Laser Scanning Microscopy In Combination Research Articles

Palps across the tree - the neuronal innervation and development of sensory head appendages in Annelida.

Polychaetes inhabit a wide variety of habitats and show a great morphological diversity. In this context, a key morphological structure for adapting to their individual lifestyles and ecological niches are the prominent head appendages. In the last years more and more studies focused on the mainly sensory annelid head appendages - namely the antennae, palps, buccal lips and cirri - to unravel the evolutionary origin and phylogeny of Annelida. Unfortunately, comparable data for most of the polychaete families are lacking so far, especially when it comes to features of the larval anterior nervous system and the related innervation and potential homology of these head appendages. In this study, we therefore use an integrative morphological approach including immunohistochemistry and confocal laser scanning microscopy in combination with histological serial sections and 3D-visualizations. With special focus on the palp-like appendages, our data provides a closer look into the development of the larval anterior nervous system and the related sensory structures of three polychaete families representing major groups of the annelid tree of life. Hence, we investigate members of the palaeoannelid Magelonidae as well as basally-branching Amphinomidae, and the pleistoannelid Spionidae forming a taxon deeply nested within Sedentaria. Our comparative data of larval and adult neuronal features support the homology of feeding-palps across the annelid tree. Furthermore, our observations show that larval palps gradually transform into the adult ones while keeping a very similar neuronal innervation pattern. Solely for Amphinomidae a loss of larval palps during ontogenesis has to be assumed. Therefore, our investigations uncover important and so far unknown details in terms of structural homology across Annelida and provide important results necessary for our understanding of annelid evolution.

Precipitation of Calcium Carbonate Inside Giant Unilamellar Vesicles Composed of Fluid-Phase Lipids.

Biomineralization of CaCO3 commonly involves the formation of amorphous CaCO3 precursor particles that are produced in a confined space surrounded by a lipid bilayer. While the influence of confinement itself has been investigated with different model systems, the impact of an enclosing continuous lipid bilayer on CaCO3 formation in a confined space is still poorly understood as appropriate model systems are rare. Here, we present a new versatile method based on droplet-based microfluidics to produce fluid-phase giant unilamellar vesicles (GUVs) in the presence of high CaCl2 concentrations. These GUVs can be readily investigated by means of confocal laser scanning microscopy in combination with bright-field microscopy, demonstrating that the formed CaCO3 particles are in conformal contact with the fluid-phase lipid bilayer and thus suggesting a strong interaction between the particle and the membrane. Atomic force microscopy adhesion studies with membrane-coated spheres on different CaCO3 crystals corroborated this notion of a strong interaction between the lipids and CaCO3.

Position, orientation, and relative quantum yield ratio determination of fluorescent nanoemitters via combined laser scanning microscopy and polarization measurements

We present a universal method of determining the position, 3D orientation, and relative quantum yield ratio (RQYR) of fluorescent nanoemitters (ZnS coated CdSe quantum dots) in a glass slide by combining laser scanning microscopy (LSM) and polarization measurements. The quantum dots were located through LSM intensity maps using azimuthal, radial, and linear incident polarizations. LSM imaging was not sufficient to determine the orientation of the quantum dots due to the isotropic absorption dipole moment. The 3D orientation was obtained through polarization measurement. By combining LSM and polarization measurements, the RQYR of a single molecule was evaluated, allowing us to compare the quantum yield of the nanoemitters.

Association of tongue brushing with the number of fungiform taste buds and taste perception: A preliminary study using confocal laser scanning microscopy in combination with a filter-paper disc method

