Effects of bromide and iodide on stalk secretion in the biofouling diatom Achnanthes longipes (Bacillariophyceae)

Extracellular polymeric substance (EPS) secretion was examined in the stalked marine diatom Achnanthes longipes Ag. in defined medium. This common biofouling diatom exhibited an absolute requirement for bromide for stalk production and substratum attachment, whereas elevated iodide concentrations in the growth medium inhibited stalk formation and adhesion. Varying EPS morphologtes resulted from altering bromide and iodide levels: pads, stalk‐pads, stalks, and no EPS. Cells showed no differences in growth with bromide or iodide concentrations, indicating that they were not physiologically stressed under conditions that impaired EPS secretion. Cells grown in elevated iodide secreted significantly more soluble extracellular carbohydrate into the medium, suggesting that the EPS was soluble and unable to be polymerized into a morphologically distinct gel. By replacing sulfate with methionine, the diatom lost its ability to form stalks even in the presence of bromide, indicating that free sulphate may be required for proper cross‐linking of stalk polymers. Lotus‐FITC, a fluorescent‐tagged lectin, preferentially labeled the EPS and, thus, was used to visualize and quantify EPS secretion along a bromide gradient in conjunction with an image analysis system. This technique demonstrated a direct correlation between the amount of bromide present in the medium and the specific EPS morphology formed. Copyright © 1995, Wiley Blackwell. All rights reserve

Identifying plant cell wall remnants in detritus of a subtropical wetland with fluorescence labeling

Sediment accretion in wetlands represents a significant carbon burial pathway. While litter studies can quantify the loss rates of plant leaf material, those studies do not provide insight into the specific cell wall polymers being retained or lost within the detrital matrix. The Everglades ecosystem has been dramatically altered due to anthropogenic eutrophication and hydrologic modifications. The results are changes in macrophyte species composition and sediment accretion- and loss- rates. To improve ecological conditions, active management strategies are re-establishing open water slough environments. A question remains about the persistence of new- and old- plant cell wall material in sediments because of active management. In this pilot project we utilized immuno-fluorescence labeling with lectins applied to plant leaf material and detrital flocculent collected from created open and control plots in the Everglades to observe the presence, absence, and overlap of specific cell wall polymers between macrophytes and detrital flocculent in increasingly recalcitrant materials that would most likely contribute to peat accumulation. The persistence and loss of specific polymers between treatment and control plots provided insight into the differing levels of recalcitrance amongst plant cell walls and their relative potential as a carbon sink. This study provides a novel method for testing for the presence and persistence of specific cell wall polymers in detritus to gain a better understanding of plant material persistence in wetland ecosystems

Social media use, connectedness, and depression in graduate students

This study will examine the relationship between social media use, sense of connectedness, and depression among graduate students. Graduate students are often disconnected from their social supports due to their program\u27s demands; additionally, the risk of depression in this population has been established in the literature. The impact of social media on depression has been investigated, and the results are discrepant. The relationship between a sense of connection, the use of social media, and depression in a graduate student population has not yet been explored. Graduate students at The Philadelphia College of Osteopathic Medicine (PCOM) and other universities studying psychology, medicine, biomedical science, or physician assistant studies will be included. Participants will be recruited through email and social media platforms. A control group of participants that do not use social media will be included. The following measures will be used: The Social Connectedness Scale-Revised (SCS-R), The Generalized Problematic Internet Use Scale 2 (GPIUS2), and The Patient Health Questionnaire-9 (PHQ-9). An inverse relationship is predicted between connectedness and depression. Participants with high scores on problematic internet use are anticipated to have elevated levels of depression and a low sense of connection compared to participants with low scores on problematic use. The subscales from the GPIUS2 are hypothesized to be predictive of participants\u27 sense of connection

Dynamics of extracellular polymeric substance (EPS) production and loss in an estuarine, diatom-dominated, microalgal biofilm over a tidal emersion-immersion period.

