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Real‐time forecasted ice information for large lakes, such as the Great Lakes, is critical for essential operations, such as ice breaking, commercial navigation, search and rescue, and oil spill response. Existing forecast products for large lake ice conditions are not available for medium‐range time horizons (5–16 days out), yet they could provide important information for decision making, particularly for ice breaking and spill responses. In addition, ice forecasts for Earth's largest lakes at these timescales could be important for Medium‐Range Weather (MRW) forecasting. However, the skill of existing operational products in predicting ice conditions at MRW timescales has not been studied. This work aims to determine how well ice forecasts from a coupled large lake hydrodynamic‐ice model perform for MRW forecast horizons. Simulations were carried out for the 2022 Great Lakes ice season, using 8 different 16‐day forecast periods. Forecast results were compared to observations of meteorology and ice conditions from the U.S. National Ice Center. Results show the MRW ice forecasts in the Great Lakes outperform persistence‐based forecasts. These findings could inform the development or extension of lake operational ice forecasting and the potential of coupling between atmospheric and large lake models at medium‐range forecast time scales. Plain Language Summary: Ice information for large lakes is critical for essential operations, such as ice breaking, commercial navigation, search and rescue, and oil spill response. Ice forecasts are currently not available in the medium‐range (5–16 days out) time scale, yet they could provide valuable information for decision making. This information could also be important for medium‐range weather forecasts. The skill of existing Great Lakes ice forecast products at medium‐range time scales is currently unknown. Our work aims to determine how well ice forecasts from an atmosphere‐lake model perform for medium‐range time scales. Simulations were carried out for the 2022 Great Lakes ice season, using 8 different 16‐day forecast periods. The modeled forecasts were compared to observations from the National Ice Center and observed meteorological conditions. Results show that the modeled forecasts outperform persistence forecasts. These findings could inform the development or extension of ice forecasting and the potential of an atmosphere‐lake model at medium‐range time scales. Key Points: Medium‐range (5‐ to 16‐day) ice forecasts show utility in the Great LakesModel ice forecasts outperform ice persistence forecastsAir temperature bias in medium‐range weather forecasts is the likely greatest contributor to ice error [ABSTRACT FROM AUTHOR]

Sulforaphane (SFN) is present in plants belonging to Cruciferae family and was first isolated from broccoli sprouts. Chemotherapeutic and anticarcinogenic properties of sulforaphane were demonstrated, however, the underlying mechanisms are not fully understood. In this study we evaluated the expression of cyclin D1 and p21 protein in SFN-treated A549 cells and correlated these results with the extent of cell death and/or cell cycle alterations, as well as determined a potential contribution of cyclin D1 to cell death. A549 cells were treated with increasing concentrations of SFN (30, 60 and 90 µM) for 24 h. Morphological and ultrastructural changes were observed using light, transmission electron microscope and videomicroscopy. Image-based cytometry was applied to evaluate the effect of SFN on apoptosis and the cell cycle. Cyclin D1 and p21 expression was determined by flow cytometry, RT-qPCR and immunofluorescence. siRNA was used to evaluate the role of cyclin D1 in the process of suforaphane-induced cell death. We found that the percentage of cyclin D1-positive cells decreased after the treatment with SFN, but at the same time mean fluorescence intensity reflecting cyclin D1 content was increased at 30 µM SFN and decreased at 60 and 90 µM SFN. Percentage of p21-positive cells increased following the treatment, with the highest increase at 60 µM SFN, at which concentration mean fluorescence intensity of this protein was also significantly increased. The 30-µM dose of SFN induced an increased G2/M phase population along with a decreased polyploid fraction of cells, which implies a functional G2/M arrest. The major mode of cell death induced by SFN was necrosis and, to a lower degree apoptosis. Transfection with cyclin D1-siRNA resulted in significantly compromised fraction of apoptotic and necrotic cells, which suggests that cyclin D1 is an important determinant of the therapeutic efficiency of SFN in the A549 cells.