ObjectiveThe aim of this study was to investigate the association of tongue brushing with the number of fungiform taste buds and taste perception using a confocal laser scanning microscopy in combination with a filter-paper disc method (FPDM). MethodsTwenty-four subjects with or without a habit of tongue brushing (11 males and 13 females, 20–46 years old) participated in this study. Nine of the 24 subjects had no habit of tongue brushing (Group 1, n=9). Fifteen subjects had a habit of tongue brushing, and the brushing regions of the tongue were as follows: central region (Group 2, n=7), or entire region (Group 3, n=8) of the tongue dorsum. Using confocal laser scanning microscopy, the average number of taste buds per fungiform papilla (FP) was counted. Taste perception was evaluated using an FPDM. These observations were performed in the midlateral region of the tongue since the distribution of fungiform papillae is large in the midlateral region compared to that in the central region. ResultsThe subjects in Group 3 showed a significantly decreased number of fungiform taste buds compared to Group 1 and Group 2. Group 3 also showed significantly higher FPDM scores than the other two groups. ConclusionsExcessive tongue brushing of the entire tongue dorsum, including the midlateral region, may have an association with the decreased number of FP and taste buds and decreased taste sensation. To avoid these conditions, instituting proper tongue brushing methods, such as limiting it to the central region of the tongue and using a light touch, is suggested and is important for the subjects who are eager to participate in tongue brushing.

Mechanism of the Peritectic Phase Transition in Fe–C and Fe–Ni Alloys under Conditions Close to Chemical and Thermal Equilibrium

In-situ studies of the peritectic phase transition were performed under conditions close to chemical and thermal equilibrium in order to investigate the governing mechanism of both the peritectic reaction and the subsequent peritectic transformation using high-temperature laser-scanning microscopy in combination with the concentric solidification technique. Experiments have been conducted in the Fe–C and Fe– Ni systems in order to determine the role of solute diffusion on the observed reaction and transformation kinetics. The peritectic reaction as such as well as the subsequent peritectic transformation can be explained by diffusion-controlled mechanisms. Observations of the triple point L/γ/δ revealed that the peritectic reaction can be described as the solidification of γ along the L/δ interface, and new insights have been gained on the localized re-melting of δ during the peritectic reaction.

Reaction patterns of wheat starch granules substituted with an anionic propylene oxide analog (POA) revealed by confocal microscopy and 3D anaglyph imaging

A technique was developed for investigating granular reaction patterns of anionic starch derivatives, utilizing the optical sectioning capability of reflectance confocal laser scanning microscopy in combination with 3D anaglyphic imaging (3D glasses required for visualization). The method facilitated improved visualization of internal granular reaction patterns by discarding optical sections comprising the highly-reacted external granule surface (generating 3D image constructs from sections constituting exclusively internal regions of granules) and minimizing confounding background signal associated with non-starch constituents (e.g., proteins). For wheat starch A-type granules substituted with a propylene oxide analog (POA), granular reaction patterns revealed that reagent reached the hilum region of granules through channels, and primarily entered the granule matrix via lateral diffusion from channel surfaces, producing relatively homogeneous reaction patterns. The homogeneous nature of POA granular reaction patterns was attributed to the relatively low reactivity of the reagent (rate of diffusion>rate of reaction).

Effect of Low Direct Current on Anaerobic Multispecies Biofilm Adhering to a Titanium Implant Surface

Peri-implantitis is caused by biofilm adhering to the implant. It has been shown that bactericidal electrolysis products are generated when a low direct current is applied to a titanium implant used as the anode. The hypothesis of this study was that low-current electrolysis would eradicate viable bacteria in a simulated subgingival multispecies biofilm adhering to a titanium implant surface. Biofilms consisting of eight anaerobic species were grown on pellicle-coated titanium discs with sand-blasted, acid-etched, large-grit (SLA; Straumann, Basel, Switzerland) surface. After 40.5 hours of growth, discs were treated with 10 mA for 10 minutes in an electrolytical setup with physiological saline and gelatin. Low direct current at discs used as the cathode caused a reduction of three to four orders of magnitude in viable counts, while no viable bacteria were recovered from anode discs (Mann-Whitney U-test, p < .01). Confocal laser scanning microscopy in combination with a live/dead stain showed biofilm detachment at the cathode and reduced viability at the anode. Electrochemical treatment of diseased implants appears to be promising and well worth investigating further.

