isolation methods ultimately inform models of memory T cell differentiation, protection, and vaccine translation, we tested their validity via parabiosis and quantitative immunofluorescence microscopy of a mouse memory CD8 T cell population. We report three major findings: lymphocyte isolation fails to recover most cells and biases against certain subsets, residents greatly outnumber recirculating cells within nonlymphoid tissues, and memory subset homing to inflammation does not conform to previously hypothesized migration patterns. These results indicate that most host cells are surveyed for reinfection by segregated residents rather than by recirculating cells that migrate throughout the blood and body. PMID:25957682

  2. Evolutionary history of the GABA transporter (GAT) group revealed by marine invertebrate GAT-1.

    PubMed

    Kinjo, Azusa; Koito, Tomoko; Kawaguchi, So; Inoue, Koji

    2013-01-01

    The GABA transporter (GAT) group is one of the major subgroups in the solute career 6 (SLC6) family of transmembrane proteins. The GAT group, which has been well studied in mammals, has 6 known members, i.e., a taurine transporter (TAUT), four GABA transporters (GAT-1, -2, -3, - 4), and a creatine transporter (CT1), which have important roles in maintaining physiological homeostasis. However, the GAT group has not been extensively investigated in invertebrates; only TAUT has been reported in marine invertebrates such as bivalves and krills, and GAT-1 has been reported in several insect species and nematodes. Thus, it is unknown how transporters in the GAT group arose during the course of animal evolution. In this study, we cloned GAT-1 cDNAs from the deep-sea mussel, Bathymodiolus septemdierum, and the Antarctic krill, Euphausia superba, whose TAUT cDNA has already been cloned. To understand the evolutionary history of the GAT group, we conducted phylogenetic and synteny analyses on the GAT group transporters of vertebrates and invertebrates. Our findings suggest that transporters of the GAT group evolved through the following processes. First, GAT-1 and CT1 arose by tandem duplication of an ancestral transporter gene before the divergence of Deuterostomia and Protostomia; next, the TAUT gene arose and GAT-3 was formed by the tandem duplication of the TAUT gene; and finally, GAT-2 and GAT-4 evolved from a GAT-3 gene by chromosomal duplication in the ancestral vertebrates. Based on synteny and phylogenetic evidence, the present naming of the GAT group members does not accurately reflect the evolutionary relationships.

  3. The swamplands of reflection: using conversation analysis to reveal the architecture of group reflection sessions.

    PubMed

    Veen, Mario; de la Croix, Anne

    2017-03-01

    Many medical schools include group reflection in their curriculum, and many researchers have considered both the concept and the outcomes of reflection. However, no research has been carried out on how 'reflective talk' is structured in the classroom. This paper describes how tutors and residents organise group reflection sessions in situ by describing an example of group reflection in medical education. Our aim is to provide an evidence base that can be used by medical educators to think about the way reflection should be included in their curriculum. We video-recorded 47 group reflection sessions of the general practice postgraduate training course at Erasmus University Medical School, Rotterdam. We used conversation analysis to unravel their overall structural organisation: the way participants organise and structure a conversation. Through micro-analysis of the moment-to-moment unfolding of group reflection, we distinguished the main building blocks that form the architecture of these sessions. We found that participants consistently oriented towards the following activity types: significant event, reason for sharing, learning issue and learning uptake. There was variation in the order of the activity types, the amount of time spent on each of them, and how they were accomplished. By studying reflection in its messy social context, we found order, commonalities and patterns that were typical of the architecture of group reflection in this setting, even if no formal structure is prescribed. In 'Exchange of Experience', the overall structural organisation consisted of activity types through which a case becomes shared, reflectable, learnable and valuable. There are essential discrepancies between cognitive reflection models and the reality of the classroom. Being conscious of this overall structural organisation can be a tool for tutors of these groups to help them navigate from one activity to another or to diagnose what is not working in the group discussion

  4. Cell dipole behaviour revealed by ECM sub-cellular geometry

    NASA Astrophysics Data System (ADS)

    Mandal, Kalpana; Wang, Irène; Vitiello, Elisa; Orellana, Laura Andreina Chacòn; Balland, Martial

    2014-12-01

    Cells sense and respond to their mechanical environment by exerting forces on their surroundings. The way forces are modulated by extra-cellular matrix (ECM) properties plays a key role in tissue homoeostasis. Using highly resolved micropatterns that constrain cells into the same square envelope but vary the adhesive geometry, here we investigate how the adhesive micro-environment affects the architecture of actin cytoskeleton and the orientation of traction forces. Our data demonstrate that local adhesive changes can trigger orientational ordering of stress fibres throughout the cell, suggesting that cells are capable of integrating information on ECM geometry at the whole-cell level. Finally, we show that cells tend to generate highly polarized force pattern, that is, unidirectional pinching, in response to adequate adhesive conditions. Hence, the geometry of adhesive environment can induce cellular orientation, a process which may have significant implications for the formation and mechanical properties of tissues.

  5. Ultrastructural observations reveal the presence of channels between cork cells.

    PubMed

    Teixeira, Rita Teresa; Pereira, Helena

    2009-12-01

    The ultrastructure of phellem cells of Quercus suber L. (cork oak) and Calotropis procera (Ait) R. Br. were analyzed using electron transmission microscopy to determine the presence or absence of plasmodesmata (PD). Different types of Q. suber cork samples were studied: one year shoots; virgin cork (first periderm), reproduction cork (traumatic periderm), and wet cork. The channel structures of PD were found in all the samples crossing adjacent cell walls through the suberin layer of the secondary wall. Calotropis phellem also showed PD crossing the cell walls of adjacent cells but in fewer numbers compared to Q. suber. In one year stems of cork oak, it was possible to follow the physiologically active PD with ribosomic accumulation next to the aperture of the channel seen in the phellogen cells to the completely obstructed channels in the dead cells that characterize the phellem tissue.

  6. ABO blood group mismatched hematopoietic stem cell transplantation.

    PubMed

    Tekgündüz, Sibel Akpınar; Özbek, Namık

    2016-02-01

    Apart from solid organ transplantations, use of ABO-blood group mismatched (ABO-mismatched) donors is acceptable in hematopoietic stem cell transplantation (HSCT) patients. About 20-40% of allogeneic HSCT recipients will receive grafts from ABO-mismatched donors. ABO incompatible HSCT procedures are associated with immediate and late consequences, including but not restricted to acute or delayed hemolytic reactions, delayed red blood cell recovery, pure red cell aplasia and graft-versus-host disease. This review summarizes the current knowledge about consequences of ABO-mismatched HSCT in terms of associated complications and will evaluate its impact on important outcome parameters of HSCT.

  7. Scanning angle interference microscopy reveals cell dynamics at the nanoscale.

    PubMed

    Paszek, Matthew J; DuFort, Christopher C; Rubashkin, Matthew G; Davidson, Michael W; Thorn, Kurt S; Liphardt, Jan T; Weaver, Valerie M

    2012-07-01

    Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.

  8. Poroelasticity of cell nuclei revealed through atomic force microscopy characterization

    NASA Astrophysics Data System (ADS)

    Wei, Fanan; Lan, Fei; Liu, Bin; Liu, Lianqing; Li, Guangyong

    2016-11-01

    With great potential in precision medical application, cell biomechanics is rising as a hot topic in biology. Cell nucleus, as the largest component within cell, not only contributes greatly to the cell's mechanical behavior, but also serves as the most vital component within cell. However, cell nucleus' mechanics is still far from unambiguous up to now. In this paper, we attempted to characterize and evaluate the mechanical property of isolated cell nuclei using Atomic Force Microscopy with a tipless probe. As indicated from typical indentation, changing loading rate and stress relaxation experiment results, cell nuclei showed significant dynamically mechanical property, i.e., time-dependent mechanics. Furthermore, through theoretical analysis, finite element simulation and stress relaxation experiment, the nature of nucleus' mechanics was better described by poroelasticity, rather than viscoelasticity. Therefore, the essence of nucleus' mechanics was clarified to be poroelastic through a sophisticated analysis. Finally, we estimated the poroelastic parameters for nuclei of two types of cells through a combination of experimental data and finite element simulation.

  9. Group 2 innate lymphoid cells in the lung.

    PubMed

    Drake, Li Yin; Kita, Hirohito

    2014-01-01

    As the first line of defense, innate immunity plays an important role in protecting the host against pathogens. Innate lymphoid cells (ILCs) are emerging as important effector cells in the innate immune system and the cell type that regulate immune and tissue homeostases. Group 2 ILCs (ILC2s) are a subset of ILCs and are characterized by their capacity to produce large quantities of type 2 cytokines and certain tissue growth factors. In animal models, lung ILC2s are involved in allergic airway inflammation induced by exposure to allergens even in the absence of CD4(+) T cells and are likely responsible for tissue repair and recovery after respiratory virus infection. ILC2s are also identified in various organs in humans, and the numbers are increased in mucosal tissues from patients with allergic disorders. Further investigations of this novel cell type will provide major conceptual advances in our understanding of the mechanisms of asthma and allergic diseases.

  10. Phylogenetic diversity of the Bacillus pumilus group and the marine ecotype revealed by multilocus sequence analysis.

    PubMed

    Liu, Yang; Lai, Qiliang; Dong, Chunming; Sun, Fengqin; Wang, Liping; Li, Guangyu; Shao, Zongze

    2013-01-01

    Bacteria closely related to Bacillus pumilus cannot be distinguished from such other species as B. safensis, B. stratosphericus, B. altitudinis and B. aerophilus simply by 16S rRNA gene sequence. In this report, 76 marine strains were subjected to phylogenetic analysis based on 7 housekeeping genes to understand the phylogeny and biogeography in comparison with other origins. A phylogenetic tree based on the 7 housekeeping genes concatenated in the order of gyrB-rpoB-pycA-pyrE-mutL-aroE-trpB was constructed and compared with trees based on the single genes. All these trees exhibited a similar topology structure with small variations. Our 79 strains were divided into 6 groups from A to F; Group A was the largest and contained 49 strains close to B. altitudinis. Additional two large groups were presented by B. safensis and B. pumilus respectively. Among the housekeeping genes, gyrB and pyrE showed comparatively better resolution power and may serve as molecular markers to distinguish these closely related strains. Furthermore, a recombinant phylogenetic tree based on the gyrB gene and containing 73 terrestrial and our isolates was constructed to detect the relationship between marine and other sources. The tree clearly showed that the bacteria of marine origin were clustered together in all the large groups. In contrast, the cluster belonging to B. safensis was mainly composed of bacteria of terrestrial origin. Interestingly, nearly all the marine isolates were at the top of the tree, indicating the possibility of the recent divergence of this bacterial group in marine environments. We conclude that B. altitudinis bacteria are the most widely spread of the B. pumilus group in marine environments. In summary, this report provides the first evidence regarding the systematic evolution of this bacterial group, and knowledge of their phylogenetic diversity will help in the understanding of their ecological role and distribution in marine environments.

  11. Phylogenetic Diversity of the Bacillus pumilus Group and the Marine Ecotype Revealed by Multilocus Sequence Analysis

    PubMed Central

    Dong, Chunming; Sun, Fengqin; Wang, Liping; Li, Guangyu; Shao, Zongze

    2013-01-01

    Bacteria closely related to Bacillus pumilus cannot be distinguished from such other species as B. safensis, B. stratosphericus, B. altitudinis and B. aerophilus simply by 16S rRNA gene sequence. In this report, 76 marine strains were subjected to phylogenetic analysis based on 7 housekeeping genes to understand the phylogeny and biogeography in comparison with other origins. A phylogenetic tree based on the 7 housekeeping genes concatenated in the order of gyrB-rpoB-pycA-pyrE-mutL-aroE-trpB was constructed and compared with trees based on the single genes. All these trees exhibited a similar topology structure with small variations. Our 79 strains were divided into 6 groups from A to F; Group A was the largest and contained 49 strains close to B. altitudinis. Additional two large groups were presented by B. safensis and B. pumilus respectively. Among the housekeeping genes, gyrB and pyrE showed comparatively better resolution power and may serve as molecular markers to distinguish these closely related strains. Furthermore, a recombinant phylogenetic tree based on the gyrB gene and containing 73 terrestrial and our isolates was constructed to detect the relationship between marine and other sources. The tree clearly showed that the bacteria of marine origin were clustered together in all the large groups. In contrast, the cluster belonging to B. safensis was mainly composed of bacteria of terrestrial origin. Interestingly, nearly all the marine isolates were at the top of the tree, indicating the possibility of the recent divergence of this bacterial group in marine environments. We conclude that B. altitudinis bacteria are the most widely spread of the B. pumilus group in marine environments. In summary, this report provides the first evidence regarding the systematic evolution of this bacterial group, and knowledge of their phylogenetic diversity will help in the understanding of their ecological role and distribution in marine environments. PMID:24244618

  12. Genetic Kinship Analyses Reveal That Gray's Beaked Whales Strand in Unrelated Groups.

    PubMed

    Patel, Selina; Thompson, Kirsten F; Santure, Anna W; Constantine, Rochelle; Millar, Craig D

    2017-06-01

    Some marine mammals are so rarely seen that their life history and social structure remain a mystery. Around New Zealand, Gray's beaked whales (Mesoplodon grayi) are almost never seen alive, yet they are a commonly stranded species. Gray's are unique among the beaked whales in that they frequently strand in groups, providing an opportunity to investigate their social organization. We examined group composition and genetic kinship in 113 Gray's beaked whales with samples collected over a 20-year period. Fifty-six individuals stranded in 19 groups (2 or more individuals), and 57 whales stranded individually. Mitochondrial control region haplotypes and microsatellite genotypes (16 loci) were obtained for 103 whales. We estimated pairwise relatedness between all pairs of individuals and average relatedness within, and between, groups. We identified 6 mother-calf pairs and 2 half-siblings, including 2 whales in different strandings 17 years and 1500 km apart. Surprisingly, none of the adults stranding together were related suggesting that groups are not formed through the retention of kin. These data suggest that both sexes may disperse from their mothers, and groups consisting of unrelated subadults are common. We also found no instances of paternity within the groups. Our results provide the first insights into dispersal, social organization, and the mating system in this rarely sighted species. Why whales strand is still unknown but, in Gray's beaked whales, the dead can tell us much about the living. © The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

    PubMed Central

    Mulhall, Hayley J.; Marchenko, Steve A.; Hoettges, Kai F.; Estrada, Laura C.; Lee, Abraham P.; Hughes, Michael P.; Flanagan, Lisa A.

    2011-01-01

    Background Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers. Methodology/Principal Findings We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates. Conclusions/Significance We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors. PMID:21980464

  14. Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells.

    PubMed

    Lawson, Devon A; Bhakta, Nirav R; Kessenbrock, Kai; Prummel, Karin D; Yu, Ying; Takai, Ken; Zhou, Alicia; Eyob, Henok; Balakrishnan, Sanjeev; Wang, Chih-Yang; Yaswen, Paul; Goga, Andrei; Werb, Zena

    2015-10-01

    Despite major advances in understanding the molecular and genetic basis of cancer, metastasis remains the cause of >90% of cancer-related mortality. Understanding metastasis initiation and progression is critical to developing new therapeutic strategies to treat and prevent metastatic disease. Prevailing theories hypothesize that metastases are seeded by rare tumour cells with unique properties, which may function like stem cells in their ability to initiate and propagate metastatic tumours. However, the identity of metastasis-initiating cells in human breast cancer remains elusive, and whether metastases are hierarchically organized is unknown. Here we show at the single-cell level that early stage metastatic cells possess a distinct stem-like gene expression signature. To identify and isolate metastatic cells from patient-derived xenograft models of human breast cancer, we developed a highly sensitive fluorescence-activated cell sorting (FACS)-based assay, which allowed us to enumerate metastatic cells in mouse peripheral tissues. We compared gene signatures in metastatic cells from tissues with low versus high metastatic burden. Metastatic cells from low-burden tissues were distinct owing to their increased expression of stem cell, epithelial-to-mesenchymal transition, pro-survival, and dormancy-associated genes. By contrast, metastatic cells from high-burden tissues were similar to primary tumour cells, which were more heterogeneous and expressed higher levels of luminal differentiation genes. Transplantation of stem-like metastatic cells from low-burden tissues showed that they have considerable tumour-initiating capacity, and can differentiate to produce luminal-like cancer cells. Progression to high metastatic burden was associated with increased proliferation and MYC expression, which could be attenuated by treatment with cyclin-dependent kinase (CDK) inhibitors. These findings support a hierarchical model for metastasis, in which metastases are initiated

  15. Complete Mitochondrial Reveals a New Phylogenetic Perspective on the Brackish Water Goby Mugilogobius Group (Teleostei: Gobiidae: Gobionellinae).

    PubMed

    Huang, Shih-Pin; Chen, I-Shiung; Jang-Liaw, Nian-Hong; Shao, Kwang-Tsao; Yung, Mana M N

    2016-10-01

    The Mugilogobius group consists of brackish water gobionellines widely distributed in the Indo-West Pacific region. Complete mitochondrial genome and morphological evidence was collected to estimate their phylogenetic relationship and taxonomic status. A total of 11 genera were sampled, including Brachygobius, Calamiana, Hemigobius, Mugilogobius, Pandaka, Pseudogobiopsis, Pseudogobius, Redigobius, Rhinogobius, Stigmatogobius, and Wuhanlinigobius, five of which were sequenced for the first time. A morphological phylogenetic tree was also reconstructed based on 35 characters. The molecular phylogenetic trees reveal that the Mugilogobius group contains four major clades. The present study also reveals that the adult male mouth size and forked sensory papillae row d can be considered as synapomorphies, and that the head pores on inter-orbital, anterior oculoscapular, and preopercular regions can be regarded as derived features among the Mugilogobius group. Furthermore, the absence of posterior oculoscapular pores may provide a clue for understanding the evolutionary history of the Mugilogobius group.

  16. Unconventional myosin traffic in cells reveals a selective actin cytoskeleton

    PubMed Central

    Brawley, Crista M.; Rock, Ronald S.

    2009-01-01

    Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin VI, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments. PMID:19478066

  17. Two sexually dimorphic cell groups in the human brain.

    PubMed

    Allen, L S; Hines, M; Shryne, J E; Gorski, R A

    1989-02-01

    A quantitative analysis of the volume of 4 cell groups in the preoptic-anterior hypothalamic area (PO-AHA) and of the supraoptic nucleus (SON) of the human brain was performed in 22 age-matched male and female individuals. We suggest the term Interstitial Nuclei of the Anterior Hypothalamus (INAH 1-4) to identify these 4 previously undescribed cell groups in the PO-AHA. While 2 INAH and the SON were not sexually dimorphic, gender-related differences were found in the other 2 cell groups. One nucleus (INAH-3) was 2.8 times larger in the male brain than in the female brain irrespective of age. The other cell group (INAH-2) was twice as large in the male brain, but also appeared to be related in women to circulating steroid hormone levels. Since the PO-AHA influences gonadotropin secretion, maternal behavior, and sexual behavior in several mammalian species, these results suggest that functional sex differences in the hypothalamus may be related to sex differences in neural structure.

  18. Identifying Two Groups of Entitled Individuals: Cluster Analysis Reveals Emotional Stability and Self-Esteem Distinction.

    PubMed

    Crowe, Michael L; LoPilato, Alexander C; Campbell, W Keith; Miller, Joshua D

    2016-12-01

    The present study hypothesized that there exist two distinct groups of entitled individuals: grandiose-entitled, and vulnerable-entitled. Self-report scores of entitlement were collected for 916 individuals using an online platform. Model-based cluster analyses were conducted on the individuals with scores one standard deviation above mean (n = 159) using the five-factor model dimensions as clustering variables. The results support the existence of two groups of entitled individuals categorized as emotionally stable and emotionally vulnerable. The emotionally stable cluster reported emotional stability, high self-esteem, more positive affect, and antisocial behavior. The emotionally vulnerable cluster reported low self-esteem and high levels of neuroticism, disinhibition, conventionality, psychopathy, negative affect, childhood abuse, intrusive parenting, and attachment difficulties. Compared to the control group, both clusters reported being more antagonistic, extraverted, Machiavellian, and narcissistic. These results suggest important differences are missed when simply examining the linear relationships between entitlement and various aspects of its nomological network.

  19. Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution

    PubMed Central

    Zhao, Chen; Pyle, Anna Marie

    2016-01-01

    Group II introns are self-splicing ribozymes that are essential in many organisms, and they are hypothesized to share a common evolutionary ancestor with the spliceosome. While structural similarity of RNA components supports this connection, it is of interest to determine whether associated protein factors also share an evolutionary heritage. Here we present the crystal structures of reverse transcriptase (RT) domains from two group II intron encoded proteins (maturases) from Roseburia intestinalis and Eubacterium rectale, obtained at 1.2 Å and 2.1 Å respectively. Their architecture is more similar to the spliceosomal Prp8 RT-like domain than to any other RTs, and they share substantial similarity with flaviviral RNA polymerases. The RT domain itself is sufficient for binding intron RNA with high affinity and specificity, and it is contained within an active RT enzyme. These studies provide a foundation for understanding structure-function relationships within group II intron–maturase complexes. PMID:27136328

  20. Blockade of maitotoxin-induced oncotic cell death reveals zeiosis

    PubMed Central

    Estacion, Mark; Schilling, William P

    2002-01-01

    Background Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca2+ influx via non-selective cation channels, 2) uptake of vital dyes via formation of large pores, and 3) release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC) in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells. Results Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 ≈ 1.9 and 0.66 μM, respectively) suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program. Conclusions Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms. PMID:11825342

  1. X-ray Crystallographic Studies Reveal That the Incorporation of Spacer Groups in Carbonic Anhydrase Inhibitors Causes Alternate Binding Modes

    SciTech Connect

    Fisher,S.; Govindasamy, L.; Boyle, N.; Agbandje-McKenna, M.; Silverman, D.; Blackburn, G.; McKenna, R.

    2006-01-01

    Human carbonic anhydrases (CAs) are well studied targets for the development of inhibitors for pharmaceutical applications. The crystal structure of human CA II has been determined in complex with two CA inhibitors (CAIs) containing conventional sulfonamide and thiadiazole moieties separated by a -CF{sub 2}- or -CHNH{sub 2}- spacer group. The structures presented here reveal that these spacer groups allow novel binding modes for the thiadiazole moiety compared with conventional CAIs.

  2. Ancestral morphology of crown-group molluscs revealed by a new Ordovician stem aculiferan.

    PubMed

    Vinther, Jakob; Parry, Luke; Briggs, Derek E G; Van Roy, Peter

    2017-02-23

    Exceptionally preserved fossils provide crucial insights into extinct body plans and organismal evolution. Molluscs, one of the most disparate animal phyla, radiated rapidly during the early Cambrian period (approximately 535-520 million years ago (Ma)). The problematic fossil taxa Halkieria and Orthrozanclus (grouped in Sachitida) have been assigned variously to stem-group annelids, brachiopods, stem-group molluscs or stem-group aculiferans (Polyplacophora and Aplacophora), but their affinities have remained controversial owing to a lack of preserved diagnostic characters. Here we describe a new early sachitid, Calvapilosa kroegeri gen. et sp. nov. from the Fezouata biota of Morocco (Early Ordovician epoch, around 478 Ma). The new taxon is characterized by the presence of a single large anterior shell plate and polystichous radula bearing a median tooth and several lateral and uncinal teeth in more than 125 rows. Its flattened body is covered by hollow spinose sclerites, and a smooth, ventral girdle flanks an extensive mantle cavity. Phylogenetic analyses resolve C. kroegeri as a stem-group aculiferan together with other single-plated forms such as Maikhanella (Siphogonuchites) and Orthrozanclus; Halkieria is recovered closer to the aculiferan crown. These genera document the stepwise evolution of the aculiferan body plan from forms with a single, almost conchiferan-like shell through two-plated taxa such as Halkieria, to the eight-plated crown-group aculiferans. C. kroegeri therefore provides key evidence concerning the long debate about the crown molluscan affinities of sachitids. This new discovery strongly suggests that the possession of only a single calcareous shell plate and the presence of unmineralised sclerites are plesiomorphic (an ancestral trait) for the molluscan crown.

  3. [Chronic prurigo revealing an angioimmunoblastic T cell lymphoma].

    PubMed

    Khaled, Aida; Sfia, Mehdi; Fazaa, Becima; Kourda, Nadia; Zermani, Rachida; Baccouche, Kamel; Ben Jilani, Sara; Kamoun, Mohamed Ridha

    2009-08-01

    Cutaneous manifestations in angio-immunoblastic T cell lymphoma (AITL) can be seen in almost 50% of patients. They are especially represented by maculo-papular pseudotoxic exanthema. The other manifestations, like nodular prurigo are rarely observed. We report a case of AITL diagnosed after an etiologic screening for chronic prurigo. The objective of our work is to stress on the possibility to diagnose a hemopathy in etiologic screening of chronic pruritus. A 45-year-old patient presented a chronic pruritus of 18 months associated with general manifestations (fever, night sweating and weight loss). Physical examination showed diffuse adenomegalies. On histology, the lymph nodes were composed of polymorphous lymphoid proliferation made of middle to large sized cells with clusters of epithelioid cells and post-capillary veinules hyperplasia. Immunohistochemical study showed T cell phenotype (CD3+). Large cells were CD30+. Latent Protein of EBV virus was not expressed. Molecular biological analysis of a lymph node showed a T cell clonal proliferation. Cutaneous biopsy showed a little dermic perivascular lymphocytic inflammatory infiltrate. The diagnosis of angio-immunoblastic T cell lymphoma was made. The abdomino-pelvian CT scanner showed multiple inter-aortico-cave lymph nodes and a splenomegaly. A polychemotherapy was initiated. In front of chronic prurigo with general manifestations, a careful etiologic screening should be done to detect internal disorders especially malignant hemopathies.

  4. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.

    PubMed

    Trapnell, Cole; Cacchiarelli, Davide; Grimsby, Jonna; Pokharel, Prapti; Li, Shuqiang; Morse, Michael; Lennon, Niall J; Livak, Kenneth J; Mikkelsen, Tarjei S; Rinn, John L

    2014-04-01

    Defining the transcriptional dynamics of a temporal process such as cell differentiation is challenging owing to the high variability in gene expression between individual cells. Time-series gene expression analyses of bulk cells have difficulty distinguishing early and late phases of a transcriptional cascade or identifying rare subpopulations of cells, and single-cell proteomic methods rely on a priori knowledge of key distinguishing markers. Here we describe Monocle, an unsupervised algorithm that increases the temporal resolution of transcriptome dynamics using single-cell RNA-Seq data collected at multiple time points. Applied to the differentiation of primary human myoblasts, Monocle revealed switch-like changes in expression of key regulatory factors, sequential waves of gene regulation, and expression of regulators that were not known to act in differentiation. We validated some of these predicted regulators in a loss-of function screen. Monocle can in principle be used to recover single-cell gene expression kinetics from a wide array of cellular processes, including differentiation, proliferation and oncogenic transformation.

  5. Revealing of Biological Activity in Crude Extracts, Seperated Fractions, Groups of Chemical Substance and Individual Compounds

    USDA-ARS?s Scientific Manuscript database

    Crude extracts, separated fractions, groups of chemical substances, and individual compounds from natural sources are all evaluated stepwise while performing purifications in in-house bioassays. In a stepwise fashion proceeding from crude extracts to fractions and on to pure compounds, decisions ar...

  6. Revealed: The spy who regulates neuroblastoma stem cells.

    PubMed

    Vora, Parvez; Venugopal, Chitra; Singh, Sheila K

    2014-11-30

    Neuroblastoma (NB), an embryonal tumour of the sympathetic nervous system, is thought to originate from undifferentiated neural crest cells and is known to exhibit extremely heterogeneous biological and clinical behaviors. Occurring in very young children, the median age at diagnosis is 17 months and it accounts for 10% of all pediatric cancer mortalities. The standard treatment regimen for patients with high-risk NB includes induction and surgery followed by isotretinoin or Accutane (13-cis retinoic acid) treatment, which is shown to induce terminal differentiation of NB cells. However, molecular regulators that maintain an undifferentiated phenotype in NB cells are still poorly understood.

  7. Spatial guilds in the Serengeti food web revealed by a Bayesian group model.

    PubMed

    Baskerville, Edward B; Dobson, Andy P; Bedford, Trevor; Allesina, Stefano; Anderson, T Michael; Pascual, Mercedes

    2011-12-01

    Food webs, networks of feeding relationships in an ecosystem, provide fundamental insights into mechanisms that determine ecosystem stability and persistence. A standard approach in food-web analysis, and network analysis in general, has been to identify compartments, or modules, defined by many links within compartments and few links between them. This approach can identify large habitat boundaries in the network but may fail to identify other important structures. Empirical analyses of food webs have been further limited by low-resolution data for primary producers. In this paper, we present a Bayesian computational method for identifying group structure using a flexible definition that can describe both functional trophic roles and standard compartments. We apply this method to a newly compiled plant-mammal food web from the Serengeti ecosystem that includes high taxonomic resolution at the plant level, allowing a simultaneous examination of the signature of both habitat and trophic roles in network structure. We find that groups at the plant level reflect habitat structure, coupled at higher trophic levels by groups of herbivores, which are in turn coupled by carnivore groups. Thus the group structure of the Serengeti web represents a mixture of trophic guild structure and spatial pattern, in contrast to the standard compartments typically identified. The network topology supports recent ideas on spatial coupling and energy channels in ecosystems that have been proposed as important for persistence. Furthermore, our Bayesian approach provides a powerful, flexible framework for the study of network structure, and we believe it will prove instrumental in a variety of biological contexts.

  8. Spatial Guilds in the Serengeti Food Web Revealed by a Bayesian Group Model

    PubMed Central

    Baskerville, Edward B.; Dobson, Andy P.; Bedford, Trevor; Allesina, Stefano; Anderson, T. Michael; Pascual, Mercedes

    2011-01-01

    Food webs, networks of feeding relationships in an ecosystem, provide fundamental insights into mechanisms that determine ecosystem stability and persistence. A standard approach in food-web analysis, and network analysis in general, has been to identify compartments, or modules, defined by many links within compartments and few links between them. This approach can identify large habitat boundaries in the network but may fail to identify other important structures. Empirical analyses of food webs have been further limited by low-resolution data for primary producers. In this paper, we present a Bayesian computational method for identifying group structure using a flexible definition that can describe both functional trophic roles and standard compartments. We apply this method to a newly compiled plant-mammal food web from the Serengeti ecosystem that includes high taxonomic resolution at the plant level, allowing a simultaneous examination of the signature of both habitat and trophic roles in network structure. We find that groups at the plant level reflect habitat structure, coupled at higher trophic levels by groups of herbivores, which are in turn coupled by carnivore groups. Thus the group structure of the Serengeti web represents a mixture of trophic guild structure and spatial pattern, in contrast to the standard compartments typically identified. The network topology supports recent ideas on spatial coupling and energy channels in ecosystems that have been proposed as important for persistence. Furthermore, our Bayesian approach provides a powerful, flexible framework for the study of network structure, and we believe it will prove instrumental in a variety of biological contexts. PMID:22219719

  9. Women’s Experience with Overactive Bladder Symptoms and Treatment: Insight Revealed from Patient Focus Groups

    PubMed Central

    Anger, Jennifer T.; Nissim, Helen A.; Le, Thuy X.; Smith, Ariana L.; Lee, Una; Sarkisian, Catherine; Litwin, Mark S.; Raz, Shlomo; Rodriguez, Larissa V.; Maliski, Sally L.

    2013-01-01

    Aims Research has focused on treatment of overactive bladder (OAB) symptoms in women with the goal of cure. The objective of this study was to assess women’s perceptions of their OAB symptoms, treatment experience, and outcomes by conducting patient focus groups. Methods Women seen in our academic center female urology referral clinics were identified by ICD-9 codes for OAB symptoms and recruited to participate in one of five focus groups, totaling 33 patients. Non-clinician moderators conducted the focus group sessions incorporating topics related to patients’ perceptions of OAB symptoms, treatments, and outcomes. Data analysis was performed using grounded theory methodology. Results Qualitative analysis yielded several preliminary themes: impact of OAB on quality of life, strategies to control wetness, medications and side effects, and triggers. The majority of focus group participants reported only a partial response to medication and other physician-recommended treatments for OAB. They therefore developed self-reliant personalized strategies to improve their quality of life. These strategies included fluid restriction, preventive toileting, and, most importantly, the use of incontinence pads. Conclusions The majority of the women who participated in the focus groups reported only a partial response to medical and other treatments for OAB. As a result, they developed personalized self-management strategies to improve their quality of life. Although most studies addressing the treatment of OAB aim at curing the condition, such a strategy may be unrealistic. Applying a chronic care model that uses a patient-centered symptom management approach to OAB may optimize patient outcomes and improve quality of life. PMID:21538495

  10. Recognition Strategies of Group 3 Innate Lymphoid Cells

    PubMed Central

    Killig, Monica; Glatzer, Timor; Romagnani, Chiara

    2014-01-01

    During the early phase of an inflammatory response, innate cells can use different strategies to sense environmental danger. These include the direct interaction of specific activating receptors with pathogen-encoded/danger molecules or the engagement of cytokine receptors by pro-inflammatory mediators produced by antigen presenting cells in the course of the infection. These general recognition strategies, which have been extensively described for innate myeloid cells, are shared by innate lymphoid cells (ILC), such as Natural Killer (NK) cells. The family of ILC has recently expanded with the discovery of group 2 (ILC2) and group 3 ILC (ILC3), which play an important role in the defense against extracellular pathogens. Although ILC3 and NK cells share some phenotypic characteristics, the recognition strategies employed by the various ILC3 subsets have been only partially characterized. In this review, we will describe and comparatively discuss how ILC3 sense environmental cues and how the triggering of different receptors may regulate their functional behavior during an immune response. PMID:24744763

  11. Brief Report: Single-Cell Analysis Reveals Cell Division-Independent Emergence of Megakaryocytes From Phenotypic Hematopoietic Stem Cells.

    PubMed

    Roch, Aline; Trachsel, Vincent; Lutolf, Matthias P

    2015-10-01

    Despite increasingly stringent methods to isolate hematopoietic stem cells (HSCs), considerable heterogeneity remains in terms of their long-term self-renewal and differentiation potential. Recently, the existence of long-lived, self-renewing, myeloid-restricted progenitors in the phenotypically defined HSC compartment has been revealed, but these cells remain poorly characterized. Here, we used an in vitro single-cell analysis approach to track the fate of 330 long-term HSCs (LT-HSC; Lin- cKit+ Sca-1+ CD150+ CD48- CD34-) cultured for 5 days under serum-free basal conditions. Our analysis revealed a highly heterogeneous behavior with approximately 15% of all phenotypic LT-HSCs giving rise to megakaryocytes (Mk). Surprisingly, in 65% of these cases, Mk development occurred in the absence of cell division. This observation suggests that myeloid-restricted progenitors may not derive directly from LT-HSCs but instead could share an identical cell surface marker repertoire.

  12. Comparison of Dolphins' Body and Brain Measurements with Four Other Groups of Cetaceans Reveals Great Diversity.

    PubMed

    Ridgway, Sam H; Carlin, Kevin P; Van Alstyne, Kaitlin R; Hanson, Alicia C; Tarpley, Raymond J

    2016-01-01

    We compared mature dolphins with 4 other groupings of mature cetaceans. With a large data set, we found great brain diversity among 5 different taxonomic groupings. The dolphins in our data set ranged in body mass from about 40 to 6,750 kg and in brain mass from 0.4 to 9.3 kg. Dolphin body length ranged from 1.3 to 7.6 m. In our combined data set from the 4 other groups of cetaceans, body mass ranged from about 20 to 120,000 kg and brain mass from about 0.2 to 9.2 kg, while body length varied from 1.21 to 26.8 m. Not all cetaceans have large brains relative to their body size. A few dolphins near human body size have human-sized brains. On the other hand, the absolute brain mass of some other cetaceans is only one-sixth as large. We found that brain volume relative to body mass decreases from Delphinidae to a group of Phocoenidae and Monodontidae, to a group of other odontocetes, to Balaenopteroidea, and finally to Balaenidae. We also found the same general trend when we compared brain volume relative to body length, except that the Delphinidae and Phocoenidae-Monodontidae groups do not differ significantly. The Balaenidae have the smallest relative brain mass and the lowest cerebral cortex surface area. Brain parts also vary. Relative to body mass and to body length, dolphins also have the largest cerebellums. Cortex surface area is isometric with brain size when we exclude the Balaenidae. Our data show that the brains of Balaenidae are less convoluted than those of the other cetaceans measured. Large vascular networks inside the cranial vault may help to maintain brain temperature, and these nonbrain tissues increase in volume with body mass and with body length ranging from 8 to 65% of the endocranial volume. Because endocranial vascular networks and other adnexa, such as the tentorium cerebelli, vary so much in different species, brain size measures from endocasts of some extinct cetaceans may be overestimates. Our regression of body length on endocranial

  13. Comparison of Dolphins' Body and Brain Measurements with Four Other Groups of Cetaceans Reveals Great Diversity

    PubMed Central

    Ridgway, Sam H.; Carlin, Kevin P.; Van Alstyne, Kaitlin R.; Hanson, Alicia C.; Tarpley, Raymond J.

    2017-01-01

    We compared mature dolphins with 4 other groupings of mature cetaceans. With a large data set, we found great brain diversity among 5 different taxonomic groupings. The dolphins in our data set ranged in body mass from about 40 to 6,750 kg and in brain mass from 0.4 to 9.3 kg. Dolphin body length ranged from 1.3 to 7.6 m. In our combined data set from the 4 other groups of cetaceans, body mass ranged from about 20 to 120,000 kg and brain mass from about 0.2 to 9.2 kg, while body length varied from 1.21 to 26.8 m. Not all cetaceans have large brains relative to their body size. A few dolphins near human body size have human-sized brains. On the other hand, the absolute brain mass of some other cetaceans is only one-sixth as large. We found that brain volume relative to body mass decreases from Delphinidae to a group of Phocoenidae and Monodontidae, to a group of other odontocetes, to Balaenopteroidea, and finally to Balaenidae. We also found the same general trend when we compared brain volume relative to body length, except that the Delphinidae and Phocoenidae-Monodontidae groups do not differ significantly. The Balaenidae have the smallest relative brain mass and the lowest cerebral cortex surface area. Brain parts also vary. Relative to body mass and to body length, dolphins also have the largest cerebellums. Cortex surface area is isometric with brain size when we exclude the Balaenidae. Our data show that the brains of Balaenidae are less convoluted than those of the other cetaceans measured. Large vascular networks inside the cranial vault may help to maintain brain temperature, and these nonbrain tissues increase in volume with body mass and with body length ranging from 8 to 65% of the endocranial volume. Because endocranial vascular networks and other adnexa, such as the tentorium cerebelli, vary so much in different species, brain size measures from endocasts of some extinct cetaceans may be overestimates. Our regression of body length on endocranial

  14. Severe immune haemolysis in a group A recipient of a group O red blood cell unit.

    PubMed

    Barjas-Castro, M L; Locatelli, M F; Carvalho, M A; Gilli, S O; Castro, V

    2003-08-01

    Haemolysis caused by passive ABO antibodies is a rare transfusional complication. We report a case of severe haemolytic reaction in a 38-year-old man (blood group A) with lymphoma who had received one red blood cell (RBC) unit group O. After transfusion of 270 mL, the patient experienced fever, dyspnoea, chills and back pain. On the following morning he was icteric and pale. Haptoglobin was inferior to 5.8 mgdL(-1), haemoglobin was not increased and lactate dehydrogenase was elevated. Haemolysis was evident on observation of the patient's post-transfusion samples. The recipient's red cells developed a positive direct antiglobulin test and Lui elution showed anti-A coated the cells. Fresh donor serum had an anti-A titre of 1024, which was not reduced by treating the serum with dithiothreitol. Donor isoagglutinin screening has been determined by microplate automated analyser and showed titre higher than 100. Physicians should be aware of the risk of haemolysis associated with ABO-passive antibodies. There is generally no agreement justifying the isoagglutinin investigation prior to transfusion. However, automated quantitative isoagglutinin determination could be part of the modern donor testing process, mainly in blood banks where identical ABO group units (platelets or phenotyped RBCs) are not available owing to limited supply.