Objective: Activation of receptor for advanced glycation end products (RAGE) has been implicated in many chronic diseases, including diabetic complications. In this study we examined the relationship between RAGE expression and the morphological changes seen in the gingiva of diabetic and periodontitis patients., Design: Gingival biopsies from 15 diabetic patients with periodontitis, 25 non diabetic patients with periodontitis and 10 healthy individuals were collected. Sections were stained with haematoxylin and eosin, and immunohistochemically to detect RAGE. Samples were examined in light and fluorescence microscopes and histomorphometric analysis was performed., Results: Increased number of inflammatory cells and changes in collagen and vasculature were observed in diabetic and non diabetic patients with periodontitis. RAGE was weakly expressed in healthy gingiva. The strongest reaction with anti-RAGE antibody was found in the gingiva of diabetic patients with periodontitis followed by the severe periodontitis patients. RAGE expression in inflammatory cells was stronger than in the epithelium. The number of inflammatory cells in the gingiva was higher in the diabetic periodontitis patients than in the non diabetic severe periodontitis patients., Conclusions: RAGE is strongly expressed in the gingiva of diabetic patients with periodontitis and with severe periodontitis alone, the latter indicating RAGE activation even in the absence of hyperglycemia. However our findings are based on relatively small sample size. With a larger patient population, some of our other findings may have reached statistical significance., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Filamentous phage as a bacteria-specific virus can be conjugated with an anticancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and rearrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells.

In this study, we have evaluated the use of ultra-sterile alginate hydrogels encapsulated with HepG2 liver cells for applications in high throughput drug screening. We have studied the cellular viability and metabolic capacity of the encapsulated cells in two different alginate structures SLM100 (G:M::40:60) and SLG100 (G:M::60:40). We have also developed protocols to characterize the encapsulated cells within the alginate structure using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Further we have studied the Phase-I/II metabolic characteristics of the encapsulated cells in monolayer and 3D culture. Our results indicate that cells encapsulated within SLM100 and SLG100 class of alginates have shown high cellular viability with >80% even after 14 days in culture. As expected, the proliferation rates of the encapsulated cells are held steady and do not proliferate within the gels. Production of liver-specific enzymes such as CYP1A1 and CYP3A4 after 14 days in culture indicates the viability and functionality of the encapsulated HepG2 cells. Phase-II Glutathione activity of the encapsulated cells were also maintained in 3D culture conditions. The encapsulated cells within the 3D gels were also capable of metabolizing the pro-drug EFC (7-ethoxy-4-trifluoromethyl coumarin) to HFC (7-hydroxy-4-trifluoromethyl) in a linear fashion over a period of time. These results have provided us with baseline results to benchmark future improvements in material and design configurations for optimal pharmacokinetic response of in vitro tissue model systems., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Filamentous bacteriophage has been proposed as a vehicle that can carry and deliver therapeutics into mammalian cells for disease treatment, thus a protocol for imaging phage-cell interaction is essential. Because high signal intensity is necessary to study the mechanism of interaction between filamentous bacteriophage and mammalian cells, it is important to optimize the procedure for fluorescence labeling of phage in order to understand such interaction. Here, we describe a procedure that gives intense fluorescence labeling and can show interactions between fd-tet bacteriophage selected from phage libraries and mammalian cells (SKBR-3 and MCF-10A). The indirect labeling of phage with dye-conjugated antibody and cytoskeleton associated proteins was significantly enhanced in the presence of a cross-linking reagent called dithiobissuccinimidylpropionate (DSP) as shown by qualitative and quantitative fluorescence microscopy. The use of DSP resulted in high signal intensity in fluorescence imaging of phage-cell complex. The DSP cross-linker is believed to preserve soluble unbound proteins for fluorescence imaging. The interaction between the phage and mammalian cells was further confirmed by scanning electron microscopy., ((c) 2009 Wiley-Liss, Inc.)

Currently, information regarding the influence of growth factors on the cytoskeleton, including G-CSF and GMCSF, remains limited. In the present study we show alterations in F-actin distribution and cell cycle progression in HL-60 promyelocytic leukemia cells, resulting from treatment with these cytokines in vitro. We found that both agents caused F-actin reorganization. Although multiple potential effects of various growth factors have been described previously, in our experimental conditions, we observed some rather subtle differences between the effects of G-CSF and GM-CSF on studied cells. The presence of these cytokines in the cell environment caused not only increased F-actin labeling in the cytoplasm, but also a weaker intensity of peripheral ring staining in comparison with control cells. In spite of the fact that HL60 cells exposed to G-CSF and GM-CSF contained different F-actin structures such as aggregates and F-actin network, the rate of actin polymerization was not significantly enhanced. Moreover, alterations were mainly related to considerable changes in the relative proportion of these different structures, what might be reflected by specific features of the differentiation process, with regard to the kind of stimulating factor used. Thus, reorganization of F-actin and other results obtained in our experimental conditions, might reflect unique characteristics of the differentiation process in HL-60 cells, involving low apoptosis frequency, the G1 to S phase transition in the cell cycle, as well as possible alternative ways of the cell death.