We studied patterns of production and loss of four different extracellular polymeric substance (EPS) fractions - colloidal carbohydrates, colloidal EPS (cEPS), hot water (HW)-extracted and hot bicarbonate (HB)-extracted fractions - and community profiles of active (RNA) bacterial communities by use of Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of reverse transcription-polymerase chain reaction amplified 16S rRNA in mudflats in the Colne Estuary, United Kingdom, over two tidal emersion-immersion cycles. Colloidal carbohydrates and intracellular storage carbohydrate (HW) increased during tidal emersion and declined during tidal cover. The dynamics of cEPS and uronic acid content were closely coupled, as were the HB fraction and HB uronic acids. Changes in monosaccharide profiles of HW and HB fractions occurred during the diel period, with some similarity between cEPS and HB fractions. Increasing enzymatic release rates of reducing sugars and increased reducing sugar content were correlated with increased concentrations of colloidal carbohydrate and cEPS during the illuminated emersion period, and with the amount of HB-extracted uronic acids (the most refractory EPS fraction measured). Loss of reducing sugars was high, with sediment concentrations far below those predicted by the measured in situ release rates, T-RFLP analysis revealed no significant shifts in the overall taxonomic composition of the active bacterial community. However, 12 of the 59 terminal restriction fragments identified showed significant changes in relative abundance during the tidal cycle. Changes in the relative abundance of three particular terminal restriction fragments (bacterial taxa) were positively correlated to the rate of extracellular hydrolysis. Losses of chlorophyll a and colloidal and cEPS (up to 50-60) occurred mainly in the first 30 min after tidal cover. About half of this may be owing to in situ degradation, with "wash away" into the water column accounting for the remainder. © 2006, by the American Society of Limnology and Oceanography, Inc

Association of T-Zone Reticular Networks and Conduits with Ectopic Lymphoid Tissues in Mice and Humans

Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node–like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin+ T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell–rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter–CXCL13–transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell–rich zone

Microscopy of bacterial translocation during small bowel obstruction and ischemia in vivo – a new animal model

BACKGROUND: Existing animal models provide only indirect information about the pathogenesis of infections caused by indigenous gastrointestinal microflora and the kinetics of bacterial translocation. The aim of this study was to develop a novel animal model to assess bacterial translocation and intestinal barrier function in vivo. METHODS: In anaesthetized male Wistar rats, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli was administered by intraluminal injection in a model of small bowel obstruction. Animals were randomly subjected to non-ischemic or ischemic bowel obstruction. Ischemia was induced by selective clamping of the terminal mesenteric vessels feeding the obstructed bowel loop. Time intervals necessary for translocation of E. coli into the submucosal stroma and the muscularis propria was assessed using intravital microscopy. RESULTS: Bacterial translocation into the submucosa and muscularis propria took a mean of 36 ± 8 min and 80 ± 10 min, respectively, in small bowel obstruction. Intestinal ischemia significantly accelerated bacterial translocation into the submucosa (11 ± 5 min, p < 0.0001) and muscularis (66 ± 7 min; p = 0.004). Green fluorescent protein-transfected E. coli were visible in frozen sections of small bowel, mesentery, liver and spleen taken two hours after E. coli administration. CONCLUSIONS: Intravital microscopy of fluorescent bacteria is a novel approach to study bacterial translocation in vivo. We have applied this technique to define minimal bacterial transit time as a functional parameter of intestinal barrier function

Quantum Dots for Tracking Dendritic Cells and Priming an Immune Response In Vitro and In Vivo

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-γ production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c+ DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 µm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-γ production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response

Murid Herpesvirus-4 Exploits Dendritic Cells to Infect B Cells

Dendritic cells (DCs) play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4), infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells

Extracellular matrix assembly in diatoms (Bacillariophyceae). IV. Ultrastructure of Achnanthes longipes and Cymbella cistula as revealed by high-pressure freezing/freeze substituton and cryo-field emission scanning electron microscopy

Extracellular matrix (ECM) polymers secreted by the diatoms Achnanthes longipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external environment. To preserve details of the highly hydrophilic ECM in the native state and to preserve, with a high degree of fidelity, the intracellular structures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high-resolution visualization of surfaces using cryo-field emission SEM (cryo-FESEM) and preservation for TEM observation of thin sections by high-pressure freezing (HPF) and freeze substitution (FS). The extracellular structures of diatoms plunge-frozen in liquid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those observed with light microscopy. Cryo-FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell-substratum and cell-cell associations and revealed details of cell attachment processes not visible using other SEM techniques or light microscopy. The layer of ECM coating the frustule and entirely encapsulating cells of A. longipes and C. cistula was shown to have a significant role in initial cell adhesion and subsequent interaction with the environment. Trails of raphe-associated ECM, generated during cell motility, were shown at high resolution and consist of anastomoses of coiled and linear strands. Cryo-FESEM revealed a sheet-like mucilage covering stalks. HPF/FS of A. longipes resulted in excellent preservation of intra- and extracellular structures comparable to previous reports for animals and higher plants and revealed several organelles not described previously. Three distinct vesicle types were identified, including a class closely associated with Golgi bodies and postulated to participate in formation of the extracellular adhesive structures. HPF/FS showed a number of continuous diatotepic layers positioned between the plasma membrane and the silicon frustule and revealed that extracellular adhesive extrusion through frustule pores during stalk production was closely related to the diatotepum. The stalks of A. longipes consist of highly organized, multilayered, fine fibrillar materials with an electron-opaque layer organized as a sheath at the stalk periphery