  15. Crystal structure of group II intron domain 1 reveals a template for RNA assembly

    SciTech Connect

    Zhao, Chen; Rajashankar, Kanagalaghatta R.; Marcia, Marco; Pyle, Anna Marie

    2015-10-26

    Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. In this paper, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Composed of domain 1 from the Oceanobacillus iheyensis group II intron (266 nucleotides), this intermediate retains native-like features but adopts a compact conformation in which the active site cleft is closed. Transition between this closed and the open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. Finally, the open state is then stabilized by sequential docking of downstream intron domains, suggesting a 'first come, first folded' strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures.

  16. Crystal structure of group II intron domain 1 reveals a template for RNA assembly

    DOE PAGES

    Zhao, Chen; Rajashankar, Kanagalaghatta R.; Marcia, Marco; ...

    2015-10-26

    Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. In this paper, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Composed of domain 1 from the Oceanobacillus iheyensis group II intron (266 nucleotides), this intermediate retains native-like features but adopts a compact conformation in which the active site cleft is closed. Transition between this closed andmore » the open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. Finally, the open state is then stabilized by sequential docking of downstream intron domains, suggesting a 'first come, first folded' strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures.« less

  17. Crystal structure of group II intron domain 1 reveals a template for RNA assembly

    PubMed Central

    Zhao, Chen; Rajashankar, Kanagalaghatta R.; Marcia, Marco; Pyle, Anna Marie

    2015-01-01

    Although the importance of large noncoding RNAs is increasingly appreciated, our understanding of their structures and architectural dynamics remains limited. In particular, we know little about RNA folding intermediates and how they facilitate the productive assembly of RNA tertiary structures. Here, we report the crystal structure of an obligate intermediate that is required during the earliest stages of group II intron folding. Comprised of intron domain 1 from the Oceanobacillus iheyensis group II intron (D1, 266 nts), this intermediate retains native-like features but adopts a compact conformation in which the active-site cleft is closed. Transition between this closed and open (native) conformation is achieved through discrete rotations of hinge motifs in two regions of the molecule. The open state is then stabilized by sequential docking of downstream intron domains, suggesting a “first comes, first folds” strategy that may represent a generalizable pathway for assembly of large RNA and ribonucleoprotein structures. PMID:26502156

  18. An information theoretic framework reveals a tunable allosteric network in group II chaperonins.

    PubMed

    Lopez, Tom; Dalton, Kevin; Tomlinson, Anthony; Pande, Vijay; Frydman, Judith

    2017-09-01

    ATP-dependent allosteric regulation of the ring-shaped group II chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows chaperonins to support robust folding over a wider range of metabolic conditions.

  19. Outskirts of Local Group Dwarf Galaxies Revealed by Subaru Hyper Suprime-Cam

    NASA Astrophysics Data System (ADS)

    Komiyama, Yutaka

    2017-03-01

    Local Group galaxies are important targets since their stellar populations can be resolved, and their properties can be investigated in detail with the help of stellar evolutionary models. The newly-built instrument for the 8.2m Subaru Telescope, Hyper Suprime-Cam (HSC), which has a 1 Giga pixel CCD camera with 1.5 degrees field of view, is the best instrument for observing Local Group galaxies. We have carried out a survey for Local Group dwarf galaxies using HSC aiming to shed light on the outskirts of these galaxies. The survey covers target galaxies out beyond the tidal radii down to a depth unexplored by previous surveys. Thanks to the high spatial resolution and high sensitivity provided by the Subaru Telescope, we are able to investigate properties such as spatial distribution and stellar population from the very center of galaxies to the outskirts. In this article, I will show results for the dwarf irregular galaxy NGC 6822 and the dwarf spheroidal galaxy Ursa Minor.

  20. Using Adolescents' Drawings to Reveal Stereotypes About Ethnic Groups in Guatemala.

    PubMed

    Ashdown, Brien K; Gibbons, Judith L; de Baessa, Yetilú; Brown, Carrie M

    2017-06-15

    It is important to identify stereotypes about indigenous people because those stereotypes influence prejudice and discrimination, both obstacles to social justice and universal human rights. The purpose of the current study was to document the stereotypes, as held by Guatemalan adolescents, of indigenous Maya people (e.g., Maya) and nonindigenous Ladinos in Guatemala (the 2 main ethnic groups in Guatemala). Guatemalan adolescents (N = 465; 38.3% female; Mage = 14.51 years; SDage = 1.81 years) provided drawings and written characteristics about indigenous Maya and nonindigenous Ladino people, which were then coded for patterns in the data. These patterns included negative stereotypes, such as the Maya being lazy and Ladina women being weak; and positive stereotypes, such as the Maya being caring and warm and Ladino men being successful. There were also interactions between the participants' own gender and ethnicity and how they depicted the target they were assigned. For example, male participants were unlikely to depict male targets of either ethnicity engaging in homemaking activities. Finally, there was evidence of in-group bias based both on gender and ethnicity. These findings suggest that perhaps because indigenous groups around the world share some common negative stereotypes, an understanding of these stereotypes will aid in decreasing prejudice and discrimination against indigenous people, could reduce intergroup conflict, and increase access to basic human rights. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  1. Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis

    PubMed Central

    Huang, Yanmei; Ainsley, Joshua A.; Reijmers, Leon G.; Jackson, F. Rob

    2013-01-01

    Abstract Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a Drosophila cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes Tdc2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity. PMID:24348200

  2. Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

    PubMed

    Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

    2015-02-01

    Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

  3. Single-Cell Reconstruction of Oxytocinergic Neurons Reveals Separate Hypophysiotropic and Encephalotropic Subtypes in Larval Zebrafish

    PubMed Central

    Gutierrez-Triana, Jose Arturo; Knerr, Boris

    2017-01-01

    Oxytocin regulates a diverse set of processes including stress, analgesia, metabolism, and social behavior. How such diverse functions are mediated by a single hormonal system is not well understood. Different functions of oxytocin could be mediated by distinct cell groups, yet it is currently unknown whether different oxytocinergic cell types exist that specifically mediate peripheral neuroendocrine or various central neuromodulatory processes via dedicated pathways. Using the Brainbow technique to map the morphology and projections of individual oxytocinergic cells in the larval zebrafish brain, we report here the existence of two main types of oxytocinergic cells: those that innervate the pituitary and those that innervate diverse brain regions. Similar to the situation in the adult rat and the adult midshipman, but in contrast to the situation in the adult trout, these two cell types are mutually exclusive and can be distinguished based on morphological and anatomical criteria. Further, our results reveal that complex oxytocinergic innervation patterns are already established in the larval zebrafish brain. PMID:28317020

  4. Focus Groups Reveal Differences in Career Experiences Between Male and Female Geoscientists

    NASA Astrophysics Data System (ADS)

    Oconnell, S.; Frey, C. D.; Holmes, M.

    2003-12-01

    We conducted twelve telephone focus groups of geoscientists to discover what motivates geoscientists to enter our field and stay in our field. There were separate male and female groups from six different professional categories: administrators, full and associate professors, non-tenure track personnel, assistant professors, post-docs and PhD candidates, Bachelor's and Master's candidates. A total of 96 geoscientists participated. Specifically, respondents were asked what initially brought them into the geosciences. Three dominant themes emerged: the subject matter itself, undergraduate experiences, and relationships. A total of 51 responses to this question related to the subject matter itself. Approximately 61 percent (31) of those responses were given by male focus group participants. Across all focus groups, participants brought up issues such as a general appreciation of the outdoors, weather, rocks, and dinosaurs. Following closely behind the general subject matter is undergraduate events. Fifty-one responses mentioned something about undergraduate experiences such as an introductory class, a laboratory experience, or field experiences. While both female and male participants discussed the role of interpersonal relationships in their decision to become a geoscientist, females were slightly more likely to bring up relevant relationships (26 times for females compared to 21 for males). These relationships varied in both groups from a parent or grandparents influence to camping trips with professors. When respondents were asked whether they had ever considered leaving the geosciences and under what circumstances, there was a striking difference between males and females: males were far less likely to have ever considered leaving. Younger males were more likely to consider leaving than older geoscientists. They feel challenged by the financial constraints of graduate school and the time constraints of academic vs. family life. Many females considered leaving at

  5. First-generation linkage map for the common frog Rana temporaria reveals sex-linkage group

    PubMed Central

    Cano, J M; Li, M-H; Laurila, A; Vilkki, J; Merilä, J

    2011-01-01

    The common frog (Rana temporaria) has become a model species in the fields of ecology and evolutionary biology. However, lack of genomic resources has been limiting utility of this species for detailed evolutionary genetic studies. Using a set of 107 informative microsatellite markers genotyped in a large full-sib family (800 F1 offspring), we created the first linkage map for this species. This partial map—distributed over 15 linkage groups—has a total length of 1698.8 cM. In line with the fact that males are the heterogametic sex in this species and a reduction of recombination is expected, we observed a lower recombination rate in the males (map length: 1371.5 cM) as compared with females (2089.8 cM). Furthermore, three loci previously documented to be sex-linked (that is, carrying male-specific alleles) in adults from the wild mapped to the same linkage group. The linkage map described in this study is one of the densest ones available for amphibians. The discovery of a sex linkage group in Rana temporaria, as well as other regions with strongly reduced male recombination rates, should help to uncover the genetic underpinnings of the sex-determination system in this species. As the number of linkage groups found (n=15) is quite close to the actual number of chromosomes (n=13), the map should provide a useful resource for further evolutionary, ecological and conservation genetic work in this and other closely related species. PMID:21587305

  6. Homing and group cohesion in Atlantic cod Gadus morhua revealed by tagging experiments.

    PubMed

    Tamdrari, H; Brêthes, J-C; Castonguay, M; Duplisea, D E

    2012-07-01

    Homing behaviour and group cohesion in Atlantic cod Gadus morhua from the northern Gulf of St Lawrence were studied based on tagging-recapture data from two periods, the 1980s and a recent period from 1996 to 2008. Two or more tags from a single tagging experiment were frequently recovered together in subsequent years. The null hypothesis was tested that the frequency of matching tag recoveries occurred by chance only through random mixing of tagged G. morhua before their recapture by the commercial fishery. The alternative hypothesis was that non-random, positive association (group cohesion) existed among tagged individuals that persisted through time and during migrations. Results show that the G. morhua population exhibits a homing behaviour, with temporal stability across seasons and years: 50% of recaptured fish in the recent period were caught <34 km from their mark site, even 3 years after release. In the 1980s, G. morhua were located at <10 km from their release site 1 year after tagging during summer and at <16 km during spring and autumn combined. Despite the increasing distance between the mark and recapture sites over time, the difference was not significant. In addition, occurrences of two or more tagged fish from the same release event that were caught together indicated a non-random association among individual fish for periods of one to several years and through migrations over several hundred kilometres. Hence G. morhua showed group cohesion in addition to site fidelity. These two interacting behaviours may be fundamental for the rebuilding and conservation of depleted fish stocks. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

  7. Kinome sequencing reveals RET G691S polymorphism in human neuroendocrine lung cancer cell lines

    PubMed Central

    Sosonkina, Nadiya; Hong, Seung-Keun; Starenki, Dmytro; Park, Jong-In

    2014-01-01

    Neuroendocrine (NE) lung tumors comprise 20–25% of all invasive lung malignancies. Currently, no effective treatments are available to cure these tumors, and it is necessary to identify a molecular alteration(s) that characterizes NE lung tumor cells. We aimed to identify a kinase mutation(s) associated with NE lung tumor by screening 517 kinase-encoding genes in human lung cancer cell lines. Our next-generation sequencing analysis of six NE lung tumor cell lines (four small cell lung cancer lines and two non-small cell lung cancer lines) and three non-NE lung tumor lines revealed various kinase mutations, including a nonsynonymous mutation in the proto-oncogene RET (c.2071G>A; p.G691S). Further evaluation of the RET polymorphism in total 15 lung cancer cell lines by capillary sequencing suggested that the frequency of the minor allele (A-allele) in NE lung tumor lines was significantly higher than its frequency in a reference population (p = 0.0001). However, no significant difference between non-NE lung tumor lines and a reference group was detected (p = 1.0). Nevertheless, neither RET expression levels were correlated with the levels of neuron-specific enolase (NSE), a key NE marker, nor vandetanib and cabozantinib, small molecule compounds that inhibit RET, affected NSE levels in lung cancer cells. Our data suggest a potential association of G691S RET polymorphism with NE lung tumor, proposing the necessity of more thorough evaluation of this possibility. The dataset of kinase mutation profiles in this report may help choosing cell line models for study of lung cancer. PMID:25530832

  8. A survey of oenophages during wine making reveals a novel group with unusual genomic characteristics.

    PubMed

    Philippe, Cécile; Jaomanjaka, Fety; Claisse, Olivier; Laforgue, Rémi; Maupeu, Julie; Petrel, Melina; Le Marrec, Claire

    2017-09-18

    Oenophages have so far been mostly isolated from red wines under malolactic fermentation (MLF), and correspond to temperate or ex-temperate phages of Oenococcus oeni. Their genomes are clustered into 4 integrase gene sequence groups, which are also related to the chromosomal integration site. Our aims were to survey the occurrence of oenophages in a broader and more diverse collection of samples than those previously explored. Active phages were isolated from 33 out of 166 samples, which mostly originated from must and MLF. Seventy one phage lysates were produced and 30% were assigned to a novel group with unusual genomic characteristics, called unk. All unk members produced similar RAPD and DNA restriction patterns, were negative by PCR for the signature sequences previously identified in the integrase and endolysin genes of oenophages, and lacked any BamHI restriction site in their genome. The data support that development of additional and novel signature genes for assessing oenophage diversity is now required. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. New patterns in human biogeography revealed by networks of contacts between linguistic groups.

    PubMed

    Capitán, José A; Bock Axelsen, Jacob; Manrubia, Susanna

    2015-03-07

    Human languages differ broadly in abundance and are distributed highly unevenly on the Earth. In many qualitative and quantitative aspects, they strongly resemble biodiversity distributions. An intriguing and previously unexplored issue is the architecture of the neighbouring relationships between human linguistic groups. Here we construct and characterize these networks of contacts and show that they represent a new kind of spatial network with uncommon structural properties. Remarkably, language networks share a meaningful property with food webs: both are quasi-interval graphs. In food webs, intervality is linked to the existence of a niche space of low dimensionality; in language networks, we show that the unique relevant variable is the area occupied by the speakers of a language. By means of a range model analogous to niche models in ecology, we show that a geometric restriction of perimeter covering by neighbouring linguistic domains explains the structural patterns observed. Our findings may be of interest in the development of models for language dynamics or regarding the propagation of cultural innovations. In relation to species distribution, they pose the question of whether the spatial features of species ranges share architecture, and eventually generating mechanism, with the distribution of human linguistic groups.

  10. New patterns in human biogeography revealed by networks of contacts between linguistic groups

    PubMed Central

    Capitán, José A.; Bock Axelsen, Jacob; Manrubia, Susanna

    2015-01-01

    Human languages differ broadly in abundance and are distributed highly unevenly on the Earth. In many qualitative and quantitative aspects, they strongly resemble biodiversity distributions. An intriguing and previously unexplored issue is the architecture of the neighbouring relationships between human linguistic groups. Here we construct and characterize these networks of contacts and show that they represent a new kind of spatial network with uncommon structural properties. Remarkably, language networks share a meaningful property with food webs: both are quasi-interval graphs. In food webs, intervality is linked to the existence of a niche space of low dimensionality; in language networks, we show that the unique relevant variable is the area occupied by the speakers of a language. By means of a range model analogous to niche models in ecology, we show that a geometric restriction of perimeter covering by neighbouring linguistic domains explains the structural patterns observed. Our findings may be of interest in the development of models for language dynamics or regarding the propagation of cultural innovations. In relation to species distribution, they pose the question of whether the spatial features of species ranges share architecture, and eventually generating mechanism, with the distribution of human linguistic groups. PMID:25632000

  11. Integrative clustering by nonnegative matrix factorization can reveal coherent functional groups from gene profile data.

    PubMed

    Brdar, Sanja; Crnojević, Vladimir; Zupan, Blaz

    2015-03-01

    Recent developments in molecular biology and techniques for genome-wide data acquisition have resulted in abundance of data to profile genes and predict their function. These datasets may come from diverse sources and it is an open question how to commonly address them and fuse them into a joint prediction model. A prevailing technique to identify groups of related genes that exhibit similar profiles is profile-based clustering. Cluster inference may benefit from consensus across different clustering models. In this paper, we propose a technique that develops separate gene clusters from each of available data sources and then fuses them by means of nonnegative matrix factorization. We use gene profile data on the budding yeast S. cerevisiae to demonstrate that this approach can successfully integrate heterogeneous datasets and yield high-quality clusters that could otherwise not be inferred by simply merging the gene profiles prior to clustering.

  12. Genomic analysis reveals hidden biodiversity within colugos, the sister group to primates

    PubMed Central

    Mason, Victor C.; Li, Gang; Minx, Patrick; Schmitz, Jürgen; Churakov, Gennady; Doronina, Liliya; Melin, Amanda D.; Dominy, Nathaniel J.; Lim, Norman T-L.; Springer, Mark S.; Wilson, Richard K.; Warren, Wesley C.; Helgen, Kristofer M.; Murphy, William J.

    2016-01-01

    Colugos are among the most poorly studied mammals despite their centrality to resolving supraordinal primate relationships. Two described species of these gliding mammals are the sole living members of the order Dermoptera, distributed throughout Southeast Asia. We generated a draft genome sequence for a Sunda colugo and a Philippine colugo reference alignment, and used these to identify colugo-specific genetic changes that were enriched in sensory and musculoskeletal-related genes that likely underlie their nocturnal and gliding adaptations. Phylogenomic analysis and catalogs of rare genomic changes overwhelmingly support the contested hypothesis that colugos are the sister group to primates (Primatomorpha), to the exclusion of treeshrews. We captured ~140 kb of orthologous sequence data from colugo museum specimens sampled across their range and identified large genetic differences between many geographically isolated populations that may result in a >300% increase in the number of recognized colugo species. Our results identify conservation units to mitigate future losses of this enigmatic mammalian order. PMID:27532052

  13. Structural insights into the LCIB protein family reveals a new group of β-carbonic anhydrases

    PubMed Central

    Jin, Shengyang; Sun, Jian; Wunder, Tobias; Tang, Desong; Cousins, Asaph B.; Sze, Siu Kwan; Mueller-Cajar, Oliver; Gao, Yong-Gui

    2016-01-01

    Aquatic microalgae have evolved diverse CO2-concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO2-fixing enzyme rubisco. The limiting CO2-inducible B protein (LCIB) is known to be essential for CCM function in Chlamydomonas reinhardtii. To assign a function to this previously uncharacterized protein family, we purified and characterized a phylogenetically diverse set of LCIB homologs. Three of the six homologs are functional carbonic anhydrases (CAs). We determined the crystal structures of LCIB and limiting CO2-inducible C protein (LCIC) from C. reinhardtii and a CA-functional homolog from Phaeodactylum tricornutum, all of which harbor motifs bearing close resemblance to the active site of canonical β-CAs. Our results identify the LCIB family as a previously unidentified group of β-CAs, and provide a biochemical foundation for their function in the microalgal CCMs. PMID:27911826

  14. Purkinje cell degeneration in mice lacking the xeroderma pigmentosum group G gene.

    PubMed

    Sun, X Z; Harada, Y N; Takahashi, S; Shiomi, N; Shiomi, T

    2001-05-15

    Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene-targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin-D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild-type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin in situ nick labeling (TUNEL)-positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg-deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells.

  15. Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR.

    PubMed Central

    van Kuppeveld, F J; Johansson, K E; Galama, J M; Kissing, J; Bölske, G; van der Logt, J T; Melchers, W J

    1994-01-01

    The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the detection of mycoplasma contamination in cell cultures was investigated. A total of 104 cell cultures were tested by using microbiological culture, DNA fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A comparison of the results obtained with these techniques revealed agreement for 95 cell cultures. Discrepant results, which were interpreted as false negative or false positive on the basis of a comparison with the results obtained with other methods, were observed with nine cell cultures. The microbiological culture technique produced false-negative results for four cell cultures. The hybridization technique produced false-negative results for two cell cultures, and for one of these cell cultures the DNA staining technique also produced a false-negative result. The PCR may have produced false-positive results for one cell culture. Ambiguous results were obtained with the remaining two cell cultures. Furthermore, the presence of contaminating bacteria interfered with the interpretation of the DNA staining results for 16 cell cultures. For the same reason the hybridization signals of nine cell cultures could not be interpreted. Our results demonstrate the drawbacks of each of the detection methods and the suitability of the PCR for the detection of mycoplasmas in cell cultures. PMID:7509584

  16. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    PubMed Central

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

  17. Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.

    PubMed

    Nagano, Takashi; Lubling, Yaniv; Stevens, Tim J; Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D; Tanay, Amos; Fraser, Peter

    2013-10-03

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.

  18. TH2, allergy and group 2 innate lymphoid cells.

    PubMed

    Licona-Limón, Paula; Kim, Lark Kyun; Palm, Noah W; Flavell, Richard A

    2013-06-01

    The initiation of type 2 immune responses by the epithelial cell-derived cytokines IL-25, IL-33 and TSLP has been an area of extensive research in the past decade. Such studies have led to the identification of a new innate lymphoid subset that produces the canonical type 2 cytokines IL-5, IL-9 and IL-13 in response to IL-25 and IL-33. These group 2 or type 2 innate lymphoid cells (ILC2 cells) represent a critical source of type 2 cytokines in vivo and serve an important role in orchestrating the type 2 response to helminths and allergens. Further characterization of ILC2 cell biology will enhance the understanding of type 2 responses and may identify new treatments for asthma, allergies and parasitic infections. Interactions between ILC2 cells and the adaptive immune system, as well as examination of potential roles for ILC2 cells in the maintenance of homeostasis, promise to be particularly fruitful areas of future research.

  19. Cell lineage tracing reveals a biliary origin of intrahepatic cholangiocarcinoma

    PubMed Central

    Guest, Rachel V; Boulter, Luke; Kendall, Timothy J; Minnis-Lyons, Sarah E; Walker, Robert; Wigmore, Stephen J; Sansom, Owen J; Forbes, Stuart J

    2014-01-01

    Intrahepatic cholangiocarcinoma (ICC) is a treatment refractory malignancy with a high mortality and an increasing incidence worldwide. Recent studies have observed that activation of Notch and AKT signalling within mature hepatocytes is able to induce the formation of tumours displaying biliary lineage markers, thereby raising the suggestion that it is hepatocytes, rather than cholangiocytes or hepatic progenitor cells that represent the cell of origin of this tumour. Here we utilise a cholangiocyte-lineage tracing system to target p53 loss to biliary epithelia and observe the appearance of labelled biliary lineage tumours in response to chronic injury. Consequent to this, up-regulation of native functional Notch signalling is observed to occur spontaneously within cholangiocytes and hepatocytes in this model as well as in human ICC. These data prove that in the context of chronic inflammation and p53 loss, frequent occurrences in human disease, biliary epithelia are a target of transformation and an origin of ICC. PMID:24310400

  20. Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells

    PubMed Central

    Invernizzi, F.; D'Amato, I.; Jensen, P.B.; Ravaglia, S.; Zeviani, M.; Tiranti, V.

    2012-01-01

    Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders. PMID:22310368

  1. Single-cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade

    PubMed Central

    2013-01-01

    Background The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt- and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet. Results We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules. Conclusions We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host. PMID:24286338

  2. Persistence of a dominant bovine lineage of group B Streptococcus reveals genomic signatures of host adaptation.

    PubMed

    Almeida, Alexandre; Alves-Barroco, Cinthia; Sauvage, Elisabeth; Bexiga, Ricardo; Albuquerque, Pedro; Tavares, Fernando; Santos-Sanches, Ilda; Glaser, Philippe

    2016-11-01

    Group B Streptococcus (GBS) is a host-generalist species, most notably causing disease in humans and cattle. However, the differential adaptation of GBS to its two main hosts, and the risk of animal to human infection remain poorly understood. Despite improvements in control measures across Europe, GBS is still one of the main causative agents of bovine mastitis in Portugal. Here, by whole-genome analysis of 150 bovine GBS isolates we discovered that a single CC61 clone is spreading throughout Portuguese herds since at least the early 1990s, having virtually replaced the previous GBS population. Mutations within an iron/manganese transporter were independently acquired by all of the CC61 isolates, underlining a key adaptive strategy to persist in the bovine host. Lateral transfer of bacteriocin production and antibiotic resistance genes also underscored the contribution of the microbial ecology and genetic pool within the bovine udder environment to the success of this clone. Compared to strains of human origin, GBS evolves twice as fast in bovines and undergoes recurrent pseudogenizations of human-adapted traits. Our work provides new insights into the potentially irreversible adaptation of GBS to the bovine environment.

  3. Dynamic renormalisation group reveals sequential mechanism of the secondary nucleation of proteins

    NASA Astrophysics Data System (ADS)

    Michaels, Thomas; Arosio, Paolo; Knowles, Tuomas

    2014-03-01

    Secondary nucleation has emerged as a key process in the self-assembly of amyloid fibrils associated with a number of neurodegenerative disorders. Secondary nucleation conceptually involves both aggregates and monomers, but a variety of ways exist, in which this process may occur. Elucidation of this complex mechanism using experimental data represents a theoretical challenge. A systematic coarse-graining procedure inspired by the renormalisation group is used to bridge the length- and timescale gaps between detailed microscopic descriptions and the processes observed in experiments. Various mechanisms of secondary nucleation are discussed at different levels of coarse graining and compact terms in the master equation are generated, that provide a single-step description of this process. This treatment is general and allows to test assumptions regarding mechanisms at the microscopic level and to filter their effect on the kinetics at the macroscopic scale. By analysing data from the polymerisation of amylin, we conclude that pre-critical nuclei in islet amyloid polypeptides stay attached to the aggregates during the process of secondary nucleation.

  4. Long-term video surveillance and automated analyses reveal arousal patterns in groups of hibernating bats

    USGS Publications Warehouse

    Hayman, David T.S.; Cryan, Paul; Fricker, Paul D.; Dannemiller, Nicholas G.

    2017-01-01

    Understanding natural behaviours is essential to determining how animals deal with new threats (e.g. emerging diseases). However, natural behaviours of animals with cryptic lifestyles, like hibernating bats, are often poorly characterized. White-nose syndrome (WNS) is an unprecedented disease threatening multiple species of hibernating bats, and pathogen-induced changes to host behaviour may contribute to mortality. To better understand the behaviours of hibernating bats and how they might relate to WNS, we developed new ways of studying hibernation across entire seasons.We used thermal-imaging video surveillance cameras to observe little brown bats (Myotis lucifugus) and Indiana bats (M. sodalis) in two caves over multiple winters. We developed new, sharable software to test for autocorrelation and periodicity of arousal signals in recorded video.We processed 740 days (17,760 hr) of video at a rate of >1,000 hr of video imagery in less than 1 hr using a desktop computer with sufficient resolution to detect increases in arousals during midwinter in both species and clear signals of daily arousal periodicity in infected M. sodalis.Our unexpected finding of periodic synchronous group arousals in hibernating bats demonstrate the potential for video methods and suggest some bats may have innate behavioural strategies for coping with WNS. Surveillance video and accessible analysis software make it now practical to investigate long-term behaviours of hibernating bats and other hard-to-study animals.

  5. Footprints reveal direct evidence of group behavior and locomotion in Homo erectus

    PubMed Central

    Hatala, Kevin G.; Roach, Neil T.; Ostrofsky, Kelly R.; Wunderlich, Roshna E.; Dingwall, Heather L.; Villmoare, Brian A.; Green, David J.; Harris, John W. K.; Braun, David R.; Richmond, Brian G.

    2016-01-01

    Bipedalism is a defining feature of the human lineage. Despite evidence that walking on two feet dates back 6–7 Ma, reconstructing hominin gait evolution is complicated by a sparse fossil record and challenges in inferring biomechanical patterns from isolated and fragmentary bones. Similarly, patterns of social behavior that distinguish modern humans from other living primates likely played significant roles in our evolution, but it is exceedingly difficult to understand the social behaviors of fossil hominins directly from fossil data. Footprints preserve direct records of gait biomechanics and behavior but they have been rare in the early human fossil record. Here we present analyses of an unprecedented discovery of 1.5-million-year-old footprint assemblages, produced by 20+ Homo erectus individuals. These footprints provide the oldest direct evidence for modern human-like weight transfer and confirm the presence of an energy-saving longitudinally arched foot in H. erectus. Further, print size analyses suggest that these H. erectus individuals lived and moved in cooperative multi-male groups, offering direct evidence consistent with human-like social behaviors in H. erectus. PMID:27403790

  6. Characterization of single disseminated prostate cancer cells reveals tumor cell heterogeneity and identifies dormancy associated pathways

    PubMed Central

    Coleman, Ilsa; Lakely, Bryce; Coleman, Roger; Larson, Sandy; Aguirre-Ghiso, Julio A.; Xia, Jing; Gulati, Roman; Nelson, Peter S.; Montgomery, Bruce; Lange, Paul; Snyder, Linda A.; Vessella, Robert L.; Morrissey, Colm

    2014-01-01

    Cancer dormancy refers to the prolonged clinical disease-free time between removal of the primary tumor and recurrence, which is common in prostate cancer (PCa), breast cancer, esophageal cancer, and other cancers. PCa disseminated tumor cells (DTC) are detected in both patients with no evidence of disease (NED) and advanced disease (ADV). However, the molecular and cellular nature of DTC is unknown. We performed a first-in-field study of single DTC transcriptomic analyses in cancer patients to identify a molecular signature associated with cancer dormancy. We profiled eighty-five individual EpCAM+/CD45− cells from the bone marrow of PCa patients with NED or ADV. We analyzed 44 DTC with high prostate-epithelial signatures, and eliminated 41 cells with high erythroid signatures and low prostate epithelial signatures. DTC were clustered into 3 groups: NED, ADV_1, and ADV_2, in which the ADV_1 group presented a distinct gene expression pattern associated with the p38 stress activated kinase pathway. Additionally, DTC from the NED group were enriched for a tumor dormancy signature associated with head and neck squamous carcinoma and breast cancer. This study provides the first clinical evidence of the p38 pathway as a potential biomarker for early recurrence and an attractive target for therapeutic intervention. PMID:25301725

  7. Metabolomic profiling reveals severe skeletal muscle group-specific perturbations of metabolism in aged FBN rats.

    PubMed

    Garvey, Sean M; Dugle, Janis E; Kennedy, Adam D; McDunn, Jonathan E; Kline, William; Guo, Lining; Guttridge, Denis C; Pereira, Suzette L; Edens, Neile K

    2014-06-01

    Mammalian skeletal muscles exhibit age-related adaptive and pathological remodeling. Several muscles in particular undergo progressive atrophy and degeneration beyond median lifespan. To better understand myocellular responses to aging, we used semi-quantitative global metabolomic profiling to characterize trends in metabolic changes between 15-month-old adult and 32-month-old aged Fischer 344 × Brown Norway (FBN) male rats. The FBN rat gastrocnemius muscle exhibits age-dependent atrophy, whereas the soleus muscle, up until 32 months, exhibits markedly fewer signs of atrophy. Both gastrocnemius and soleus muscles were analyzed, as well as plasma and urine. Compared to adult gastrocnemius, aged gastrocnemius showed evidence of reduced glycolytic metabolism, including accumulation of glycolytic, glycogenolytic, and pentose phosphate pathway intermediates. Pyruvate was elevated with age, yet levels of citrate and nicotinamide adenine dinucleotide were reduced, consistent with mitochondrial abnormalities. Indicative of muscle atrophy, 3-methylhistidine and free amino acids were elevated in aged gastrocnemius. The monounsaturated fatty acids oleate, cis-vaccenate, and palmitoleate also increased in aged gastrocnemius, suggesting altered lipid metabolism. Compared to gastrocnemius, aged soleus exhibited far fewer changes in carbohydrate metabolism, but did show reductions in several glycolytic intermediates, fumarate, malate, and flavin adenine dinucleotide. Plasma biochemicals showing the largest age-related increases included glycocholate, heme, 1,5-anhydroglucitol, 1-palmitoleoyl-glycerophosphocholine, palmitoleate, and creatine. These changes suggest reduced insulin sensitivity in aged FBN rats. Altogether, these data highlight skeletal muscle group-specific perturbations of glucose and lipid metabolism consistent with mitochondrial dysfunction in aged FBN rats.

  8. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    NASA Astrophysics Data System (ADS)

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  9. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    DOE PAGES

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; ...

    2016-01-21

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. Here, we present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity.more » High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. Moreover, we observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Finally, our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.« less

  10. Single cell activity reveals direct electron transfer in methanotrophic consortia

    NASA Astrophysics Data System (ADS)

    McGlynn, Shawn E.; Chadwick, Grayson L.; Kempes, Christopher P.; Orphan, Victoria J.

    2015-10-01

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

  11. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    SciTech Connect

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Urban, Volker S.; Ohl, Michael

    2016-01-21

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. Here, we present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. Moreover, we observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Finally, our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  12. Oogonial biometry and phylogenetic analyses of the Pythium vexans species group from woody agricultural hosts in South Africa reveal distinct groups within this taxon.

    PubMed

    Spies, Christoffel F J; Mazzola, Mark; Botha, Wilhelm J; Van Der Rijst, Marieta; Mostert, Lizel; Mcleod, Adéle

    2011-02-01

    Pythium vexans fits into the internal transcribed spacer (ITS) clade K sensu Lévesque & De Cock (2004). Within clade K, P. vexans forms a distinct clade containing two enigmatic species, Pythium indigoferae and Pythium cucurbitacearum of which no ex-type strains are available. In South Africa, as well as in other regions of the world, P. vexans isolates are known to be heterogeneous in their ITS sequences and may consist of more than one species. This study aimed to investigate the diversity of South African P. vexans isolates, mainly from grapevines, but also citrus and apple using (i) phylogenetic analyses of the ITS, cytochrome c oxidase (cox) I, cox II, and β-tubulin regions and (ii) seven biometric oogonial parameters. Each of the phylogenies clustered P. vexans isolates into a single well-supported clade, distinct from other clade K species. The β-tubulin region was phylogenetically uninformative regarding the P. vexans group. The ITS phylogeny and combined cox I and II phylogenies, although each revealing several P. vexans subclades, were incongruent. One of the most striking incongruences was the presence of one cox subclade that contained two distinct ITS subclades (Ib and IV). Three groups (A-C) were subjectively identified among South African P. vexans isolates using (i) phylogenetic clades (ITS and cox), (ii) univariate analysis of oogonial diameters, and (iii) multivariate analyses of biometric oogonial parameters. Group A is considered to be P. vexans s. str. since it contained the P. vexans CBS reference strain from Van der Plaats-Niterink (1981). This group had significantly smaller oogonial diameters than group B and C isolates. Group B contained the isolates from ITS subclades Ib and IV, which formed a single cox subclade. The ITS subclade IV isolates were all sexually sterile or produced mainly abortive oospores, as opposed to the sexually fertile subclade Ib isolates, and may thus represent a distinct assemblage within group B. Although ITS

  13. Proteomic profiling reveals dopaminergic regulation of progenitor cell functions of goldfish radial glial cells in vitro.

    PubMed

    Xing, Lei; Martyniuk, Christopher J; Esau, Crystal; Da Fonte, Dillon F; Trudeau, Vance L

    2016-07-20

    Radial glial cells (RGCs) are stem-like cells found in the developing and adult central nervous system. They function as both a scaffold to guide neuron migration and as progenitor cells that support neurogenesis. Our previous study revealed a close anatomical relationship between dopamine neurons and RGCs in the telencephalon of female goldfish. In this study, label-free proteomics was used to identify the proteins in a primary RGC culture and to determine the proteome response to the selective dopamine D1 receptor agonist SKF 38393 (10μM), in order to better understand dopaminergic regulation of RGCs. A total of 689 unique proteins were identified in the RGCs and these were classified into biological and pathological pathways. Proteins such as nucleolin (6.9-fold) and ependymin related protein 1 (4.9-fold) were increased in abundance while proteins triosephosphate isomerase (10-fold) and phosphoglycerate dehydrogenase (5-fold) were decreased in abundance. Pathway analysis revealed that proteins that consistently changed in abundance across biological replicates were related to small molecules such as ATP, lipids and steroids, hormones, glucose, cyclic AMP and Ca(2+). Sub-network enrichment analysis suggested that estrogen receptor signaling, among other transcription factors, is regulated by D1 receptor activation. This suggests that these signaling pathways are correlated to dopaminergic regulation of radial glial cell functions. Most proteins down-regulated by SKF 38393 were involved in cell cycle/proliferation, growth, death, and survival, which suggests that dopamine inhibits the progenitor-related processes of radial glial cells. Examples of differently expressed proteins including triosephosphate isomerase, nucleolin, phosphoglycerate dehydrogenase and capping protein (actin filament) muscle Z-line beta were validated by qPCR and western blot, which were consistent with MS/MS data in the direction of change. This is the first study to characterize the RGC

  14. Phenotypic Profiling Reveals that Candida albicans Opaque Cells Represent a Metabolically Specialized Cell State Compared to Default White Cells.

    PubMed

    Ene, Iuliana V; Lohse, Matthew B; Vladu, Adrian V; Morschhäuser, Joachim; Johnson, Alexander D; Bennett, Richard J

    2016-11-22

    The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state. Epigenetic transitions are an important mechanism by which microbes adapt to external stimuli. For Candida albicans, such transitions are crucial for adaptation to complex, fluctuating environments, and therefore contribute to its success as a human pathogen. The white-opaque switch

  15. The neural stem cell lineage reveals novel relationships among spermatogonial germ stem cells and other pluripotent stem cells.

    PubMed

    Teichert, Anouk-Martine; Pereira, Schreiber; Coles, Brenda; Chaddah, Radha; Runciman, Susan; Brokhman, Irina; van der Kooy, Derek

    2014-04-01

    The embryonic stem cell (ESC) derived from the inner cell mass is viewed as the core pluripotent cell (PC) type from which all other cell types emanate. This familiar perspective derives from an embryological time line in which PCs are ordered according to their time of appearance. However, this schema does not take into account their potential for interconversion, thereby excluding this critical quality of PCs. The persistence of bona fide pluripotent adult stem cells has garnered increasing attention in recent years. Adult pluripotent spermatogonial germ stem cells (aSGSCs) arise from primordial germ cells (pGCs) that emerge from the epiblast during gastrulation. Adult definitive neural stem cells (dNSCs) arise clonally from pluripotent embryonic primitive neural stem cells (pNSCs), which can also be derived clonally from ESCs. To test for stem cell-type convertibility, we employed differentiation in the clonal lineage from ESCs to pNSCs to dNSCs, and revealed the relationships and lineage positioning among various PC populations, including spermatogonial germ cells (aSGSCs), epiblast-derived stem cells (Epi-SCs) and the bFGF, Activin, and BIO-derived stem cell (FAB-SC). Adult, murine aSGSCs assumed a 'pseudo-ESC' state in vitro, and then differentiated into dNSCs, but not pNSCs. Similarly, Epi-SCs and FAB-SCs only gave rise to dNSCs and not to pNSCs. The results of these experiments suggest a new pluripotency lineage model describing the relationship(s) among PCs that better reflects the transitions between these cell types in vitro.