Influence of taxol, microtubular poison, has been studied on distribution of actin. K-562 and HL-60 cells were treated with taxol in a range of concentrations 0.02-10 microM for 72 hours. The reorganization of F-actin was dependent on its dose. Phalloidin conjugated to TRITC was used to evaluate actin distribution by classical fluorescence and confocal microscopy. Actin was visualized at ultrastructural level by using a postembedding streptavidin-gold method. The treatment of K-562 and HL-60 cells with 2-10 microM of taxol resulted in an increase of F- actin in the cytoplasm, with intense labeling as a ring close to surface of the cell. In HL-60 cells a concentration of F- actin at the site of apoptotic bodies was often observed. Immunogold labeling of actin was localized in the nuclei and cytoplasm in control cells and cells treated with all doses of taxol. At higher doses, compaction of chromatin in the nucleus with strong actin labeling was observed. These observations at the ultrastructural level suggest actin involvement in chromatin reorganization during the process of apoptosis. The present study demonstrated a dose dependent reorganization of actin after treatment with taxol.

Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis. The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdB-treated HeLa cells. Caspase-3 activation was subsequently confirmed in TcdB-intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB-induced cell death, but did not alter the time-course of cytopathic effects. A similar effect was also observed in MCF-7 cells, which are deficient in caspase-3 activity. The time-course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1-556). Unlike TcdB treated cells, intoxication with TcdB1-556 or expression of TcdB1-556 in a transfected cell line, did not stimulate caspase-3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB1-556 treated cells did not demonstrate caspase-3 activation these cells were apoptotic as determined by differential annexin-V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase-independent apoptosis as a result of substrate inactivation and may evoke caspase-dependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.

To investigate the mechanisms of protrusion in vertebrate cells, the primary event in cell motility, human fibroblasts were treated with neomycin, an inhibitor of the phosphatidylinositol cycle, to induce protrusion. Changes in cell motility and the cytoskeleton were examined by video, fluorescence, scanning electron, and confocal microscopy and by cytofluorometry. Protrusion in neomycin-treated human fibroblasts is correlated with a transient overall decrease in F-actin followed by an increase in F-actin at the leading edge of the protruding lamella. In growing lamellae, F-actin is organized in a marginal band at the leading edge. Although actin is present in the lamella behind the leading edge, very little of it is F-actin. Scanning electron microscopy of detergent-extracted cells reveals a band of dense filaments at the leading edge, corresponding to the marginal band of F-actin seen in fluorescently labeled cells, and a sparse population of short, fragmented filaments, in the rest of the lamella. Gelsolin is colocalized with F-actin in the marginal band and is also present in the lamella where F-actin is largely absent. The data support the hypothesis that the protrusion is initiated by the breakdown of cortical actin filaments, possibly mediated by gelsolin, whereas expansion of the protrusion requires de novo polymerization of actin filaments at the leading edge., (Copyright 2001 Wiley-Liss, Inc.)

The organization of actin, tubulin, and vimentin was studied in protruding lamellae of human fibroblasts induced by the aminoglycoside antibiotic neomycin, an inhibitor of the phosphatidylinositol cycle. Neomycin stimulates the simultaneous protrusion of lamellae in all treated cells, and the lamellae remain extended for about 15-20 min, before gradually withdrawing. The pattern and distribution of actin, tubulin, and vimentin during neomycin stimulation were analyzed by fluorescence and electron microscopy. F-actin in the newly formed lamellae is localized in a marginal band at the leading edge. Tubulin is colocalized with F-actin in the marginal band, but the newly formed lamellae are initially devoid of microtubules. Over a period of 10 to 20 min after the addition of neomycin, microtubules grow into the lamellae from the adjacent cytoplasm, while the intensity of tubulin staining of the marginal band decreases. Distribution of vimentin remains unchanged in neomycin-treated cells and vimentin filaments do not enter the new protrusions. Treatment of cells with colchicine and Taxol do not inhibit neomycin-induced protrusion but protrusions are no longer localized at the ends of cell processes and occur all around the cell periphery. We conclude that actin filaments are the major component of the cytoskeleton involved in generating protrusions. Microtubules and, possibly, intermediate filaments control the pattern of protrusions by their interaction with actin filaments., (Copyright 1998 Academic Press.)