Growth and characterization of Escherichia coli DH5α biofilm on concrete surfaces as a protective layer against microbiologically influenced concrete deterioration (MICD)

Biofilms of selected bacteria strains were previously used on metal coupons as a protective layer against microbiologically influenced corrosion of metals. Unlike metal surfaces, concrete surfaces present a hostile environment for growing a protective biofilm. The main objective of this research was to investigate whether a beneficial biofilm can be successfully grown on mortar surfaces. Escherichia coli DH5α biofilm was grown on mortar surfaces for 8days, and the structure and characteristics of the biofilm were studied using advanced microscopy techniques such as scanning electron microscopy and confocal laser scanning microscopy in combination with fluorescence in situ hybridization, live/dead, extracellular polymer staining, ATP analysis, and membrane filtration. A biofilm layer with a varying thickness of 20-40μm was observed on the mortar surface. The distribution of live and dead bacteria and extracellular polymers varied with depth. The density of the live population near the mortar surface was the lowest. The bacteria reached their highest density at three fourths of the biofilm depth and then decreased again near the biofilm-liquid interface. Overall, the results indicated a healthy biofilm growth in the chosen growth period of 8days, and it is expected that longer growth periods would lead to formation of a more resistant biofilm with more coverage of mortar surfaces.

Effects of Long Exposure to Low Temperatures on Nitrifying Biofilm and Biomass in Wastewater Treatment

Attached growth biological treatment systems are a promising solution to ammonia removal in cold-temperature climates. Environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy in combination with fluorescent in situ hybridization (FISH) was used to investigate the effects of 4 months of exposure to 4 degrees C on nitrifying biofilm and biomass. These molecular and microscopic methods were modified to minimize loss of mass and distortion of in situ perspectives. Environmental scanning electron microscopy revealed that nitrifying biofilm did not exhibit significant changes in volume with exposure to 4 degrees C. Confocal laser scanning microscopy in combination with FISH showed that the number of ammonia-oxidizing bacteria (AOB) cells present in the biofilm was statistically consistent during exposure to 4 degrees C. The RNA content of AOB cells remained sufficient for FISH enumeration. The number of nitrite-oxidizing bacteria cells remained steady during exposure to 4 degrees C; however, the RNA content of the cells appeared to decrease with exposure to 4 degrees C, thereby preventing their enumeration using FISH.

English

With two-photon and confocal laser scanning microscopy in combination with fluorescent probes Hoechst 33342, 2&cent;,7&cent;-dichlorofluorescin diacetate (DCFH-DA) and Fluo 3-AM, we simultaneously observed arsenic trioxide (As2O3)-induced changes in nuclear morphology, reactive oxygen species (ROS) and intracellular calcium concentration [Ca2+]i&nbsp;within human skin squamous carcinoma cells (Colo-16 cells). Our results indicated that As2O3&nbsp;induced [Ca2+]i&nbsp;elevation and ROS production within Colo-16 cells, and both [Ca2+]i&nbsp;elevation and ROS production were involved in the apoptosis of Colo-16 cells. These results suggested that two-photon and confocal laser scanning microscopy might provide a real-time, sensitive and noninvasive method for simultaneously multi-parameter observation of As2O3-induced apoptosis at the single cell level. &nbsp; Key words:&nbsp;Two-photon laser scanning microscopy, confocal laser scanning microscopy, human skin squamous carcinoma cells (Colo-16 cells), arsenic trioxide, apoptosis.

Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

BackgroundCoagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.MethodsCoagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.ResultsMost coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.ConclusionWe conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.

Protozoan impact on bacterial biofilm formation

Protozoan impact on bacterial biofilm formation Confocal laser scanning microscopy in combination with digital image analysis was used to assess the impact of protozoa on bacterial colonisation of surfaces. Bacterial biofilms were developed from activated sludge in microscope flow cells and were exposed to the grazing pressure of protozoa. The protozoan community from healthy activated sludge and a culture of flagellate Bodo saltans were used as grazers. Experiments comprised 48-h incubations in 3 treatment variants: bacteria with protozoa, bacteria with protozoa added after some time and bacteria without protozoa. When necessary, the elimination of protozoa from the inoculum was carried out with cycloheximide and NiSO4. Experiments demonstrated that protozoa from healthy activated sludge initially disturbed the biofilm development but later they could stimulate its growth. Similar results could be established in the experiment with Bodo saltans (inoculum: 1000 cells/ml), however differences were not statistically significant. The finding that protozoa support biofilm development during specific stages may be relevant for biofilm studies with mixed environmental biofilm communities.