  16. Anchoring groups for dye-sensitized solar cells.

    PubMed

    Zhang, Lei; Cole, Jacqueline M

    2015-02-18

    The dyes in dye-sensitized solar cells (DSSCs) require one or more chemical substituents that can act as an anchor, enabling their adsorption onto a metal oxide substrate. This adsorption provides a means for electron injection, which is the process that initiates the electrical circuit in a DSSC. Understanding the structure of various DSSC anchors and the search for new anchors are critical factors for the development of improved DSSCs. Traditionally, carboxylic acid and cyanoacrylic acid groups are employed as dye anchors in DSSCs. In recent years, novel anchor groups have emerged, which make a larger pool of materials available for DSSC dyes, and their associated physical and chemical characteristics offer interesting effects at the interface between dye and metal oxide. This review focuses especially on the structural aspects of these novel dye anchors for TiO2-based DSSCs, including pyridine, phosphonic acid, tetracyanate, perylene dicarboxylic acid anhydride, 2-hydroxylbenzonitrile, 8-hydroxylquinoline, pyridine-N-oxide, hydroxylpyridium, catechol, hydroxamate, sulfonic acid, acetylacetanate, boronic acid, nitro, tetrazole, rhodanine, and salicylic acid substituents. We anticipate that further exploration and understanding of these new types of anchoring groups for TiO2 substrates will not only contribute to the development of advanced DSSCs, but also of quantum dot-sensitized solar cells, water splitting systems, and other self-assembled monolayer-based technologies.

  17. Phenotypic Profiling Reveals that Candida albicans Opaque Cells Represent a Metabolically Specialized Cell State Compared to Default White Cells

    PubMed Central

    Ene, Iuliana V.; Lohse, Matthew B.; Vladu, Adrian V.; Morschhäuser, Joachim; Johnson, Alexander D.

    2016-01-01

    ABSTRACT The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state. PMID:27879329

  18. Metabolic profiling reveals key metabolic features of renal cell carcinoma.

    PubMed

    Catchpole, Gareth; Platzer, Alexander; Weikert, Cornelia; Kempkensteffen, Carsten; Johannsen, Manfred; Krause, Hans; Jung, Klaus; Miller, Kurt; Willmitzer, Lothar; Selbig, Joachim; Weikert, Steffen

    2011-01-01

    Recent evidence suggests that metabolic changes play a pivotal role in the biology of cancer and in particular renal cell carcinoma (RCC). Here, a global metabolite profiling approach was applied to characterize the metabolite pool of RCC and normal renal tissue. Advanced decision tree models were applied to characterize the metabolic signature of RCC and to explore features of metastasized tumours. The findings were validated in a second independent dataset. Vitamin E derivates and metabolites of glucose, fatty acid, and inositol phosphate metabolism determined the metabolic profile of RCC. α-tocopherol, hippuric acid, myoinositol, fructose-1-phosphate and glucose-1-phosphate contributed most to the tumour/normal discrimination and all showed pronounced concentration changes in RCC. The identified metabolic profile was characterized by a low recognition error of only 5% for tumour versus normal samples. Data on metastasized tumours suggested a key role for metabolic pathways involving arachidonic acid, free fatty acids, proline, uracil and the tricarboxylic acid cycle. These results illustrate the potential of mass spectroscopy based metabolomics in conjunction with sophisticated data analysis methods to uncover the metabolic phenotype of cancer. Differentially regulated metabolites, such as vitamin E compounds, hippuric acid and myoinositol, provide leads for the characterization of novel pathways in RCC.

  19. Do ABO Blood Group Antigens Hamper the Therapeutic Efficacy of Mesenchymal Stromal Cells?

    PubMed Central

    Moll, Guido; Hult, Annika; von Bahr, Lena; Alm, Jessica J.; Heldring, Nina; Hamad, Osama A.; Stenbeck-Funke, Lillemor; Larsson, Stella; Teramura, Yuji; Roelofs, Helene; Nilsson, Bo; Fibbe, Willem E.; Olsson, Martin L.; Le Blanc, Katarina

    2014-01-01

    Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens – genetically governed antigenic determinants – at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals. PMID:24454787

  20. Group 2 Innate Lymphoid Cells in Health and Disease

    PubMed Central

    Kim, Brian S.; Artis, David

    2015-01-01

    Group 2 innate lymphoid cells (ILC2s) play critical roles in anti-helminth immunity, airway epithelial repair, and metabolic homeostasis. Recently, these cells have also emerged as key players in the development of allergic inflammation at multiple barrier surfaces. ILC2s arise from common lymphoid progenitors in the bone marrow, are dependent on the transcription factors RORα, GATA3, and TCF-1, and produce the type 2 cytokines interleukin (IL)-4, IL-5, IL-9, and/or IL-13. The epithelial cell–derived cytokines IL-25, IL-33, and TSLP regulate the activation and effector functions of ILC2s, and recent studies suggest that their responsiveness to these cytokines and other factors may depend on their tissue environment. In this review, we focus on recent advances in our understanding of the various factors that regulate ILC2 function in the context of immunity, inflammation, and tissue repair across multiple organ systems. PMID:25573713

  1. Expression of blood group antigens on red cell microvesicles.

    PubMed

    Oreskovic, R T; Dumaswala, U J; Greenwalt, T J

    1992-01-01

    The purpose of this study was to determine whether epitopes of the A, B, D, Fya, M, N, S, s, and K blood group antigens are present on microvesicle membranes shed by red cells during storage. Vesicles were isolated from outdated units of blood having and lacking the specified antigens. Diluted antisera were absorbed with fixed quantities of vesicles from red cells with the test antigen and red cells lacking that antigen (controls). The adsorbed and unadsorbed antisera were titrated and scored by using panel cells from persons known to be heterozygous for all the non-AB antigens. The mean titration scores following adsorption with the vesicles from A, B, D, M+N-, M-N+, S+s-, S-s+, and Fy(a+b-) units were appreciably lower than the control scores (0, 0, 3, 2, 2, 0, 4, and 4 vs. 19, 23, 34, 13, 12, 16, 18, and 29, respectively), which indicated the presence of these epitopes on the membrane of shed vesicles. The results following adsorption with K:1,2 vesicles were equivocal.

  2. An integrative approach to phylogeny reveals patterns of environmental distribution and novel evolutionary relationships in a major group of ciliates

    PubMed Central

    Sun, Ping; Clamp, John; Xu, Dapeng; Huang, Bangqin; Shin, Mann Kyoon

    2016-01-01

    Peritrichs are a major group of ciliates with worldwide distribution. Yet, its internal phylogeny remains unresolved owing to limited sampling. Additionally, ecological distributions of peritrichs are poorly known. We performed substantially expanded phylogenetic analyses of peritrichs that incorporated SSU rDNA sequences of samples collected from three continents, revealing a number of new relationships between and within major lineages that greatly challenged the classic view of the group. Interrogation of a dataset comprising new environmental sequences from an estuary and the open ocean generated with high throughput sequencing and clone libraries plus putative environmental peritrich sequences at Genbank, produced a comprehensive tree of peritrichs from a variety of habitats and revealed unique ecological distribution patterns of several lineages for the first time. Also, evidence of adaptation to extreme environments in the Astylozoidae clade greatly broadened the phylogenetic range of peritrichs capable of living in extreme environments. Reconstruction of ancestral states revealed that peritrichs may have transitioned repeatedly from freshwater to brackish/marine/hypersaline environments. This work establishes a phylogenetic framework for more mature investigations of peritrichs in the future, and the approach used here provides a model of how to elucidate evolution in the context of ecological niches in any lineage of microbial eukaryotes. PMID:26880590

  3. An integrative approach to phylogeny reveals patterns of environmental distribution and novel evolutionary relationships in a major group of ciliates.

    PubMed

    Sun, Ping; Clamp, John; Xu, Dapeng; Huang, Bangqin; Shin, Mann Kyoon

    2016-02-16

    Peritrichs are a major group of ciliates with worldwide distribution. Yet, its internal phylogeny remains unresolved owing to limited sampling. Additionally, ecological distributions of peritrichs are poorly known. We performed substantially expanded phylogenetic analyses of peritrichs that incorporated SSU rDNA sequences of samples collected from three continents, revealing a number of new relationships between and within major lineages that greatly challenged the classic view of the group. Interrogation of a dataset comprising new environmental sequences from an estuary and the open ocean generated with high throughput sequencing and clone libraries plus putative environmental peritrich sequences at Genbank, produced a comprehensive tree of peritrichs from a variety of habitats and revealed unique ecological distribution patterns of several lineages for the first time. Also, evidence of adaptation to extreme environments in the Astylozoidae clade greatly broadened the phylogenetic range of peritrichs capable of living in extreme environments. Reconstruction of ancestral states revealed that peritrichs may have transitioned repeatedly from freshwater to brackish/marine/hypersaline environments. This work establishes a phylogenetic framework for more mature investigations of peritrichs in the future, and the approach used here provides a model of how to elucidate evolution in the context of ecological niches in any lineage of microbial eukaryotes.

  4. Organization of ventrolateral periolivary cells of the cat superior olive as revealed by PEP-19 immunocytochemistry and Nissl stain.

    PubMed

    Spirou, G A; Berrebi, A S

    1996-04-22

    Ventrolateral periolivary cell groups, through their descending projections to the cochlear nucleus (CN) and local projections to principal nuclei of the superior olive, may participate in brainstem mechanisms mediating such tasks as signal detection in noisy environments and sound localization. Understanding the function of these cell groups can be improved by increased knowledge of the organization of their synaptic inputs in relation to their cellular characteristics. Immunocytochemistry for PEP-19 (a putative calcium binding protein) reveals four patterns of immunolabeling within the ventrolateral periolivary region. Three of the patterns, which have distinct fiber and punctate labeling characteristics, help to define three subdivisions of the lateral nucleus of the trapezoid body (LNTB). The fourth pattern defines two other nuclei, the anterolateral periolivary nucleus (rostral) and the posterior periolivary nucleus (caudal), which display many immunoreactive cell bodies but little fiber and punctate labeling. One of the subdivisions of the LNTB contains large PEP-19 immunolabeled puncta arranged in pericellular nests. Analysis of Nissl-stained sections reveals a neuronal population that resembles globular cells of the ventral cochlear nucleus (VCN) and which colocalizes with pericellular nests of large immunolabeled puncta. Cell counts reveal that roughly 10,000 neurons constitute the cat ventrolateral periolivary region, 9,000 of which are found in the LNTB. Three-dimensional reconstructions of auditory brainstem nuclei clarify the complex spatial relationships among these structures.

  5. The Occurrence of Intercellular Bridges in Groups of Cells Exhibiting Synchronous Differentiation

    PubMed Central

    Fawcett, Don W.; Ito, Susumu; Slautterback, David

    1959-01-01

    A previous electron microscopic study of the cat testis revealed that spermatids derived from the same spermatogonium are joined together by intercellular bridges. The present paper records the observation of similar connections between spermatocytes and between spermatids in Hydra, fruit-fly, opossum, pigeon, rat, hamster, guinea pig, rabbit, monkey, and man. In view of these findings, it is considered likely that a syncytial relationship within groups of developing male germ cells is of general occurrence and is probably responsible for their synchronous differentiation. When clusters of spermatids, freshly isolated from the germinal epithelium are observed by phase contrast microscopy, the constrictions between the cellular units of the syncytium disappear and the whole group coalesces into a spherical multinucleate mass. The significance of this observation in relation to the occurrence of abnormal spermatozoa in semen and the prevalence of multinucleate giant cells in pathological testes is discussed. In the ectoderm of Hydra, the clusters of cnidoblasts that arise from proliferation of interstitial cells are also connected by intercellular bridges. The development of nematocysts within these groups of conjoined cells is precisely synchronized. Both in the testis of vertebrates and the ectoderm of Hydra, a syncytium results from incomplete cytokinesis in the proliferation of relatively undifferentiated cells. The intercellular bridges between daughter cells are formed when the cleavage furrow encounters the spindle remnant and is arrested by it. The subsequent dissolution of the spindle filaments establishes free communication between the cells. The discovery of intercellular bridges in the two unrelated tissues discussed here suggests that a similar syncytial relationship may be found elsewhere in nature where groups of cells of common origin differentiate synchronously. PMID:13664686

  6. Activated group 3 innate lymphoid cells promote T-cell–mediated immune responses

    PubMed Central

    von Burg, Nicole; Chappaz, Stéphane; Baerenwaldt, Anne; Horvath, Edit; Bose Dasgupta, Somdeb; Ashok, Devika; Pieters, Jean; Tacchini-Cottier, Fabienne; Rolink, Antonius; Acha-Orbea, Hans; Finke, Daniela

    2014-01-01

    Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1β stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4+ T-cell responses in vitro. The cognate interaction of ILC3s and CD4+ T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3–CD4+ T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4+ T-cell immune responses. PMID:25136120

  7. Characterization of a YAC-1 mouse cell receptor for group B coxsackieviruses.

    PubMed Central

    Hsu, K H; Crowell, R L

    1989-01-01

    A receptor on YAC-1 cells, a mouse T-lymphoma cell line, bound all six serotypes of the group B coxsackieviruses (CVB). In addition, the cells produced infectious virus. Each of the CVB competed for the same receptor on YAC-1 cells. CVB3 bound relatively slowly to YAC-1 cells (k = 4 x 10(-11) min-1 cell-1), and there were only 500 attachment sites per cell. A rabbit antiserum prepared against the HeLa cell receptor protein Rp-a specifically inhibited the binding of CVB1 and CVB3. A virus-receptor complex with CVB3 could be isolated from detergent (0.5% sodium deoxycholate, 1% Triton X-100)-solubilized YAC-1 plasma membranes. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the iodinated virus-receptor complex revealed a band with the same mobility as Rp-a. The results suggested that the YAC-1 receptor for CVB resembles that of the HeLa cell receptor. Images PMID:2724420

  8. Anchoring Groups for Dye-sensitized Solar Cells.

    SciTech Connect

    Zhang, Lei; Cole, Jacqueline M.

    2015-02-18

    The dyes in dye-sensitized solar cells (DSSCs) require one or more chemical substituents that can act as an anchor, enabling their adsorption onto a metal oxide substrate. This adsorption provides a means for electron injection, which is the process that initiates the electrical circuit in a DSSC. Understanding the structure of various DSSC anchors and the search for new anchors are critical factors for the development of improved DSSCs. Traditionally, carboxylic acid and cyanoacrylic acid groupsare employed as dye anchors in DSSCs. In recent years, novel anchor groups have emerged, which make a larger pool of materials available for DSSC dyes, and their associated physical and chemical characteristics offer interesting effects at the interface between dye and metal oxide. This review focuses especially on the structural aspects of these novel dye anchors for TiO2-based DSSCs, including pyridine, phosphonic acid, tetracyanate, perylene dicarboxylic acid anhydride, 2-hydroxylbenzonitrile, 8-hydroxylquinoline, pyridine-N-oxide, hydroxylpyridium, catechol, hydroxamate, sulfonic acid, acetylacetanate, boronic acid, nitro, tetrazole, rhodanine, and salicylic acid substituents. We anticipate that further exploration and understanding of these new types of anchoring groups for TiO2 substrates will not only contribute to the development of advanced DSSCs, but also of quantum dot-sensitized solar cells, water splitting systems, and other self-assembled monolayer-based technologies.

  9. Growth conditions of 0-group plaice Pleuronectes platessa in the western Wadden Sea as revealed by otolith microstructure analysis

    NASA Astrophysics Data System (ADS)

    Cardoso, Joana F. M. F.; Freitas, Vânia; de Paoli, Hélène; Witte, Johannes IJ.; van der Veer, Henk W.

    2016-05-01

    Growth studies based on population-based growth estimates are limited by the fact that they do not take into account differences in age/size structure within the population. To overcome these problems, otolith microstructure analysis is often used to estimate individual growth. Here, we analyse growth of 0-group plaice in the western Wadden Sea in two years: a year preceded by a mild winter (1995) and a year preceded by a severe winter (1996). Growth was analysed by combining information on individual growth based on otolith analysis with predictions of maximum growth (= under optimal food conditions) based on a Dynamic Energy Budget model. Otolith analysis revealed that settlement occurred earlier in 1995 than in 1996. In both years, one main cohort was found, followed by a group of late settlers. No differences in mean length-at-age were found between these groups. DEB modelling suggested that growth was not maximal during the whole growing season: realized growth (the fraction of maximum growth realized by 0-group plaice) declined in the summer, although this decline was relatively small. In addition, late settling individuals exhibited lower realized growth than individuals from the main cohort. This study confirms that growth conditions for 0-group plaice are not optimal and that a growth reduction occurs in summer, as suggested in previous studies.

  10. BOLD delay times using group delay in sickle cell disease

    NASA Astrophysics Data System (ADS)

    Coloigner, Julie; Vu, Chau; Bush, Adam; Borzage, Matt; Rajagopalan, Vidya; Lepore, Natasha; Wood, John

    2016-03-01

    Sickle cell disease (SCD) is an inherited blood disorder that effects red blood cells, which can lead to vasoocclusion, ischemia and infarct. This disease often results in neurological damage and strokes, leading to morbidity and mortality. Functional Magnetic Resonance Imaging (fMRI) is a non-invasive technique for measuring and mapping the brain activity. Blood Oxygenation Level-Dependent (BOLD) signals contain also information about the neurovascular coupling, vascular reactivity, oxygenation and blood propagation. Temporal relationship between BOLD fluctuations in different parts of the brain provides also a mean to investigate the blood delay information. We used the induced desaturation as a label to profile transit times through different brain areas, reflecting oxygen utilization of tissue. In this study, we aimed to compare blood flow propagation delay times between these patients and healthy subjects in areas vascularized by anterior, middle and posterior cerebral arteries. In a group comparison analysis with control subjects, BOLD changes in these areas were found to be almost simultaneous and shorter in the SCD patients, because of their increased brain blood flow. Secondly, the analysis of a patient with a stenosis on the anterior cerebral artery indicated that signal of the area vascularized by this artery lagged the MCA signal. These findings suggest that sickle cell disease causes blood propagation modifications, and that these changes could be used as a biomarker of vascular damage.

  11. [Diffuse interstitial lung disease revealing adult T-Cell leukemia/lymphoma (HTLV-1)].

    PubMed

    Bouanani, Nouama; Lamchahab, Mouna

    2016-01-01

    Adult T-Cell Leukemia/Lymphoma is a tumoral proliferation of activated mature T lymphoid cells whose causative agent is a retrovirus known as Human T-cell leukemia virus type 1. This virus rarely causes inflammatory bronchioloalveolar disorders. We report the case of a patient hospitalized with diffuse interstitial lung disease and whose etiological assessment revealed adult T-Cell Leukemia/Lymphoma (HTLV-1).

  12. Expression of blood group genes by mesenchymal stem cells

    PubMed Central

    Schäfer, Richard; Schnaidt, Martina; Klaffschenkel, Roland A.; Siegel, Georg; Schüle, Michael; Rädlein, Maria Anna; Hermanutz-Klein, Ursula; Ayturan, Miriam; Buadze, Marine; Gassner, Christoph; Danielyan, Lusine; Kluba, Torsten; Northoff, Hinnak; Flegel, Willy A.

    2011-01-01

    Incompatible blood group antigens are highly immunogenic and can cause graft rejections. Focusing on distinct carbohydrate- and protein-based membrane structures, defined by blood group antigens, we investigated human bone marrow-derived mesenchymal stem cells (MSCs) cultured in human serum. The presence of H (CD173), ABO, RhD, RhCE, RhAG, Kell, urea transporter type B (SLC14A1, previously known as JK), and Duffy antigen receptor of chemokines (DARC) was evaluated at the levels of genome, transcriptome and antigen. Fucosyltransferase-1 (FUT1), RHCE, KEL, SLC14A1 (JK) and DARC mRNA were transcribed in MSCs. FUT1 mRNA transcription was lost during differentiation. The mRNA transcription of SLC14A1 (JK) decreased during chondrogenic differentiation, while that of DARC increased during adipogenic differentiation. All MSCs synthesized SLC14A1 (JK) but no DARC protein. However, none of the protein antigens tested occurred on the surface, indicating a lack of associated protein function in the membrane. As A and B antigens are neither expressed nor adsorbed, concerns of ABO compatibility with human serum supplements during culture are alleviated. The H antigen expression by GD2dim+ MSCs identified two distinct MSC subpopulations and enabled their isolation. We hypothesize that GD2dim+H+ MSCs retain a better “stemness”. Because immunogenic blood group antigens are lacking, they cannot affect MSC engraftment in vivo, which is promising for clinical applications. PMID:21418181

  13. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

    PubMed Central

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

    2016-01-01

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  14. Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

    PubMed

    Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris; Kiessling, Ann A

    2016-01-15

    Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that

  15. Single Cell Phenotyping Reveals Heterogeneity among Haematopoietic Stem Cells Following Infection.

    PubMed

    MacLean, Adam L; Smith, Maia A; Liepe, Juliane; Sim, Aaron; Khorshed, Reema; Rashidi, Narges M; Scherf, Nico; Krinner, Axel; Roeder, Ingo; Lo Celso, Cristina; Stumpf, Michael Ph

    2017-08-22

    The haematopoietic stem cell (HSC) niche provides essential micro-environmental cues for the production and maintenance of HSCs within the bone marrow. During inflammation, haematopoietic dynamics are perturbed, but it is not known whether changes to the HSC-niche interaction occur as a result. We visualise HSCs directly in vivo, enabling detailed analysis of the 3D niche dynamics and migration patterns in murine bone marrow following Trichinella spiralis infection. Spatial statistical analysis of these HSC trajectories reveals two distinct modes of HSC behaviour: (i) a pattern of revisiting previously explored space, and (ii) a pattern of exploring new space. Whereas HSCs from control donors predominantly follow pattern (i), those from infected mice adopt both strategies. Using detailed computational analyses of cell migration tracks and life-history theory, we show that the increased motility of HSCs following infection can, perhaps counterintuitively, enable mice to cope better in deteriorating HSC-niche micro-environments following infection. This article is protected by copyright. All rights reserved. © 2017 AlphaMed Press.

  16. NK Cells Alleviate Lung Inflammation by Negatively Regulating Group 2 Innate Lymphoid Cells.

    PubMed

    Bi, Jiacheng; Cui, Lulu; Yu, Guang; Yang, Xiaolu; Chen, Youhai; Wan, Xiaochun

    2017-04-15

    Group 2 innate lymphoid cells (ILC2s) play an important role in orchestrating type II immune responses. However, the cellular mechanisms of group 2 innate lymphoid cell regulation remain poorly understood. In this study, we found that activated NK cells inhibited the proliferation of, as well as IL-5 and IL-13 production by, ILC2s in vitro via IFN-γ. In addition, in a murine model of ILC2 expansion in the liver, polyinosinic-polycytidylic acid, an NK cell-activating agent, inhibited ILC2 proliferation, IL-5 and IL-13 production, and eosinophil recruitment. Such effects of polyinosinic-polycytidylic acid were abrogated in NK cell-depleted mice and in IFN-γ-deficient mice. Adoptively transferring wild-type NK cells into NK cell-depleted mice resulted in fewer ILC2s induced by IL-33 compared with the transfer of IFN-γ-deficient NK cells. Importantly, during the early stage of papain- or bleomycin-induced lung inflammation, depletion of NK cells resulted in increased ILC2 numbers and enhanced cytokine production by ILC2s, as well as aggravated eosinophilia and goblet cell hyperplasia. Collectively, these data show that NK cells negatively regulate ILC2s during the early stage of lung inflammation, which represents the novel cellular interaction between two family members of ILCs. Copyright © 2017 by The American Association of Immunologists, Inc.

  17. Long-term microfluidic tracking of coccoid cyanobacterial cells reveals robust control of division timing.

    PubMed

    Yu, Feiqiao Brian; Willis, Lisa; Chau, Rosanna Man Wah; Zambon, Alessandro; Horowitz, Mark; Bhaya, Devaki; Huang, Kerwyn Casey; Quake, Stephen R

    2017-02-14

    Cyanobacteria are important agents in global carbon and nitrogen cycling and hold great promise for biotechnological applications. Model organisms such as Synechocystis sp. and Synechococcus sp. have advanced our understanding of photosynthetic capacity and circadian behavior, mostly using population-level measurements in which the behavior of individuals cannot be monitored. Synechocystis sp. cells are small and divide slowly, requiring long-term experiments to track single cells. Thus, the cumulative effects of drift over long periods can cause difficulties in monitoring and quantifying cell growth and division dynamics. To overcome this challenge, we enhanced a microfluidic cell-culture device and developed an image analysis pipeline for robust lineage reconstruction. This allowed simultaneous tracking of many cells over multiple generations, and revealed that cells expand exponentially throughout their cell cycle. Generation times were highly correlated for sister cells, but not between mother and daughter cells. Relationships between birth size, division size, and generation time indicated that cell-size control was inconsistent with the "sizer" rule, where division timing is based on cell size, or the "timer" rule, where division occurs after a fixed time interval. Instead, single cell growth statistics were most consistent with the "adder" rule, in which division occurs after a constant increment in cell volume. Cells exposed to light-dark cycles exhibited growth and division only during the light period; dark phases pause but do not disrupt cell-cycle control. Our analyses revealed that the "adder" model can explain both the growth-related statistics of single Synechocystis cells and the correlation between sister cell generation times. We also observed rapid phenotypic response to light-dark transitions at the single cell level, highlighting the critical role of light in cyanobacterial cell-cycle control. Our findings suggest that by monitoring the growth

  18. Intra- and inter-group coordination patterns reveal collective behaviors of football players near the scoring zone.

    PubMed

    Duarte, Ricardo; Araújo, Duarte; Freire, Luís; Folgado, Hugo; Fernandes, Orlando; Davids, Keith

    2012-12-01

    This study examined emergent coordination processes in collective patterns of behavior in 3 vs 3 sub-phases of the team sport of association football near the scoring zone. We identified coordination tendencies for the centroid (i.e., team center) and surface area (i.e., occupied space) of each sub-group of performers (n=20 plays). We also compared these kinematic variables at three key moments of play using mixed-model ANOVAs. The centroids demonstrated a strong symmetric relation that described the coordinated attacking/defending actions of performers in this sub-phase of play. Conversely, analysis of the surface area of each team did not reveal a clear coordination pattern between sub-groups. But the difference in the occupied area between the attacking and defending sub-groups significantly increased over time. Findings emphasized that major changes in sub-group behaviors occurred just before an assisted pass was made (i.e., leading to a loss of stability in the 3 vs 3 sub-phases).

  19. Functional and phenotypic heterogeneity of group 3 innate lymphoid cells.

    PubMed

    Melo-Gonzalez, Felipe; Hepworth, Matthew R

    2017-03-01

    Group 3 innate lymphoid cells (ILC3), defined by expression of the transcription factor retinoid-related orphan receptor γt, play key roles in the regulation of inflammation and immunity in the gastrointestinal tract and associated lymphoid tissues. ILC3 consist largely of two major subsets, NCR(+) ILC3 and LTi-like ILC3, but also demonstrate significant plasticity and heterogeneity. Recent advances have begun to dissect the relationship between ILC3 subsets and to define distinct functional states within the intestinal tissue microenvironment. In this review we discuss the ever-expanding roles of ILC3 in the context of intestinal homeostasis, infection and inflammation - with a focus on comparing and contrasting the relative contributions of ILC3 subsets. © 2016 The Authors. Immunology published by John Wiley & Sons Ltd.

  20. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation.

    PubMed

    Shalek, Alex K; Satija, Rahul; Shuga, Joe; Trombetta, John J; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S; Gaublomme, Jellert T; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P; Regev, Aviv

    2014-06-19

    High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a 'core' module of antiviral genes is expressed very early by a few 'precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced 'peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

  1. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation

    NASA Astrophysics Data System (ADS)

    Shalek, Alex K.; Satija, Rahul; Shuga, Joe; Trombetta, John J.; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S.; Gaublomme, Jellert T.; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P.; Regev, Aviv

    2014-06-01

    High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a `core' module of antiviral genes is expressed very early by a few `precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced `peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

  2. Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells

    NASA Astrophysics Data System (ADS)

    Dvir, Liron; Nissim, Ronen; Alvarez-Elizondo, Martha B.; Weihs, Daphne

    2015-04-01

    Metastasis formation is a major cause of mortality in cancer patients and includes tumor cell relocation to distant organs. A metastatic cell invades through other cells and extracellular matrix by biochemical attachment and mechanical force application. Force is used to move on or through a 2- or 3-dimensional (3D) environment, respectively, or to penetrate a 2D substrate. We have previously shown that even when a gel substrate is impenetrable, metastatic breast cancer cells can still indent it by applying force. Cells typically apply force through the acto-myosin network, which is mechanically connected to the nucleus. We develop a 3D image-analysis to reveal relative locations of the cell elements, and show that as cells apply force to the gel, a coordinated process occurs that involves cytoskeletal remodeling and repositioning of the nucleus. Our approach shows that the actin and microtubules reorganize in the cell, bringing the actin to the leading edge of the cell. In parallel, the nucleus is transported behind the actin, likely by the cytoskeleton, into the indentation dimple formed in the gel. The nucleus volume below the gel surface correlates with indentation depth, when metastatic breast cancer cells indent gels deeply. However, the nucleus always remains above the gel in benign cells, even when small indentations are observed. Determining mechanical processes during metastatic cell invasion can reveal how cells disseminate in the body and can uncover targets for diagnosis and treatment.

  3. Interleukin-5-producing group 2 innate lymphoid cells control eosinophilia induced by interleukin-2 therapy.

    PubMed

    Van Gool, Frédéric; Molofsky, Ari B; Morar, Malika M; Rosenzwajg, Michelle; Liang, Hong-Erh; Klatzmann, David; Locksley, Richard M; Bluestone, Jeffrey A

    2014-12-04

    Interleukin (IL)-2 promotes regulatory T-cell development and function, and treatment with IL-2 is being tested as therapy for some autoimmune diseases. However, patients receiving IL-2 treatment also experience eosinophilia due to an unknown mechanism. Here, we show that patients receiving low-dose IL-2 have elevated levels of serum IL-5, and this correlates with their degree of eosinophilia. In mice, low-dose IL-2-anti-IL-2 antibody complexes drove group 2 innate lymphoid cells (ILC2) to produce IL-5 and proliferate. Using genetic approaches in mice, we demonstrate that activation of ILC2 was responsible for the eosinophilia observed with IL-2 therapy. These observations reveal a novel cellular network that is activated during IL-2 treatment. A better understanding of the cross talk between these cell populations may lead to more effective targeting of IL-2 to treat autoimmune disease. © 2014 by The American Society of Hematology.

  4. Role of group 3 innate lymphoid cells during experimental otitis media in a rat model.

    PubMed

    Cho, Chang Gun; Gong, Sung Ho; Kim, Hee-Bok; Song, Jae-Jun; Park, Joo Hyun; Lim, Yun-Sung; Park, Seok-Won

    2016-09-01

    The objective of this study was to evaluate the role of group 3 innate lymphoid cells (ILC3) in the middle ear (ME) mucosal response to bacterial infection in a rat model. To confirm the role of ILC3 in bacterially induced otitis media (OM), the serum concentrations of IL-17 and IL-22 were determined by ELISA, and the tissue expression of IL-17 and IL-22 in infected ME mucosa was assessed by immunohistochemical staining. Immunohistochemical staining of specific cell surface markers was also assessed to confirm the origin of the cells expressing IL-17 and IL-22. Twenty Sprague-Dawley rats were used in the surgically-induced animal model of OM. OM was induced by inoculation of non-typeable Haemophilus influenzae into the ME cavity of the rats. The rats were divided into four experimental groups: three infected groups and one control group. Infected groups were subdivided into sets of 5 rats, one for each of the three time points (1, 4 and 7 days post-inoculation). For determination of rat IL-17 and IL-22 levels in infected rats and control rats, infected or control ME mucosa sections were analyzed by immunohistochemistry with specific antibodies directed against IL-17 and IL-22. Immunohistochemical staining for CD3, RORγt, and NKp46 were also conducted on the samples to confirm the origin of cells expressing IL-17 and IL-22. IL-17 and IL-22 serum concentrations were significantly increased in the infected rats compared to control rats. Immunohistochemical staining revealed increased IL-17 and IL-22 expressions in all infected ME mucosae from the first day after inoculation. In addition, the results of tissue staining for the specific surface markers were negative for CD3 and NKp46, but were highly positive for RORγt. IL-17 and IL-22 revealed their association with the bacterially induced proliferative and hyperplastic responses of ME mucosa, which are characteristic features in pathogenesis of OM. Surface marker examination showed that the source cells for IL-17

  5. COMPARISON OF OUTCOMES BETWEEN BLOOD GROUP O AND NON-GROUP O PREMATURE NEONATES RECEIVING RED CELL TRANSFUSIONS

    PubMed Central

    Boral, Leonard I.; Staubach, Zane G.; de Leeuw, Reny; MacIvor, Duncan C.; Kryscio, Richard; Bada, Henrietta S.

    2015-01-01

    Background At some institutions all babies requiring red blood cell (RBC) transfusions in neonatal intensive care units (NICUs) receive group O RBCs. Although transfused O plasma is minimized in packed RBCs, small amounts of residual anti-A, anti-B and anti-A, B in group O packed RBCs may bind to the corresponding A and B antigens of non-group O RBCs, possibly hemolyzing their native RBCs and thereby releasing free hemoglobin theoretically resulting in hypercoagulability and promoting bacterial growth from free iron. Study Design and Methods Transfused group O and non- group O premature infants in the University of Kentucky Children’s Hospital NICU database were compared for a number of severity markers to determine if transfused non-group O patients had worse outcomes than those of group O. Results 724 neonates in this sample of NICU babies received at least one blood component. There were no significant differences between group O and non-group O babies with regard to final disposition or complications. Conclusions This reassuring finding validates the longstanding neonatal transfusion practice of using group O packed red cells for NICU babies of all blood groups. However, because a recent study shows increased mortality from NEC in AB neonates receiving only group O RBC and suggests a change in neonatal transfusion practice to ABO group specific red cells, more studies may be warranted PMID:24225743

  6. Morphological and molecular characterization of three Agaricus species from tropical Asia (Pakistan, Thailand) reveals a new group in section Xanthodermatei.

    PubMed

    Thongklang, Naritsada; Nawaz, Rizwana; Khalid, Abdul N; Chen, Jie; Hyde, Kevin D; Zhao, Ruilin; Parra, Luis A; Hanif, Muhammad; Moinard, Magalie; Callac, Philippe

    2014-01-01

    The genus Agaricus is known for its medicinal and edible species but also includes toxic species that belong to section Xanthodermatei. Previous phylogenetic reconstruction for temperate species, based on sequence data of nuc rRNA gene (rDNA) internal transcribed spacers (ITS), has revealed two major groups in this section and a possible third lineage for A. pseudopratensis. Recent research in Agaricus has shown that classifications need improving with the addition of tropical taxa. In this study we add new tropical collections to section Xanthodermatei. We describe three species from collections made in Pakistan and Thailand and include them in a larger analysis using all available ITS data for section Xanthodermatei. Agaricus bisporiticus sp. nov. and A. fuscopunctatus sp. nov. are introduced based on molecular and morphological studies, whereas A. microvolvatulus is recorded for the first time in Asia. Specimens from Thailand however have a much larger pileus than the type specimens from Congo. In maximum likelihood (ML) and maximum parsimony (MP) phylogenetic analyses these three species cluster with A. pseudopratensis from the Mediterranean area and A. murinocephalus recently described from Thailand. In Agaricus section Xanthodermatei this new group is monophyletic and receives low bootstrap support whereas the two previously known groups receive strong support. Within the new group, the most closely related species share some traits, but we did not find any unifying morphological character; however the five species of the group share a unique short nucleotide sequence. Two putatively toxic species of section Xanthodermatei are now recognized in Pakistan and six in Thailand.

  7. Murine cell-mediated immune response recognizes an enterovirus group-specific antigen(s).

    PubMed Central

    Beck, M A; Tracy, S M

    1989-01-01

    Splenocytes taken from mice inoculated with coxsackievirus B3 (CVB3) (Nancy) developed an in vitro proliferative response against CVB3 antigen. This response could not be detected earlier than 8 days postinoculation but could be detected up to 28 days after exposure to CB3. CVB3-sensitized splenocytes responded not only to the CVB3 antigen but to other enteroviruses as well. This response was found to be enterovirus specific in that no response was detected to a non-enteroviral picornavirus, encephalomyocarditis virus, or to an unrelated influenza virus. The generation of a splenocyte population capable of responding to an enterovirus group antigen(s) was not limited to inoculation of mice with CVB3, as similar responses were generated when mice were inoculated with CVB2. Cell subset depletions revealed that the major cell type responding to the enterovirus group antigen(s) was the CD4+ T cell. Current evidence suggests that the group antigen(s) resides in the structural proteins of the virus, since spleen cells from mice inoculated with a UV-inactivated, highly purified preparation of CVB3 virions also responded in vitro against enteroviral antigens. PMID:2476566

  8. Research Resource: RNA-Seq Reveals Unique Features of the Pancreatic β-Cell Transcriptome

    PubMed Central

    Ku, Gregory M.; Kim, Hail; Vaughn, Ian W.; Hangauer, Matthew J.; Myung Oh, Chang

    2012-01-01

    The pancreatic β-cell is critical for the maintenance of glycemic control. Knowing the compendium of genes expressed in β-cells will further our understanding of this critical cell type and may allow the identification of future antidiabetes drug targets. Here, we report the use of next-generation sequencing to obtain nearly 1 billion reads from the polyadenylated RNA of islets and purified β-cells from mice. These data reveal novel examples of β-cell-specific splicing events, promoter usage, and over 1000 long intergenic noncoding RNA expressed in mouse β-cells. Many of these long intergenic noncoding RNA are β-cell specific, and we hypothesize that this large set of novel RNA may play important roles in β-cell function. Our data demonstrate unique features of the β-cell transcriptome. PMID:22915829

  9. Tumor Mutational Load and Immune Parameters across Metastatic Renal Cell Carcinoma Risk Groups.

    PubMed

    de Velasco, Guillermo; Miao, Diana; Voss, Martin H; Hakimi, A Ari; Hsieh, James J; Tannir, Nizar M; Tamboli, Pheroze; Appleman, Leonard J; Rathmell, W Kimryn; Van Allen, Eliezer M; Choueiri, Toni K

    2016-10-01

    Patients with metastatic renal cell carcinoma (mRCC) have better overall survival when treated with nivolumab, a cancer immunotherapy that targets the immune checkpoint inhibitor programmed cell death 1 (PD-1), rather than everolimus (a chemical inhibitor of mTOR and immunosuppressant). Poor-risk mRCC patients treated with nivolumab seemed to experience the greatest overall survival benefit, compared with patients with favorable or intermediate risk, in an analysis of the CheckMate-025 trial subgroup of the Memorial Sloan Kettering Cancer Center (MSKCC) prognostic risk groups. Here, we explore whether tumor mutational load and RNA expression of specific immune parameters could be segregated by prognostic MSKCC risk strata and explain the survival seen in the poor-risk group. We queried whole-exome transcriptome data in renal cell carcinoma patients (n = 54) included in The Cancer Genome Atlas who ultimately developed metastatic disease or were diagnosed with metastatic disease at presentation and did not receive immune checkpoint inhibitors. Nonsynonymous mutational load did not differ significantly by the MSKCC risk group, nor was the expression of cytolytic genes-granzyme A and perforin-or selected immune checkpoint molecules different across MSKCC risk groups. In conclusion, this analysis revealed that mutational load and expression of markers of an active tumor microenvironment did not correlate with MSKCC risk prognostic classification in mRCC. Cancer Immunol Res; 4(10); 820-2. ©2016 AACR.

  10. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin.

