Quantitation of cell proliferation,colony formation,and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers

Summary A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

Life cycle size dynamics in Didymosphenia geminata (Bacillariophyceae)

Didymosphenia geminata has received a great deal of attention in the last 25 years, and considerable effort has gone into determining the origin, ecological impact, and economic consequences of its invasive behavior. While environmental conditions are a controlling influence in distribution, the extreme success of the species may be tied to its basic biology and life history. Little is known, however, about population dynamics, size restoration and reproduction of D. geminata. The objective of this study was to determine the temporal patterns in cell size frequency, size restoration strategy, and synchronization of life cycles between populations in close proximity. We implemented FlowCam technology to measure the length of more than 100,000 D. geminata cells from two sites in South Boulder Creek, Colorado over 1 year. We applied finite mixture modeling to uncover temporal patterns in size distribution. Our results show that collections of D. geminata exhibited a complex, multimodal size distribution, almost always containing four overlapping age cohorts. We failed to observe direct visual evidence of the sexual phase. Multiple abrupt and directional shifts in size distribution, however, were documented providing conclusive evidence of cell size restoration. Lastly, nodules in close proximity were asynchronous with respect to size frequency profiles and size diminution, highlighting the relevance of spatial heterogeneity in in situ diatom size dynamics. This study is the first to document the complexity of diatom cell size distribution in a lotic system, size restoration in D. geminata, and the variability in rates of size reduction at microhabitat spatial scales.     

Kinetic model of Proteus mirabilis swarm colony development

 Proteus mirabilis colonies display striking symmetry and periodicity. Based on experimental observations of cellular differentiation and group motility, a kinetic model has been developed to describe the swarmer cell differentiation-dedifferentiation cycle and the spatial evolution of swimmer and swarmer cells during Proteus mirabilis swarm colony development. A key element of the model is the age dependence of swarmer cell behaviour, in particular specifying a minimal age for motility and maximum age for septation and dedifferentiation to swimmer cells. Density thresholds for collective motility by mature swarmer cells serve to synchronize the movements of distinct swarmer cell groups and thus help provide temporal coherence to colony expansion cycles. Numerical computations show that the model fits experimental data by generating a complete swarming plus consolidation cycle period that is robust to changes in parameters which affect other aspects of swarmer cell migration and colony development. The kinetic equations underlying this model provide a different mathematical basis for a temporal oscillator from reaction-diffusion partial differential equations. The modelling shows that Proteus colony geometries arise as a consequence of macroscopic rules governing collective motility. Thus, in this case, pattern formation results from the operation of an adaptive bacterial system for spreading on solid substrates, not as an independent biological function. Kinetic models similar to this one may be applicable to periodic phenomena displayed by other biological systems with differentiated components of defined lifetimes. Received 3 July 1996; received in revised form 9 December 1996     

Not like Botryllus: indiscriminate post-metamorphic fusion in a compound ascidian

Fusion trials between metamorphs of the aplousobranch compound ascidian Diplosoma listerianum indicated that chimera formation was not dependent on relatedness. Similar, high rates of union were observed between full siblings, half siblings, unrelated individuals from the same population, and individuals from two geographically distant localities. This is in contrast to the well-studied ascidian genus Botryllus, in which a highly polymorphic allorecognition system governing the fusion–non-fusion reaction (colony specificity) largely limits fusion to close relatives. Fusion in Botryllus establishes a vascular chimera throughout which stem cells may circulate, promoting cell lineage competition between the fusion partners. The restriction of fusion to close kin in Botryllus is thought to reduce the inclusive fitness costs of competitive interactions between cell lineages within the chimera. In contrast to Botryllus, modules (zooids) of a D. listerianum colony are not interlinked by blood vessels, seemingly precluding the exchange of stem cells. The apparent absence of strict colony specificity in D. listerianum is thus in keeping with the predictions of the Botryllus model for the maintenance of allorecognition polymorphism. However, colony specificity has been reported in other species of aplousobranch ascidian that also lack a common vascular system. In these, the threat of migrating blood-borne stem cells cannot be responsible for the presence of colony specificity. One possibility, requiring experimental investigation, is that stem cells could perhaps migrate between zooids by another route, such as through the matrix of the colonial tunic. Even in the absence of stem cell exchange, cheating on the costs of colony maintenance and defence could also produce selective forces favouring colony specificity. In compound ascidians, this could involve unequal contribution to extrazooidal structures, principally the tunic and related tissues. This consideration seems potentially relevant to the lack of discrimination during fusion in D. listerianum, since extrazooidal somatic investment in this species appears minimal, severely limiting the scope for this other form of cheating. The various possible modes of exploitative interaction between fused colonies are not mutually exclusive, and offer fundamentally similar explanations for colony specificity. If none of them can be shown to occur in non-botryllid species possessing colony specificity, the generality of the Botryllus model may require re-evaluation.     