Viral ecology of organic and inorganic particles in aquatic systems: avenues for further research.

Viral abundance and processes in the water column and sediments are well studied for some systems; however, we know relatively little about virus-host interactions on particles and how particles influence these interactions. Here we review virus-prokaryote interactions on inorganic and organic particles in the water column. Profiting from recent methodological progress, we show that confocal laser scanning microscopy in combination with lectin and nucleic acid staining is one of the most powerful methods to visualize the distribution of viruses and their hosts on particles such as organic aggregates. Viral abundance on suspended matter ranges from 105 to 1011 ml-1. The main factors controlling viral abundance are the quality, size and age of aggregates and the exposure time of viruses to aggregates. Other factors such as water residence time likely act indirectly. Overall, aggregates appear to play a role of viral scavengers or reservoirs rather than viral factories. Adsorption of viruses to organic aggregates or inorganic particles can stimulate growth of the free-living prokaryotic community, e.g. by reducing viral lysis. Such mechanisms can affect microbial diversity, food web structure and biogeochemical cycles. Viral lysis of bacterio- and phytoplankton influences the formation and fate of aggregates and can, for example, result in a higher stability of algal flocs. Thus, viruses also influence carbon export; however, it is still not clear whether they short-circuit or prime the biological pump. Throughout this review, emphasis has been placed on defining general problems and knowledge gaps in virus-particle interactions and on providing avenues for further research, particularly those linked to global change.

Cellular Interaction Between Human Mesenchymal Stem Cells and Hematopoietic Stem Cells in 2D- and 3D-Culture-Systems.

Abstract 1442Poster Board I-465We previously characterized the interaction between human hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) in a 2D-coculture-system and demonstrated that MSC-feeder-layer is capable of maintaining “stemness” in HSC. In order to develop a more physiological in vitro surrogate model for the stem cell niche, we established a 3D-coculture-system using MSC-spheroids. In this model system we analyzed the role of major adhesion molecules such as N-cadherin and the CXCR4/SDF-1 axis upon migration, adhesion and proliferation of HSC.MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood by magnetic cell sorting of CD34+ mononuclear cells. MSC were cultured as “hanging drops” with initially around 500 cells. Alternatively, MSC were seeded on a layer of 1% agarose in 96 well plates (500-1000 cells per well). In both cases after 24-48h forming of spheroid-like cell aggregations could be observed. Formation of spheroids was obviously independent upon different MSC culture conditions. MSC-spheroids were cocultured with HSC and continuously monitored with time-lapse microscopy. After 24-48 hours of cocultivation, we stained the cellular contacts with a panel of antibodies specific for various components of tight, gap and adherens junctions. Using advanced confocal laser scanning microscopy in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC and MSC. Additionally, thin sections of the spheroids were analyzed by electron microscopy.In 2D-cocultures, intercellular connections between HSC and MSC are mainly characterized by podia formation of the HSC linking to the adjacent MSC. At the intimate contact zone to the MSC, the cytoplasmic plaque proteins alpha- and beta-catenin, as well as the transmembrane glycoprotein N-cadherin could be identified. Upon 3D-coculture of HSCs with MSC-spheroids, HSCs showed directed locomotion towards MSC-spheroids. After 12h several HSC showed directed locomotion towards MSC-spheroids and connected to the outer cell layer by podia formation. At the contact zone cadherin-catenin-based junctions could be identified. After additional 12-24h a further integration of HSC into the inner mass of the spheroid was observed. Cell division kinetic of HSC was increased when cocultured with MSC and the rate of CD34+ cells remained higher compared to monoculturing of HSC in the same culture-medium. Culturing HSC on MSC feeder-layer resulted in a reduced CXCR4 expression, possibly due to an internalization triggered by direct cell contact to MSC.This novel 3D-setup of an in vitro surrogate model for the human stem cell niche provides deeper inside into the cellular interaction between HSC and stromal cells and demonstrates that direct cellular contact influences cell division kinetics and CXCR4 expression of HSC. DisclosuresNo relevant conflicts of interest to declare.