To determine the optimal procedures to preserve cytoskeletal and other macromolecular structures for microscopic studies we have evaluated the effects of various methods to extract cultured cells. In this report, we compare results using different fixatives, crosslinking reagents, and permeabilization methods on (1) the labeling of cells for fluorescence microscopy with phalloidin or antibody against tubulin; and (2) the morphological preservation of macromolecular structures for scanning electron microscopy. Maximal labeling of F-actin with phalloidin was obtained by fixing cells in 4% paraformaldehyde (PFA) and labeling the unextracted cells with methanolic phalloidin, whereas maximal labeling of tubulin required prefixation with either PFA or the bifunctional protein crosslinking reagent, dithiobis (succinimidylpropionate) (DSP) and extraction with ethanol or Triton in a high salt buffer. However, for both qualitative and quantitative light and electron microscopic studies of intracellular macromolecular structures, prefixation with DSP and extracting with Triton X-100 in a stabilizing buffer is the overall method of choice for both labeling and morphological studies. Although other methods provide maximal labeling or preservation of specific structures, this method provides excellent preservation of morphological structure while allowing proteins to be preserved and labeled by specific probes.

In this paper we describe an experimental investigation of the mechanism of motility of vertebrate cells. Human glioma cells were treated with neomycin, an inhibitor of the phosphatidylinositol cycle; and changes in cell motility and the cytoskeleton were examined by video, fluorescence, and scanning electron microscopy and by cytofluorometry. Neomycin stimulates a single protrusion of lamellipodia from the cell margin, which is correlated with an initial rapid decrease in the amount of F-actin throughout the cell, especially at the cell edge; the fragmentation of actin filaments within the lamellipodia; and the subsequent de novo polymerization of F-actin in a marginal band at the leading edge of lamellipodia. Changes in F-actin are paralleled by changes in the distribution and amount of gelsolin. These results support the hypothesis that protrusion is initiated by the gelsolin-mediated severing and subsequent depolymerization of cortical actin filaments, which weakens the cell cortex, allowing hydrostatic or gel osmotic pressure to force the cell margin to protrude. The accompanying polymerization of filaments actin at the leading edge of the protrusion may stabilize the protrusion and support its expansion.

The addition of platelet-derived growth factor (PDGF) to serum-starved fibroblasts induces increased motility, formation of lamellipodia, increased ruffling activity, and actin ring structures associated with dorsal ruffles. Involvement of the phosphatidylinositol cycle (PI-cycle) in these morphological changes was investigated by observing the effects of neomycin, an inhibitor of the PI-cycle, on cultured human foreskin fibroblasts. The role of actin in the changes was investigated by using cytochalasin D (CD). Actin in detergent-extracted cells was labelled with TRITC-phalloidin and examined with fluorescence microscopy. Using PDGF and neomycin simultaneously potentiated lamellipodia formation, ruffling activity, as well as the number of cells with actin rings. Furthermore, neomycin by itself induced morphological changes similar to those induced by PDGF. Quantitation of actin rings showed dose and time dependency for PDGF and neomycin respectively, with a maximal number of cells containing rings after 15 min of exposure to either 3.5 mM neomycin or 10 ng PDGF/ml. Comparing the two substances, PDGF induced ring formation in a greater number of cells. These processes were inhibited by the presence of CD. PDGF- and neomycin-induced changes in the actin cytoskeleton were also observed in human embryonic lung fibroblasts, human glial cells, and embryonic mouse fibroblasts, all of which are known to express PDGF-receptors. In conclusion, the present study indicates that an increased turnover of the PI-cycle is not essential for the changes in actin organization induced by PDGF.

Electron microscopic study of the breast tumors were carried out on 67 patients. There were 7 intraductal carcinomas and 60 infiltrating ductal carcinomas of the breast. Observations were made on the morphological characteristics of neoplastic cells, myoepithelial cells and basal lamina. Cells organelles in the neoplastic cells are similar in both types of ductal carcinomas. Nuclei are predominantly large and irregular with poor dense chromatin. Often a lot of filaments are around the nuclei. Myoepithelial cells in the infiltrating ductal carcinomas are ultrastructurally the same as seen in the intraductal carcinomas, but they seems to be less numerously than in the intraductal carcinomas. In the intraductal carcinomas of the breast basal lamina is irregular and from time to time is disrupt. In the infiltrating ductal carcinomas basal lamina is usually absent, but sometimes we can find its short fragments.