    PubMed

    Zhu, Lixin; Hatakeyama, Jason; Zhang, Bing; Makdisi, Joy; Ender, Cody; Forte, John G

    2009-02-01

    ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland.

  11. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

    PubMed

    Tan, Haiyan; Yang, Kai; Li, Yuxin; Shaw, Timothy I; Wang, Yanyan; Blanco, Daniel Bastardo; Wang, Xusheng; Cho, Ji-Hoon; Wang, Hong; Rankin, Sherri; Guy, Cliff; Peng, Junmin; Chi, Hongbo

    2017-03-21

    The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

  12. Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

    PubMed

    Shin, Jaehoon; Berg, Daniel A; Zhu, Yunhua; Shin, Joseph Y; Song, Juan; Bonaguidi, Michael A; Enikolopov, Grigori; Nauen, David W; Christian, Kimberly M; Ming, Guo-li; Song, Hongjun

    2015-09-03

    Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

  13. Cell-material interactions revealed via material techniques of surface patterning.

    PubMed

    Yao, Xiang; Peng, Rong; Ding, Jiandong

    2013-10-04

    Cell-material interactions constitute a key fundamental topic in biomaterials study. Various cell cues and matrix cues as well as soluble factors regulate cell behaviors on materials. These factors are coupled with each other as usual, and thus it is very difficult to unambiguously elucidate the role of each regulator. The recently developed material techniques of surface patterning afford unique ways to reveal the underlying science. This paper reviews the pertinent material techniques to fabricate patterns of microscale and nanoscale resolutions, and corresponding cell studies. Some issues are emphasized, such as cell localization on patterned surfaces of chemical contrast, and effects of cell shape, cell size, cell-cell contact, and seeding density on differentiation of stem cells. Material cues to regulate cell adhesion, cell differentiation and other cell events are further summed up. Effects of some physical properties, such as surface topography and matrix stiffness, on cell behaviors are also discussed; nanoscaled features of substrate surfaces to regulate cell fate are summarized as well. The pertinent work sheds new insight into the cell-material interactions, and is stimulating for biomaterial design in regenerative medicine, tissue engineering, and high-throughput detection, diagnosis, and drug screening. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Data-driven modeling reveals cell behaviors controlling self-organization during Myxococcus xanthus development.

    PubMed

    Cotter, Christopher R; Schüttler, Heinz-Bernd; Igoshin, Oleg A; Shimkets, Lawrence J

    2017-06-06

    Collective cell movement is critical to the emergent properties of many multicellular systems, including microbial self-organization in biofilms, embryogenesis, wound healing, and cancer metastasis. However, even the best-studied systems lack a complete picture of how diverse physical and chemical cues act upon individual cells to ensure coordinated multicellular behavior. Known for its social developmental cycle, the bacterium Myxococcus xanthus uses coordinated movement to generate three-dimensional aggregates called fruiting bodies. Despite extensive progress in identifying genes controlling fruiting body development, cell behaviors and cell-cell communication mechanisms that mediate aggregation are largely unknown. We developed an approach to examine emergent behaviors that couples fluorescent cell tracking with data-driven models. A unique feature of this approach is the ability to identify cell behaviors affecting the observed aggregation dynamics without full knowledge of the underlying biological mechanisms. The fluorescent cell tracking revealed large deviations in the behavior of individual cells. Our modeling method indicated that decreased cell motility inside the aggregates, a biased walk toward aggregate centroids, and alignment among neighboring cells in a radial direction to the nearest aggregate are behaviors that enhance aggregation dynamics. Our modeling method also revealed that aggregation is generally robust to perturbations in these behaviors and identified possible compensatory mechanisms. The resulting approach of directly combining behavior quantification with data-driven simulations can be applied to more complex systems of collective cell movement without prior knowledge of the cellular machinery and behavioral cues.

  15. Characterization of Streptokinases from Group A Streptococci Reveals a Strong Functional Relationship That Supports the Coinheritance of Plasminogen-binding M Protein and Cluster 2b Streptokinase*

    PubMed Central

    Zhang, Yueling; Liang, Zhong; Hsueh, Hsing-Tse; Ploplis, Victoria A.; Castellino, Francis J.

    2012-01-01

    Group A streptococcus (GAS) strains secrete the protein streptokinase (SK), which functions by activating host human plasminogen (hPg) to plasmin (hPm), thus providing a proteolytic framework for invasive GAS strains. The types of SK secreted by GAS have been grouped into two clusters (SK1 and SK2) and one subcluster (SK2a and SK2b). SKs from cluster 1 (SK1) and cluster 2b (SK2b) display significant evolutionary and functional differences, and attempts to relate these properties to GAS skin or pharynx tropism and invasiveness are of great interest. In this study, using four purified SKs from each cluster, new relationships between plasminogen-binding group A streptococcal M (PAM) protein and SK2b have been revealed. All SK1 proteins efficiently activated hPg, whereas all subclass SK2b proteins only weakly activated hPg in the absence of PAM. Surface plasmon resonance studies revealed that the lower affinity of SK2b to hPg served as the basis for the attenuated activation of hPg by SK2b. Binding of hPg to either human fibrinogen (hFg) or PAM greatly enhanced activation of hPg by SK2b but minimally influenced the already effective activation of hPg by SK1. Activation of hPg in the presence of GAS cells containing PAM demonstrated that PAM is the only factor on the surface of SK2b-expressing cells that enabled the direct activation of hPg by SK2b. As the binding of hPg to PAM is necessary for hPg activation by SK2b, this dependence explains the coinherant relationship between PAM and SK2b and the ability of these particular strains to generate the proteolytic activity that disrupts the innate barriers that limit invasiveness. PMID:23086939

  16. The early chemical enrichment histories of two Sculptor group dwarf galaxies as revealed by RR lyrae variables

    SciTech Connect

    Yang, Soung-Chul; Kim, Sang Chul; Kyeong, Jaemann; Wagner-Kaiser, Rachel; Sarajedini, Ata

    2014-03-20

    We present the results of our analysis of the RR Lyrae (RRL) variable stars detected in two transition-type dwarf galaxies (dTrans), ESO294-G010 and ESO410-G005 in the Sculptor group, which is known to be one of the closest neighboring galaxy groups to our Local Group. Using deep archival images from the Advanced Camera for Surveys on board the Hubble Space Telescope, we have identified a sample of RRL candidates in both dTrans galaxies (219 RRab (RR0) and 13 RRc (RR1) variables in ESO294-G010; 225 RRab and 44 RRc stars in ESO410-G005). The metallicities of the individual RRab stars are calculated via the period-amplitude-[Fe/H] relation derived by Alcock et al. This yields mean metallicities of ([Fe/H]){sub ESO294} = –1.77 ± 0.03 and ([Fe/H]){sub ESO410} = –1.64 ± 0.03. The RRL metallicity distribution functions (MDFs) are investigated further via simple chemical evolution models; these reveal the relics of the early chemical enrichment processes for these two dTrans galaxies. In the case of both galaxies, the shapes of the RRL MDFs are well described by pre-enrichment models. This suggests two possible channels for the early chemical evolution for these Sculptor group dTrans galaxies: (1) the ancient stellar populations of our target dwarf galaxies might have formed from the star forming gas which was already enriched through 'prompt initial enrichment' or an 'initial nucleosynthetic spike' from the very first massive stars, or (2) this pre-enrichment state might have been achieved by the end products from more evolved systems of their nearest neighbor, NGC 55. We also study the environmental effects of the formation and evolution of our target dTrans galaxies by comparing their properties with those of 79 volume limited (D {sub ☉} < 2 Mpc) dwarf galaxy samples in terms of the luminosity-metallicity relation and the H I gas content. The presence of these RRL stars strongly supports the idea that although the Sculptor Group galaxies have a considerably

  17. Differential gene expression analysis by RNA-seq reveals the importance of actin cytoskeletal proteins in erythroleukemia cells

    PubMed Central

    Fernández-Calleja, Vanessa; Hernández, Pablo; Schvartzman, Jorge B.; García de Lacoba, Mario

    2017-01-01

    Development of drug resistance limits the effectiveness of anticancer treatments. Understanding the molecular mechanisms triggering this event in tumor cells may lead to improved therapeutic strategies. Here we used RNA-seq to compare the transcriptomes of a murine erythroleukemia cell line (MEL) and a derived cell line with induced resistance to differentiation (MEL-R). RNA-seq analysis identified a total of 596 genes (Benjamini–Hochberg adjusted p-value < 0.05) that were differentially expressed by more than two-fold, of which 81.5% (486/596) of genes were up-regulated in MEL cells and 110 up-regulated in MEL-R cells. These observations revealed that for some genes the relative expression of mRNA amount in the MEL cell line has decreased as the cells acquired the resistant phenotype. Clustering analysis of a group of genes showing the highest differential expression allowed identification of a sub-group among genes up-regulated in MEL cells. These genes are related to the organization of the actin cytoskeleton network. Moreover, the majority of these genes are preferentially expressed in the hematopoietic lineage and at least three of them, Was (Wiskott Aldrich syndrome), Btk (Bruton’s tyrosine kinase) and Rac2, when mutated in humans, give rise to severe hematopoietic deficiencies. Among the group of genes that were up-regulated in MEL-R cells, 16% of genes code for histone proteins, both canonical and variants. A potential implication of these results on the blockade of differentiation in resistant cells is discussed. PMID:28663935

  18. Generation of 2,000 breast cancer metabolic landscapes reveals a poor prognosis group with active serotonin production

    PubMed Central

    Leoncikas, Vytautas; Wu, Huihai; Ward, Lara T.; Kierzek, Andrzej M.; Plant, Nick J.

    2016-01-01

    A major roadblock in the effective treatment of cancers is their heterogeneity, whereby multiple molecular landscapes are classified as a single disease. To explore the contribution of cellular metabolism to cancer heterogeneity, we analyse the Metabric dataset, a landmark genomic and transcriptomic study of 2,000 individual breast tumours, in the context of the human genome-scale metabolic network. We create personalized metabolic landscapes for each tumour by exploring sets of active reactions that satisfy constraints derived from human biochemistry and maximize congruency with the Metabric transcriptome data. Classification of the personalized landscapes derived from 997 tumour samples within the Metabric discovery dataset reveals a novel poor prognosis cluster, reproducible in the 995-sample validation dataset. We experimentally follow mechanistic hypotheses resulting from the computational study and establish that active serotonin production is a major metabolic feature of the poor prognosis group. These data support the reconsideration of concomitant serotonin-specific uptake inhibitors treatment during breast cancer chemotherapy. PMID:26813959

  19. Large-cell Monte Carlo renormalization group for percolation

    NASA Astrophysics Data System (ADS)

    Reynolds, Peter J.; Stanley, H. Eugene; Klein, W.

    1980-02-01

    We obtain the critical parameters for the site-percolation problem on the square lattice to a high degree of accuracy (comparable to that of series expansions) by using a Monte Carlo position-space renormalization-group procedure directly on the site-occupation probability. Our method involves calculating recursion relations using progressively larger lattice rescalings, b. We find smooth sequences for the value of the critical percolation concentration pc(b) and for the scaling powers yp(b) and yh(b). Extrapolating these sequences to the limit b-->∞ leads to quite accurate numerical predictions. Further, by considering other weight functions or "rules" which also embody the essential connectivity feature of percolation, we find that the numerical results in the infinite-cell limit are in fact "rule independent." However, the actual fashion in which this limit is approached does depend upon the rule chosen. A connection between extrapolation of our renormalization-group results and finite-size scaling is made. Furthermore, the usual finite-size scaling arguments lead to independent estimates of pc and yp. Combining both the large-cell approach and the finite-size scaling results, we obtain yp=0.7385+/-0.0080 and yh=1.898+/-0.003. Thus we find αp=-0.708+/-0.030, βp=0.138(+0.006,-0.005), γp=2.432+/-0.035, δp=18.6+/-0.6, νp=1.354+/-0.015, and 2-ηp=1.796+/-0.006. The site-percolation threshold is found for the square lattice at pc=0.5931+/-0.0006. We note that our calculated value of νp is in considerably better agreement with the proposal of Klein et al. that νp=ln3ln(32)≅1.3548, than with den Nijs' recent conjecture, which predicts νp=43. However, our results cannot entirely rule out the latter possibility.

  20. Variation in Carbohydrates between Cancer and Normal Cell Membranes Revealed by Super‐Resolution Fluorescence Imaging

    PubMed Central

    Chen, Junling; Liu, Tianzhou; Gao, Jing; Gao, Lan; Zhou, Lulu; Cai, Mingjun; Shi, Yan; Xiong, Wenyong; Jiang, Junguang

    2016-01-01

    Carbohydrate alterations on cell membranes are associated with various cancer processes, including tumorigenesis, malignant transformation, and tumor dissemination. However, variations in the distributions of cancer‐associated carbohydrates are unclear at the molecular level. Herein, direct stochastic optical reconstruction microscopy is used to reveal that seven major types of carbohydrates tended to form obvious clusters on cancer cell membranes compared with normal cell membranes (both cultured and primary cells), and most types of carbohydrates present a similar distributed characteristic on various cancer cells (e.g., HeLa and Os‐Rc‐2 cells). Significantly, sialic acid is found to distribute in larger‐sized clusters with a higher cluster coverage percentage on various cancer cells than normal cells. These findings on the aberrant distributions of cancer‐associated carbohydrates can potentially serve as novel diagnostic and therapeutic targets, as well as making a contribution to clarify how abnormal glycosylations of membrane glycoconjugates participate in tumorigenesis and metastasis. PMID:27981014

  1. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.

    PubMed

    Gokce, Ozgun; Stanley, Geoffrey M; Treutlein, Barbara; Neff, Norma F; Camp, J Gray; Malenka, Robert C; Rothwell, Patrick E; Fuccillo, Marc V; Südhof, Thomas C; Quake, Stephen R

    2016-07-26

    The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

  2. Systematic perturbation of cytoskeletal function reveals a linear scaling relationship between cell geometry and fitness.

    PubMed

    Monds, Russell D; Lee, Timothy K; Colavin, Alexandre; Ursell, Tristan; Quan, Selwyn; Cooper, Tim F; Huang, Kerwyn Casey

    2014-11-20

    Diversification of cell size is hypothesized to have occurred through a process of evolutionary optimization, but direct demonstrations of causal relationships between cell geometry and fitness are lacking. Here, we identify a mutation from a laboratory-evolved bacterium that dramatically increases cell size through cytoskeletal perturbation and confers a large fitness advantage. We engineer a library of cytoskeletal mutants of different sizes and show that fitness scales linearly with respect to cell size over a wide physiological range. Quantification of the growth rates of single cells during the exit from stationary phase reveals that transitions between "feast-or-famine" growth regimes are a key determinant of cell-size-dependent fitness effects. We also uncover environments that suppress the fitness advantage of larger cells, indicating that cell-size-dependent fitness effects are subject to both biophysical and metabolic constraints. Together, our results highlight laboratory-based evolution as a powerful framework for studying the quantitative relationships between morphology and fitness.

  3. Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

    PubMed Central

    Sato, Sachiko; Rancourt, Ann; Sato, Yukiko; Satoh, Masahiko S.

    2016-01-01

    Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells. PMID:27003384

  4. ENDIVE PLANTLETS FROM FREELY SUSPENDED CELLS AND CELL GROUPS GROWN IN VITRO.

    PubMed

    VASIL, I K; HILDEBRANDT, A C; RIKER, A J

    1964-10-02

    Callus tissue derived from mature embryos of the endive, Cichorium endivia Linn. (family Compositae) grows and develops chlorophyll on a completely defined nutrient medium. The tissue breaks up into a thick suspentsion of cells and cell groups in a liquid medium kept in a flask on a shaker. Gradually, many small round masses of tissue, designated here as embryoids, are formed; these become differentiated and organized to form numnerous small plantlets having typical curled and fringed green leaves and roots.

  5. Dynamic migration and cell-cell interactions of early reprogramming revealed by high resolution time-lapse imaging

    PubMed Central

    Megyola, Cynthia M.; Gao, Yuan; Teixeira, Alexandra M.; Cheng, Jijun; Heydari, Kartoosh; Cheng, Ee-chun; Nottoli, Timothy; Krause, Diane S.; Lu, Jun; Guo, Shangqin

    2014-01-01

    Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multi-color time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as five minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct 2-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate the requirement of E-cadherin for proper cellular interactions from an early stage of reprogramming, including the 2-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution. PMID:23335078

  6. Continuous single cell imaging reveals sequential steps of plasmacytoid dendritic cell development from common dendritic cell progenitors

    PubMed Central

    Dursun, Ezgi; Endele, Max; Musumeci, Andrea; Failmezger, Henrik; Wang, Shu-Hung; Tresch, Achim; Schroeder, Timm; Krug, Anne B.

    2016-01-01

    Functionally distinct plasmacytoid and conventional dendritic cells (pDC and cDC) shape innate and adaptive immunity. They are derived from common dendritic cell progenitors (CDPs) in the murine bone marrow, which give rise to CD11c+ MHCII− precursors with early commitment to DC subpopulations. In this study, we dissect pDC development from CDP into an ordered sequence of differentiation events by monitoring the expression of CD11c, MHC class II, Siglec H and CCR9 in CDP cultures by continuous single cell imaging and tracking. Analysis of CDP genealogies revealed a stepwise differentiation of CDPs into pDCs in a part of the CDP colonies. This developmental pathway involved an early CD11c+ SiglecH− pre-DC stage and a Siglec H+ CCR9low precursor stage, which was followed rapidly by upregulation of CCR9 indicating final pDC differentiation. In the majority of the remaining CDP pedigrees however the Siglec H+ CCR9low precursor state was maintained for several generations. Thus, although a fraction of CDPs transits through precursor stages rapidly to give rise to a first wave of pDCs, the majority of CDP progeny differentiate more slowly and give rise to longer lived precursor cells which are poised to differentiate on demand. PMID:27892478

  7. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.

    PubMed

    Shinohara, Naoki; Uyama, Toru; Jin, Xing-Hua; Tsuboi, Kazuhito; Tonai, Takeharu; Houchi, Hitoshi; Ueda, Natsuo

    2011-11-01

    A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

  8. A simple engineered platform reveals different modes of tumor-microenvironmental cell interaction

    PubMed Central

    Zhang, Chentian; Shenk, Elizabeth M; Blaha, Laura C; Ryu, Byungwoo; Alani, Rhoda M; Cabodi, Mario; Wong, Joyce Y

    2016-01-01

    How metastatic cancer lesions survive and grow in secondary locations is not fully understood. There is a growing appreciation for the importance of tumor components, i.e. microenvironmental cells, in this process. Here, we used a simple microfabricated dual cell culture platform with a 500 μm gap to assess interactions between two different metastatic melanoma cell lines (1205Lu isolated from a lung lesion established through a mouse xenograft; and WM852 derived from a stage III metastatic lesion of skin) and microenvironmental cells derived from either skin (fibroblasts), lung (epithelial cells) or liver (hepatocytes). We observed differential bi-directional migration between microenvironmental cells and melanoma, depending on the melanoma cell line. Lung epithelial cells and skin fibroblasts, but not hepatocytes, stimulated higher 1205Lu migration than without microenvironmental cells; in the opposite direction, 1205Lu cells induced hepatocytes to migrate, but had no effect on skin fibroblasts and slightly inhibited lung epithelial cells. In contrast, none of the microenvironments had a significant effect on WM852; in this case, skin fibroblasts and hepatocytes—but not lung epithelial cells—exhibited directed migration toward WM852. These observations reveal significant effects a given microenvironmental cell line has on the two different melanoma lines, as well as how melanoma effects different microenvironmental cell lines. Our simple platform thus has potential to provide complex insights into different strategies used by cancerous cells to survive in and colonize metastatic sites. PMID:26716792

  9. Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group.

    PubMed

    Nunoura, Takuro; Takaki, Yoshihiro; Kakuta, Jungo; Nishi, Shinro; Sugahara, Junichi; Kazama, Hiromi; Chee, Gab-Joo; Hattori, Masahira; Kanai, Akio; Atomi, Haruyuki; Takai, Ken; Takami, Hideto

    2011-04-01

    The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.

  10. Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group

    PubMed Central

    Nunoura, Takuro; Takaki, Yoshihiro; Kakuta, Jungo; Nishi, Shinro; Sugahara, Junichi; Kazama, Hiromi; Chee, Gab-Joo; Hattori, Masahira; Kanai, Akio; Atomi, Haruyuki; Takai, Ken; Takami, Hideto

    2011-01-01

    The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the ‘Thaumarchaeota’ and ‘Korarchaeota’. Here, we show the genome sequence of Candidatus ‘Caldiarchaeum subterraneum’ that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea. PMID:21169198

  11. Population-based resequencing revealed an ancestral winter group of cultivated flax: implication for flax domestication processes

    PubMed Central

    Fu, Yong-Bi

    2012-01-01

    Cultivated flax (Linum usitatissimum L.) is the earliest oil and fiber crop and its early domestication history may involve multiple events of domestication for oil, fiber, capsular indehiscence, and winter hardiness. Genetic studies have demonstrated that winter cultivated flax is closely related to oil and fiber cultivated flax and shows little relatedness to its progenitor, pale flax (L. bienne Mill.), but winter hardiness is one major characteristic of pale flax. Here, we assessed the genetic relationships of 48 Linum samples representing pale flax and four trait-specific groups of cultivated flax (dehiscent, fiber, oil, and winter) through population-based resequencing at 24 genomic regions, and revealed a winter group of cultivated flax that displayed close relatedness to the pale flax samples. Overall, the cultivated flax showed a 27% reduction of nucleotide diversity when compared with the pale flax. Recombination frequently occurred at these sampled genomic regions, but the signal of selection and bottleneck was relatively weak. These findings provide some insight into the impact and processes of flax domestication and are significant for expanding our knowledge about early flax domestication, particularly for winter hardiness. PMID:22822439

  12. Structure of the polycomb group protein PCGF1 in complex with BCOR reveals basis for binding selectivity of PCGF homologs.

    PubMed

    Junco, Sarah E; Wang, Renjing; Gaipa, John C; Taylor, Alexander B; Schirf, Virgil; Gearhart, Micah D; Bardwell, Vivian J; Demeler, Borries; Hart, P John; Kim, Chongwoo A

    2013-04-02

    Polycomb-group RING finger homologs (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6) are critical components in the assembly of distinct Polycomb repression complex 1 (PRC1)-related complexes. Here, we identify a protein interaction domain in BCL6 corepressor, BCOR, which binds the RING finger- and WD40-associated ubiquitin-like (RAWUL) domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals that (1) PUFD binds to the same surfaces as observed for a different Polycomb group RAWUL domain and (2) the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly.

  13. Conditional clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells.

    PubMed

    Reynolds, S D; Hong, K U; Giangreco, A; Mango, G W; Guron, C; Morimoto, Y; Stripp, B R

    2000-06-01

    The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of associated pulmonary neuroendocrine (PNE) cells. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for development of small cell lung carcinoma, mechanisms regulating expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. We have previously used a Clara cell-specific toxicant, naphthalene, to induce airway injury in mice and have demonstrated that naphthalene-resistant Clara cells, characterized by their expression of Clara cell secretory protein (CCSP), and PNE cells contribute to airway repair and associated hyperplasia of NEBs. This study was conducted to define the contribution of NEB-associated CCSP-expressing progenitor cells to PNE cell hyperplasia after Clara cell ablation. Transgenic (CCtk) mice were generated in which herpes simplex virus thymidine kinase was expressed within all CCSP-expressing cells of the conducting airway epithelium through the use of transcriptional regulatory elements from the mouse CCSP promoter. Chronic administration of ganciclovir (GCV) to CCtk transgenic mice resulted in selective ablation of CCSP-expressing cells within conducting airways. Proliferation and hyperplasia of PNE cells occurred in the absence of detectable proliferation among any other residual airway epithelial cell populations. These results demonstrate that PNE cells function as a self-renewing progenitor population and that NEB-associated Clara cells are not necessary for PNE cell hyperplasia.

  14. Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum.

    PubMed

    Choi, Eunyoung; Roland, Joseph T; Barlow, Brittney J; O'Neal, Ryan; Rich, Amy E; Nam, Ki Taek; Shi, Chanjuan; Goldenring, James R

    2014-11-01

    The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of the stomach as well as in over 50% of antral glands. MIST1 expressing chief cells were predominantly observed in the body although individual glands of the antrum also showed MIST1 expressing chief cells. While classically described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed type glands containing both parietal cells and G cells throughout the antrum. Enteroendocrine cells show distinct patterns of localisation in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  15. A muscle stem cell for every muscle: variability of satellite cell biology among different muscle groups

    PubMed Central

    Randolph, Matthew E.; Pavlath, Grace K.

    2015-01-01

    The human body contains approximately 640 individual skeletal muscles. Despite the fact that all of these muscles are composed of striated muscle tissue, the biology of these muscles and their associated muscle stem cell populations are quite diverse. Skeletal muscles are affected differentially by various muscular dystrophies (MDs), such that certain genetic mutations specifically alter muscle function in only a subset of muscles. Additionally, defective muscle stem cells have been implicated in the pathology of some MDs. The biology of muscle stem cells varies depending on the muscles with which they are associated. Here we review the biology of skeletal muscle stem cell populations of eight different muscle groups. Understanding the biological variation of skeletal muscles and their resident stem cells could provide valuable insight into mechanisms underlying the susceptibility of certain muscles to myopathic disease. PMID:26500547

  16. A muscle stem cell for every muscle: variability of satellite cell biology among different muscle groups.

    PubMed

    Randolph, Matthew E; Pavlath, Grace K

    2015-01-01

    The human body contains approximately 640 individual skeletal muscles. Despite the fact that all of these muscles are composed of striated muscle tissue, the biology of these muscles and their associated muscle stem cell populations are quite diverse. Skeletal muscles are affected differentially by various muscular dystrophies (MDs), such that certain genetic mutations specifically alter muscle function in only a subset of muscles. Additionally, defective muscle stem cells have been implicated in the pathology of some MDs. The biology of muscle stem cells varies depending on the muscles with which they are associated. Here we review the biology of skeletal muscle stem cell populations of eight different muscle groups. Understanding the biological variation of skeletal muscles and their resident stem cells could provide valuable insight into mechanisms underlying the susceptibility of certain muscles to myopathic disease.

  17. Lineage tracing quantification reveals symmetric stem cell division in Drosophila male germline stem cells.

    PubMed

    Salzmann, Viktoria; Inaba, Mayu; Cheng, Jun; Yamashita, Yukiko M

    2013-12-01

    In the homeostatic state, adult stem cells divide either symmetrically to increase the stem cell number to compensate stem cell loss, or asymmetrically to maintain the population while producing differentiated cells. We have investigated the mode of stem cell division in the testes of Drosophila melanogaster by lineage tracing and confirm the presence of symmetric stem cell division in this system. We found that the rate of symmetric division is limited to 1-2% of total germline stem cell (GSC) divisions, but it increases with expression of a cell adhesion molecule, E-cadherin, or a regulator of the actin cytoskeleton, Moesin, which may modulate adhesiveness of germ cells to the stem cell niche. Our results indicate that the decision regarding asymmetric vs. symmetric division is a dynamically regulated process that contributes to tissue homeostasis, responding to the needs of the tissue.

  18. Substantial genetic substructuring in southeastern and alpine Australia revealed by molecular phylogeography of the Egernia whitii (Lacertilia: Scincidae) species group.

    PubMed

    Chapple, David G; Keogh, J Scott; Hutchinson, Mark N

    2005-04-01

    Palaeoclimatic events and biogeographical processes since the mid-Tertiary are believed to have strongly influenced the evolution and distribution of the terrestrial vertebrate fauna of southeastern Australia. We examined the phylogeography of the temperate-adapted members of the Egernia whitii species group, a group of skinks that comprise both widespread low- to mid-elevation (E. whitii) and montane-restricted species (Egernia guthega, Egernia montana), in order to obtain important insights into the influence of past biogeographical processes on the herpetofauna of southeastern Australia. Sequence data were obtained from all six temperate-adapted species within the E. whitii species group, and specifically from across the distributional ranges of E. whitii, E. guthega and E. montana. We targeted a fragment of the ND4 mitochondrial gene (696 bp) and analysed the data using maximum likelihood and Bayesian methods. Our data reveal a deep phylogeographical break in the east Gippsland region of Victoria between 'northern' (Queensland, New South Wales, Australian Capital Territory) and 'southern' (Victoria, Tasmania, South Australia) populations of E. whitii. This divergence appears to have occurred during the late Miocene-Pliocene, with the Gippsland basin possibly forming a geographical barrier to dispersal. Substantial structuring within both the 'northern' and the 'southern' clades is consistent with the effects of Plio-Pleistocene glacial-interglacial cycles. Pleistocene glacial cycles also appear to have shaped the phylogeographical patterns observed in the alpine species, E. guthega and E. montana. We used our results to examine the biogeographical process that led to the origin and subsequent diversification of the lowland and alpine herpetofauna of southeastern Australia.

  19. Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

    PubMed Central

    Gu, Bin; Zhang, Jiarong; Wu, Ying; Zhang, Xinzong; Tan, Zhou; Lin, Yuanji; Huang, Xiao; Chen, Liangbiao; Yao, Kangshou; Zhang, Ming

    2011-01-01

    Background It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. Methods and Principal Findings Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. Conclusions/Significance Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells. PMID:21559292

  20. Usage of Murine T-cell Hybridoma Cells as Responder Cells Reveals Interference of Helicobacter Pylori with Human Dendritic Cell-mediated Antigen Presentation

    PubMed Central

    Fehlings, Michael; Drobbe, Lea; Beigier-Bompadre, Macarena; Viveros, Pablo Renner; Moos, Verena; Schneider, Thomas; Meyer, Thomas F.; Aebischer, Toni; Ignatius, Ralf

    2016-01-01

    Direct effects of Helicobacter pylori (H. pylori) on human CD4+ T-cells hamper disentangling a possible bacterial-mediated interference with major histocompatibility complex class II (MHC-II)-dependent antigen presentation to these cells. To overcome this limitation, we employed a previously described assay, which enables assessing human antigen-processing cell function by using murine T-cell hybridoma cells restricted by human leukocyte antigen (HLA) alleles. HLA-DR1+ monocyte-derived dendritic cells were exposed to H. pylori and pulsed with the antigen 85B from Mycobacterium tuberculosis (M. tuberculosis). Interleukin-2 (IL-2) secretion by AG85Baa97-112-specific hybridoma cells was then evaluated as an integral reporter of cognate antigen presentation. This methodology enabled revealing of interference of H. pylori with the antigen-presenting capacity of human dendritic cells. PMID:27980859

  1. Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells.

    PubMed

    Yu, Tao; Wang, Yongtao; Zhang, Huizhen; Johnson, Caroline H; Jiang, Yiming; Li, Xiangjun; Wu, Zeming; Liu, Tian; Krausz, Kristopher W; Yu, Aiming; Gonzalez, Frank J; Huang, Min; Bi, Huichang

    2016-06-01

    Mycoplasma contamination is a common problem in cell culture and can alter cellular functions. Since cell metabolism is either directly or indirectly involved in every aspect of cell function, it is important to detect changes to the cellular metabolome after mycoplasma infection. In this study, liquid chromatography mass spectrometry (LC/MS)-based metabolomics was used to investigate the effect of mycoplasma contamination on the cellular metabolism of human pancreatic carcinoma cells (PANC-1). Multivariate analysis demonstrated that mycoplasma contamination induced significant metabolic changes in PANC-1 cells. Twenty-three metabolites were identified and found to be involved in arginine and purine metabolism and energy supply. This study demonstrates that mycoplasma contamination significantly alters cellular metabolite levels, confirming the compelling need for routine checking of cell cultures for mycoplasma contamination, particularly when used for metabolomics studies. Graphical abstract Metabolomics reveals mycoplasma contamination changes the metabolome of PANC-1 cells.

  2. Single-cell messenger RNA sequencing reveals rare intestinal cell types.

    PubMed

    Grün, Dominic; Lyubimova, Anna; Kester, Lennart; Wiebrands, Kay; Basak, Onur; Sasaki, Nobuo; Clevers, Hans; van Oudenaarden, Alexander

    2015-09-10

    Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID we then applied the algorithm to ex vivo-isolated Lgr5-positive stem cells and their direct progeny. We find that Lgr5-positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5

  3. Single-cell analysis reveals lineage segregation in early post-implantation mouse embryos.

    PubMed

    Wen, Jing; Zeng, Yanwu; Fang, Zhuoqing; Gu, Junjie; Ge, Laixiang; Tang, Fan; Qu, Zepeng; Hu, Jing; Cui, Yaru; Zhang, Kunshan; Wang, Junbang; Li, Siguang; Sun, Yi; Jin, Ying

    2017-03-15

    The mammalian post-implantation embryo has been extensively investigated at the tissue level. However, to unravel the molecular basis for the cell-fate plasticity and determination, it is essential to study the characteristics of individual cells. Especially, the individual definitive endoderm (DE) cells have not been characterized in vivo. Here, we report gene expression patterns in single cells freshly isolated from mouse embryos on days 5.5 and 6.5. Initial transcriptome data from 124 single cells yielded signature genes for the epiblast, visceral endoderm, and extra-embryonic ectoderm and revealed a unique distribution pattern of fibroblast growth factor (Fgf) ligands and receptors. Further analysis indicated that early-stage epiblast cells do not segregate into lineages of the major germ layers. Instead, some cells began to diverge from epiblast cells, displaying molecular features of the pre-mesendoderm by expressing higher levels of mesendoderm markers and lower levels of Sox3 transcripts. Analysis of single-cell high-throughput quantitative RT-PCR data from 441 cells identified a late stage of the day 6.5 embryo in which mesoderm and DE cells emerge, with many of them coexpressing Oct4 and Gata6. Analysis of single-cell RNA-seq data from 112 cells of the late-stage day 6.5 embryos revealed differentially expressed signaling genes and networks of transcription factors that might underlie the segregation of the mesoderm and DE lineages. Moreover, we discovered a subpopulation of mesoderm cells that possess molecular features of the extraembryonic mesoderm. This study provides fundamental insight into the molecular basis for lineage segregation in post-implantation mouse embryos.

  4. Global gene expression profiling reveals similarities and differences among mouse pluripotent stem cells of different origins and strains

    PubMed Central

    Sharova, Lioudmila V.; Sharov, Alexei A.; Piao, Yulan; Shaik, Nabeebi; Sullivan, Terry; Stewart, Colin L.; Hogan, Brigid L.M.; Ko, Minoru S.H.

    2007-01-01

    Pluripotent stem cell lines with similar phenotypes can be derived from both blastocysts (embryonic stem cells, ESC) and primordial germ cells (embryonic germ cells, EGC). Here, we present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6). Even in the differentiation-promoting conditions, these pluripotent cells showed the same general trends of gene expression changes regardless of their origin and genetic background. These data indicate that ESCs and EGCs are indistinguishable based on global gene expression patterns alone. On the other hand, a detailed comparison between a group of ESC lines and a group of EGC lines identified 20 signature genes whose average expression levels were consistently higher in ESC lines, and 84 signature genes whose average expression levels were consistently higher in EGC lines, irrespective of mouse strains. Similar analysis identified 250 signature genes whose average expression levels were consistently higher in a group of 129 cell lines, and 337 signature genes whose average expression levels were consistently higher in a group of C57BL/6 cell lines. Although none of the genes was exclusively expressed in either ESCs versus EGCs or 129 versus C57BL/6, in combination these signature genes provide a reliable separation and identification of each cell type. Differentiation-promoting conditions also revealed some minor differences between the cell

  5. Polycomb group protein ezh2 controls actin polymerization and cell signaling.

    PubMed

    Su, I-hsin; Dobenecker, Marc-Werner; Dickinson, Ephraim; Oser, Matthew; Basavaraj, Ashwin; Marqueron, Raphael; Viale, Agnes; Reinberg, Danny; Wülfing, Christoph; Tarakhovsky, Alexander

    2005-05-06

    Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.

  6. A Quorum-Sensing Factor in Vegetative Dictyostelium Discoideum Cells Revealed by Quantitative Migration Analysis

    PubMed Central

    Golé, Laurent; Rivière, Charlotte; Hayakawa, Yoshinori; Rieu, Jean-Paul

    2011-01-01

    Background Many cells communicate through the production of diffusible signaling molecules that accumulate and once a critical concentration has been reached, can activate or repress a number of target genes in a process termed quorum sensing (QS). In the social amoeba Dictyostelium discoideum, QS plays an important role during development. However little is known about its effect on cell migration especially in the growth phase. Methods and Findings To investigate the role of cell density on cell migration in the growth phase, we use multisite timelapse microscopy and automated cell tracking. This analysis reveals a high heterogeneity within a given cell population, and the necessity to use large data sets to draw reliable conclusions on cell motion. In average, motion is persistent for short periods of time (), but normal diffusive behavior is recovered over longer time periods. The persistence times are positively correlated with the migrated distances. Interestingly, the migrated distance decreases as well with cell density. The adaptation of cell migration to cell density highlights the role of a secreted quorum sensing factor (QSF) on cell migration. Using a simple model describing the balance between the rate of QSF generation and the rate of QSF dilution, we were able to gather all experimental results into a single master curve, showing a sharp cell transition between high and low motile behaviors with increasing QSF. Conclusion This study unambiguously demonstrates the central role played by QSF on amoeboid motion in the growth phase. PMID:22073217

  7. Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves.

    PubMed

    Digiuni, Simona; Berne-Dedieu, Annik; Martinez-Torres, Cristina; Szecsi, Judit; Bendahmane, Mohammed; Arneodo, Alain; Argoul, Françoise

    2015-05-05

    Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions.

  8. Optomechanical properties of cancer cells revealed by light-induced deformation and quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Kastl, Lena; Budde, Björn; Isbach, Michael; Rommel, Christina; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

  9. Multifunctional cells of mouse anterior pituitary reveal a striking sexual dimorphism

    PubMed Central

    Núñez, Lucía; Villalobos, Carlos; Senovilla, Laura; García-Sancho, Javier

    2003-01-01

    The existence of cells storing and secreting two different anterior pituitary (AP) hormones (polyhormonal cells) or responding to several hypothalamic releasing hormones (HRHs) (multiresponsive cells) has been reported previously. These multifunctional cells could be involved in paradoxical secretion (AP hormone secretion evoked by a non-corresponding HRH) and transdifferentiation (phenotypic switch between mature cell types without cell division). Despite their putative physiological relevance, a comprehensive characterization of multifunctional AP cells is lacking. Here we combine calcium imaging (to assess responses to the four HRHs) and multiple sequential immunoassay of the six AP hormones in the same individual cells to perform a complete phenotypic characterization of mouse AP cells. Polyhormonal and multiresponsive cells were identified within all five AP cell types. They were scarce in the more abundant cell types, somatotropes and lactotropes, but quite frequent in corticotropes and gonadotropes. Cells with mixed phenotypes were the rule rather than the exception in thyrotropes, where 56–83 % of the cells stored two to five different hormones. Multifunctional AP cells were much more abundant in females than in males, indicating that the hormonal changes associated with the sexual cycle may promote transdifferentiation. As the phenotypic analysis was performed here after stimulation with HRHs, the fraction of polyhormonal cells might have been underestimated. With this limitation, the polyhormonal cells detected here responded to the HRHs less than the monohormonal ones, suggesting that they might contribute less than expected a priori to paradoxical secretion. Overall, our results reveal a striking sexual dimorphism, the female pituitary being much more plastic than the male pituitary. PMID:12730343

  10. Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies.