Human Hematopoietic Stem Cells and Leukemic Cells Form Cadherin-Catenin Based Junctional Complexes with Mesenchymal Stromal Cells

The interaction between human hematopoietic stem cells (HSC) and their niche plays a key role in regulating maintenance of “stemness” and differentiation. We have demonstrated that a feeder layer of human mesenchymal stromal cells (MSC) can serve as a surrogate model for the niche for human HSC. We could also show, MSC are intimately connected to one another by a novel kind of adhering junction, consisting of villiformto-vermiform cell projections (processus adhaerentes). With this background, we have analyzed the intercellular junctional complexes between HSC and MSC. In comparison, we also studied the cell-cell contacts between leukemia cells (LC) and MSC.MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood. Leukemia cells that were CD34+ were obtained from bone marrow aspirates from patients suffering from acute myeloid leukemia at the time point of initial diagnosis. After 24–48 hours of co-cultivation, we stained the cellular contacts with a panel of antibodies specific for various components of tight, gap and adherens junctions. Using advanced confocal laser scanning microscopy in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC/MSC as well as between LC/MSC. To examine the specific function of N-cadherin, we analyzed the effect of siRNA knock down of N-cadherin in MSC upon co-cultures of HSC and MSC.Intercellular connections between HSC and MSC are mainly characterized by podia formation of the HSC linking to the adjacent MSC. At the intimate contact zone to the MSC, we have identified the cytoplasmic plaque proteins alpha- and beta-catenin, co-localized with the transmembrane glycoprotein N-cadherin. Additionally, we compared these findings with a similar setting consisting of human LC co-cultured with feeder-layer of MSC. Our results demonstrated that in comparison to HSC, the proportion of leukemia cells adherent to the feeder-layer is significantly lower and podia formation is less frequent (ratio 1:3). However, the mechanism of adhesion through cadherin-catenin-complex has remained the same. At a functional level, we found that siRNA knock down of N-cadherin in MSC resulted in decreased adhesion of HSC to MSC and in a reduction of cell divisions of HSC. These results confirm that direct cellular contact via N-cadherin-based junctions is essential for homing and adhesion of HSC to the cellular niche and subsequently for the regulation of self-renewal versus differentiation in HSC.

Human Hematopoietic and Mesenchymal Stem Cells Are Interconnected by Cadherin-Catenin Based Junctions.

The direct cell-cell contact between human hematopoietic stem cells (HSC) and their niche has been shown to control stem cell fate by regulating self-renewal versus differentiation. In previous studies we have demonstrated gap and adherens junctions arranged in complex villiform, vermiform, intercalating protrusions and invaginations among mesenchymal stem cells (MSC). Specific cadherin-catenin-molecules, among other junction proteins, were found in these contact systems. Using MSC-feeder-layer as an in vitro surrogate model for the hematopoietic stem cell niche, we have analyzed the intercellular junctional complexes between HSC and MSC in this study. Additionally, we characterized cell-cell-connections between leukemia stem cells (LSC) and MSC. MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood. Leukemia stem cells were obtained from bone marrow aspirates from patients suffering from acute myeloid leukemia either at the time of initial diagnosis or at relapse after chemotherapy. After 24–48 hours of co-cultivation, we have stained the cell-cell contacts with a panel of antibodies specific for various components of tight, gap and adherens junctions including alpha-/beta-catenin, N-cadherin, Cadherin 11, E-Cadherin, p120, Vinculin and Connexin 43. Using advanced confocal laser scanning microscopy in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC/MSC as well as between LSC/MSC. We have confirmed previous observations that HSC demonstrated directed locomotion towards a MSC gradient. HSC then migrated around the MSC and some of them are then interconnected by podia formation to the MSC. These podia are directly linked to the MSC feeder layer and at the intimate contact zone we have identified N-cadherin as well as alpha-/beta-catenin as the main junction proteins. The specific function of this cadherin/catenin-complex is probably associated with redistribution of beta-catenin and hence to self-renewing division, similar to the findings in stem cells of drosophila model systems. Functional analyses are currently underway to confirm this hypothesis. We have also compared these findings with a similar setting consisting of human LSC co-cultured with MSC-feeder-layer.