Molecular characterization and embryonic expression of innexins in the leech Hirudo medicinalis

Gap junctions are direct intercellular channels that permit the passage of ions and small signaling molecules. The temporal and spatial regulation of gap junctional communication is, thus, one mechanism by which cell interactions, and hence cell properties and cell fate, may be regulated during development. The nervous system of the leech, Hirudo medicinalis, is a particularly advantageous system in which to study developmental mechanisms involving gap junctions because interactions between identified cells may be studied in vivo in both the embryo and the adult. As in most invertebrates, gap junctions in the leech are composed of innexin proteins, which are distantly related to the vertebrate pannexins and are encoded by a multi-gene family. We have cloned ten novel leech innexins and describe the expression of these, plus two other previously reported members of this gene family, in the leech embryo between embryonic days 6 and 12, a period during which the main features of the central nervous system are established. Four innexins are expressed in neurons and two in glia, while several innexins are expressed in the excretory, circulatory, and reproductive organs. Of particular interest is Hm-inx6, whose expression appears to be restricted to the characterized S cell and two other neurons putatively identified as presynaptic to this cell. Two other innexins also show highly restricted expressions in neurons and may be developmentally regulated. Electronic Supplementary Material Supplementary material is available for this article at .     

Cell shape in the algaPediastrum (Hydrodictyaceae; Chlorophyta)

Summary Species ofPediastrum, a genus in which the colonies assemble from aggregating zoospores, differ in the number and form of prongs on peripheral cells and the amount of space between cells of the colony; cell shape appears to be genetically based. Peripheral cells of theP. boryanum colony, for example, have two prongs per cell;P. simplex has one prong per cell. Prong extension is suppressed in the interior cells ofP. boryanum, but prong sites have been reported in scanning electron micrographs of the cell walls. A mutant unicellular strain in which cells of the colony separate after attaining typical form reveals several prong sites (6 or more) in each cell. Multiple suppressed prong sites are evident inP. simplex cells as well. Polyeders, 4- and 5-pronged unicells, occur in the life cycle ofP. simplex. Based on these observations and a recent report byMarchant (1979) of a microtubule organizing center associated with the prongs, it is suggested that several microtubule organizing centers are to be found in zoospores ofPediastrum species and may be related to species differences in cell shape.Research supported in part by Argonne Center for Educational Affairs, U.S. Department of Energy, under contract No. W-31-109-ENG-38.     

The AP‐2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast

In metazoans the AP‐2 complex has a well‐defined role in clathrin‐mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co‐ immunoprecipitation between the fission yeast AP‐2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4‐64 uptake. In an otherwise wild‐type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP‐2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP‐2 in endocytosis has been conserved throughout evolution.     

Genotypic variability following fusion in the invasive colonial tunicate Didemnum vexillum

Fusion to form a chimera has been documented in many marine invertebrate taxa, including poriferans, cnidarians, bryozoans, and colonial ascidians. Allogenic interactions in chimeric ascidian colonies vary widely across taxonomic groups but are poorly characterized in the invasive colonial ascidian Didemnum vexillum. The moderate level of discrimination expressed in the fusion–rejection response of D. vexillum suggests that there is some integration of cells beyond the fusion line in a chimeric colony. We tracked the shifts in representation of microsatellite alleles between fused colonies of D. vexillum to elucidate the extent of genotypic integration in fused colonies and the patterns of changes to the genotypic composition of colonies immediately following chimera formation. By genotyping colonies before and after fusion, we found that allogeneic fusion in D. vexillum may lead to genotypic changes beyond the visible fusion line. Alleles from one colony were found in multiple tissue samples in the chimera 7–10 days after fusion had occurred. In some instances, alleles that were in a single colony prior to fusion were lost following fusion. We observed multiple patterns of allelic change, including both the unidirectional transfer and reciprocal exchange of alleles between fused colonies. Our findings suggest that tissue or cells are exchanged following allogeneic fusion between colonies of D. vexillum and that the genotypic composition of chimeric colonies may be fluid.     