    PubMed

    Bakker, Bjorn; Taudt, Aaron; Belderbos, Mirjam E; Porubsky, David; Spierings, Diana C J; de Jong, Tristan V; Halsema, Nancy; Kazemier, Hinke G; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline S J M; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M; Colomé-Tatché, Maria; Foijer, Floris

    2016-05-31

    Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number changes affecting all lymphoma cells. This suggests that chromosome instability is somehow suppressed in the aneuploid lymphomas or that selection for frequently lost/gained chromosomes out-competes the CIN-imposed mis-segregation. To distinguish between these explanations and to examine karyotype dynamics in chromosome instable lymphoma, we use a newly developed single-cell whole genome sequencing (scWGS) platform that provides a complete and unbiased overview of copy number variations (CNV) in individual cells. To analyse these scWGS data, we develop AneuFinder, which allows annotation of copy number changes in a fully automated fashion and quantification of CNV heterogeneity between cells. Single-cell sequencing and AneuFinder analysis reveals high levels of copy number heterogeneity in chromosome instability-driven murine T-cell lymphoma samples, indicating ongoing chromosome instability. Application of this technology to human B cell leukaemias reveals different levels of karyotype heterogeneity in these cancers. Our data show that even though aneuploid tumours select for particular and recurring chromosome combinations, single-cell analysis using AneuFinder reveals copy number heterogeneity. This suggests ongoing chromosome instability that other platforms fail to detect. As chromosome instability might drive tumour evolution, karyotype analysis using single-cell sequencing technology could become an essential tool for cancer treatment stratification.

  11. Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group

    PubMed Central

    Amro, Ahmad; Mentis, Andreas; Pratlong, Francine; Dedet, Jean-Pierre; Votypka, Jan; Volf, Petr; Ozensoy Toz, Seray; Kuhls, Katrin; Schönian, Gabriele; Soteriadou, Ketty

    2012-01-01

    Background New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. Methodology/Principal Findings The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. Conclusion The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring

  12. Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls

    PubMed Central

    Sun, Yuliang; Juzenas, Kevin

    2017-01-01

    Abstract Immunofluorescence microscopy (IFM) and immunogold transmission electron microscopy (TEM) are the two main techniques commonly used to detect polysaccharides in plant cell walls. Both are important in localizing cell wall polysaccharides, but both have major limitations, such as low resolution in IFM and restricted sample size for immunogold TEM. In this study, we have developed a robust technique that combines immunocytochemistry with scanning electron microscopy (SEM) to study cell wall polysaccharide architecture in xylem cells at high resolution over large areas of sample. Using multiple cell wall monoclonal antibodies (mAbs), this immunogold SEM technique reliably localized groups of hemicellulosic and pectic polysaccharides in the cell walls of five different xylem structures (vessel elements, fibers, axial and ray parenchyma cells, and tyloses). This demonstrates its important advantages over the other two methods for studying cell wall polysaccharide composition and distribution in these structures. In addition, it can show the three-dimensional distribution of a polysaccharide group in the vessel lateral wall and the polysaccharide components in the cell wall of developing tyloses. This technique, therefore, should be valuable for understanding the cell wall polysaccharide composition, architecture and functions of diverse cell types. PMID:28398585

  13. Calcium Imaging Reveals Coordinated Simple Spike Pauses in Populations of Cerebellar Purkinje Cells.

    PubMed

    Ramirez, Jorge E; Stell, Brandon M

    2016-12-20

    The brain's control of movement is thought to involve coordinated activity between cerebellar Purkinje cells. The results reported here demonstrate that somatic Ca(2+) imaging is a faithful reporter of Na(+)-dependent "simple spike" pauses and enables us to optically record changes in firing rates in populations of Purkinje cells in brain slices and in vivo. This simultaneous calcium imaging of populations of Purkinje cells reveals a striking spatial organization of pauses in Purkinje cell activity between neighboring cells. The source of this organization is shown to be the presynaptic gamma-Aminobutyric acid producing (GABAergic) network, and blocking ionotropic gamma-Aminobutyric acid receptor (GABAARs) abolishes the synchrony. These data suggest that presynaptic interneurons synchronize (in)activity between neighboring Purkinje cells, and thereby maximize their effect on downstream targets in the deep cerebellar nuclei. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy.

    PubMed

    Jaros, Josef; Petrov, Michal; Tesarova, Marketa; Hampl, Ales

    2017-01-01

    Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.

  15. Global Survey of Cell Death Mechanisms Reveals Metabolic Regulation of Ferroptosis

    PubMed Central

    Shimada, Kenichi; Skouta, Rachid; Kaplan, Anna; Yang, Wan Seok; Hayano, Miki; Dixon, Scott J.; Brown, Lewis M.; Valenzuela, Carlos A.; Wolpaw, Adam J.

    2016-01-01

    Apoptosis is known as programmed cell death. Some non-apoptotic cell death is increasingly recognized as genetically controlled, or ‘regulated’. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. Here, we surveyed the landscape of pharmacologically-accessible cell death mechanisms. Of 56 caspase-independent lethal compounds, modulatory profiling revealed ten inducing three types of regulated non-apoptotic cell death. Lead optimization of one of the ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis occurs when the lipid repair enzyme GPX4 is inhibited. We found that FIN56 promotes degradation of GPX4. We performed chemoproteomics to reveal that FIN56 also binds to and activates squalene synthase, an enzyme involved in the cholesterol synthesis, in a manner independent of GPX4 degradation. These discoveries reveal that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes. PMID:27159577

  16. Clonal Dynamics Reveal Two Distinct Populations of Basal Cells in Slow-Turnover Airway Epithelium

    PubMed Central

    Watson, Julie K.; Rulands, Steffen; Wilkinson, Adam C.; Wuidart, Aline; Ousset, Marielle; Van Keymeulen, Alexandra; Göttgens, Berthold; Blanpain, Cédric; Simons, Benjamin D.; Rawlins, Emma L.

    2015-01-01

    Summary Epithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells. PMID:26119728

  17. Differences in Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells

    PubMed Central

    Curto, Pedro; Simões, Isaura; Riley, Sean P.; Martinez, Juan J.

    2016-01-01

    Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (Rickettsia conorii) and Rocky Mountain spotted fever (Rickettsia rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and Rickettsia montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated

  18. Transcriptional profile of TB antigen-specific T cells reveals novel multifunctional features1

    PubMed Central

    Arlehamn, Cecilia Lindestam; Seumois, Gregory; Gerasimova, Anna; Huang, Charlie; Fu, Zheng; Yue, Xiaojing; Sette, Alessandro; Vijayanand, Pandurangan; Peters, Bjoern

    2014-01-01

    In latent tuberculosis infection (LTBI) spread of the bacteria is contained by a persistent immune response, which includes CD4+ T cells as important contributors. Here we show that TB-specific CD4+ T cells have a characteristic chemokine expression signature (CCR6+CXCR3+CCR4−), and that the overall number of these cells is significantly increased in LTBI donors compared to healthy subjects. We have comprehensively characterized the transcriptional signature of CCR6+CXCR3+CCR4− cells and find significant differences to conventional Th1, Th17 and Th2 cells, but no major changes between healthy and LTBI donors. CCR6+CXCR3+CCR4− cells display linage-specific signatures of both Th1 and Th17 cells, but also have a unique gene expression program including genes associated with susceptibility to TB, enhanced T cell activation, enhanced cell survival, and induction of a cytotoxic program akin to CTL cells. Overall, the gene expression signature of CCR6+CXCR3+CCR4− cells reveals characteristics important for controlling latent TB infections. PMID:25092889

  19. Branching process deconvolution algorithm reveals a detailed cell-cycle transcription program.

    PubMed

    Guo, Xin; Bernard, Allister; Orlando, David A; Haase, Steven B; Hartemink, Alexander J

    2013-03-05

    Due to cell-to-cell variability and asymmetric cell division, cells in a synchronized population lose synchrony over time. As a result, time-series measurements from synchronized cell populations do not reflect the underlying dynamics of cell-cycle processes. Here, we present a branching process deconvolution algorithm that learns a more accurate view of dynamic cell-cycle processes, free from the convolution effects associated with imperfect cell synchronization. Through wavelet-basis regularization, our method sharpens signal without sharpening noise and can remarkably increase both the dynamic range and the temporal resolution of time-series data. Although applicable to any such data, we demonstrate the utility of our method by applying it to a recent cell-cycle transcription time course in the eukaryote Saccharomyces cerevisiae. Our method more sensitively detects cell-cycle-regulated transcription and reveals subtle timing differences that are masked in the original population measurements. Our algorithm also explicitly learns distinct transcription programs for mother and daughter cells, enabling us to identify 82 genes transcribed almost entirely in early G1 in a daughter-specific manner.

  20. Myf5 haploinsufficiency reveals distinct cell fate potentials for adult skeletal muscle stem cells.

    PubMed

    Gayraud-Morel, Barbara; Chrétien, Fabrice; Jory, Aurélie; Sambasivan, Ramkumar; Negroni, Elisa; Flamant, Patricia; Soubigou, Guillaume; Coppée, Jean-Yves; Di Santo, James; Cumano, Ana; Mouly, Vincent; Tajbakhsh, Shahragim

    2012-04-01

    Skeletal muscle stem cell fate in adult mice is regulated by crucial transcription factors, including the determination genes Myf5 and Myod. The precise role of Myf5 in regulating quiescent muscle stem cells has remained elusive. Here we show that most, but not all, quiescent satellite cells express Myf5 protein, but at varying levels, and that resident Myf5 heterozygous muscle stem cells are more primed for myogenic commitment compared with wild-type satellite cells. Paradoxically however, heterotypic transplantation of Myf5 heterozygous cells into regenerating muscles results in higher self-renewal capacity compared with wild-type stem cells, whereas myofibre regenerative capacity is not altered. By contrast, Pax7 haploinsufficiency does not show major modifications by transcriptome analysis. These observations provide a mechanism linking Myf5 levels to muscle stem cell heterogeneity and fate by exposing two distinct and opposing phenotypes associated with Myf5 haploinsufficiency. These findings have important implications for how stem cell fates can be modulated by crucial transcription factors while generating a pool of responsive heterogeneous cells.

  1. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    SciTech Connect

    Resch, Michael G.; Donohoe, Byron; Ciesielski, Peter; Nill, Jennifer; McKinney, Kellene; Mittal, Ashutosh; Katahira, Rui; Himmel, Michael; Biddy, Mary; Beckham, Gregg; Decker, Steve

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution.

  2. Single-Cell (Meta-)Genomics of a Dimorphic Candidatus Thiomargarita nelsonii Reveals Genomic Plasticity

    PubMed Central

    Flood, Beverly E.; Fliss, Palmer; Jones, Daniel S.; Dick, Gregory J.; Jain, Sunit; Kaster, Anne-Kristin; Winkel, Matthias; Mußmann, Marc; Bailey, Jake

    2016-01-01

    The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group

  3. Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells

    PubMed Central

    Lee, Timothy K.; Meng, Kevin; Shi, Handuo; Huang, Kerwyn Casey

    2016-01-01

    The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis. PMID:27774981

  4. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.

    PubMed

    Deng, Qiaolin; Ramsköld, Daniel; Reinius, Björn; Sandberg, Rickard

    2014-01-10

    Expression from both alleles is generally observed in analyses of diploid cell populations, but studies addressing allelic expression patterns genome-wide in single cells are lacking. Here, we present global analyses of allelic expression across individual cells of mouse preimplantation embryos of mixed background (CAST/EiJ × C57BL/6J). We discovered abundant (12 to 24%) monoallelic expression of autosomal genes and that expression of the two alleles occurs independently. The monoallelic expression appeared random and dynamic because there was considerable variation among closely related embryonic cells. Similar patterns of monoallelic expression were observed in mature cells. Our allelic expression analysis also demonstrates the de novo inactivation of the paternal X chromosome. We conclude that independent and stochastic allelic transcription generates abundant random monoallelic expression in the mammalian cell.

  5. Dissecting the Tumor Myeloid Compartment Reveals Rare Activating Antigen Presenting Cells, Critical for T cell Immunity

    PubMed Central

    Broz, Miranda; Binnewies, Mikhail; Boldajipour, Bijan; Nelson, Amanda; Pollock, Joshua; Erle, David; Barczak, Andrea; Rosenblum, Michael; Daud, Adil; Barber, Diane; Amigorena, Sebastian; van’t Veer, Laura J.; Sperling, Anne; Wolf, Denise; Krummel, Matthew F.

    2014-01-01

    SUMMARY It is well understood that antigen-presenting cells (APC) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human tumor biopsies, we have delineated the intratumoral dendritic-cell (DC) populations as distinct from macrophage populations. Within these, CD103+ DCs are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent upon IRF8, Zbtb46 and Batf3 transcription factors and generated by GM-CSF and Flt3L cytokines. Regressing tumors have higher proportions of these cells, T-cell dependent immune clearance relies upon them, and abundance of their transcripts in human tumors correlates with clinical outcome. This cell type presents opportunities for prognostic and therapeutic approaches across multiple cancer types. PMID:25446897

  6. Heterogeneity of neural progenitor cells revealed by enhancers in the nestin gene

    PubMed Central

    Yaworsky, Paul J.; Kappen, Claudia

    2014-01-01

    Using transgenic embryos, we have identified two distinct CNS progenitor cell-specific enhancers, each requiring the cooperation of at least two independent regulatory sites, within the second intron of the rat nestin gene. One enhancer is active throughout the developing CNS while the other is specifically active in the ventral midbrain. These experiments demonstrate that neural progenitor cells in the midbrain constitute a unique subpopulation based upon their ability to activate the midbrain regulatory elements. Our finding of differential enhancer activity from a gene encoding a structural protein reveals a previously unrecognized diversity in neural progenitor cell populations. PMID:9917366

  7. Stringent comparative sequence analysis reveals SOX10 as a putative inhibitor of glial cell differentiation.

    PubMed

    Gopinath, Chetna; Law, William D; Rodríguez-Molina, José F; Prasad, Arjun B; Song, Lingyun; Crawford, Gregory E; Mullikin, James C; Svaren, John; Antonellis, Anthony

    2016-11-07

    The transcription factor SOX10 is essential for all stages of Schwann cell development including myelination. SOX10 cooperates with other transcription factors to activate the expression of key myelin genes in Schwann cells and is therefore a context-dependent, pro-myelination transcription factor. As such, the identification of genes regulated by SOX10 will provide insight into Schwann cell biology and related diseases. While genome-wide studies have successfully revealed SOX10 target genes, these efforts mainly focused on myelinating stages of Schwann cell development. We propose that less-biased approaches will reveal novel functions of SOX10 outside of myelination. We developed a stringent, computational-based screen for genome-wide identification of SOX10 response elements. Experimental validation of a pilot set of predicted binding sites in multiple systems revealed that SOX10 directly regulates a previously unreported alternative promoter at SOX6, which encodes a transcription factor that inhibits glial cell differentiation. We further explored the utility of our computational approach by combining it with DNase-seq analysis in cultured Schwann cells and previously published SOX10 ChIP-seq data from rat sciatic nerve. Remarkably, this analysis enriched for genomic segments that map to loci involved in the negative regulation of gliogenesis including SOX5, SOX6, NOTCH1, HMGA2, HES1, MYCN, ID4, and ID2. Functional studies in Schwann cells revealed that: (1) all eight loci are expressed prior to myelination and down-regulated subsequent to myelination; (2) seven of the eight loci harbor validated SOX10 binding sites; and (3) seven of the eight loci are down-regulated upon repressing SOX10 function. Our computational strategy revealed a putative novel function for SOX10 in Schwann cells, which suggests a model where SOX10 activates the expression of genes that inhibit myelination during non-myelinating stages of Schwann cell development. Importantly, the

  8. Nuclear Motility in Glioma Cells Reveals a Cell-Line Dependent Role of Various Cytoskeletal Components

    PubMed Central

    Kiss, Alexa; Horvath, Peter; Rothballer, Andrea; Kutay, Ulrike; Csucs, Gabor

    2014-01-01

    Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns - thereby forced into a bipolar morphology - displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved. PMID:24691067

  9. Single-cell RNAseq reveals cell adhesion molecule profiles in electrophysiologically defined neurons

    PubMed Central

    Földy, Csaba; Darmanis, Spyros; Aoto, Jason; Malenka, Robert C.; Quake, Stephen R.; Südhof, Thomas C.

    2016-01-01

    In brain, signaling mediated by cell adhesion molecules defines the identity and functional properties of synapses. The specificity of presynaptic and postsynaptic interactions that is presumably mediated by cell adhesion molecules suggests that there exists a logic that could explain neuronal connectivity at the molecular level. Despite its importance, however, the nature of such logic is poorly understood, and even basic parameters, such as the number, identity, and single-cell expression profiles of candidate synaptic cell adhesion molecules, are not known. Here, we devised a comprehensive list of genes involved in cell adhesion, and used single-cell RNA sequencing (RNAseq) to analyze their expression in electrophysiologically defined interneurons and projection neurons. We compared the cell type-specific expression of these genes with that of genes involved in transmembrane ion conductances (i.e., channels), exocytosis, and rho/rac signaling, which regulates the actin cytoskeleton. Using these data, we identified two independent, developmentally regulated networks of interacting genes encoding molecules involved in cell adhesion, exocytosis, and signal transduction. Our approach provides a framework for a presumed cell adhesion and signaling code in neurons, enables correlating electrophysiological with molecular properties of neurons, and suggests avenues toward understanding synaptic specificity. PMID:27531958

  10. Single-cell RNAseq reveals cell adhesion molecule profiles in electrophysiologically defined neurons.

    PubMed

    Földy, Csaba; Darmanis, Spyros; Aoto, Jason; Malenka, Robert C; Quake, Stephen R; Südhof, Thomas C

    2016-08-30

    In brain, signaling mediated by cell adhesion molecules defines the identity and functional properties of synapses. The specificity of presynaptic and postsynaptic interactions that is presumably mediated by cell adhesion molecules suggests that there exists a logic that could explain neuronal connectivity at the molecular level. Despite its importance, however, the nature of such logic is poorly understood, and even basic parameters, such as the number, identity, and single-cell expression profiles of candidate synaptic cell adhesion molecules, are not known. Here, we devised a comprehensive list of genes involved in cell adhesion, and used single-cell RNA sequencing (RNAseq) to analyze their expression in electrophysiologically defined interneurons and projection neurons. We compared the cell type-specific expression of these genes with that of genes involved in transmembrane ion conductances (i.e., channels), exocytosis, and rho/rac signaling, which regulates the actin cytoskeleton. Using these data, we identified two independent, developmentally regulated networks of interacting genes encoding molecules involved in cell adhesion, exocytosis, and signal transduction. Our approach provides a framework for a presumed cell adhesion and signaling code in neurons, enables correlating electrophysiological with molecular properties of neurons, and suggests avenues toward understanding synaptic specificity.

  11. Genetically Induced Cell Death in Bulge Stem Cells Reveals Their Redundancy for Hair and Epidermal Regeneration

    PubMed Central

    Driskell, Iwona; Oeztuerk-Winder, Feride; Humphreys, Peter; Frye, Michaela

    2015-01-01

    Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of Setd8 eliminated the contribution of bulge cells to hair follicle regeneration through inhibition of cell division and induction of cell death, but the growth and morphology of hair follicles were unaffected. Furthermore, ablation of Setd8 in the hair follicle bulge blocked the contribution of K19-postive stem cells to wounded epidermis, but the wound healing process was unaltered. Our data indicate that quiescent bulge stem cells are dispensable for hair follicle regeneration and epidermal injury in the short term and support the hypothesis that quiescent and cycling stem cell populations are equipotent. Stem Cells 2015;33:988–998 PMID:25447755

  12. Whole population cell analysis of a landmark-rich mammalian epithelium reveals multiple elongation mechanisms

    PubMed Central

    Economou, Andrew D.; Brock, Lara J.; Cobourne, Martyn T.; Green, Jeremy B. A.

    2013-01-01

    Tissue elongation is a fundamental component of developing and regenerating systems. Although localised proliferation is an important mechanism for tissue elongation, potentially important contributions of other elongation mechanisms, specifically cell shape change, orientated cell division and cell rearrangement, are rarely considered or quantified, particularly in mammalian systems. Their quantification, together with proliferation, provides a rigorous framework for the analysis of elongation. The mammalian palatal epithelium is a landmark-rich tissue, marked by regularly spaced ridges (rugae), making it an excellent model in which to analyse the contributions of cellular processes to directional tissue growth. We captured confocal stacks of entire fixed mouse palate epithelia throughout the mid-gestation growth period, labelled with membrane, nuclear and cell proliferation markers and segmented all cells (up to ∼20,000 per palate), allowing the quantification of cell shape and proliferation. Using the rugae as landmarks, these measures revealed that the so-called growth zone is a region of proliferation that is intermittently elevated at ruga initiation. The distribution of oriented cell division suggests that it is not a driver of tissue elongation, whereas cell shape analysis revealed that both elongation of cells leaving the growth zone and apico-basal cell rearrangements do contribute significantly to directional growth. Quantitative comparison of elongation processes indicated that proliferation contributes most to elongation at the growth zone, but cell shape change and rearrangement contribute as much as 40% of total elongation. We have demonstrated the utility of an approach to analysing the cellular mechanisms underlying tissue elongation in mammalian tissues. It should be broadly applied to higher-resolution analysis of links between genotypes and malformation phenotypes. PMID:24173805

  13. An efficient method that reveals both the dendrites and the soma mosaics of retinal ganglion cells.

    PubMed

    Zhan, X J; Troy, J B

    1997-03-01

    A method of using neurobiotin to stain both the dendrites and the soma mosaics of retinal ganglion cells in fresh retinae is described. This method is simple to use and efficient in revealing morphological details for a large number of retinal ganglion cells. It has five advantages over currently available staining methods. (1) It stains all ganglion cells in the whole retina or in a selected retinal area, permitting ganglion cell distributions across the retina to be obtained. (2) It reveals cell dendrites in great detail, especially in regions outside the area centralis. The dendritic field mosaics and, therefore the dendritic field coverage factors, of different ganglion cell types across the whole retina can be obtained easily. (3) It works reliably, efficiently, and does not require the expensive set-up or the pains-taking work needed when staining cells through intracellular injection. (4) It works under both in vivo and in vitro settings, permitting the use of retinae from animals sacrificed for other purposes and the use of postmortem human retinae. (5) The end product of the visualization process is optically dark and electron dense, permitting specimens to be examined under both light and electron microscopes.

  14. MRI reveals slow clearance of dead cell transplants in mouse forelimb muscles.

    PubMed

    Zhang, Yanhui; Zhang, Hongyan; Ding, Lijun; Zhang, Hailu; Zhang, Pengli; Jiang, Haizhen; Tan, Bo; Deng, Zongwu

    2017-10-01

    A small molecule tetraazacyclododecane-1,4,7,10-tetraacetic acid (Gd‑DOTA)4‑TPP agent is used to label human mesenchymal stem cells (hMSCs) via electroporation (EP). The present study assessed the cytotoxicity of cell labeling, in addition to its effect on cell differentiation potential. There were no significant adverse effects on cell viability or differentiation induced by either EP or cellular uptake of (Gd‑DOTA)4‑TPP. Labeled live and dead hMSCs were transplanted into mouse forelimb muscles. T2‑weighted magnetic resonance imaging (MRI) was used to track the in vivo fate of the cell transplants. The labeling and imaging strategy allowed long term tracking of the cell transplants and unambiguous distinguishing of the cell transplants from their surrounding tissues. Cell migration was observed for live hMSCs injected into subcutaneous tissues, however not for either live or dead hMSCS injected into limb muscles. A slow clearance process occurred of the dead cell transplants in the limb muscular tissue. The MRI results therefore reveal that the fate and physiological activities of cell transplants depend on the nature of their host tissue.

  15. Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers.

    PubMed

    Agirre, Xabier; Castellano, Giancarlo; Pascual, Marien; Heath, Simon; Kulis, Marta; Segura, Victor; Bergmann, Anke; Esteve, Anna; Merkel, Angelika; Raineri, Emanuele; Agueda, Lidia; Blanc, Julie; Richardson, David; Clarke, Laura; Datta, Avik; Russiñol, Nuria; Queirós, Ana C; Beekman, Renée; Rodríguez-Madoz, Juan R; San José-Enériz, Edurne; Fang, Fang; Gutiérrez, Norma C; García-Verdugo, José M; Robson, Michael I; Schirmer, Eric C; Guruceaga, Elisabeth; Martens, Joost H A; Gut, Marta; Calasanz, Maria J; Flicek, Paul; Siebert, Reiner; Campo, Elías; Miguel, Jesús F San; Melnick, Ari; Stunnenberg, Hendrik G; Gut, Ivo G; Prosper, Felipe; Martín-Subero, José I

    2015-04-01

    While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

  16. Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers

    PubMed Central

    Castellano, Giancarlo; Pascual, Marien; Heath, Simon; Kulis, Marta; Segura, Victor; Bergmann, Anke; Esteve, Anna; Merkel, Angelika; Raineri, Emanuele; Agueda, Lidia; Blanc, Julie; Richardson, David; Clarke, Laura; Datta, Avik; Russiñol, Nuria; Queirós, Ana C.; Beekman, Renée; Rodríguez-Madoz, Juan R.; José-Enériz, Edurne San; Fang, Fang; Gutiérrez, Norma C.; García-Verdugo, José M.; Robson, Michael I.; Schirmer, Eric C.; Guruceaga, Elisabeth; Martens, Joost H.A.; Gut, Marta; Calasanz, Maria J.; Flicek, Paul; Siebert, Reiner; Campo, Elías; Miguel, Jesús F. San; Melnick, Ari; Stunnenberg, Hendrik G.; Gut, Ivo G.

    2015-01-01

    While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM. PMID:25644835

  17. Traumatic brain injury reveals novel cell lineage relationships within the subventricular zone

    PubMed Central

    Thomsen, Gretchen M.; Le Belle, Janel E.; Harnisch, Jessica A.; Mc Donald, Whitney; Hovda, David A.; Sofroniew, Michael V.; Kornblum, Harley I.; Harris, Neil G.

    2014-01-01

    The acute response of the rodent subventricular zone (SVZ) to traumatic brain injury (TBI) involves a physical expansion through increased cell proliferation. However, the cellular underpinnings of these changes are not well understood. Our analyses have revealed that there are two distinct transit-amplifying cell populations that respond in opposite ways to injury. Mash1+ transit-amplifying cells are the primary SVZ cell type that is stimulated to divide following TBI. In contrast, the EGFR+ population, which has been considered to be a functionally equivalent progenitor population to Mash1+ cells in the uninjured brain, becomes significantly less proliferative after injury. Although normally quiescent GFAP+ stem cells are stimulated to divide in SVZ ablation models, we found that the GFAP+ stem cells do not divide more after TBI. We found, instead, that TBI results in increased numbers of GFAP+/EGFR+ stem cells via non-proliferative means—potentially through the dedifferentiation of progenitor cells. EGFR+ progenitors from injured brains only were competent to revert to a stem cell state following brief exposure to growth factors. Thus, our results demonstrate previously unknown changes in lineage relationships that differ from conventional models and likely reflect an adaptive response of the SVZ to maintain endogenous brain repair after TBI. PMID:24835668

  18. Molecular Integrative Clustering of Asian Gastric Cell Lines Revealed Two Distinct Chemosensitivity Clusters

    PubMed Central

    Choong, Meng Ling; Tan, Shan Ho; Tan, Tuan Zea; Manesh, Sravanthy; Ngo, Anna; Yong, Jacklyn W. Y.; Yang, Henry He; Lee, May Ann

    2014-01-01

    Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy. PMID:25343454

  19. Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility

    PubMed Central

    Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

    2014-01-01

    A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4+ T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis. PMID:24997565

  20. Differentiation of Human Dental Stem Cells Reveal a Role for microRNA-218

    PubMed Central

    Gay, Isabel; Cavender, Adriana; Peto, David; Sun, Zhao; Speer, Aline; Cao, Huojun; Amendt, Brad A.

    2013-01-01

    Background Regeneration of the lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in the periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data is available on micro-RNAs (miRNAs) activity behind human-derived dental stem cells. Methods In this study, we isolated periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and gingival stem cells (GSCs) from extracted third molars; human bone marrow stem cells (BMSCs) were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. A miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. Results RUNX2 expression demonstrated the successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human dental stem cells (DSCs). DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. Conclusions These data reveal a miRNA regulated pathway for the differentiation of human DSCs and a select network of human microRNAs that control DSC osteogenic differentiation. PMID:23662917

  1. Fourier transform infrared microspectroscopy reveals biochemical changes associated with glioma stem cell differentiation.

    PubMed

    Kenig, Saša; Bedolla, Diana E; Birarda, Giovanni; Faoro, Valentina; Mitri, Elisa; Vindigni, Alessandro; Storici, Paola; Vaccari, Lisa

    2015-12-01

    According to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem cells - a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection. Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content of stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative, labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics. The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipids is higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stem cells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogen level increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of the identified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Although it was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with the increasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a space of differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocol in clinical practice to estimate the content of stem cells in a tumor sample. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Single cell transcriptome analysis of muscle satellite cells reveals widespread transcriptional heterogeneity.

    PubMed

    Cho, Dong Seong; Doles, Jason D

    2017-09-08

    Tissue specific stem cells are indispensable contributors to adult tissue maintenance, repair, and regeneration. In skeletal muscle, satellite cells (SCs) are the resident muscle stem cell population and are required to maintain skeletal muscle homeostasis throughout life. Increasing evidence suggests that SCs are a heterogeneous cell population with substantial biochemical and functional diversity. A major limitation in the field is an incomplete understanding of the nature and extent of this cellular heterogeneity. Single cell analyses are well suited to addressing this issue, especially when coupled to unbiased profiling paradigms such as high throughout RNA sequencing. We performed single cell RNA sequencing (scRNA-seq) on freshly isolated muscle satellite cells and found a surprising degree of heterogeneity at multiple levels, from muscle-specific transcripts to the broader SC transcriptome. We leveraged several comparative bioinformatics techniques and found that individual SCs enrich for unique transcript clusters. We propose that these gene expression "fingerprints" may contribute to observed functional SC diversity. Overall, these studies underscore the importance of several established SC signaling pathways/processes on a single cell level, implicate novel regulators of SC heterogeneity, and lay the groundwork for further investigation into SC heterogeneity in health and disease. Copyright © 2017. Published by Elsevier B.V.

  3. Genetically induced cell death in bulge stem cells reveals their redundancy for hair and epidermal regeneration.

    PubMed

    Driskell, Iwona; Oeztuerk-Winder, Feride; Humphreys, Peter; Frye, Michaela

    2015-03-01

    Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of Setd8 eliminated the contribution of bulge cells to hair follicle regeneration through inhibition of cell division and induction of cell death, but the growth and morphology of hair follicles were unaffected. Furthermore, ablation of Setd8 in the hair follicle bulge blocked the contribution of K19-postive stem cells to wounded epidermis, but the wound healing process was unaltered. Our data indicate that quiescent bulge stem cells are dispensable for hair follicle regeneration and epidermal injury in the short term and support the hypothesis that quiescent and cycling stem cell populations are equipotent.

  4. Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

    PubMed

    Zheng, Chunhong; Zheng, Liangtao; Yoo, Jae-Kwang; Guo, Huahu; Zhang, Yuanyuan; Guo, Xinyi; Kang, Boxi; Hu, Ruozhen; Huang, Julie Y; Zhang, Qiming; Liu, Zhouzerui; Dong, Minghui; Hu, Xueda; Ouyang, Wenjun; Peng, Jirun; Zhang, Zemin

    2017-06-15

    Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8(+) T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8(+) T cells and Tregs and represses the CD8(+) T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Live Cell Imaging Reveals Structural Associations between the Actin and Microtubule Cytoskeleton in Arabidopsis [W] [OA

    PubMed Central

    Sampathkumar, Arun; Lindeboom, Jelmer J.; Debolt, Seth; Gutierrez, Ryan; Ehrhardt, David W.; Ketelaar, Tijs; Persson, Staffan

    2011-01-01

    In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells. PMID:21693695

  6. Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq.

    PubMed

    Chan, Sunny Sun-Kin; Chan, Howe H W; Kyba, Michael

    2016-06-03

    Mesp1 is a transcription factor that promotes differentiation of pluripotent cells into different mesoderm lineages including hematopoietic, cardiac and skeletal myogenic. This occurs via at least two transient cell populations: a common hematopoietic/cardiac progenitor population and a common cardiac/skeletal myogenic progenitor population. It is not established whether Mesp1-induced mesoderm cells are intrinsically heterogeneous, or are simply capable of multiple lineage decisions. In the current study, we applied single-cell RNA-seq to analyze Mesp1+ mesoderm. Initial whole transcriptome analysis showed a surprising homogeneity among Mesp1-induced mesoderm cells. However, this apparent global homogeneity masked an intrinsic heterogeneity revealed by interrogating a panel of early mesoderm patterning factors. This approach enabled discovery of subpopulations primed for hematopoietic or cardiac development. These studies demonstrate the heterogeneic nature of Mesp1+ mesoderm.

  7. Patterns of kinship in groups of free-living sperm whales (Physeter macrocephalus) revealed by multiple molecular genetic analyses.

    PubMed

    Richard, K R; Dillon, M C; Whitehead, H; Wright, J M

    1996-08-06

    Mature female sperm whales (Physeter macrocephalus) live in socially cohesive groups of 10-30, which include immature animals of both sexes, and within which there is communal care of the young. We examined kinship in such groups using analyses of microsatellite DNA, mitochondrial DNA sequence, and sex-linked markers on samples of sloughed skin collected noninvasively from animals in three groups off the coast of Ecuador. Social groups were defined through photographic identification of individuals. Each group contained about 26 members, mostly female (79%). Relatedness was greater within groups, as compared to between groups. Particular mitochondrial haplotypes were characteristic of groups, but all groups contained more than one haplotype. The data are generally consistent with each group being comprised of several matrillines from which males disperse at about the age of 6 years. There are indications of paternal relatedness among grouped individuals with different mitochondrial haplotypes, suggesting long-term associations between different matrilines.

  8. Patterns of kinship in groups of free-living sperm whales (Physeter macrocephalus) revealed by multiple molecular genetic analyses.

    PubMed Central

    Richard, K R; Dillon, M C; Whitehead, H; Wright, J M

    1996-01-01

    Mature female sperm whales (Physeter macrocephalus) live in socially cohesive groups of 10-30, which include immature animals of both sexes, and within which there is communal care of the young. We examined kinship in such groups using analyses of microsatellite DNA, mitochondrial DNA sequence, and sex-linked markers on samples of sloughed skin collected noninvasively from animals in three groups off the coast of Ecuador. Social groups were defined through photographic identification of individuals. Each group contained about 26 members, mostly female (79%). Relatedness was greater within groups, as compared to between groups. Particular mitochondrial haplotypes were characteristic of groups, but all groups contained more than one haplotype. The data are generally consistent with each group being comprised of several matrillines from which males disperse at about the age of 6 years. There are indications of paternal relatedness among grouped individuals with different mitochondrial haplotypes, suggesting long-term associations between different matrilines. PMID:8710951

  9. Polycomb Group Protein Ezh2 Regulates Hepatic Progenitor Cell Proliferation and Differentiation in Murine Embryonic Liver

    PubMed Central

    Ueno, Yasuharu; Nakata, Susumu; Obana, Yuta; Sekine, Keisuke; Zheng, Yun-Wen; Takebe, Takanori; Isono, Kyoichi; Koseki, Haruhiko; Taniguchi, Hideki

    2014-01-01

    In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. Enhancer of zeste homolog 2 (Ezh2), a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which trigger the gene suppression. In the present study, we investigated a role of Ezh2 in the regulation of the expanding hepatic progenitor population in vivo. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of Ezh2, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from in vivo fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion in vivo, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3. PMID:25153170

  10. Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone

    SciTech Connect

    Carl, M.; Robbins, F.; Hartzman, R.J.; Dasch, G.A.

    1987-12-15

    Cytolytic human T cells clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii, as measured by /sup 51/Cr-release from target cells. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.

  11. Enantiodivergent Fluorination of Allylic Alcohols: Data Set Design Reveals Structural Interplay between Achiral Directing Group and Chiral Anion.

    PubMed

    Neel, Andrew J; Milo, Anat; Sigman, Matthew S; Toste, F Dean

    2016-03-23

    Enantioselectivity values represent relative rate measurements that are sensitive to the structural features of the substrates and catalysts interacting to produce them. Therefore, well-designed enantioselectivity data sets are information rich and can provide key insights regarding specific molecular interactions. However, if the mechanism for enantioselection varies throughout a data set, these values cannot be easily compared. This premise, which is the crux of free energy relationships, exposes a challenging issue of identifying mechanistic breaks within multivariate correlations. Herein, we describe an approach to addressing this problem in the context of a chiral phosphoric acid catalyzed fluorination of allylic alcohols using aryl boronic acids as transient directing groups. By designing a data set in which both the phosphoric and boronic acid structures were systematically varied, key enantioselectivity outliers were identified and analyzed. A mechanistic study was executed to reveal the structural origins of these outliers, which was consistent with the presence of several mechanistic regimes within the data set. While 2- and 4-substituted aryl boronic acids favored the (R)-enantiomer with most of the studied catalysts, meta-alkoxy substituted aryl boronic acids resulted in the (S)-enantiomer when used in combination with certain (R)-phosphoric acids. We propose that this selectivity reversal is the result of a lone pair-π interaction between the substrate ligated boronic acid and the phosphate. On the basis of this proposal, a catalyst system was identified, capable of producing either enantiomer in high enantioselectivity (77% (R)-2 to 92% (S)-2) using the same chiral catalyst by subtly changing the structure of the achiral boronic acid.