Exovesicles from Human Activated Dendritic Cells Fuse with Resting Dendritic Cells, Allowing Them to Present Alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Confocal microscopy analysis of native, full length and B-domain deleted coagulation factor VIII trafficking in mammalian cells.

In mammalian cells, factor VIII (FVIII) secretion depends upon its interaction with chaperones of the endoplasmic reticulum (ER) and requires a unique ATP-dependent step to dissociate aggregates formed within the ER. To further elucidate mechanisms which might account for the inefficient secretion of recombinant FVIII (rFVIII), we have analyzed the pathways of recombinant full length (rFVIII-FL) and B-domain deleted (rFVIII Delta B) FVIII and compared these to the secretion route of native FVIII in primary hepatocytes. Using confocal laser scanning microscopy in combination with a pulse chase of a known secretion marker, we describe the trafficking route of FVIII, which upon release from the ER--where it colocalizes with calnexin--is transported to the Golgi complex in vesicular-tubular transport complexes (VTCs) which could be further identified as being COP I coated. However, a large portion of rFVIII is retained in the ER and additionally in structures which could not be assigned to the ER, Golgi complex or intermediate compartment. Moderate BiP transcription levels indicate that this observed retention of FVIII does not reflect cellular stress due to an overexpression of FVIII-protein in transduced cells. Moreover, a pulse of newly synthesized rFVIII protein is released within 4 hrs, indicating that once rFVIII is released from the ER there is no further limitation to its secretion. Our data provide new details about the secretory route of FVIII, which may ultimately help to identify factors currently limiting the efficient and physiological expression of FVIII in gene therapy and manufacture.

Simultaneous multi-parameter observation of Harringtonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca(2+)](i)) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug-treating living cells at the level of single cell.

Neochlamydia hartmannellae gen. nov., sp. nov. (Parachlamydiaceae), an endoparasite of the amoeba Hartmannella vermiformis.

Free-living amoebae are increasingly being recognized to serve as vehicles of dispersal for various bacterial human pathogens and as hosts for a variety of obligate bacterial endocytobionts. Several Chlamydia-like Acanthamoeba endocytobionts constituting the recently proposed family Parachlamydiaceae are of special interest as potential human pathogens. In this study coccoid bacterial endocytobionts of a Hartmannella vermiformis isolate were analysed. Infection of H. vermiformis with these bacteria resulted in prevention of cyst formation and subsequent host-cell lysis. Transfection experiments demonstrated that the parasites were not capable of propagating within other closely related free-living amoebae but were able to infect the distantly related species Dictyostelium discoideum. Electron microscopy of the parasites revealed typical morphological characteristics of the Chlamydiales, including the existence of a Chlamydia-like life-cycle, but indicated that these endocytobionts, in contrast to Chlamydia species, do not reside within a vacuole. Comparative 16S rRNA sequence analysis showed that the endocytobiont of H. vermiformis, classified as Neochlamydia hartmannellae gen. nov., sp. nov., is affiliated to the family Parachlamydiaceae. Confocal laser scanning microscopy in combination with fluorescence in situ hybridization using rRNA-targeted oligonucleotide probes confirmed the intracellular localization of the parasites and demonstrated the absence of other bacterial species within the Hartmannella host. These findings extend our knowledge of the phylogenetic diversity of the Parachlamydiaceae and demonstrate for the first time that these endocytobionts can naturally develop within amoebae of the genus Hartmannella.