Identification of a complex genetic network underlying Saccharomyces cerevisiae colony morphology

When grown on solid substrates, different microorganisms often form colonies with very specific morphologies. Whereas the pioneers of microbiology often used colony morphology to discriminate between species and strains, the phenomenon has not received much attention recently. In this study, we use a genome‐wide assay in the model yeast Saccharomyces cerevisiae to identify all genes that affect colony morphology. We show that several major signalling cascades, including the MAPK, TORC, SNF1 and RIM101 pathways play a role, indicating that morphological changes are a reaction to changing environments. Other genes that affect colony morphology are involved in protein sorting and epigenetic regulation. Interestingly, the screen reveals only few genes that are likely to play a direct role in establishing colony morphology, with one notable example being FLO11, a gene encoding a cell‐surface adhesin that has already been implicated in colony morphology, biofilm formation, and invasive and pseudohyphal growth. Using a series of modified promoters for fine‐tuning FLO11 expression, we confirm the central role of Flo11 and show that differences in FLO11 expression result in distinct colony morphologies. Together, our results provide a first comprehensive look at the complex genetic network that underlies the diversity in the morphologies of yeast colonies.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Nonlinearity in the Growth of Bacterial Colonies: Conditions and Causes

The universally recognized kinetic model of colony growth, introduced by Pirt, predicts a linear increase of colony size. The linearity follows from the assumption that the colony expands through the growth of only such cells that are located immediately behind the moving colony front, in the so-called peripheral zone of constant width and density. In this work, Pirt's model was tested on two bacteria—Alcaligenes sp. and Pseudomonas fluorescens—having markedly distinct cultural properties and grown on an agarized medium with pyruvate. The colony size dynamics was followed for different densities of the inoculum, ranging from a single cell to a microdroplet of bacterial suspension (10⁵–10⁶ cells), and for different depths of the agar layer, determining the amount of available substrate. A linear growth mode was observed only with P. fluorescens and only in the case of growth from a microdroplet. When originating from a single cell, colonies of both organisms displayed nonlinear growth with a distinct peak of K _r (the rate of colony radius increase) occurring after 2–3 days of growth. The growth of P. fluorescens colonies showed virtually no dependence on the depth of the agarized medium, whereas the rate of colony size increase of Alcaligenes sp. turned out to be directly related to the medium layer thickness. The departure from linearity is consistently explained by a new kinetic scheme stipulating a possible contribution to the colony growth not only of peripheral cells but also (much more distinct in Alcaligenes) of cells at the colony center. The colony growth dynamics is determined not only by the concentration of the limiting substrate but also by the amount of autoinhibitor, the synthesis of which is governed by the age of cells. The distinctions of growth from a single cell and microdroplet could also originate as a result of dissociation into the R- and S-forms and competition between the corresponding subpopulations for oxygen and the common substrate.     

Polysaccharides from Phellinus linteus inhibit cell growth and invasion and induce apoptosis in HepG2 human hepatocellular carcinoma cells

It has been demonstrated that the medicinal mushroom Phellinus linteus (PL), which consists mainly of polysaccharides, possesses antitumor and immunomodulatory properties in vivo and in vitro. The mechanism, however, by which PL inhibits growth and invasive behavior of HepG2 cells remains poorly understood. Here we demonstrated that PL inhibited proliferation and colony formation of HepG2 and that the growth inhibition of HepG2 cells was mediated by S-phase cell cycle arrest. PL also markedly inhibited cancer cell adhesion and invasion of the extracellular matrix. Additionally, we demonstrated that PL-induced apoptosis was associated with a reduction in B-cell lymphoma 2 levels and an increase in the release of cytochrome c. These results suggest that PL exerts a direct antitumor effect by initiating apoptosis and cell cycle blockade in HepG2 cells.     