  12. Comparative 16S rRNA Analysis of Lake Bacterioplankton Reveals Globally Distributed Phylogenetic Clusters Including an Abundant Group of Actinobacteria

    PubMed Central

    Glöckner, Frank Oliver; Zaichikov, Evgeny; Belkova, Natalia; Denissova, Ludmilla; Pernthaler, Jakob; Pernthaler, Annelie; Amann, Rudolf

    2000-01-01

    In a search for cosmopolitan phylogenetic clusters of freshwater bacteria, we recovered a total of 190 full and partial 16S ribosomal DNA (rDNA) sequences from three different lakes (Lake Gossenköllesee, Austria; Lake Fuchskuhle, Germany; and Lake Baikal, Russia). The phylogenetic comparison with the currently available rDNA data set showed that our sequences fall into 16 clusters, which otherwise include bacterial rDNA sequences of primarily freshwater and soil, but not marine, origin. Six of the clusters were affiliated with the α, four were affiliated with the β, and one was affiliated with the γ subclass of the Proteobacteria; four were affiliated with the Cytophaga-Flavobacterium-Bacteroides group; and one was affiliated with the class Actinobacteria (formerly known as the high-G+C gram-positive bacteria). The latter cluster (hgcI) is monophyletic and so far includes only sequences directly retrieved from aquatic environments. Fluorescence in situ hybridization (FISH) with probes specific for the hgcI cluster showed abundances of up to 1.7 × 105 cells ml−1 in Lake Gossenköllesee, with strong seasonal fluctuations, and high abundances in the two other lakes investigated. Cell size measurements revealed that Actinobacteria in Lake Gossenköllesee can account for up to 63% of the bacterioplankton biomass. A combination of phylogenetic analysis and FISH was used to reveal 16 globally distributed sequence clusters and to confirm the broad distribution, abundance, and high biomass of members of the class Actinobacteria in freshwater ecosystems. PMID:11055963

  13. Turnover of microbial groups and cell components in soil: 13C analysis of cellular biomarkers

    NASA Astrophysics Data System (ADS)

    Gunina, Anna; Dippold, Michaela; Glaser, Bruno; Kuzyakov, Yakov

    2017-01-01

    Microorganisms regulate the carbon (C) cycle in soil, controlling the utilization and recycling of organic substances. To reveal the contribution of particular microbial groups to C utilization and turnover within the microbial cells, the fate of 13C-labelled glucose was studied under field conditions. Glucose-derived 13C was traced in cytosol, amino sugars and phospholipid fatty acid (PLFA) pools at intervals of 3, 10 and 50 days after glucose addition into the soil. 13C enrichment in PLFAs ( ˜ 1.5 % of PLFA C at day 3) was an order of magnitude greater than in cytosol, showing the importance of cell membranes for initial C utilization. The 13C enrichment in amino sugars of living microorganisms at day 3 accounted for 0.57 % of total C pool; as a result, we infer that the replacement of C in cell wall components is 3 times slower than that of cell membranes. The C turnover time in the cytosol (150 days) was 3 times longer than in PLFAs (47 days). Consequently, even though the cytosol pool has the fastest processing rates compared to other cellular compartments, intensive recycling of components here leads to a long C turnover time. Both PLFA and amino-sugar profiles indicated that bacteria dominated in glucose utilization. 13C enrichment decreased with time for bacterial cell membrane components, but it remained constant or even increased for filamentous microorganisms. 13C enrichment of muramic acid was the 3.5 times greater than for galactosamine, showing a more rapid turnover of bacterial cell wall components compared to fungal. Thus, bacteria utilize a greater proportion of low-molecular-weight organic substances, whereas filamentous microorganisms are responsible for further C transformations. Thus, tracing 13C in cellular compounds with contrasting turnover rates elucidated the role of microbial groups and their cellular compartments in C utilization and recycling in soil. The results also reflect that microbial C turnover is not restricted to the death or

  14. Inducible lineage tracing of Pax7-descendant cells reveals embryonic origin of adult satellite cells

    PubMed Central

    Lepper, Christoph; Fan, Chen-Ming

    2011-01-01

    We have generated a mouse strain carrying a Cre-ERT2 knock-in allele at the Pax7 locus, the Pax7CE allele (Lepper et al., 2009). Combining Pax7CE and the R26RLacZ Cre reporter allele, here we describe temporal-specific tamoxifen (tmx)-inducible lineage tracing of embryonic Pax7-expressing cells. In particular, we focus on the somitic lineage. Tmx-inducible Cre activity directed by the Pax7CE allele is similar to the endogenous Pax7 expression pattern. The somitic Pax7-expressing cells selectively marked at embryonic day 9.5 (E9.5) give rise to dorsal dermis and brown adipose tissue, in addition to dorsal aspects of trunk muscles and the diaphragm muscle. However, they do not contribute to ventral body wall and limb muscles. After E12.5, marked Pax7-expressing cells become lineage restricted to muscles. Descendants of these early marked Pax7-expressing cells begin to occupy sublaminal positions associated with the myofibers around E16.5, characteristic of embryonic satellite cells. Furthermore, they contribute to adult myofibers and regeneration competent satellite cells in the tibialis anterior muscle, providing evidence that some adult satellite cells are of embryonic origin. PMID:20641127

  15. Single-Cell mRNA Profiling Reveals Cell-Type Specific Expression of Neurexin Isoforms

    PubMed Central

    Fuccillo, Marc V.; Földy, Csaba; Gökce, Özgün; Rothwell, Patrick E.; Sun, Gordon L.; Malenka, Robert C.; Südhof, Thomas C.

    2016-01-01

    Summary Neurexins are considered central organizers of synapse architecture that are implicated in neuropsychiatric disorders. Expression of neurexins in hundreds of alternatively spliced isoforms suggested that individual neurons might exhibit a cell type-specific neurexin expression pattern (a neurexin code). To test this hypothesis, we quantified the single-cell levels of neurexin isoforms and other trans-synaptic cell-adhesion molecules by microfluidics-based RT-PCR. We show that the neurexin repertoire displays pronounced cell-type specificity that is remarkably consistent within each type of neuron. Furthermore, we uncovered region-specific regulation of neurexin transcription and splice-site usage. Finally, we demonstrate that the transcriptional profiles of neurexins can be altered in an experience-dependent fashion by exposure to a drug of abuse. Our data provide evidence of cell type-specific expression patterns of multiple neurexins at the single-cell level, and suggest that expression of synaptic cell-adhesion molecules overlaps with other key features of cellular identity and diversity. PMID:26182417

  16. Inducible lineage tracing of Pax7-descendant cells reveals embryonic origin of adult satellite cells.

    PubMed

    Lepper, Christoph; Fan, Chen-Ming

    2010-07-01

    We have generated a mouse strain carrying a Cre-ER(T2) knock-in allele at the Pax7 locus, the Pax7(CE) allele (Lepper et al., 2009, Nature 460:627-631). Combining Pax7(CE) and the R26R(LacZ) Cre reporter allele, here we describe temporal-specific tamoxifen (tmx)-inducible lineage tracing of embryonic Pax7-expressing cells. In particular, we focus on the somitic lineage. Tmx-inducible Cre activity directed by the Pax7(CE) allele is similar to the endogenous Pax7 expression pattern. The somitic Pax7-expressing cells selectively marked at embryonic day 9.5 (E9.5) give rise to dorsal dermis and brown adipose tissue, in addition to dorsal aspects of trunk muscles and the diaphragm muscle. However, they do not contribute to ventral body wall and limb muscles. After E12.5, marked Pax7-expressing cells become lineage restricted to muscles. Descendants of these early marked Pax7-expressing cells begin to occupy sublaminal positions associated with the myofibers around E16.5, characteristic of embryonic satellite cells. Furthermore, they contribute to adult myofibers and regeneration competent satellite cells in the tibialis anterior muscle, providing evidence that some adult satellite cells are of embryonic origin. (c) 2010 Wiley-Liss, Inc.

  17. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

    PubMed Central

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  18. Power-Law Modeling of Cancer Cell Fates Driven by Signaling Data to Reveal Drug Effects.

    PubMed

    Zhang, Fan; Wu, Min; Kwoh, Chee Keong; Zheng, Jie

    2016-01-01

    Extracellular signals are captured and transmitted by signaling proteins inside a cell. An important type of cellular responses to the signals is the cell fate decision, e.g., apoptosis. However, the underlying mechanisms of cell fate regulation are still unclear, thus comprehensive and detailed kinetic models are not yet available. Alternatively, data-driven models are promising to bridge signaling data with the phenotypic measurements of cell fates. The traditional linear model for data-driven modeling of signaling pathways has its limitations because it assumes that the a cell fate is proportional to the activities of signaling proteins, which is unlikely in the complex biological systems. Therefore, we propose a power-law model to relate the activities of all the measured signaling proteins to the probabilities of cell fates. In our experiments, we compared our nonlinear power-law model with the linear model on three cancer datasets with phosphoproteomics and cell fate measurements, which demonstrated that the nonlinear model has superior performance on cell fates prediction. By in silico simulation of virtual protein knock-down, the proposed model is able to reveal drug effects which can complement traditional approaches such as binding affinity analysis. Moreover, our model is able to capture cell line specific information to distinguish one cell line from another in cell fate prediction. Our results show that the power-law data-driven model is able to perform better in cell fate prediction and provide more insights into the signaling pathways for cancer cell fates than the linear model.

  19. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

    PubMed

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis.

  20. Bimodal dynamics of granular organelles in primary renin-expressing cells revealed using TIRF microscopy.

    PubMed

    Buckley, Charlotte; Dun, Alison R; Peter, Audrey; Bellamy, Christopher; Gross, Kenneth W; Duncan, Rory R; Mullins, John J

    2017-01-01

    Renin is the initiator and rate-limiting factor in the renin-angiotensin blood pressure regulation system. Although renin is not exclusively produced in the kidney, in nonmurine species the synthesis and secretion of the active circulatory enzyme is confined almost exclusively to the dense core granules of juxtaglomerular (JG) cells, where prorenin is processed and stored for release via a regulated pathway. Despite its importance, the structural organization and regulation of granules within these cells is not well understood, in part due to the difficulty in culturing primary JG cells in vitro and the lack of appropriate cell lines. We have streamlined the isolation and culture of primary renin-expressing cells suitable for high-speed, high-resolution live imaging using a Percoll gradient-based procedure to purify cells from RenGFP(+) transgenic mice. Fibronectin-coated glass coverslips proved optimal for the adhesion of renin-expressing cells and facilitated live cell imaging at the plasma membrane of primary renin cells using total internal reflection fluorescence microscopy (TIRFM). To obtain quantitative data on intracellular function, we stained mixed granule and lysosome populations with Lysotracker Red and stimulated cells using 100 nM isoproterenol. Analysis of membrane-proximal acidic granular organelle dynamics and behavior within renin-expressing cells revealed the existence of two populations of granular organelles with distinct functional responses following isoproterenol stimulation. The application of high-resolution techniques for imaging JG and other specialized kidney cells provides new opportunities for investigating renal cell biology. Copyright © 2017 the American Physiological Society.

  1. Power-Law Modeling of Cancer Cell Fates Driven by Signaling Data to Reveal Drug Effects

    PubMed Central

    Zhang, Fan; Wu, Min; Kwoh, Chee Keong; Zheng, Jie

    2016-01-01

    Extracellular signals are captured and transmitted by signaling proteins inside a cell. An important type of cellular responses to the signals is the cell fate decision, e.g., apoptosis. However, the underlying mechanisms of cell fate regulation are still unclear, thus comprehensive and detailed kinetic models are not yet available. Alternatively, data-driven models are promising to bridge signaling data with the phenotypic measurements of cell fates. The traditional linear model for data-driven modeling of signaling pathways has its limitations because it assumes that the a cell fate is proportional to the activities of signaling proteins, which is unlikely in the complex biological systems. Therefore, we propose a power-law model to relate the activities of all the measured signaling proteins to the probabilities of cell fates. In our experiments, we compared our nonlinear power-law model with the linear model on three cancer datasets with phosphoproteomics and cell fate measurements, which demonstrated that the nonlinear model has superior performance on cell fates prediction. By in silico simulation of virtual protein knock-down, the proposed model is able to reveal drug effects which can complement traditional approaches such as binding affinity analysis. Moreover, our model is able to capture cell line specific information to distinguish one cell line from another in cell fate prediction. Our results show that the power-law data-driven model is able to perform better in cell fate prediction and provide more insights into the signaling pathways for cancer cell fates than the linear model. PMID:27764199

  2. Single neuron transcriptome analysis can reveal more than cell type classification

    PubMed Central

    Harbom, Lise J.; Chronister, William D.

    2016-01-01

    A recent single cell mRNA sequencing study by Dueck et al. compares neuronal transcriptomes to the transcriptomes of adipocytes and cardiomyocytes. Single cell ‘omic approaches such as those used by the authors are at the leading edge of molecular and biophysical measurement. Many groups are currently employing single cell sequencing approaches to understand cellular heterogeneity in cancer and during normal development. These single cell approaches also are beginning to address long‐standing questions regarding nervous system diversity. Beyond an innate interest in cataloging cell type diversity in the brain, single cell neuronal diversity has important implications for neurotypic neural circuit function and for neurological disease. Herein, we review the authors’ methods and findings, which most notably include evidence of unique expression profiles in some single neurons. PMID:26749010

  3. Extracellular Matrix-dependent Pathways in Colorectal Cancer Cell Lines Reveal Potential Targets for Anticancer Therapies.

    PubMed

    Stankevicius, Vaidotas; Vasauskas, Gintautas; Noreikiene, Rimante; Kuodyte, Karolina; Valius, Mindaugas; Suziedelis, Kestutis

    2016-09-01

    Cancer cells grown in a 3D culture are more resistant to anticancer therapy treatment compared to those in a monolayer 2D culture. Emerging evidence has suggested that the key reasons for increased cell survival could be gene expression changes in cell-extracellular matrix (ECM) interaction-dependent manner. Global gene-expression changes were obtained in human colorectal carcinoma HT29 and DLD1 cell lines between 2D and laminin-rich (lr) ECM 3D growth conditions by gene-expression microarray analysis. The most significantly altered functional categories were revealed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The microarray data revealed that 841 and 1190 genes were differentially expressed in colorectal carcinoma DLD1 and HT29 cells. KEGG analysis indicated that the most significantly altered categories were cell adhesion, mitogen-activated protein kinase and immune response. Our results indicate altered pathways related to cancer development and progression and suggest potential ECM-regulated targets for the development of anticancer therapies. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  4. An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

    PubMed

    Benitez, Cecil M; Qu, Kun; Sugiyama, Takuya; Pauerstein, Philip T; Liu, Yinghua; Tsai, Jennifer; Gu, Xueying; Ghodasara, Amar; Arda, H Efsun; Zhang, Jiajing; Dekker, Joseph D; Tucker, Haley O; Chang, Howard Y; Kim, Seung K

    2014-10-01

    The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

  5. An Integrated Cell Purification and Genomics Strategy Reveals Multiple Regulators of Pancreas Development

    PubMed Central

    Benitez, Cecil M.; Qu, Kun; Sugiyama, Takuya; Pauerstein, Philip T.; Liu, Yinghua; Tsai, Jennifer; Gu, Xueying; Ghodasara, Amar; Arda, H. Efsun; Zhang, Jiajing; Dekker, Joseph D.; Tucker, Haley O.; Chang, Howard Y.; Kim, Seung K.

    2014-01-01

    The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus. PMID:25330008

  6. A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination.

    PubMed

    Taylor, Marcus J; Husain, Kabir; Gartner, Zev J; Mayor, Satyajit; Vale, Ronald D

    2017-03-23

    A T cell mounts an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Hematopoietic Signaling Mechanism Revealed From a Stem/Progenitor Cell Cistrome

    PubMed Central

    Hewitt, Kyle J.; Kim, Duk Hyoung; Devadas, Prithvia; Sanalkumar, Prathibha; Zuo, Chandler; Sanalkumar, Rajendran; Johnson, Kirby D.; Kang, Yoon-A; Kim, Jin-Soo; Dewey, Colin N.; Keles, Sunduz; Bresnick, Emery H.

    2015-01-01

    SUMMARY Thousands of cis-elements in genomes are predicted to have vital functions. While conservation, activity in surrogate assays, polymorphisms, and disease mutations provide functional clues, deletion from endogenous loci constitutes the gold-standard test. A GATA-2-binding, Gata2 intronic cis-element (+9.5) required for hematopoietic stem cell genesis in mice is mutated in a human immunodeficiency syndrome. As +9.5 is the only cis-element known to mediate stem cell genesis, we devised a strategy to identify functionally comparable enhancers (“+9.5-like”) genome-wide. Gene editing revealed +9.5-like activity to mediate GATA-2 occupancy, chromatin opening, and transcriptional activation. A +9.5-like element resided in Samd14, which encodes a protein of unknown function. Samd14 increased hematopoietic progenitor levels/activity, promoted signaling by a pathway vital for hematopoietic stem/progenitor cell regulation (Stem Cell Factor/c-Kit), and c-Kit rescued Samd14 loss-of-function phenotypes. Thus, the hematopoietic stem/progenitor cell cistrome revealed a mediator of a signaling pathway that has broad importance for stem/progenitor cell biology. PMID:26073540

  8. Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells

    PubMed Central

    Grover, Amit; Sanjuan-Pla, Alejandra; Thongjuea, Supat; Carrelha, Joana; Giustacchini, Alice; Gambardella, Adriana; Macaulay, Iain; Mancini, Elena; Luis, Tiago C.; Mead, Adam; Jacobsen, Sten Eirik W.; Nerlov, Claus

    2016-01-01

    Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis. PMID:27009448

  9. Lassa Virus Cell Entry Reveals New Aspects of Virus-Host Cell Interaction.

    PubMed

    Torriani, Giulia; Galan-Navarro, Clara; Kunz, Stefan

    2017-02-15

    Viral entry represents the first step of every viral infection and is a determinant for the host range and disease potential of a virus. Here, we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa virus, providing novel insights into the complex virus-host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.

  10. Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA CDR1as in Hepatocellular Carcinoma Cells.

    PubMed

    Yang, Xue; Xiong, Qian; Wu, Ying; Li, Siting; Ge, Feng

    2017-09-20

    Circular RNAs (circRNAs), a class of widespread endogenous RNAs, play crucial roles in diverse biological processes and are potential biomarkers in diverse human diseases and cancers. Cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), an oncogenic circRNA, is involved in human tumorigenesis and is dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CDR1as functions in HCC remain unclear. Here we explored the functions of CDR1as and searched for CDR1as-regulated proteins in HCC cells. A quantitative proteomics strategy was employed to globally identify CDR1as-regulated proteins in HCC cells. In total, we identified 330 differentially expressed proteins (DEPs) upon enhanced CDR1as expression in HepG2 cells, indicating that they could be proteins regulated by CDR1as. Bioinformatic analysis revealed that many DEPs were involved in cell proliferation and the cell cycle. Further functional studies of epidermal growth factor receptor (EGFR) found that CDR1as exerts its effects on cell proliferation at least in part through the regulation of EGFR expression. We further confirmed that CDR1as could inhibit the expression of microRNA-7 (miR-7). EGFR is a validated target of miR-7; therefore, CDR1as may exert its function by regulating EGFR expression via targeting miR-7 in HCC cells. Taken together, we revealed novel functions and underlying mechanisms of CDR1as in HCC cells. This study serves as the first proteome-wide analysis of a circRNA-regulated protein in cells and provides a reliable and highly efficient method for globally identifying circRNA-regulated proteins.

  11. Integrated Metabolomics and Transcriptomics Reveal Enhanced Specialized Metabolism in Medicago truncatula Root Border Cells1[OPEN

    PubMed Central

    Watson, Bonnie S.; Bedair, Mohamed F.; Urbanczyk-Wochniak, Ewa; Huhman, David V.; Yang, Dong Sik; Allen, Stacy N.; Li, Wensheng; Tang, Yuhong; Sumner, Lloyd W.

    2015-01-01

    Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased β-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4′-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4′-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously. PMID:25667316

  12. The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

    PubMed Central

    Braet, Filip; Spector, Ilan; Shochet, Nava; Crews, Phillip; Higa, Tatsuo; Menu, Eline; de Zanger, Ronald; Wisse, Eddie

    2002-01-01

    Background Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied. Results Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers. Conclusion (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs. PMID:11914125

  13. Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

    SciTech Connect

    Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

    2009-02-01

    The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.

  14. Principles for the morphological characterization of transmitter-identified nerve cell groups.

    PubMed

    Agnati, L F; Fuxe, K; Zini, I; Benfenati, F; Hökfelt, T; de Mey, J

    1982-07-01

    Principles for a morphometric description of transmitter identified nerve cell groups have been introduced, exemplified on the 5-HT nerve cell group of nucleus raphe dorsalis (B7 cell group), using immunocytochemical procedures to visualize 5-HT. Both cell body parameters (mean diameter, mean perimeter, mean area, shape factor) and cell group parameters (number of cells, mean free distance among cells, volume fraction, gravity centre, major axis slope with respect to the midline) have been considered. These parameters have been obtained by the use of a semiautomatic image analyzer (Kontron MOP AMO2) plugged into an Apple II computer. By the use of this system and of suitable computer programs, it is possible to give a Cartesian representation of 5-HT nerve cell bodies in a coronal section under study. The present work has mainly one aim. To detect whether subgroups exist within a transmitter-identified cell group. Two approaches have been introduced to obtain, on objective grounds, evidence whether or not a group consists of subgroups. The first of these approaches is based on differences in cell body density within the cell group, while the second approach is based on frequency distribution analysis. This second approach is mainly sensitive to shape changes of the cell group.

  15. Phenothiazine-sensitized organic solar cells: effect of dye anchor group positioning on the cell performance.

    PubMed

    Hart, Aaron S; K C, Chandra Bikram; Subbaiyan, Navaneetha K; Karr, Paul A; D'Souza, Francis

    2012-11-01

    Effect of positioning of the cyanoacrylic acid anchoring group on ring periphery of phenothiazine dye on the performance of dye-sensitized solar cells (DSSCs) is reported. Two types of dyes, one having substitution on the C-3 aromatic ring (Type 1) and another through the N-terminal (Type 2), have been synthesized for this purpose. Absorption and fluorescence studies have been performed to visualize the effect of substitution pattern on the spectral coverage and electrochemical studies to monitor the tuning of redox levels. B3LYP/6-31G* studies are performed to visualize the frontier orbital location and their significance in charge injection when surface modified on semiconducting TiO₂. New DSSCs have been built on nanocrystalline TiO₂ according to traditional two-electrode Grätzel solar cell setup with a reference cell based on N719 dye for comparison. The lifetime of the adsorbed phenothiazine dye is found to be quenched significantly upon immobilizing on TiO₂ suggesting charge injection from excited dye to semiconducting TiO₂. The performances of the cells are found to be prominent for solar cells made out of Type 1 dyes compared to Type 2 dyes. This trend has been rationalized on the basis of spectral, electrochemical, computational, and electrochemical impedance spectroscopy results.

  16. In vivo fluorescence imaging reveals the promotion of mammary tumorigenesis by mesenchymal stromal cells.

    PubMed

    Ke, Chien-Chih; Liu, Ren-Shyan; Suetsugu, Atsushi; Kimura, Hiroaki; Ho, Jennifer H; Lee, Oscar K; Hoffman, Robert M

    2013-01-01

    Mesenchymal stromal cells (MSCs) are multipotent adult stem cells which are recruited to the tumor microenvironment (TME) and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.

  17. Children's Oncology Group's 2013 blueprint for research: stem cell transplantation.

    PubMed

    Grupp, Stephan A; Dvorak, Christopher C; Nieder, Michael L; Levine, John E; Wall, Donna A; Langholz, Bryan; Pulsipher, Michael A

    2013-06-01

    The role of SCT in pediatric oncology has continued to evolve with the introduction of new therapeutic agents and immunological insights into cancer. COG has focused its efforts on the study of hematopoietic stem cell transplantation in the treatment of pediatric malignancies in several major multi-institutional Phase II and Phase III studies. These studies include addressing the impact of allogenicity in ALL (ASCT0431), and establishing autologous stem cell transplant as the standard of care in neuroblastoma. Reducing transplant-associated toxicity was addressed in the ASCT0521 study, where the TNFα inhibitor etanercept was tested for the treatment of idiopathic pneumonia syndrome. Impact of cell dose was explored in the single versus tandem umbilical cord blood study CTN-0501, in close collaboration with the BMT-CTN.

  18. Physical Forces Shape Group Identity of Swimming Pseudomonas putida Cells

    PubMed Central

    Espeso, David R.; Martínez-García, Esteban; de Lorenzo, Víctor; Goñi-Moreno, Ángel

    2016-01-01

    The often striking macroscopic patterns developed by motile bacterial populations on agar plates are a consequence of the environmental conditions where the cells grow and spread. Parameters such as medium stiffness and nutrient concentration have been reported to alter cell swimming behavior, while mutual interactions among populations shape collective patterns. One commonly observed occurrence is the mutual inhibition of clonal bacteria when moving toward each other, which results in a distinct halt at a finite distance on the agar matrix before having direct contact. The dynamics behind this phenomenon (i.e., intolerance to mix in time and space with otherwise identical others) has been traditionally explained in terms of cell-to-cell competition/cooperation regarding nutrient availability. In this work, the same scenario has been revisited from an alternative perspective: the effect of the physical mechanics that frame the process, in particular the consequences of collisions between moving bacteria and the semi-solid matrix of the swimming medium. To this end, we set up a simple experimental system in which the swimming patterns of Pseudomonas putida were tested with different geometries and agar concentrations. A computational analysis framework that highlights cell-to-medium interactions was developed to fit experimental observations. Simulated outputs suggested that the medium is compressed in the direction of the bacterial front motion. This phenomenon generates what was termed a compression wave that goes through the medium preceding the swimming population and that determines the visible high-level pattern. Taken together, the data suggested that the mechanical effects of the bacteria moving through the medium created a factual barrier that impedes to merge with neighboring cells swimming from a different site. The resulting divide between otherwise clonal bacteria is thus brought about by physical forces—not genetic or metabolic programs. PMID

  19. Transcriptomic comparison between Brassica oleracea and rice (Oryza sativa) reveals diverse modulations on cell death in response to Sclerotinia sclerotiorum.

    PubMed

    Mei, Jiaqin; Ding, Yijuan; Li, Yuehua; Tong, Chaobo; Du, Hai; Yu, Yang; Wan, Huafan; Xiong, Qing; Yu, Jingyin; Liu, Shengyi; Li, Jiana; Qian, Wei

    2016-09-20

    Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops, but not in rice. The leaves of a rice line, a partial resistant (R) and a susceptible (S) Brassica oleracea pool that bulked from a resistance-segregating F2 population were employed for transcriptome sequencing before and after inoculation by S. sclerotiorum for 6 and 12 h. Distinct transcriptome profiles were revealed between B. oleracea and rice in response to S. sclerotiorum. Enrichment analyses of GO and KEGG indicated an enhancement of antioxidant activity in the R B. oleracea and rice, and histochemical staining exhibited obvious lighter reactive oxygen species (ROS) accumulation and cell death in rice and the R B. oleracea as compared to that in the S B. oleracea. Significant enhancement of Ca(2+) signalling, a positive regulator of ROS and cell death, were detected in S B. oleracea after inoculation, while it was significantly repressed in the R B. oleracea group. Obvious difference was detected between two B. oleracea groups for WRKY transcription factors, particularly for those regulating cell death. These findings suggest diverse modulations on cell death in host in response to S. sclerotiorum. Our study provides useful insight into the resistant mechanism to S. sclerotiorum.

  20. Transcriptomic comparison between Brassica oleracea and rice (Oryza sativa) reveals diverse modulations on cell death in response to Sclerotinia sclerotiorum

    PubMed Central

    Mei, Jiaqin; Ding, Yijuan; Li, Yuehua; Tong, Chaobo; Du, Hai; Yu, Yang; Wan, Huafan; Xiong, Qing; Yu, Jingyin; Liu, Shengyi; Li, Jiana; Qian, Wei

    2016-01-01

    Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops, but not in rice. The leaves of a rice line, a partial resistant (R) and a susceptible (S) Brassica oleracea pool that bulked from a resistance-segregating F2 population were employed for transcriptome sequencing before and after inoculation by S. sclerotiorum for 6 and 12 h. Distinct transcriptome profiles were revealed between B. oleracea and rice in response to S. sclerotiorum. Enrichment analyses of GO and KEGG indicated an enhancement of antioxidant activity in the R B. oleracea and rice, and histochemical staining exhibited obvious lighter reactive oxygen species (ROS) accumulation and cell death in rice and the R B. oleracea as compared to that in the S B. oleracea. Significant enhancement of Ca2+ signalling, a positive regulator of ROS and cell death, were detected in S B. oleracea after inoculation, while it was significantly repressed in the R B. oleracea group. Obvious difference was detected between two B. oleracea groups for WRKY transcription factors, particularly for those regulating cell death. These findings suggest diverse modulations on cell death in host in response to S. sclerotiorum. Our study provides useful insight into the resistant mechanism to S. sclerotiorum. PMID:27647523

  1. Necrotic enlargement of cone photoreceptor cells and the release of high-mobility group box-1 in retinitis pigmentosa

    PubMed Central

    Murakami, Y; Ikeda, Y; Nakatake, S; Tachibana, T; Fujiwara, K; Yoshida, N; Notomi, S; Nakao, S; Hisatomi, T; Miller, J W; Vavvas, DG; Sonoda, KH; Ishibashi, T

    2015-01-01

    Retinitis pigmentosa (RP) refers to a group of inherited retinal degenerations resulting form rod and cone photoreceptor cell death. The rod cell death due to deleterious genetic mutations has been shown to occur mainly through apoptosis, whereas the mechanisms and features of the secondary cone cell death have not been fully elucidated. Our previous study showed that the cone cell death in rd10 mice, an animal model of RP, involves necrotic features and is partly mediated by the receptor interacting protein kinase. However, the relevancy of necrotic cone cell death in human RP patients remains unknown. In the present study, we showed that dying cone cells in rd10 mice exhibited cellular enlargement, along with necrotic changes such as cellular swelling and mitochondrial rupture. In human eyes, live imaging of cone cells by adaptive optics scanning laser ophthalmoscopy revealed significantly increased percentages of enlarged cone cells in the RP patients compared with the control subjects. The vitreous of the RP patients contained significantly higher levels of high-mobility group box-1, which is released extracellularly associated with necrotic cell death. These findings suggest that necrotic enlargement of cone cells is involved in the process of cone degeneration, and that necrosis may be a novel target to prevent or delay the loss of cone-mediated central vision in RP. PMID:27551484

  2. Identification of essential Alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems

    PubMed Central

    Curtis, Patrick D.; Brun, Yves V.

    2014-01-01

    Summary The cell cycle of Caulobacter crescentus is controlled by a complex signaling network that coordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism’s different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non-essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism’s essential gene pool is specific to that organism. PMID:24975755

  3. Functional plant cell wall design revealed by the Raman imaging approach.

    PubMed

    Richter, Stephan; Müssig, Jörg; Gierlinger, Notburga

    2011-04-01

    Using the Raman imaging approach, the optimization of the plant cell wall design was investigated on the micron level within different tissue types at different positions of a Phormium tenax leaf. Pectin and lignin distribution were visualized and the cellulose microfibril angle (MFA) of the cell walls was determined. A detailed analysis of the Raman spectra extracted from the selected regions, allowed a semi-quantitative comparison of the chemical composition of the investigated tissue types on the micron level. The cell corners of the parenchyma revealed almost pure pectin and the cell wall an amount of 38-49% thereof. Slight lignification was observed in the parenchyma and collenchyma in the top of the leaf and a high variability (7-44%) in the sclerenchyma. In the cell corners and in the cell wall of the sclerenchymatic fibres surrounding the vascular tissue, the highest lignification was observed, which can act as a barrier and protection of the vascular tissue. In the sclerenchyma high variable MFA (4°-40°) was detected, which was related with lignin variability. In the primary cell walls a constant high MFA (57°-58°) was found together with pectin. The different plant cell wall designs on the tissue and microlevel involve changes in chemical composition as well as cellulose microfibril alignment and are discussed and related according to the development and function.

  4. Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

    NASA Astrophysics Data System (ADS)

    Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

    2015-03-01

    Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction.

  5. New wrinkling substrate assay reveals traction force fields of leader and follower cells undergoing collective migration.

    PubMed

    Yokoyama, Sho; Matsui, Tsubasa S; Deguchi, Shinji

    2017-01-22

    Physical forces play crucial roles in coordinating collective migration of epithelial cells, but details of such force-related phenomena remain unclear partly due to the lack of robust methodologies to probe the underlying force fields. Here we develop a method for fabricating silicone substrates that detect cellular traction forces with a high sensitivity. Specifically, a silicone elastomer is exposed to oxygen plasma under heating. Removal of the heat shrinks the substrate so as to reduce its critical buckling strain in a spatially uniform manner. Thus, even small cellular traction forces can be visualized as micro-wrinkles that are reversibly emerged on the substrate in a direction orthogonal to the applied forces. Using this technique, we show that so-called leader cells in MDCK-II cell clusters exert significant magnitudes of traction forces distinct from those of follower cells. We reveal that the direction of traction forces is highly correlated with the long axis of the local, individual cells within clusters. These results suggest that the force fields in collective migration of MDCK-II cells are predominantly determined locally at individual cell scale rather than globally at the whole cell cluster scale. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Cell lineage tracing in the developing enteric nervous system: superstars revealed by experiment and simulation

    PubMed Central

    Cheeseman, Bevan L.; Zhang, Dongcheng; Binder, Benjamin J.; Newgreen, Donald F.; Landman, Kerry A.

    2014-01-01

    Cell lineage tracing is a powerful tool for understanding how proliferation and differentiation of individual cells contribute to population behaviour. In the developing enteric nervous system (ENS), enteric neural crest (ENC) cells move and undergo massive population expansion by cell division within self-growing mesenchymal tissue. We show that single ENC cells labelled to follow clonality in the intestine reveal extraordinary and unpredictable variation in number and position of descendant cells, even though ENS development is highly predictable at the population level. We use an agent-based model to simulate ENC colonization and obtain agent lineage tracing data, which we analyse using econometric data analysis tools. In all realizations, a small proportion of identical initial agents accounts for a substantial proportion of the total final agent population. We term these individuals superstars. Their existence is consistent across individual realizations and is robust to changes in model parameters. This inequality of outcome is amplified at elevated proliferation rate. The experiments and model suggest that stochastic competition for resources is an important concept when understanding biological processes which feature high levels of cell proliferation. The results have implications for cell-fate processes in the ENS. PMID:24501272

  7. Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human β Cell Function.

    PubMed

    Arda, H Efsun; Li, Lingyu; Tsai, Jennifer; Torre, Eduardo A; Rosli, Yenny; Peiris, Heshan; Spitale, Robert C; Dai, Chunhua; Gu, Xueying; Qu, Kun; Wang, Pei; Wang, Jing; Grompe, Markus; Scharfmann, Raphael; Snyder, Michael S; Bottino, Rita; Powers, Alvin C; Chang, Howard Y; Kim, Seung K

    2016-05-10

    Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. After birth, islet cell growth and function are dynamically regulated; however, establishing these age-dependent changes in humans has been challenging. Here, we describe a multimodal strategy for isolating pancreatic endocrine and exocrine cells from children and adults to identify age-dependent gene expression and chromatin changes on a genomic scale. These profiles revealed distinct proliferative and functional states of islet α cells or β cells and histone modifications underlying age-dependent gene expression changes. Expression of SIX2 and SIX3, transcription factors without prior known functions in the pancreas and linked to fasting hyperglycemia risk, increased with age specifically in human islet β cells. SIX2 and SIX3 were sufficient to enhance insulin content or secretion in immature β cells. Our work provides a unique resource to study human-specific regulators of islet cell maturation and function. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Cell lineage tracing in the developing enteric nervous system: superstars revealed by experiment and simulation.

    PubMed

    Cheeseman, Bevan L; Zhang, Dongcheng; Binder, Benjamin J; Newgreen, Donald F; Landman, Kerry A

    2014-04-06

    Cell lineage tracing is a powerful tool for understanding how proliferation and differentiation of individual cells contribute to population behaviour. In the developing enteric nervous system (ENS), enteric neural crest (ENC) cells move and undergo massive population expansion by cell division within self-growing mesenchymal tissue. We show that single ENC cells labelled to follow clonality in the intestine reveal extraordinary and unpredictable variation in number and position of descendant cells, even though ENS development is highly predictable at the population level. We use an agent-based model to simulate ENC colonization and obtain agent lineage tracing data, which we analyse using econometric data analysis tools. In all realizations, a small proportion of identical initial agents accounts for a substantial proportion of the total final agent population. We term these individuals superstars. Their existence is consistent across individual realizations and is robust to changes in model parameters. This inequality of outcome is amplified at elevated proliferation rate. The experiments and model suggest that stochastic competition for resources is an important concept when understanding biological processes which feature high levels of cell proliferation. The results have implications for cell-fate processes in the ENS.

  9. Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids.

    PubMed

    Schmitz, Alexander; Fischer, Sabine C; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H K

    2017-03-03

    Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid's size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 10(5) to 1 × 10(6 )cells/mm(3). Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture.

  10. Ribosome Profiling Reveals a Cell-Type-Specific Translational Landscape in Brain Tumors

    PubMed Central

    Gonzalez, Christian; Sims, Jennifer S.; Hornstein, Nicholas; Mela, Angeliki; Garcia, Franklin; Lei, Liang; Gass, David A.; Amendolara, Benjamin; Bruce, Jeffrey N.

    2014-01-01

    Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5′-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation. PMID:25122893

  11. Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids

    PubMed Central

    Schmitz, Alexander; Fischer, Sabine C.; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H. K.

    2017-01-01

    Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid’s size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 105 to 1 × 106 cells/mm3. Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture. PMID:28255161

  12. Genome-Wide Profiling of Pluripotent Cells Reveals a Unique Molecular Signature of Human Embryonic Germ Cells

    PubMed Central

    Pashai, Nikta; Hao, Haiping; All, Angelo; Gupta, Siddharth; Chaerkady, Raghothama; De Los Angeles, Alejandro; Gearhart, John D.; Kerr, Candace L.

    2012-01-01

    Human embryonic germ cells (EGCs) provide a powerful model for identifying molecules involved in the pluripotent state when compared to their progenitors, primordial germ cells (PGCs), and other pluripotent stem cells. Microarray and Principal Component Analysis (PCA) reveals for the first time that human EGCs possess a transcription profile distinct from PGCs and other pluripotent stem cells. Validation with qRT-PCR confirms that human EGCs and PGCs express many pluripotency-associated genes but with quantifiable differences compared to pluripotent embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and embryonal carcinoma cells (ECCs). Analyses also identified a number of target genes that may be potentially associated with their unique pluripotent states. These include IPO7, MED7, RBM26, HSPD1, and KRAS which were upregulated in EGCs along with other pluripotent stem cells when compared to PGCs. Other potential target genes were also found which may contribute toward a primed ESC-like state. These genes were exclusively up-regulated in ESCs, IPSCs and ECCs including PARP1, CCNE1, CDK6, AURKA, MAD2L1, CCNG1, and CCNB1 which are involved in cell cycle regulation, cellular metabolism and DNA repair and replication. Gene classification analysis also confirmed that the distinguishing feature of EGCs compared to ESCs, ECCs, and IPSCs lies primarily in their genetic contribution to cellular metabolism, cell cycle, and cell adhesion. In contrast, several genes were found upregulated in PGCs which may help distinguish their unipotent state including HBA1, DMRT1, SPANXA1, and EHD2. Together, these findings provide the first glimpse into a unique genomic signature of human germ cells and pluripotent stem cells and provide genes potentially involved in defining different states of germ-line pluripotency. PMID:22737227

  13. An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability

    PubMed Central

    Schütte, Judith; Wang, Huange; Antoniou, Stella; Jarratt, Andrew; Wilson, Nicola K; Riepsaame, Joey; Calero-Nieto, Fernando J; Moignard, Victoria; Basilico, Silvia; Kinston, Sarah J; Hannah, Rebecca L; Chan, Mun Chiang; Nürnberg, Sylvia T; Ouwehand, Willem H; Bonzanni, Nicola; de Bruijn, Marella FTR; Göttgens, Berthold

    2016-01-01

    Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes. DOI: http://dx.doi.org/10.7554/eLife.11469.001 PMID:26901438

  14. Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

    PubMed

    Schmidt, Jens C; Zaug, Arthur J; Cech, Thomas R

    2016-08-25

    Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Dichotomy of cellular inhibition by small-molecule inhibitors revealed by single-cell analysis

    NASA Astrophysics Data System (ADS)

    Vogel, Robert M.; Erez, Amir; Altan-Bonnet, Grégoire

    2016-09-01

    Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings.

  16. Dichotomy of cellular inhibition by small-molecule inhibitors revealed by single-cell analysis

    PubMed Central

    Vogel, Robert M.; Erez, Amir; Altan-Bonnet, Grégoire

    2016-01-01

    Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings. PMID:27687249

  17. Single-cell analyses to reveal hematopoietic stem cell fate decisions.

    PubMed

    Lunger, Ilaria; Fawaz, Malak; Rieger, Michael A

    2017-08-01

    Hematopoietic stem cells (HSCs) are the best studied adult stem cells with enormous clinical value. Most of our knowledge about their biology relies on assays at the single HSC level. However, only the recent advances in developing new single cell technologies allowed the elucidation of the complex regulation of HSC fate decision control. This Review will focus on current attempts to investigate individual HSCs at molecular and functional levels. The advantages of these technologies leading to groundbreaking insights into hematopoiesis will be highlighted, and the challenges facing these technologies will be discussed. The importance of combining molecular and functional assays to enlighten regulatory networks of HSC fate decision control, ideally at high temporal resolution, becomes apparent for future studies. © 2017 Federation of European Biochemical Societies.

  18. Chemokine receptor co-expression reveals aberrantly distributed TH effector memory cells in GPA patients.

    PubMed

    Lintermans, Lucas L; Rutgers, Abraham; Stegeman, Coen A; Heeringa, Peter; Abdulahad, Wayel H

    2017-06-14

    Persistent expansion of circulating CD4(+) effector memory T cells (TEM) in patients with granulomatosis with polyangiitis (GPA) suggests their fundamental role in disease pathogenesis. Recent studies have shown that distinct functional CD4(+) TEM cell subsets can be identified based on expression patterns of chemokine receptors. The current study aimed to determine different CD4(+) TEM cell subsets based on chemokine receptor expression in peripheral blood of GPA patients. Identification of particular circulating CD4(+) TEM cells subsets may reveal distinct contributions of specific CD4(+) TEM subsets to the disease pathogenesis in GPA. Peripheral blood of 63 GPA patients in remission and 42 age- and sex-matched healthy controls was stained immediately after blood withdrawal with fluorochrome-conjugated antibodies for cell surface markers (CD3, CD4, CD45RO) and chemokine receptors (CCR4, CCR6, CCR7, CRTh2, CXCR3) followed by flow cytometry analysis. CD4(+) TEM memory cells (CD3(+)CD4(+)CD45RO(+)CCR7(-)) were gated, and the expression patterns of chemokine receptors CXCR3(+)CCR4(-)CCR6(-)CRTh2(-), CXCR3(-)CCR4(+)CCR6(-)CRTh2(+), CXCR3(-)CCR4(+)CCR6(+)CRTh2(-), and CXCR3(+)CCR4(-)CCR6(+)CRTh2(-) were used to distinguish TEM1, TEM2, TEM17, and TEM17.1 cells, respectively. The percentage of CD4(+) TEM cells was significantly increased in GPA patients in remission compared to HCs. Chemokine receptor co-expression analysis within the CD4(+) TEM cell population demonstrated a significant increase in the proportion of TEM17 cells with a concomitant significant decrease in the TEM1 cells in GPA patients compared to HC. The percentage of TEM17 cells correlated negatively with TEM1 cells in GPA patients. Moreover, the circulating proportion of TEM17 cells showed a positive correlation with the number of organs involved and an association with the tendency to relapse in GPA patients. Interestingly, the aberrant distribution of TEM1 and TEM17 cells is modulated in CMV

  19. Electron and carbon balances in microbial fuel cells reveal temporary bacterial storage behavior during electricity generation.

    PubMed

    Freguia, Stefano; Rabaey, Korneel; Yuan, Zhiguo; Keller, Jürg

    2007-04-15

    Microbial fuel cells (MFCs) are emerging as a novel technology with a great potential to reduce the costs of wastewater treatment. Their most studied application is organic carbon removal. One of the parameters commonly used to quantify the performance of these cells is the Coulombic efficiency, i.e., the electron recovery as electricity from the removed substrate. However, the "inefficiencies" of the process have never been fully identified. This study presents a method that uses the combination of electrochemical monitoring, chemical analysis, and a titration and off-gas analysis (TOGA) sensor to identify and quantify the sources of electron loss. The method was used successfully to close electron, carbon, and proton balances in acetate and glucose fed microbial fuel cells. The method revealed that in the case that a substrate is loaded as pulses carbon is stored inside the cells during initial high substrate conditions and consumed during starvation, with up to 57% of the current being generated after depletion of the external carbon source. Nile blue staining of biomass samples revealed lipophilic inclusions during high substrate conditions, thus confirming the storage of polymeric material in the bacterial cells. The method also allows for indirect measurement of growth yields, which ranged from 0 to 0.54 g biomass-C formed per g substrate-C used, depending on the type of substrate and the external resistance of the circuit.

  20. Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

    PubMed Central

    Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

    2016-01-01

    Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

  1. Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism

    PubMed Central

    Özdemir, Emre; McKinney, John D.

    2015-01-01

    ABSTRACT ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. PMID:25691591

  2. Charge transport in CdTe solar cells revealed by conductive tomographic atomic force microscopy

    DOE PAGES

    Luria, Justin; Kutes, Yasemin; Moore, Andrew; ...

    2016-09-26

    Polycrystalline photovoltaics comprising cadmium telluride (CdTe) represent a growing portion of the solar cell market, yet the physical picture of charge transport through the meso-scale grain morphology remains a topic of debate. It is unknown how thin film morphology affects the transport of electron-hole pairs. Accordingly this study is the first to generate three dimensional images of photocurrent throughout a thin-film solar cell, revealing the profound influence of grain boundaries and stacking faults on device efficiency.

  3. Intravital imaging reveals new ancillary mechanisms co-opted by cancer cells to drive tumor progression

    PubMed Central

    Lucas, Morghan C.; Timpson, Paul

    2016-01-01

    Intravital imaging is providing new insights into the dynamics of tumor progression in native tissues and has started to reveal the layers of complexity found in cancer. Recent advances in intravital imaging have allowed us to look deeper into cancer behavior and to dissect the interactions between tumor cells and the ancillary host niche that promote cancer development. In this review, we provide an insight into the latest advances in cancer biology achieved by intravital imaging, focusing on recently discovered mechanisms by which tumor cells manipulate normal tissue to facilitate disease progression. PMID:27239290

  4. Effect of different agents onto multidrug resistant cells revealed by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Boutin, C.; Roche, Y.; Jaffiol, R.; Millot, J.-M.; Millot, C.; Plain, J.; Deturche, R.; Jeannesson, P.; Manfait, M.; Royer, P.

    Fluorescence correlation spectroscopy (FCS), which is a sensitive and non invasive technique, has been used to characterize the plasma membrane fluidity and heterogeneity of multidrug resistant living cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been analyzed. Firstly, we reveal a modification of plasma membrane microviscosity according to the addition of a fluidity modulator, benzyl alcohol. In the other hand, revertant such as verapamil and cyclosporin-A appears to act more specifically on the slow diffusion sites as microdomains.

  5. Functional Heterogeneity of Embryonic Stem Cells Revealed through Translational Amplification of an Early Endodermal Transcript

    PubMed Central

    Canham, Maurice A.; Sharov, Alexei A.; Ko, Minoru S. H.; Brickman, Joshua M.

    2010-01-01

    ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically “undifferentiated” cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V+S+), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly

  6. Epithelial cells captured from ductal carcinoma in situ reveal a gene expression signature associated with progression to invasive breast cancer

    PubMed Central

    Abuázar, Carolina Sens; de Toledo Osorio, Cynthia Aparecida Bueno; Pinilla, Mabel Gigliola; da Silva, Sabrina Daniela; Camargo, Anamaria Aranha; Silva, Wilson Araujo; e Ferreira, Elisa Napolitano; Brentani, Helena Paula; Carraro, Dirce Maria

    2016-01-01

    Breast cancer biomarkers that can precisely predict the risk of progression of non-invasive ductal carcinoma in situ (DCIS) lesions to invasive disease are lacking. The identification of molecular alterations that occur during the invasion process is crucial for the discovery of drivers of transition to invasive disease and, consequently, biomarkers with clinical utility. In this study, we explored differences in gene expression in mammary epithelial cells before and after the morphological manifestation of invasion, i.e., early and late stages, respectively. In the early stage, epithelial cells were captured from both pre-invasive lesions with distinct malignant potential [pure DCIS as well as the in situ component that co-exists with invasive breast carcinoma lesions (DCIS-IBC)]; in the late stage, epithelial cells were captured from the two distinct morphological components of the same sample (in situ and invasive components). Candidate genes were identified using cDNA microarray and rapid subtractive hybridization (RaSH) cDNA libraries and validated by RT-qPCR assay using new samples from each group. These analyses revealed 26 genes, including 20 from the early and 6 from the late stage. The expression profile based on the 20 genes, marked by a preferential decrease in expression level towards invasive phenotype, discriminated the majority of DCIS samples. Thus, this study revealed a gene expression signature with the potential to predict DCIS progression and, consequently, provides opportunities to tailor treatments for DCIS patients. PMID:27708222

  7. Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins

    PubMed Central

    CRUZ*, ISA N.; COLEY*, HELEN M.; KRAMER, HOLGER B.; MADHURI, THUMULURU KAVITAH; SAFUWAN, NUR A.M.; ANGELINO, ANA RITA; YANG, MIN

    2016-01-01

    Background: Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear. Materials and Methods: A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes. Results: A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease. Conclusion: Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer. *These Authors contributed equally to this study. PMID:28031236

  8. Distinct outcomes of CRL–Nedd8 pathway inhibition reveal cancer cell plasticity

    PubMed Central

    Rulina, Anastasia V; Mittler, Frédérique; Obeid, Patricia; Gerbaud, Sophie; Guyon, Laurent; Sulpice, Eric; Kermarrec, Frédérique; Assard, Nicole; Dolega, Monika E; Gidrol, Xavier; Balakirev, Maxim Y

    2016-01-01

    Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/β-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR–ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR–ERG interaction. PMID:27906189

  9. Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

    PubMed

    Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-02-06

    Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy.

  10. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  11. Epigenetic landscapes reveal transcription factors that regulate CD8(+) T cell differentiation.

    PubMed

    Yu, Bingfei; Zhang, Kai; Milner, J Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P; Pereira, Renata M; Crotty, Shane; Chang, John T; Pipkin, Matthew E; Wang, Wei; Goldrath, Ananda W

    2017-03-13

    Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8(+) T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8(+) T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8(+) T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8(+) T cell differentiation.

  12. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging

    PubMed Central

    Yan, Jing; Sharo, Andrew G.; Stone, Howard A.; Wingreen, Ned S.; Bassler, Bonnie L.

    2016-01-01

    Biofilms are surface-associated bacterial communities that are crucial in nature and during infection. Despite extensive work to identify biofilm components and to discover how they are regulated, little is known about biofilm structure at the level of individual cells. Here, we use state-of-the-art microscopy techniques to enable live single-cell resolution imaging of a Vibrio cholerae biofilm as it develops from one single founder cell to a mature biofilm of 10,000 cells, and to discover the forces underpinning the architectural evolution. Mutagenesis, matrix labeling, and simulations demonstrate that surface adhesion-mediated compression causes V. cholerae biofilms to transition from a 2D branched morphology to a dense, ordered 3D cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture in V. cholerae biofilms, and this growth pattern is controlled by a single gene, rbmA. Competition analyses reveal that the dense growth mode has the advantage of providing the biofilm with superior mechanical properties. Our single-cell technology can broadly link genes to biofilm fine structure and provides a route to assessing cell-to-cell heterogeneity in response to external stimuli. PMID:27555592

  13. A single-cell imaging screen reveals multiple effects of secreted small molecules on bacteria

    PubMed Central

    Salje, Jeanne

    2014-01-01

    Bacteria cells exist in close proximity to other cells of both the same and different species. Bacteria secrete a large number of different chemical species, and the local concentrations of these compounds at the surfaces of nearby cells may reach very high levels. It is fascinating to imagine how individual cells might sense and respond to the complex mix of signals at their surface. However, it is difficult to measure exactly what the local environmental composition looks like, or what the effects of individual compounds on nearby cells are. Here, an electron microscopy imaging screen was designed that would detect morphological changes induced by secreted small molecules. This differs from conventional approaches by detecting structural changes in individual cells rather than gene expression or growth rate changes at the population level. For example, one of the changes detected here was an increase in outer membrane vesicle production, which does not necessarily correspond to a change in gene expression. This initial study focussed on Pseudomonas aeruginosa, Escherichia coli, and Burkholderia dolosa, and revealed an intriguing range of effects of secreted small molecules on cells both within and between species. PMID:24910069

  14. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  15. Microfluidic Platform for Studying Chemotaxis of Adhesive Cells Revealed a Gradient-Dependent Migration and Acceleration of Cancer Stem Cells.

    PubMed

    Zou, Heng; Yue, Wanqing; Yu, Wai-Kin; Liu, Dandan; Fong, Chi-Chun; Zhao, Jianlong; Yang, Mengsu

    2015-07-21

    Recent studies reveal that solid tumors consist of heterogeneous cells with distinct phenotypes and functions. However, it is unclear how different subtypes of cancer cells migrate under chemotaxis. Here, we developed a microfluidic device capable of generating multiple stable gradients, culturing cells on-chip, and monitoring single cell migratory behavior. The microfluidic platform was used to study gradient-induced chemotaxis of lung cancer stem cell (LCSC) and differentiated LCSC (dLCSC) in real time. Our results showed the dynamic and differential response of both LCSC and dLCSC to chemotaxis, which was regulated by the β-catenin dependent Wnt signaling pathway. The microfluidic analysis showed that LCSC and dLCSC from the same origin behaved differently in the same external stimuli, suggesting the importance of cancer cell heterogeneity. We also observed for the first time the acceleration of both LCSC and dLCSC during chemotaxis caused by increasing local concentration in different gradients, which could only be realized through the microfluidic approach. The capability to analyze single cell chemotaxis under spatially controlled conditions provides a novel analytical platform for the study of cellular microenvironments and cancer cell metastasis.

  16. Sensitivity Analysis of the NPM-ALK Signalling Network Reveals Important Pathways for Anaplastic Large Cell Lymphoma Combination Therapy

    PubMed Central

    Buetti-Dinh, Antoine; O’Hare, Thomas

    2016-01-01

    A large subset of anaplastic large cell lymphoma (ALCL) patients harbour a somatic aberration in which anaplastic lymphoma kinase (ALK) is fused to nucleophosmin (NPM) resulting in a constitutively active signalling fusion protein, NPM-ALK. We computationally simulated the signalling network which mediates pathological cell survival and proliferation through NPM-ALK to identify therapeutically targetable nodes through which it may be possible to regain control of the tumourigenic process. The simulations reveal the predominant role of the VAV1-CDC42 (cell division control protein 42) pathway in NPM-ALK-driven cellular proliferation and of the Ras / mitogen-activated ERK kinase (MEK) / extracellular signal-regulated kinase (ERK) cascade in controlling cell survival. Our results also highlight the importance of a group of interleukins together with the Janus kinase 3 (JAK3) / signal transducer and activator of transcription 3 (STAT3) signalling in the development of NPM-ALK derived ALCL. Depending on the activity of JAK3 and STAT3, the system may also be sensitive to activation of protein tyrosine phosphatase-1 (SHP1), which has an inhibitory effect on cell survival and proliferation. The identification of signalling pathways active in tumourigenic processes is of fundamental importance for effective therapies. The prediction of alternative pathways that circumvent classical therapeutic targets opens the way to preventive approaches for countering the emergence of cancer resistance. PMID:27669408

  17. Early transcriptional and epigenetic regulation of CD8(+) T cell differentiation revealed by single-cell RNA sequencing.

    PubMed

    Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E; He, Zhaoren; Aigner, Stefan; Metz, Patrick J; Yu, Bingfei; Wehrens, Ellen J; Lopez, Justine; Kim, Stephanie H; Zuniga, Elina I; Goldrath, Ananda W; Chang, John T; Yeo, Gene W

    2017-04-01

    During microbial infection, responding CD8(+) T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA-sequencing approach and analyzed individual CD8(+) T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants that controlled the fate specification of CD8(+) T lymphocytes. Our findings suggest a model for the differentiation of terminal effector cells initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power and necessity of single-cell approaches.

  18. Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA-seq

    PubMed Central

    Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E.; He, Zhaoren; Aigner, Stefan; Metz, Patrick J.; Yu, Bingfei; Wehrens, Ellen J.; Lopez, Justine; Kim, Stephanie H.; Zuniga, Elina I.; Goldrath, Ananda W.; Chang, John T.; Yeo, Gene W.

    2017-01-01

    SUMMARY During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants controlling CD8+ T lymphocyte fate specification. These findings suggest a model of terminal effector cell differentiation initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, highlighting the power and necessity of single-cell approaches. PMID:28218746

  19. Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent Metabolic Fingerprints in Perfusion Culture.

    PubMed

    Karst, Daniel J; Steinhoff, Robert F; Kopp, Marie R G; Serra, Elisa; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

    2016-12-20

    Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 10(6) cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 10(6) cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016.

  20. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    PubMed Central

    Cordeiro, Ingrid R.; Lopes, Daiana V.; Abreu, José G.; Carneiro, Katia; Rossi, Maria I. D.; Brito, José M.

    2015-01-01

    ABSTRACT Human adipose-derived stromal cells (hADSC) are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1) regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues. PMID:26319582

  1. Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

    PubMed Central

    Miyazaki, Kaoru; Maruyama, Tetsuo; Masuda, Hirotaka; Yamasaki, Akiko; Uchida, Sayaka; Oda, Hideyuki; Uchida, Hiroshi; Yoshimura, Yasunori

    2012-01-01

    Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue

  2. Reproductive isolation revealed in preliminary crossbreeding experiments using field collected Triatoma dimidiata (Hemiptera: Reduviidae) from three ITS–2 defined groups

    PubMed Central

    García, Mauricio; Menes, Marianela; Dorn, Patricia L.; Monroy, Carlota; Richards, Bethany; Panzera, Francisco; Bustamante, Dulce María

    2013-01-01

    Triatoma dimidiata, a Chagas disease vector distributed in Mexico, Central America, Colombia, Venezuela, Peru and Ecuador, has been studied using genetic markers and four groups have been defined by ITS–2 sequences: 1A, 1B, 2 and 3. To gather evidence on the divergence and reproductive isolation among T. dimidiata ITS–2 groups, we carried out 15 crossbreeding experiments with field–collected sylvan and domestic T. dimidiata from Guatemala where three groups are found: 1A, 2 and 3. Reciprocal crosses between individuals from groups 1A and 2, and a cross between group 2 individuals from different habitats, produced an average 129.78±42.29 eggs with hatching success ranging from 31.6% to 90.1%. The offspring of these crosses reached the adult stage, and crosses between F1 insects produced eggs. These results suggest that there are no pre– or post– zygotic reproductive barriers between groups 1A and 2, or within group 2. Crosses between group 3 females and males from groups 1A or 2 produced on average 85.67±30.26 eggs and none of them hatched. These results support the existence of pre– zygotic barriers between T. dimidiata group 3 and groups 1A and 2. The group 3 individuals were collected in sylvatic environments in Yaxha, Peten, Guatemala. Previously, distinct chromosomal characteristics (cytotype 3) were described in individuals from this population. Based on this evidence we suggest that this population is divergent at the species level from other T. dimidiata populations. PMID:24041592

  3. Reproductive isolation revealed in preliminary crossbreeding experiments using field collected Triatoma dimidiata (Hemiptera: Reduviidae) from three ITS-2 defined groups.

    PubMed

    García, Mauricio; Menes, Marianela; Dorn, Patricia L; Monroy, Carlota; Richards, Bethany; Panzera, Francisco; Bustamante, Dulce María

    2013-12-01

    Triatoma dimidiata, a Chagas disease vector distributed in Mexico, Central America, Colombia, Venezuela, Peru and Ecuador, has been studied using genetic markers and four groups have been defined by ITS-2 sequences: 1A, 1B, 2 and 3. To gather evidence on the divergence and reproductive isolation among T. dimidiata ITS-2 groups, we carried out 15 crossbreeding experiments with field-collected sylvan and domestic T. dimidiata from Guatemala where three groups are found: 1A, 2 and 3. Reciprocal crosses between individuals from groups 1A and 2, and a cross between group 2 individuals from different habitats, produced an average 129.78±42.29 eggs with hatching success ranging from 31.6 to 90.1%. The offspring of these crosses reached the adult stage, and crosses between F1 insects produced eggs. These results suggest that there are no pre- or post-zygotic reproductive barriers between groups 1A and 2, or within group 2. Crosses between group 3 females and males from groups 1A or 2 produced on average 85.67±30.26 eggs and none of them hatched. These results support the existence of pre-zygotic barriers between T. dimidiata group 3 and groups 1A and 2. The group 3 individuals were collected in sylvatic environments in Yaxha, Peten, Guatemala. Previously, distinct chromosomal characteristics (cytotype 3) were described in individuals from this population. Based on this evidence we suggest that this population is divergent at the species level from other T. dimidiata populations. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny

    PubMed Central

    Davis, Felicity M.; Lloyd-Lewis, Bethan; Harris, Olivia B.; Kozar, Sarah; Winton, Douglas J.; Muresan, Leila; Watson, Christine J.

    2016-01-01

    The mammary gland undergoes cycles of growth and regeneration throughout reproductive life, a process that requires mammary stem cells (MaSCs). Whilst recent genetic fate-mapping studies using lineage-specific promoters have provided valuable insights into the mammary epithelial hierarchy, the true differentiation potential of adult MaSCs remains unclear. To address this, herein we utilize a stochastic genetic-labelling strategy to indelibly mark a single cell and its progeny in situ, combined with tissue clearing and 3D imaging. Using this approach, clones arising from a single parent cell could be visualized in their entirety. We reveal that clonal progeny contribute exclusively to either luminal or basal lineages and are distributed sporadically to branching ducts or alveoli. Quantitative analyses suggest that pools of unipotent stem/progenitor cells contribute to adult mammary gland development. Our results highlight the utility of tracing a single cell and reveal that progeny of a single proliferative MaSC/progenitor are dispersed throughout the epithelium. PMID:27779190

  5. Mammary Stem Cell Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation

    PubMed Central

    Zhang, Zheng; Christin, John R.; Wang, Chunhui; Ge, Kai; Oktay, Maja H.; Guo, Wenjun

    2016-01-01

    SUMMARY Cancer genomics have provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC) organoid-based approach for rapid generation of somatic GEMMs (genetically engineered mouse models). By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K-targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study established a robust in vivo platform for functional cancer genomics and discovered functional breast cancer mutations. PMID:27653681

  6. African American Adolescents with Sickle Cell Disease: Support Groups and Psychological Well-Being.

    ERIC Educational Resources Information Center

    Gardner, Marilyn M.; Telfair, Joseph

    1999-01-01

    Studied the impact of support groups on the psychological well-being of adolescents with sickle cell disease (SCD). Response of 79 adolescent SCD group members show that psychological well-being was best predicted by fewer physical symptoms and greater satisfaction with the group. Findings suggest the beneficial effects of SCD support groups. (SLD)

  7. African American Adolescents with Sickle Cell Disease: Support Groups and Psychological Well-Being.

    ERIC Educational Resources Information Center

    Gardner, Marilyn M.; Telfair, Joseph

    1999-01-01

    Studied the impact of support groups on the psychological well-being of adolescents with sickle cell disease (SCD). Response of 79 adolescent SCD group members show that psychological well-being was best predicted by fewer physical symptoms and greater satisfaction with the group. Findings suggest the beneficial effects of SCD support groups. (SLD)

  8. Oxidant Signaling in Cells Revealed by Single Rare-Earth Based Nanoparticle Imaging

    NASA Astrophysics Data System (ADS)

    Bouzigues, Cedric; Abdesselem, Mouna; Ramodiharilafy, Rivo; Gacoin, Thierry; Tharaux, Pierre-Louis; Alexandrou, Antigoni

    The spatio-temporal organization of signaling pathways controls the cell response. Reactive oxygen species (ROS) are second messengers involved in the control of numerous normal and pathological processes and their local concentration is thus tightly regulated. However, the dynamics of ROS production and organization is mostly unknown, due to the lack of efficient probes. We developed single ROS sensitive Eu3+-doped nanoparticle imaging to quantitatively probed the intracellular ROS response. We revealed specific temporal patterns of ROS production under different types of stimulation (PDGF and ET-1) and quantitatively identified mechanisms of transactivation, which notably control the dynamics of the cell response. By using a microfluidic system, we apply spatially controlled stimulations and displayed the maintenance of asymmetric ROS concentration in the cell under a PDGF gradient. We then developed a ratiometric method using a nanoparticle mix, to quantitatively detect ROS with a 500 ms temporal resolution. We thus elucidate molecular mechanisms responsible for the control of the oxidant production kinetics. Altogether, our results reveal regulation mechanisms controlling ROS spatio-temporal organization, which can be crucial for the buildup of the cell response.

  9. Molecular analysis of aggressive renal cell carcinoma with unclassified histology reveals distinct subsets

    PubMed Central

    Chen, Ying-Bei; Xu, Jianing; Skanderup, Anders Jacobsen; Dong, Yiyu; Brannon, A. Rose; Wang, Lu; Won, Helen H.; Wang, Patricia I.; Nanjangud, Gouri J.; Jungbluth, Achim A.; Li, Wei; Ojeda, Virginia; Hakimi, A. Ari; Voss, Martin H.; Schultz, Nikolaus; Motzer, Robert J.; Russo, Paul; Cheng, Emily H.; Giancotti, Filippo G.; Lee, William; Berger, Michael F.; Tickoo, Satish K.; Reuter, Victor E.; Hsieh, James J.

    2016-01-01

    Renal cell carcinomas with unclassified histology (uRCC) constitute a significant portion of aggressive non-clear cell renal cell carcinomas that have no standard therapy. The oncogenic drivers in these tumours are unknown. Here we perform a molecular analysis of 62 high-grade primary uRCC, incorporating targeted cancer gene sequencing, RNA sequencing, single-nucleotide polymorphism array, fluorescence in situ hybridization, immunohistochemistry and cell-based assays. We identify recurrent somatic mutations in 29 genes, including NF2 (18%), SETD2 (18%), BAP1 (13%), KMT2C (10%) and MTOR (8%). Integrated analysis reveals a subset of 26% uRCC characterized by NF2 loss, dysregulated Hippo–YAP pathway and worse survival, whereas 21% uRCC with mutations of MTOR, TSC1, TSC2 or PTEN and hyperactive mTORC1 signalling are associated with better clinical outcome. FH deficiency (6%), chromatin/DNA damage regulator mutations (21%) and ALK translocation (2%) distinguish additional cases. Altogether, this study reveals distinct molecular subsets for 76% of our uRCC cohort, which could have diagnostic and therapeutic implications. PMID:27713405

  10. Simultaneous transport of different localized mRNA species revealed by live-cell imaging.

    PubMed

    Lange, Susanne; Katayama, Yoshihiko; Schmid, Maria; Burkacky, Ondrej; Bräuchle, Christoph; Lamb, Don C; Jansen, Ralf-Peter

    2008-08-01

    Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.

  11. Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

    PubMed Central

    Bolisetty, Mohan; Kursawe, Romy; Sun, Lili; Sivakamasundari, V.; Kycia, Ina

    2017-01-01

    Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type–specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis. PMID:27864352

  12. Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state.

    PubMed

    Verfaillie, Annelien; Imrichova, Hana; Atak, Zeynep Kalender; Dewaele, Michael; Rambow, Florian; Hulselmans, Gert; Christiaens, Valerie; Svetlichnyy, Dmitry; Luciani, Flavie; Van den Mooter, Laura; Claerhout, Sofie; Fiers, Mark; Journe, Fabrice; Ghanem, Ghanem-Elias; Herrmann, Carl; Halder, Georg; Marine, Jean-Christophe; Aerts, Stein

    2015-04-09

    Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading. Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event. This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively. Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors. Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma. Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance.

  13. Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state

    PubMed Central

    Verfaillie, Annelien; Imrichova, Hana; Atak, Zeynep Kalender; Dewaele, Michael; Rambow, Florian; Hulselmans, Gert; Christiaens, Valerie; Svetlichnyy, Dmitry; Luciani, Flavie; Van den Mooter, Laura; Claerhout, Sofie; Fiers, Mark; Journe, Fabrice; Ghanem, Ghanem-Elias; Herrmann, Carl; Halder, Georg; Marine, Jean-Christophe; Aerts, Stein

    2015-01-01

    Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading. Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event. This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively. Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors. Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma. Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance. PMID:25865119

  14. Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover.

    PubMed

    Cambridge, Sidney B; Gnad, Florian; Nguyen, Chuong; Bermejo, Justo Lorenzo; Krüger, Marcus; Mann, Matthias

    2011-12-02

    The turnover of each protein in the mammalian proteome is a functionally important characteristic. Here, we employed high-resolution mass spectrometry to quantify protein dynamics in nondividing mammalian cells. The ratio of externally supplied versus endogenous amino acids to de novo protein synthesis was about 17:1. Using subsaturating SILAC labeling, we obtained accurate turnover rates of 4106 proteins in HeLa and 3528 proteins in C2C12 cells. Comparison of these human and mouse cell lines revealed a highly significant turnover correlation of protein orthologs and thus high species conservation. Functionally, we observed statistically significant trends for the turnover of phosphoproteins and gene ontology categories that showed extensive covariation between mouse and human. Likewise, the members of some protein complexes, such as the proteasome, have highly similar turnover rates. The high species conservation and the low complex variances thus imply great regulatory fine-tuning of protein turnover.

  15. Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.

    PubMed

    Flodby, Per; Kim, Yong Ho; Beard, LaMonta L; Gao, Danping; Ji, Yanbin; Kage, Hidenori; Liebler, Janice M; Minoo, Parviz; Kim, Kwang-Jin; Borok, Zea; Crandall, Edward D

    2016-09-01

    Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which the β1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump β3 subunit expression and β2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump β1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell-specific roles in the lung.

  16. TNFRSF13B hemizygosity reveals TACI haploinsufficiency at later stages of B-cell development

    PubMed Central

    Romberg, Neil; Virdee, Manmeet; Chamberlain, Nicolas; Oe, Tyler; Schickel, Jean-Nicolas; Perkins, Tiffany; Cantaert, Tineke; Rachid, Rima; Rosengren, Sally; Palazzo, Regina; Geha, Raif; Cunningham-Rundles, Charlotte; Meffre, Eric

    2015-01-01

    Background Heterozygous C104R or A181E TNFRSF13B mutations impair the removal of autoreactive B cells, weaken B-cell activation and convey to common variable immune deficiency (CVID) patients an increased risk for autoimmunity. How mutant TACI influences wildtype TACI function is unclear; different models suggest either a dominant-negative effect or haploinsufficiency. Objective We investigated potential TACI haploinsufficiency by analyzing antibody-deficient Smith-Magenis Syndrome (SMS) patients, who possess only one TNFRSF13B allele and antibody-deficient patients carrying one c.204insA TNFRSF13B null mutation. Methods We tested the reactivity of antibodies isolated from single B cells from SMS patients and patients with a c.204insA TNFRSF13B mutation and compared them with counterparts from CVID patients with heterozygous C104R or A181E TNFRSF13B missense mutations. We also assessed if loss of a TNFRSF13B allele induced haploinsufficiency in naïve and memory B cells recapitulate abnormal immunological features typical of CVID patients with heterozygous TNFRSF13B missense mutations. Results We found loss of a TNFRSF13B allele does not impact TACI expression, activation responses, or establishment of central B-cell tolerance in naïve B cells. Additionally, SMS patients and patients with a c.204insA TNFRSF13B mutation display normal Treg function and peripheral B-cell tolerance. The lack of a TNFRSF13B allele did result in decreased TACI expression on memory B cells, resulting in impaired activation and antibody secretion. Conclusion TNFRSF13B hemizygosity does not recapitulate autoimmune features of CVID-associated C104R and A181E TNFRSF13B mutations, which likely encode dominant-negative products, but instead reveals selective TACI haploinsufficiency at later stages of B-cell development. PMID:26100089

  17. Clonogenic assay of type a influenza viruses reveals noninfectious cell-killing (apoptosis-inducing) particles.

    PubMed

    Ngunjiri, John M; Sekellick, Margaret J; Marcus, Philip I

    2008-03-01

    Clonogenic (single-cell plating) assays were used to define and quantify subpopulations of two genetically closely related variants of influenza virus A/TK/OR/71 that differed primarily in the size of the NS1 gene product; they expressed a full-size (amino acids [aa] 1 to 230) or truncated (aa 1 to 124) NS1 protein. Monolayers of Vero cells were infected with different amounts of virus, monodispersed, and plated. Cell survival curves were generated from the fraction of cells that produced visible colonies as a function of virus multiplicity. The exponential loss of colony-forming capacity at low multiplicities demonstrated that a single virus particle sufficed to kill a cell. The ratios of cell-killing particles (CKP) to plaque-forming particles (PFP) were 1:1 and 7:1 in populations of variants NS1(1-124) and NS1(1-230), respectively. This study revealed a new class of particles in influenza virus populations-noninfectious CKP. Both infectious and noninfectious CKP were 6.3 times more resistant to UV radiation than PFP activity. Based on UV target theory, a functional polymerase subunit was implicated in a rate-limiting step in cell killing. Since influenza viruses kill cells by apoptosis (programmed cell death), CKP are functionally apoptosis-inducing particles. Noninfectious CKP are present in excess of PFP in virus populations with full-size NS1 and induce apoptosis that is temporally delayed and morphologically different than that initiated by infectious CKP present in the virus population expressing truncated NS1. The identification and quantification of both infectious and noninfectious CKP defines new phenotypes in influenza virus populations and presents a challenge to determine their role in regulating infectivity, pathogenesis, and vaccine efficacy.

  18. Association of ActA to Peptidoglycan Revealed by Cell Wall Proteomics of Intracellular Listeria monocytogenes*

    PubMed Central

    García-del Portillo, Francisco; Calvo, Enrique; D'Orazio, Valentina; Pucciarelli, M. Graciela

    2011-01-01

    Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that colonizes the cytosol of eukaryotic cells. Recent transcriptomic studies have revealed that intracellular L. monocytogenes alter expression of genes encoding envelope components. However, no comparative global analysis of this cell wall remodeling process is yet known at the protein level. Here, we used high resolution mass spectrometry to define the cell wall proteome of L. monocytogenes growing inside epithelial cells. When compared with extracellular bacteria growing in a nutrient-rich medium, a major difference found in the proteome was the presence of the actin assembly-inducing protein ActA in peptidoglycan purified from intracellular bacteria. ActA was also identified in the peptidoglycan of extracellular bacteria growing in a chemically defined minimal medium. In this condition, ActA maintains its membrane anchoring domain and promotes efficient bacterial entry into nonphagocytic host cells. Unexpectedly, Internalin-A, which mediates entry of extracellular L. monocytogenes into eukaryotic cells, was identified at late infection times (6 h) as an abundant protein in the cell wall of intracellular bacteria. Other surface proteins covalently bound to the peptidoglycan, as Lmo0514 and Lmo2085, were detected exclusively in intracellular and extracellular bacteria, respectively. Altogether, these data provide the first insights into the changes occurring at the protein level in the L. monocytogenes cell wall as the pathogen transits from the extracellular environment to an intracytosolic lifestyle inside eukaryotic cells. Some of these changes include alterations in the relative amount and the mode of association of certain surface proteins. PMID:21846725

  19. Revealing the sequence of interactions of PuroA peptide with Candida albicans cells by live-cell imaging

    NASA Astrophysics Data System (ADS)

    Shagaghi, Nadin; Bhave, Mrinal; Palombo, Enzo A.; Clayton, Andrew H. A.

    2017-03-01

    To determine the mechanism(s) of action of antimicrobial peptides (AMPs) it is desirable to provide details of their interaction kinetics with cellular, sub-cellular and molecular targets. The synthetic peptide, PuroA, displays potent antimicrobial activities which have been attributed to peptide-induced membrane destabilization, or intracellular mechanisms of action (DNA-binding) or both. We used time-lapse fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM) to directly monitor the localization and interaction kinetics of a FITC- PuroA peptide on single Candida albicans cells in real time. Our results reveal the sequence of events leading to cell death. Within 1 minute, FITC-PuroA was observed to interact with SYTO-labelled nucleic acids, resulting in a noticeable quenching in the fluorescence lifetime of the peptide label at the nucleus of yeast cells, and cell-cycle arrest. A propidium iodide (PI) influx assay confirmed that peptide translocation itself did not disrupt the cell membrane integrity; however, PI entry occurred 25-45 minutes later, which correlated with an increase in fractional fluorescence of pores and an overall loss of cell size. Our results clarify that membrane disruption appears to be the mechanism by which the C. albicans cells are killed and this occurs after FITC-PuroA translocation and binding to intracellular targets.

  20. Revealing the sequence of interactions of PuroA peptide with Candida albicans cells by live-cell imaging

    PubMed Central

    Shagaghi, Nadin; Bhave, Mrinal; Palombo, Enzo A.; Clayton, Andrew H. A.

    2017-01-01

    To determine the mechanism(s) of action of antimicrobial peptides (AMPs) it is desirable to provide details of their interaction kinetics with cellular, sub-cellular and molecular targets. The synthetic peptide, PuroA, displays potent antimicrobial activities which have been attributed to peptide-induced membrane destabilization, or intracellular mechanisms of action (DNA-binding) or both. We used time-lapse fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM) to directly monitor the localization and interaction kinetics of a FITC- PuroA peptide on single Candida albicans cells in real time. Our results reveal the sequence of events leading to cell death. Within 1 minute, FITC-PuroA was observed to interact with SYTO-labelled nucleic acids, resulting in a noticeable quenching in the fluorescence lifetime of the peptide label at the nucleus of yeast cells, and cell-cycle arrest. A propidium iodide (PI) influx assay confirmed that peptide translocation itself did not disrupt the cell membrane integrity; however, PI entry occurred 25–45 minutes later, which correlated with an increase in fractional fluorescence of pores and an overall loss of cell size. Our results clarify that membrane disruption appears to be the mechanism by which the C. albicans cells are killed and this occurs after FITC-PuroA translocation and binding to intracellular targets. PMID:28252014

  1. Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells

    NASA Technical Reports Server (NTRS)

    Hu, Shaohua; Chen, Jianxin; Fabry, Ben; Numaguchi, Yasushi; Gouldstone, Andrew; Ingber, Donald E.; Fredberg, Jeffrey J.; Butler, James P.; Wang, Ning

    2003-01-01

    We describe a novel synchronous detection approach to map the transmission of mechanical stresses within the cytoplasm of an adherent cell. Using fluorescent protein-labeled mitochondria or cytoskeletal components as fiducial markers, we measured displacements and computed stresses in the cytoskeleton of a living cell plated on extracellular matrix molecules that arise in response to a small, external localized oscillatory load applied to transmembrane receptors on the apical cell surface. Induced synchronous displacements, stresses, and phase lags were found to be concentrated at sites quite remote from the localized load and were modulated by the preexisting tensile stress (prestress) in the cytoskeleton. Stresses applied at the apical surface also resulted in displacements of focal adhesion sites at the cell base. Cytoskeletal anisotropy was revealed by differential phase lags in X vs. Y directions. Displacements and stresses in the cytoskeleton of a cell plated on poly-L-lysine decayed quickly and were not concentrated at remote sites. These data indicate that mechanical forces are transferred across discrete cytoskeletal elements over long distances through the cytoplasm in the living adherent cell.

  2. Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

    PubMed

    Zdravkovic, Tamara; Nazor, Kristopher L; Larocque, Nicholas; Gormley, Matthew; Donne, Matthew; Hunkapillar, Nathan; Giritharan, Gnanaratnam; Bernstein, Harold S; Wei, Grace; Hebrok, Matthias; Zeng, Xianmin; Genbacev, Olga; Mattis, Aras; McMaster, Michael T; Krtolica, Ana; Valbuena, Diana; Simón, Carlos; Laurent, Louise C; Loring, Jeanne F; Fisher, Susan J

    2015-12-01

    Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines. © 2015. Published by The Company of Biologists Ltd.

  3. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation

    NASA Astrophysics Data System (ADS)

    Gentleman, Eileen; Swain, Robin J.; Evans, Nicholas D.; Boonrungsiman, Suwimon; Jell, Gavin; Ball, Michael D.; Shean, Tamaryn A. V.; Oyen, Michelle L.; Porter, Alexandra; Stevens, Molly M.

    2009-09-01

    An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their counterparts in the healthy native tissue. It remains unclear, however, whether cells used in bone regeneration applications produce a material that mimics the structural and compositional complexity of native bone. By applying multivariate analysis techniques to micro-Raman spectra of mineralized nodules formed in vitro, we reveal cell-source-dependent differences in interactions between multiple bone-like mineral environments. Although osteoblasts and adult stem cells exhibited bone-specific biological activities and created a material with many of the hallmarks of native bone, the `bone nodules' formed from embryonic stem cells were an order of magnitude less stiff, and lacked the distinctive nanolevel architecture and complex biomolecular and mineral composition noted in the native tissue. Understanding the biological mechanisms of bone formation in vitro that contribute to cell-source-specific materials differences may facilitate the development of clinically successful engineered bone.