DWARF50 (D50), a rice (Oryza sativa L.) gene encoding inositol polyphosphate 5‐phosphatase,is required for proper development of intercalary meristem

Rice internodes are vital for supporting high‐yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell‐producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5‐phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization.     

Model investigation of central regulatory contour of gene net of <Emphasis Type="Italic">D. melanogaster</Emphasis> macrochaete morphogenesis

Morphogenesis of drosophila macrochaete functioning as mechanoreceptors includes several steps, each of which has their own genetic support described in terms of gene nets. Mechanoreceptor develops from one parental cell (Sensory Organ Precursor cell—SOP cell), the determination of which has a critical role in macrochaete development. The highest content of AS-C proneural proteins with respect to surrounding cells that initiate a neural way of cellular development and by means of it mechanoreceptor morphogenesis is typical for SOP cell. The key object of gene net providing parental cell determination consists of gene complex achaete-scute (AS-C). This complex activity is controlled by central regulatory contour (CRC). Besides AS-C, CRC includes the following genes: hairy, senseless (sens), charlatan (chn), scratch (scrt), daughterless (da), extramacrochaete (emc), and groucho (gro). The system of direct relation and feedback and induction and repression relations between CRC components are realized via the coding by these genes proteins. A mathematical model of CRC functioning as a regulator of proneural AS-C protein content in SOP cell determining successful passing of the main phase of morphogenesis of D. melanogaster mechanoreceptor is discussed.     

A tRF-5a fragment that regulates radiation resistance of colorectal cancer cells by targeting MKNK1

Radiotherapy serves as a crucial strategy in the treatment of colorectal cancer (CRC). However, its efficacy is often hindered by the challenge of radiation resistance. Although the literature suggests that some tRNA-derived small RNAs (tsRNAs) are associated with various cancers, studies reporting the relationship of tsRNAs with cancer cell radiosensitivity have not been published yet. In our study, we utilized tsRNAs sequencing to predict differentially expressed tsRNAs in two CRC cells and their radioresistant cells, and 10 tsRNAs with significant differences in expression were validated by qPCR. The target genes of tRF-16-7X9PN5D were predicted and verified by the bioinformatics, dual-luciferase reporter gene assay and western blotting analyses. Wound healing, colony formation, transwell invasion and CCK-8 assays were performed to detect the effects of tRF-16-7X9PN5D on cell function and radiosensitivity. Western blotting evaluated the relationship between tRF-16-7X9PN5D and the MKNK-eIF4E axis. Our findings demonstrated that tRF-16-7X9PN5D expression was substantially downregulated in radioresistant CRC cells. Furthermore, tRF-16-7X9PN5D could promote CRC cells' ability to proliferate, migrate, invade and obtain radiation resistance by targeting MKNK1. Finally, tRF-16-7X9PN5D could regulate eIF4E phosphorylation via MKNK1. This investigation indicated that tRF-16-7X9PN5D has an essential regulatory role in the radiation resistance of CRC by directly targeting MKNK1, and may be a new pathway for regulating the CRC radiosensitivity.     

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Cell lysis and molecular delivery in confluent monolayers of PtK₂ cells are achieved by the delivery of 6 ns, λ = 532 nm laser pulses via a 40×, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3 kDa) FITC‐conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time‐resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam‐induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses τ_{w, max} > 190 ± 20 kPa are immediately lysed while cells exposed to τ_{w, max} > 18 ± 2 kPa are necrotic and subsequently detach. Cells exposed to τ_{w, max} in the range 8–18 kPa are viable and successfully optoporated with 3 kDa Dextran molecules. Cells exposed to τ_{w, max} < 8 ± 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam‐induced shear stresses and subsequent cellular outcome. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Suburban sprawl: environmental features affect colony social and spatial structure in the black carpenter ant,Camponotus pennsylvanicus