  4. Revealing dynamic processes of materials in liquids using liquid cell transmission electron microscopy.

    PubMed

    Niu, Kai-Yang; Liao, Hong-Gang; Zheng, Haimei

    2012-12-20

    The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

  5. Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells

    NASA Technical Reports Server (NTRS)

    Hu, Shaohua; Chen, Jianxin; Fabry, Ben; Numaguchi, Yasushi; Gouldstone, Andrew; Ingber, Donald E.; Fredberg, Jeffrey J.; Butler, James P.; Wang, Ning

    2003-01-01

    We describe a novel synchronous detection approach to map the transmission of mechanical stresses within the cytoplasm of an adherent cell. Using fluorescent protein-labeled mitochondria or cytoskeletal components as fiducial markers, we measured displacements and computed stresses in the cytoskeleton of a living cell plated on extracellular matrix molecules that arise in response to a small, external localized oscillatory load applied to transmembrane receptors on the apical cell surface. Induced synchronous displacements, stresses, and phase lags were found to be concentrated at sites quite remote from the localized load and were modulated by the preexisting tensile stress (prestress) in the cytoskeleton. Stresses applied at the apical surface also resulted in displacements of focal adhesion sites at the cell base. Cytoskeletal anisotropy was revealed by differential phase lags in X vs. Y directions. Displacements and stresses in the cytoskeleton of a cell plated on poly-L-lysine decayed quickly and were not concentrated at remote sites. These data indicate that mechanical forces are transferred across discrete cytoskeletal elements over long distances through the cytoplasm in the living adherent cell.

  6. A systems approach reveals distinct metabolic strategies among the NCI-60 cancer cell lines

    PubMed Central

    Aurich, Maike K.; Fleming, Ronan M. T.; Thiele, Ines

    2017-01-01

    The metabolic phenotype of cancer cells is reflected by the metabolites they consume and by the byproducts they release. Here, we use quantitative, extracellular metabolomic data of the NCI-60 panel and a novel computational method to generate 120 condition-specific cancer cell line metabolic models. These condition-specific cancer models used distinct metabolic strategies to generate energy and cofactors. The analysis of the models’ capability to deal with environmental perturbations revealed three oxotypes, differing in the range of allowable oxygen uptake rates. Interestingly, models based on metabolomic profiles of melanoma cells were distinguished from other models through their low oxygen uptake rates, which were associated with a glycolytic phenotype. A subset of the melanoma cell models required reductive carboxylation. The analysis of protein and RNA expression levels from the Human Protein Atlas showed that IDH2, which was an essential gene in the melanoma models, but not IDH1 protein, was detected in normal skin cell types and melanoma. Moreover, the von Hippel-Lindau tumor suppressor (VHL) protein, whose loss is associated with non-hypoxic HIF-stabilization, reductive carboxylation, and promotion of glycolysis, was uniformly absent in melanoma. Thus, the experimental data supported the predicted role of IDH2 and the absence of VHL protein supported the glycolytic and low oxygen phenotype predicted for melanoma. Taken together, our approach of integrating extracellular metabolomic data with metabolic modeling and the combination of different network interrogation methods allowed insights into the metabolism of cells. PMID:28806730

  7. DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus

    PubMed Central

    2013-01-01

    Background DNA methylation is of pivotal importance during development. Previous genome-wide studies identified numerous differentially methylated regions upon differentiation of stem cells, many of them associated with transcriptional start sites. Results We present the first genome-wide, single-base-resolution view into DNA methylation dynamics during differentiation of a mammalian epithelial stem cell: the mouse small intestinal Lgr5+ stem cell. Very little change was observed at transcriptional start sites and our data suggest that differentiation-related genes are already primed for expression in the stem cell. Genome-wide, only 50 differentially methylated regions were identified. Almost all of these loci represent enhancers driving gene expression in the differentiated part of the small intestine. Finally, we show that binding of the transcription factor Tcf4 correlates with hypo-methylation and demonstrate that Tcf4 is one of the factors contributing to formation of differentially methylated regions. Conclusions Our results reveal limited DNA methylation dynamics during small intestine stem cell differentiation and an impact of transcription factor binding on shaping the DNA methylation landscape during differentiation of stem cells in vivo. PMID:23714178

  8. Stochastic simulation of notch signaling reveals novel factors that mediate the differentiation of neural stem cells.

    PubMed

    Tzou, Wen-Shyong; Lo, Ying-Tsang; Pai, Tun-Wen; Hu, Chin-Hwa; Li, Chung-Hao

    2014-07-01

    Notch signaling controls cell fate decisions and regulates multiple biological processes, such as cell proliferation, differentiation, and apoptosis. Computational modeling of the deterministic simulation of Notch signaling has provided important insight into the possible molecular mechanisms that underlie the switch from the undifferentiated stem cell to the differentiated cell. Here, we constructed a stochastic model of a Notch signaling model containing Hes1, Notch1, RBP-Jk, Mash1, Hes6, and Delta. mRNA and protein were represented as a discrete state, and 334 reactions were employed for each biochemical reaction using a graphics processing unit-accelerated Gillespie scheme. We employed the tuning of 40 molecular mechanisms and revealed several potential mediators capable of enabling the switch from cell stemness to differentiation. These effective mediators encompass different aspects of cellular regulations, including the nuclear transport of Hes1, the degradation of mRNA (Hes1 and Notch1) and protein (Notch1), the association between RBP-Jk and Notch intracellular domain (NICD), and the cleavage efficiency of the NICD. These mechanisms overlap with many modifiers that have only recently been discovered to modulate the Notch signaling output, including microRNA action, ubiquitin-mediated proteolysis, and the competitive binding of the RBP-Jk-DNA complex. Moreover, we identified the degradation of Hes1 mRNA and nuclear transport of Hes1 as the dominant mechanisms that were capable of abolishing the cell state transition induced by other molecular mechanisms.

  9. [Revealing the chemical changes of tea cell wall induced by anthracnose with confocal Raman microscopy].

    PubMed

    Li, Xiao-li; Luo, Liu-bin; Hu, Xiao-qian; Lou, Bing-gan; He, Yong

    2014-06-01

    Healthy tea and tea infected by anthracnose were first studied by confocal Raman microscopy to illustrate chemical changes of cell wall in the present paper. Firstly, Raman spectra of both healthy and infected sample tissues were collected with spatial resolution at micron-level, and ultrastructure of healthy and infected tea cells was got from scanning electron microscope. These results showed that there were significant changes in Raman shift and Raman intensity between healthy and infected cell walls, indicating that great differences occurred in chemical compositions of cell walls between healthy and infected samples. In details, intensities at many Raman bands which were closely associated with cellulose, pectin, esters were reduced after infection, revealing that the content of chemical compounds such as cellulose, pectin, esters was decreased after infection. Subsequently, chemical imaging of both healthy and infected tea cell walls were realized based on Raman fingerprint spectra of cellulose and microscopic spatial structure. It was found that not only the content of cellulose was reduced greatly after infection, but also the ordered structure of cellulose was destroyed by anthracnose infection. Thus, confocal Raman microscopy was shown to be a powerful tool to detect the chemical changes in cell wall of tea caused by anthracnose without any chemical treatment or staining. This research firstly applied confocal Raman microscopy in phytopathology for the study of interactive relationship between host and pathogen, and it will also open a new way for intensive study of host-pathogen at cellular level.

  10. Plasmacytoid dendritic cells promote HIV-1–induced group 3 innate lymphoid cell depletion

    PubMed Central

    Zhang, Zheng; Cheng, Liang; Zhao, Juanjuan; Li, Guangming; Zhang, Liguo; Chen, Weiwei; Nie, Weiming; Reszka-Blanco, Natalia J.; Wang, Fu-Sheng; Su, Lishan

    2015-01-01

    Group 3 innate lymphoid cells (ILC3s) have demonstrated roles in promoting antibacterial immunity, maintaining epithelial barrier function, and supporting tissue repair. ILC3 alterations are associated with chronic inflammation and inflammatory disease; however, the characteristics and relevant regulatory mechanisms of this cell population in HIV-1 infection are poorly understood due in part to a lack of a robust model. Here, we determined that functional human ILC3s develop in lymphoid organs of humanized mice and that persistent HIV-1 infection in this model depletes ILC3s, as observed in chronic HIV-1–infected patients. In HIV-1–infected mice, effective antiretroviral therapy reversed the loss of ILC3s. HIV-1–dependent reduction of ILC3s required plasmacytoid dendritic cells (pDCs), IFN-I, and the CD95/FasL pathway, as targeted depletion or blockade of these prevented HIV-1–induced ILC3 depletion in vivo and in vitro, respectively. Finally, we determined that HIV-1 infection induces CD95 expression on ILC3s via a pDC- and IFN-I–dependent mechanism that sensitizes ILC3s to undergo CD95/FasL-mediated apoptosis. We conclude that chronic HIV-1 infection depletes ILC3s through pDC activation, induction of IFN-I, and CD95-mediated apoptosis. PMID:26301812

  11. Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

    PubMed

    Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

    2016-12-02

    Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

  12. Nonaqueous titration of amino groups in polymeric matrix of plant cell walls.

    PubMed

    Meychik, N R; Nikolaeva, Yu I; Ermakov, I P

    2009-08-01

    Nonaqueous titration was used for detection of free amino groups in the polymeric matrix of plant cell walls. The content of amino groups varied in the range 0.54-0.91 and total nitrogen in the range 1.0-4.2 mmol per gram dry mass of cell walls depending on the plant species. However, these data on the high content of free amino groups do not correlate with the present day concept that the nitrogen fraction in charged amino groups in plant cell wall proteins, which are assumed to be mainly amino groups of lysine and arginine residues, is about 10%. It is supposed that most detected free amino groups belong to the hydroxy-amino acids hydroxyproline and tyrosine that can be bound at the hydroxyl group with the carbohydrate part of glycoprotein or another structural cell wall polymer.

  13. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation

    PubMed Central

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J.; Zhang, Jianyi; Ge, Ying

    2015-01-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function. PMID:26033914

  14. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

    PubMed

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J; Zhang, Jianyi; Ge, Ying

    2015-08-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

  15. A cross-reactive antigen of thymus and skin epithelial cells common with the polysaccharide of group A streptococci.

    PubMed

    Lyampert, I M; Beletskaya, L V; Borodiyuk, N A; Gnezditskaya, E V; Rassokhina, I I; Danilova, T A

    1976-07-01

    Investigation of antibodies to the specific determinant of streptococcal group A polysaccharide in indirect immunofluorescence experiments has revealed the existence of a cross-reactive antigen in the epithelial cells of the thymus and skin. This CR antigen is contained by the epithelial cells of man and animals of different species. It has been demonstrated in all the individuals studied including animals producing antibodies to the polysaccharide of Group A streptococci. The principal cause of autoimmune thymitis characteristic of rheumatic fever and other autoimmune processes is probably damage done to the thymus by autoantibodies resulting from immunization with microbial cross-reactive antigens shared by the thymus. Reaction of the autoantibodies with thymic antigens may affect the immunosuppressive function of the thymus and the maturation process of suppressor T cells. These events probably constitute the basic stage in the development of an autoimmune process.

  16. Quantitative microspectroscopic imaging reveals viral and cellular RNA helicase interactions in live cells.

    PubMed

    Corby, M J; Stoneman, Michael R; Biener, Gabriel; Paprocki, Joel D; Kolli, Rajesh; Raicu, Valerica; Frick, David N

    2017-07-07

    Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR (i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. The Macronuclear Genome of Stentor coeruleus Reveals Tiny Introns in a Giant Cell.

    PubMed

    Slabodnick, Mark M; Ruby, J Graham; Reiff, Sarah B; Swart, Estienne C; Gosai, Sager; Prabakaran, Sudhakaran; Witkowska, Ewa; Larue, Graham E; Fisher, Susan; Freeman, Robert M; Gunawardena, Jeremy; Chu, William; Stover, Naomi A; Gregory, Brian D; Nowacki, Mariusz; Derisi, Joseph; Roy, Scott W; Marshall, Wallace F; Sood, Pranidhi

    2017-02-20

    The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor [1, 2] is a heterotrichous ciliate distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan [3], were attracted to the system because of its regenerative abilities-if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2, 3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor's cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Plasma membrane-cell wall connections: roles in mitosis and cytokinesis revealed by plasmolysis of Tradescantia virginiana leaf epidermal cells.

    PubMed

    Cleary, A L

    2001-01-01

    Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions--cortical actin patch--and in the establishment and maintenance of the division site--preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 micrograms of cytochalasin B per ml but inhibited by 10 microM oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.

  19. Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.

    PubMed Central

    Jehuda-Cohen, T; Slade, B A; Powell, J D; Villinger, F; De, B; Folks, T M; McClure, H M; Sell, K W; Ahmed-Ansari, A

    1990-01-01

    Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals. Images PMID:2111024

  20. Modelling epigenetic regulation of gene expression in 12 human cell types reveals combinatorial patterns of cell-type-specific genes.

    PubMed

    Lu, Yiming; Qu, Wubin; Min, Bo; Liu, Zheyan; Chen, Changsheng; Zhang, Chenggang

    2014-06-01

    The maintenance of the diverse cell types in a multicellular organism is one of the fundamental mysteries of biology. Modelling the dynamic regulatory relationships between the histone modifications and the gene expression across the diverse cell types is essential for the authors to understand the mechanisms of the epigenetic regulation. Here, the authors thoroughly assessed the histone modification enrichment profiles at the promoters and constructed quantitative models between the histone modification abundances and the gene expression in 12 human cell types. The author's results showed that the histone modifications at the promoters exhibited remarkably cell-type-dependent variability in the cell-type-specific (CTS) genes. They demonstrated that the variable profiles of the modifications are highly predictive for the dynamic changes of the gene expression across all the cell types. Their findings revealed the close relationship between the combinatorial patterns of the histone modifications and the CTS gene expression. They anticipate that the findings and the methods they used in this study could provide useful information for the future studies of the regulatory roles of the histone modifications in the CTS genes.

  1. Responses of innate immune cells to group A Streptococcus

    PubMed Central

    Fieber, Christina; Kovarik, Pavel

    2014-01-01

    Group A Streptococcus (GAS), also called Streptococcus pyogenes, is a Gram-positive beta-hemolytic human pathogen which causes a wide range of mostly self-limiting but also several life-threatening diseases. Innate immune responses are fundamental for defense against GAS, yet their activation by pattern recognition receptors (PRRs) and GAS-derived pathogen-associated molecular patterns (PAMPs) is incompletely understood. In recent years, the use of animal models together with the powerful tools of human molecular genetics began shedding light onto the molecular mechanisms of innate immune defense against GAS. The signaling adaptor MyD88 was found to play a key role in launching the immune response against GAS in both humans and mice, suggesting that PRRs of the Toll-like receptor (TLR) family are involved in sensing this pathogen. The specific TLRs and their ligands have yet to be identified. Following GAS recognition, induction of cytokines such as TNF and type I interferons (IFNs), leukocyte recruitment, phagocytosis, and the formation of neutrophil extracellular traps (NETs) have been recognized as key events in host defense. A comprehensive knowledge of these mechanisms is needed in order to understand their frequent failure against GAS immune evasion strategies. PMID:25325020

  2. Responses of innate immune cells to group A Streptococcus.

    PubMed

    Fieber, Christina; Kovarik, Pavel

    2014-01-01

    Group A Streptococcus (GAS), also called Streptococcus pyogenes, is a Gram-positive beta-hemolytic human pathogen which causes a wide range of mostly self-limiting but also several life-threatening diseases. Innate immune responses are fundamental for defense against GAS, yet their activation by pattern recognition receptors (PRRs) and GAS-derived pathogen-associated molecular patterns (PAMPs) is incompletely understood. In recent years, the use of animal models together with the powerful tools of human molecular genetics began shedding light onto the molecular mechanisms of innate immune defense against GAS. The signaling adaptor MyD88 was found to play a key role in launching the immune response against GAS in both humans and mice, suggesting that PRRs of the Toll-like receptor (TLR) family are involved in sensing this pathogen. The specific TLRs and their ligands have yet to be identified. Following GAS recognition, induction of cytokines such as TNF and type I interferons (IFNs), leukocyte recruitment, phagocytosis, and the formation of neutrophil extracellular traps (NETs) have been recognized as key events in host defense. A comprehensive knowledge of these mechanisms is needed in order to understand their frequent failure against GAS immune evasion strategies.

  3. Metabolomics Analysis Reveals that AICAR Affects Glycerolipid, Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells.

    PubMed

    ElAzzouny, Mahmoud A; Evans, Charles R; Burant, Charles F; Kennedy, Robert T

    2015-01-01

    AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using 13C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival.

  4. Metabolomics Analysis Reveals that AICAR Affects Glycerolipid, Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells

    PubMed Central

    ElAzzouny, Mahmoud A.; Evans, Charles R.; Burant, Charles F; Kennedy, Robert T.

    2015-01-01

    AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using 13C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival. PMID:26107620

  5. Charge transport in CdTe solar cells revealed by conductive tomographic atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Luria, Justin; Kutes, Yasemin; Moore, Andrew; Zhang, Lihua; Stach, Eric A.; Huey, Bryan D.

    2016-11-01

    The influence of microstructural defects on the device properties in CdTe remains largely unknown. This is partly because characterization techniques have been unable to image electrical pathways throughout three-dimensional grains and grain boundaries with nanoscale resolution. Here, we employ a conductive and tomographic variation of atomic force microscopy to study charge transport at the nanoscale in a functioning thin-film solar cell with 12.3% efficiency. Images of electric current collected through the device thickness reveal spatially dependent short-circuit and open-circuit performance, and confirm that grain boundaries are preferential pathways for electron transport. Results on samples with and without cadmium chloride treatment reveal little difference in grain structure at the microscale, with samples without treatment showing almost no photocurrent either at planar defects or at grain boundaries. Our results supports an energetically orthogonal transport system of grain boundaries and interconnected planar defects as contributing to optimal solar cell performance, contrary to the conventional wisdom of the deleterious role of planar defects on polycrystalline thin-film solar cells.

  6. Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation.

    PubMed

    Wang, Maojun; Wang, Pengcheng; Tu, Lili; Zhu, Sitao; Zhang, Lin; Li, Zhonghua; Zhang, Qinghua; Yuan, Daojun; Zhang, Xianlong

    2016-05-19

    Epigenetic modifications are highlighted for their great importance in regulating plant development, but their function associated with single-cell differentiation remains undetermined. Here, we used the cotton fibre, which is the epidermal hair on the cotton ovule, as a model to investigate the regulatory role of DNA methylation in cell differentiation. The level of CHH (H = A, T, or C) DNA methylation level was found to increase during fibre development, accompanied by a decrease in RNA-directed DNA methylation (RdDM). Examination of nucleosome positioning revealed a gradual transition from euchromatin to heterochromatin for chromatin dynamics in developing fibres, which could shape the DNA methylation landscape. The observed increase in DNA methylation in fibres, compared with other ovule tissue, was demonstrated to be mediated predominantly by an active H3K9me2-dependent pathway rather than the RdDM pathway, which was inactive. Furthermore, integrated multi-omics analyses revealed that dynamic DNA methylation played a role in the regulation of lipid biosynthesis and spatio-temporal modulation of reactive oxygen species during fibre differentiation. Our study illustrates two divergent pathways mediating a continuous increase of DNA methylation and also sheds further light on the epigenetic basis for single-cell differentiation in plants. These data and analyses are made available to the wider research community through a comprehensive web portal. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Live-cell CRISPR imaging in plants reveals dynamic telomere movements.

    PubMed

    Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F; Puchta, Holger; Houben, Andreas

    2017-08-01

    Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  8. Efficient isolation and proteomic analysis of cell plasma membrane proteins in gastric cancer reveal a novel differentiation and progression related cell surface marker, R-cadherin.

    PubMed

    Chen, Bo; Luo, Qi-Cong; Chen, Jian-Bo; Lin, Li-E; Luo, Ming-Xu; Ren, Hong-Yue; Chen, Pei-Qiong; Shi, Lian-Guo

    2016-09-01

    Cell plasma membrane proteins, playing a crucial role in cell malignant transformation and development, were the main targets of tumor detection and therapy. In this study, CyDye/biotin double-labeling proteomic approach was adopted to profile the membrane proteome of gastric cancer cell line BGC-823 and paired immortalized gastric epithelial cell GES-1. Real-time PCR, Western blotting, and immunohistochemical staining were used to validate the differential expression of a novel identified cell surface marker R-cadherin in gastric cancer cells and tissues. Clinicopathological study and survival analysis were performed to estimate its roles in tumor progression and outcome prediction. Real-time PCR and Western blotting showed that the expression level of R-cadherin in gastric cancer were significantly lower than non-cancerous epithelial cell and tissues. Clinicopathological study indicated that R-cadherin was dominantly expressed on cell surface of normal gastric epithelium, and its expression deletion in gastric cancer tissues was associated with tumor site, differentiation, lymph node metastasis, and pTNM (chi-square test, P < 0.05). Those patients with R-cadherin positive expression displayed better overall survivals than negative expression group (log-rank test, P = 0.000). Cox multivariate survival analysis revealed lacking the expression of R-cadherin was a main independent predictor for poor clinical outcome in gastric cancer (RR = 5.680, 95 % CI 2.250-14.341, P < 0.01). We have established a fundamental membrane proteome database for gastric cancer and identified R-cadherin as a tumor differentiation and progression-related cell surface marker of gastric cancer. Lacking the expression of R-cadherin indicates poor prognosis in patients with gastric cancer.

  9. Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

    PubMed Central

    Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-01-01

    Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma. PMID:27732954

  10. Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis.

    PubMed

    Malmstrom, Rex R; Rodrigue, Sébastien; Huang, Katherine H; Kelly, Libusha; Kern, Suzanne E; Thompson, Anne; Roggensack, Sara; Berube, Paul M; Henn, Matthew R; Chisholm, Sallie W

    2013-01-01

    Prochlorococcus is the numerically dominant photosynthetic organism throughout much of the world's oceans, yet little is known about the ecology and genetic diversity of populations inhabiting tropical waters. To help close this gap, we examined natural Prochlorococcus communities in the tropical Pacific Ocean using a single-cell whole-genome amplification and sequencing. Analysis of the gene content of just 10 single cells from these waters added 394 new genes to the Prochlorococcus pan-genome--that is, genes never before seen in a Prochlorococcus cell. Analysis of marker genes, including the ribosomal internal transcribed sequence, from dozens of individual cells revealed several representatives from two uncultivated clades of Prochlorococcus previously identified as HNLC1 and HNLC2. While the HNLC clades can dominate Prochlorococcus communities under certain conditions, their overall geographic distribution was highly restricted compared with other clades of Prochlorococcus. In the Atlantic and Pacific oceans, these clades were only found in warm waters with low Fe and high inorganic P levels. Genomic analysis suggests that at least one of these clades thrives in low Fe environments by scavenging organic-bound Fe, a process previously unknown in Prochlorococcus. Furthermore, the capacity to utilize organic-bound Fe appears to have been acquired horizontally and may be exchanged among other clades of Prochlorococcus. Finally, one of the single Prochlorococcus cells sequenced contained a partial genome of what appears to be a prophage integrated into the genome.

  11. Systematic proteomic analysis of human hepotacellular carcinoma cells reveals molecular pathways and networks involved in metastasis.

    PubMed

    Yu, Yanyan; Shen, Huali; Yu, Hongxiu; Zhong, Fan; Zhang, Yang; Zhang, Chen; Zhao, Jian; Li, Hong; Chen, Jie; Liu, Yinkun; Yang, Pengyuan

    2011-06-01

    Systematic proteomic studying of the mechanism of hepatocellular carcinoma (HCC) metastasis remains challenging. We performed comparative proteomic and pathway analysis of four human metastatic HCC cell lines to identify metastasis-associated proteins. These HCC cell lines had a similar genetic background but with an increasing potential of metastasis. Using a combination of two dimensional electrophoresis (2-DE) and MALDI-TOF mass spectrometry, a total of 125 proteins and their post-translational modification forms or isoforms were found to be differentially expressed in the cell lines. Among them, 29 were gradually up-regulated whereas 17 were down-regulated with increasing metastatic potential. Instead of a traditional single-gene readout, global bioinformatics analysis was carried out, which revealed that the glycolysis pathway was the most significantly enriched pathway. The heat shock proteins (HSPs) centered and NF-kappaB centered networks were also enriched in the result, which may imply the key function of inflaming on metastasis. Meanwhile, knockdown of HDGF, an up-regulated protein and a target of NF-kappaB, induced cell apoptosis in the metastatic HCC cells. This work provides a demonstration that a combination of bioinformatics and comparative proteomics can help in finding out potential biomarkers associated with HCC metastasis on the level of pathways.

  12. Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation.

    PubMed

    Verboogen, Daniëlle Rianne José; González Mancha, Natalia; Ter Beest, Martin; van den Bogaart, Geert

    2017-05-19

    SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution.

  13. Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell-wall structure and assembly

    PubMed Central

    Desmarais, Samantha M.; De Pedro, Miguel A.; Cava, Felipe; Huang, Kerwyn Casey

    2013-01-01

    The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation, and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell-wall synthesis and cell growth. High Performance Liquid Chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques. PMID:23679048

  14. Dynamics of Natural Killer Cell Receptor Revealed by Quantitative Analysis of Photoswitchable Protein

    NASA Astrophysics Data System (ADS)

    Pageon, Sophie V.; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G.; Davis, Daniel M.

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies which rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report a novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, following a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 +- 0.06 micron^2/s) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse and remains surprisingly stable there. Unexpectedly however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected inter-synaptic exchange of protein.

  15. Amyloid plaque structure and cell surface interactions of β-amyloid fibrils revealed by electron tomography

    PubMed Central

    Han, Shen; Kollmer, Marius; Markx, Daniel; Claus, Stephanie; Walther, Paul; Fändrich, Marcus

    2017-01-01

    The deposition of amyloid fibrils as plaques is a key feature of several neurodegenerative diseases including in particular Alzheimer’s. This disease is characterized, if not provoked, by amyloid aggregates formed from Aβ peptide that deposit inside the brain or are toxic to neuronal cells. We here used scanning transmission electron microscopy (STEM) to determine the fibril network structure and interactions of Aβ fibrils within a cell culture model of Alzheimer’s disease. STEM images taken from the formed Aβ amyloid deposits revealed three main types of fibril network structures, termed amorphous meshwork, fibril bundle and amyloid star. All three were infiltrated by different types of lipid inclusions from small-sized exosome-like structures (50–100 nm diameter) to large-sized extracellular vesicles (up to 300 nm). The fibrils also presented strong interactions with the surrounding cells such that fibril bundles extended into tubular invaginations of the plasma membrane. Amyloid formation in the cell model was previously found to have an intracellular origin and we show here that it functionally destroys the integrity of the intracellular membranes as it leads to lysosomal leakage. These data provide a mechanistic link to explain why intracellular fibril formation is toxic to the cell. PMID:28240273

  16. Amyloid plaque structure and cell surface interactions of β-amyloid fibrils revealed by electron tomography.

    PubMed

    Han, Shen; Kollmer, Marius; Markx, Daniel; Claus, Stephanie; Walther, Paul; Fändrich, Marcus

    2017-02-27

    The deposition of amyloid fibrils as plaques is a key feature of several neurodegenerative diseases including in particular Alzheimer's. This disease is characterized, if not provoked, by amyloid aggregates formed from Aβ peptide that deposit inside the brain or are toxic to neuronal cells. We here used scanning transmission electron microscopy (STEM) to determine the fibril network structure and interactions of Aβ fibrils within a cell culture model of Alzheimer's disease. STEM images taken from the formed Aβ amyloid deposits revealed three main types of fibril network structures, termed amorphous meshwork, fibril bundle and amyloid star. All three were infiltrated by different types of lipid inclusions from small-sized exosome-like structures (50-100 nm diameter) to large-sized extracellular vesicles (up to 300 nm). The fibrils also presented strong interactions with the surrounding cells such that fibril bundles extended into tubular invaginations of the plasma membrane. Amyloid formation in the cell model was previously found to have an intracellular origin and we show here that it functionally destroys the integrity of the intracellular membranes as it leads to lysosomal leakage. These data provide a mechanistic link to explain why intracellular fibril formation is toxic to the cell.

  17. Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis

    PubMed Central

    Malmstrom, Rex R; Rodrigue, Sébastien; Huang, Katherine H; Kelly, Libusha; Kern, Suzanne E; Thompson, Anne; Roggensack, Sara; Berube, Paul M; Henn, Matthew R; Chisholm, Sallie W

    2013-01-01

    Prochlorococcus is the numerically dominant photosynthetic organism throughout much of the world's oceans, yet little is known about the ecology and genetic diversity of populations inhabiting tropical waters. To help close this gap, we examined natural Prochlorococcus communities in the tropical Pacific Ocean using a single-cell whole-genome amplification and sequencing. Analysis of the gene content of just 10 single cells from these waters added 394 new genes to the Prochlorococcus pan-genome—that is, genes never before seen in a Prochlorococcus cell. Analysis of marker genes, including the ribosomal internal transcribed sequence, from dozens of individual cells revealed several representatives from two uncultivated clades of Prochlorococcus previously identified as HNLC1 and HNLC2. While the HNLC clades can dominate Prochlorococcus communities under certain conditions, their overall geographic distribution was highly restricted compared with other clades of Prochlorococcus. In the Atlantic and Pacific oceans, these clades were only found in warm waters with low Fe and high inorganic P levels. Genomic analysis suggests that at least one of these clades thrives in low Fe environments by scavenging organic-bound Fe, a process previously unknown in Prochlorococcus. Furthermore, the capacity to utilize organic-bound Fe appears to have been acquired horizontally and may be exchanged among other clades of Prochlorococcus. Finally, one of the single Prochlorococcus cells sequenced contained a partial genome of what appears to be a prophage integrated into the genome. PMID:22895163

  18. Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells.

    PubMed

    Reichel, Jonathan; Chadburn, Amy; Rubinstein, Paul G; Giulino-Roth, Lisa; Tam, Wayne; Liu, Yifang; Gaiolla, Rafael; Eng, Kenneth; Brody, Joshua; Inghirami, Giorgio; Carlo-Stella, Carmelo; Santoro, Armando; Rahal, Daoud; Totonchy, Jennifer; Elemento, Olivier; Cesarman, Ethel; Roshal, Mikhail

    2015-02-12

    Classical Hodgkin lymphoma (cHL) is characterized by sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells amid reactive host background, complicating the acquisition of neoplastic DNA without extensive background contamination. We overcame this limitation by using flow-sorted HRS and intratumor T cells and optimized low-input exome sequencing of 10 patient samples to reveal alterations in genes involved in antigen presentation, chromosome integrity, transcriptional regulation, and ubiquitination. β-2-microglobulin (B2M) is the most commonly altered gene in HRS cells, with 7 of 10 cases having inactivating mutations that lead to loss of major histocompatibility complex class I (MHC-I) expression. Enforced wild-type B2M expression in a cHL cell line restored MHC-I expression. In an extended cohort of 145 patients, the absence of B2M protein in the HRS cells was associated with lower stage of disease, younger age at diagnosis, and better overall and progression-free survival. B2M-deficient cases encompassed most of the nodular sclerosis subtype cases and only a minority of mixed cellularity cases, suggesting that B2M deficiency determines the tumor microenvironment and may define a major subset of cHL that has more uniform clinical and morphologic features. In addition, we report previously unknown genetic alterations that may render selected patients sensitive to specific targeted therapies. © 2015 by The American Society of Hematology.

  19. The T cell IFT20 interactome reveals new players in immune synapse assembly

    PubMed Central

    Galgano, Donatella; Onnis, Anna; Galvagni, Federico; Acuto, Oreste

    2017-01-01

    ABSTRACT Sustained signalling at the immune synapse (IS) requires the synaptic delivery of recycling endosome-associated T cell antigen receptors (TCRs). IFT20, a component of the intraflagellar transport system, controls TCR recycling to the IS as a complex with IFT57 and IFT88. Here, we used quantitative mass spectrometry to identify additional interaction partners of IFT20 in Jurkat T cells. In addition to IFT57 and IFT88, the analysis revealed new binding partners, including IFT54 (also known as TRAF3IP1), GMAP-210 (also known as TRIP11), Arp2/3 complex subunit-3 (ARPC3), COP9 signalosome subunit-1 (CSN1, also known as GPS1) and ERGIC-53 (also known as LMAN1). A direct interaction between IFT20 and both IFT54 and GMAP-210 was confirmed in pulldown assays. Confocal imaging of antigen-specific conjugates using T cells depleted of these proteins by RNA interference showed that TCR accumulation and phosphotyrosine signalling at the IS were impaired in the absence of IFT54, ARPC3 or ERGIC-53. Similar to in IFT20-deficient T cells, this defect resulted from a reduced ability of endosomal TCRs to polarize to the IS despite a correct translocation of the centrosome towards the antigen-presenting cell contact. Our data underscore the traffic-related role of an IFT20 complex that includes components of the intracellular trafficking machinery in IS assembly. PMID:28154159

  20. Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Daosen; Feng, Yanyan; Zhang, Qinnan; Su, Xin; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

    2015-04-01

    Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

  1. High Glucose-Induced PC12 Cell Death by Increasing Glutamate Production and Decreasing Methyl Group Metabolism

    PubMed Central

    Chen, Minjiang; Zheng, Hong; Wei, Tingting; Wang, Dan; Xia, Huanhuan; Zhao, Liangcai; Ji, Jiansong

    2016-01-01

    Objective. High glucose- (HG-) induced neuronal cell death is responsible for the development of diabetic neuropathy. However, the effect of HG on metabolism in neuronal cells is still unclear. Materials and Methods. The neural-crest derived PC12 cells were cultured for 72 h in the HG (75 mM) or control (25 mM) groups. We used NMR-based metabolomics to examine both intracellular and extracellular metabolic changes in HG-treated PC12 cells. Results. We found that the reduction in intracellular lactate may be due to excreting more lactate into the extracellular medium under HG condition. HG also induced the changes of other energy-related metabolites, such as an increased succinate and creatine phosphate. Our results also reveal that the synthesis of glutamate from the branched-chain amino acids (isoleucine and valine) may be enhanced under HG. Increased levels of intracellular alanine, phenylalanine, myoinositol, and choline were observed in HG-treated PC12 cells. In addition, HG-induced decreases in intracellular dimethylamine, dimethylglycine, and 3-methylhistidine may indicate a downregulation of methyl group metabolism. Conclusions. Our metabolomic results suggest that HG-induced neuronal cell death may be attributed to a series of metabolic changes, involving energy metabolism, amino acids metabolism, osmoregulation and membrane metabolism, and methyl group metabolism. PMID:27413747

  2. Tissue distribution of blood group membrane proteins beyond red cells: evidence from cDNA libraries.

    PubMed

    Rojewski, Markus T; Schrezenmeier, Hubert; Flegel, Willy A

    2006-08-01

    The proteins of blood group systems are expressed on red blood cells (RBC) by definition. We searched nucleotide databases of human expressed sequence tags (EST) to collate the distribution of 22 distinct membrane proteins in cells and tissues other than RBC. The documented blood group genes are: MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, John-Milton-Hagen and Gill. The genes were grouped according to their overall and their relative expression in embryo and adults. We describe the distribution of EST in cells, tissues and cell lines with a focus on non-RBC tissues.

  3. Genetic Characterization of Turkish Snake Melon (Cucumis melo L. subsp. melo flexuosus Group) Accessions Revealed by SSR Markers.

    PubMed

    Solmaz, Ilknur; Kacar, Yildiz Aka; Simsek, Ozhan; Sari, Nebahat

    2016-08-01

    Snake melon is an important cucurbit crop especially in the Southeastern and the Mediterranean region of Turkey. It is consumed as fresh or pickled. The production is mainly done with the local landraces in the country. Turkey is one of the secondary diversification centers of melon and possesses valuable genetic resources which have different morphological characteristics in case of snake melon. Genetic diversity of snake melon genotypes collected from different regions of Turkey and reference genotypes obtained from World Melon Gene Bank in Avignon-France was examined using 13 simple sequence repeat (SSR) markers. A total of 69 alleles were detected, with an average of 5.31 alleles per locus. The polymorphism information content of SSR markers ranged from 0.19 to 0.57 (average 0.38). Based on cluster analysis, two major groups were defined. The first major group included only one accession (61), while the rest of all accessions grouped in the second major group and separated into different sub-clusters. Based on SSR markers, cluster analysis indicated that considerably high genetic variability exists among the examined accessions; however, Turkish snake melon accessions were grouped together with the reference snake melon accessions.

  4. Morphology and COI barcodes reveal four new species in the lycieus group of Calisto (Lepidoptera, Nymphalidae, Satyrinae).

    PubMed

    Pérez-Asso, Antonio R; Núñez-Aguila, Rayner; Genaro, Julio A

    2016-09-26

    The predominantly Greater Antillean endemic genus Calisto Hübner, 1823 is highly diversified on several islands being more species rich on Hispaniola. We conducted expeditions during five years in the Dominican Republic resulting in new findings related with lyceius species group. Material belonging to this group was examined following the traditional morphological characters employed in genus taxonomy, and the COI barcode sequences obtained were analyzed through different approaches: Neighbor Joining clustering, ABGD, Maximum Likelihood (ML), and Bayesian Inference (BI). Analysis yielded 12 groups representing putative species: eight corresponding to previously named ones and four new species which are described in the present work: C. mariposa sp. nov., C. azua sp. nov., C. victori sp. nov., and C. samana sp. nov. The results also confirmed a single taxonomic entity within C. pulchella Lathy and the conspecific nature of C. franciscoi Gali and C. hendersoni. A dichotomic key for identification of species within the group is also given. Both phylogenetic reconstruction methods (ML and BI) employing molecular data achieved similar results with the relationships among the majority of taxa being supported by some ecological and morphological features. The exceptions were C. zangis Fabricius, C. raburni Gali, and C. pulchella, grouped together in a weakly supported clade. These species possess a highly differentiated adult and immature morphology which indicates an earlier divergence.

  5. Bcl11b is essential for group 2 innate lymphoid cell development

    PubMed Central

    Oliphant, Christopher J.; Englezakis, Alexandros; Yu, Yong; Clare, Simon; Rodewald, Hans-Reimer; Belz, Gabrielle; Liu, Pentao; Fallon, Padraic G.

    2015-01-01

    Group 2 innate lymphoid cells (ILC2s) are often found associated with mucosal surfaces where they contribute to protective immunity, inappropriate allergic responses, and tissue repair. Although we know they develop from a common lymphoid progenitor in the bone marrow (BM), the specific lineage path and transcriptional regulators that are involved are only starting to emerge. After ILC2 gene expression analysis we investigated the role of Bcl11b, a factor previously linked to T cell commitment, in ILC2 development. Using combined Bcl11b-tom and Id2-gfp reporter mice, we show that Bcl11b is expressed in ILC2 precursors in the BM and maintained in mature ILC2s. In vivo deletion of Bcl11b, by conditional tamoxifen-induced depletion or by Bcl11b−/− fetal liver chimera reconstitution, demonstrates that ILC2s are wholly dependent on Bcl11b for their development. Notably, in the absence of Bcl11b there is a concomitant expansion of the RORγt+ ILC3 population, suggesting that Bcl11b may negatively regulate this lineage. Using Nippostrongylus brasiliensis infection, we reveal that the absence of Bcl11b leads to impaired worm expulsion, caused by a deficit in ILC2s, whereas Citrobacter rodentium infection is cleared efficiently. These data clearly establish Bcl11b as a new factor in the differentiation of ILC2s. PMID:25964370