1. In social insects, the number of nests that a colony inhabits may have important consequences for colony genetic structure, the number of queens, sex allocation, foraging efficiency, and nestmate recognition. Within the ants, colonies may either occupy a single nest (monodomy) or may be organised into a complex network of nests and trails, a condition known as polydomy. 2. The current study is a large‐scale, long‐term, comprehensive field examination of various features of colony social and spatial structure in the facultatively polydomous black carpenter ant, Camponotus pennsylvanicus (DeGeer). The study examined the density, persistence, and the spatiotemporal distribution of colonies across a gradient of land disturbance associated with urban development. The temporal and spatial pattern of nest use was compared between fragmented landscapes where nesting sites were interspersed among human‐built structures (urban plots) and less disturbed landscapes with higher tree density (suburban plots). In addition, nesting site fidelity and changes in colony spatial structure were monitored over 7 years. 3. Long‐term monitoring and extensive sampling over a large spatial area allowed the first comprehensive insight into the spatiotemporal dynamics of colony and population structure in C. pennsylvanicus. A total of 1113 trees were inspected over 233 ha. Camponotus pennsylvanicus were active on 348 of the 1113 trees (31%) and these represented 182 distinct colonies. The colonisation rate remained relatively stable over 7 years suggesting that an equilibrium point had been reached. Relative to the suburban plots, tree density was 65% lower in the urban plots. The proportion of trees colonised by C. pennsylvanicus was significantly higher in the urban plots suggesting that intraspecific competition for nesting sites may be especially high in areas with lower tree density. Colony spatial structure also differed significantly between habitats and a higher incidence of monodomy was observed in the urban environment. The average number of trees per colony across all subplots was 1.95 (range 1–4) indicating that C. pennsylvanicus are weakly polydomous. 4. The composite picture that emerges for C. pennsylvanicus colonies in the urban habitat is a chain reaction of events: (i) the urban habitat has a lower tree density, (ii) lower tree density results in higher tree colonisation rate, (iii) higher tree colonisation rate results in simpler colony spatial structure (i.e. higher incidence of monodomy), and (iv) simpler colony spatial structure results in numerically smaller colonies. Long‐term monitoring of the spatiotemporal pattern of nest site use in selected colonies revealed a unique trend. While worker counts in selected colonies remained relatively stable throughout the course of the study, colony spatial structure changed considerably with 28% of colonies experiencing a change. Furthermore, the likelihood of detecting a change in colony spatial structure increased with the amount of time passing from the initial inspection. 5. In conclusion, tree density has a significant effect on a number of important colony features in C. pennsylvanicus. Besides tree density, other environmental features such as human‐built structures cause habitat fragmentation and may act as natural barriers to worker dispersal and/or foraging. Such barriers may ultimately affect the social and/or spatial structure at both the colony and the population level.     

Human umbilical cord blood-derived stromal cells: Multifaceted regulators of megakaryocytopoiesis

It has been demonstrated that stromal cell precursors exist in human umbilical cord blood. After being cultured in vitro, these cells are called human umbilical cord blood-derived stromal cells (hUCBDSCs). However, the role of hUCBDSCs in hematopoiesis is still unclear. We have previously shown that hUCBDSCs are superior to human bone marrow stromal cells (hBMSCs) at enhancing the expansion of megakaryocyte colony forming units (CFU-Meg). Based on this observation, we postulated that hUCBDSCs might promote megakaryocytopoiesis. To test this hypothesis, we developed a megakaryocyte/hUCBDSC co-culture model and a hematopoietic microenvironment injury model in nude mice. We explored the ability and mechanisms by which hUCBDSCs promoted the proliferation of megakaryocytes in vitro, and we also explored their capacity to restore the hematopoietic microenvironment in vivo. As expected, hUCBDSCs were more effective than hBMSCs at enhancing the proliferation of megakaryocyte lines from HEL cells and restoring megakaryocytopoiesis in a hematopoietic microenvironment injury model in nude mice. Thrombopoietin (TPO) and stromal cell derived factor-1 (SDF-1) are two of the key factors underlying this capacity. We also found that gap junction intercellular communication (GJIC) between HEL cells and hUCBDSCs might be partially absent. Our data provide the first evidence that hUCBDSCs play a regulatory role during megakaryocytopoiesis, which might be important for designing treatments for patients with megakaryocytic injury.     

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5′ and 3′ deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the β-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9×10⁻⁴, whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8×10⁻⁴. The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.