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Understanding mechanotransduction in adipocytes is important for research of obesity and related diseases. We cultured 3T3-L1 preadipocytes on elastic substrata and applied static tensile strains of 12% to the substrata while inducing differentiation. Using an image processing method, we monitored lipid production for a period of 3–4 wk. The ratio of %-lipid area per field of view (FOV) in the stretched over nonstretched cultures was significantly greater than unity (P < 0.05), reaching ∼1.8 on average starting from experimental day ∼10. The superior coverage of the FOV by lipids in the stretched cultures was due to significantly greater sizes of lipid droplets (LDs) with respect to nonstretched cultures, starting from experimental day ∼10 (P < 0.05), and due to significantly more LDs per cell between days ∼10 and ∼17 (P < 0.05). The statically stretched cells also differentiated significantly faster than the nonstretched cells within the first ∼10 days (P < 0.05). Adding peroxisome proliferator-activated receptor-γ (PPARγ) antagonist did not change these trends, as the %-lipid area per FOV in the stretched cultures that received this treatment was still significantly greater than in the nonstretched cultures without the PPARγ antagonist (14.44 ± 1.96% vs. 10.21 ± 3%; P < 0.05). Hence, the accelerated adipogenesis in the stretched cultures was not mediated through PPARγ. Nonetheless, inhibiting the MEK/MAPK signaling pathway reduced the extent of adipogenesis in the stretched cultures (13.53 ± 5.63%), bringing it to the baseline level of the nonstretched cultures without the MEK inhibitor (10.21 ± 3.07%). Our results hence demonstrate that differentiation of adipocytes can be enhanced by sustained stretching, which activates the MEK signaling pathway.
- mechanotransduction
- obesity
- tensile strain
- 3T3-L1 preadipocytes
- differentiation
obesity is one of the major preventable causes of morbidity and mortality, and its prevalence is rising in adults as well as in children (16, 29, 53). There are numerous reports in the literature regarding the contribution of obesity to the risks for developing type 2 diabetes, hypertension, atherosclerosis, and hyperlipidemia (29, 51); this motivates research regarding the etiology of obesity, which, at a cell scale, importantly involves characterization of specific stimuli that promote synthesis of cytosolic lipids.
Adipose tissue is a complex organ, which, other than storing energy in the form of triglycerides (which is essential for metabolism of glucose), is able to secret hormones and cytokines that regulate systemic metabolism and homeostasis (9, 29, 51, 76). The increased adipose tissue mass in obesity is due to hypertrophy and hyperplasia of adipocytes. The number of adipocytes can increase through mitosis (mostly in the preadipocyte phase) or through differentiation of additional preadipocytes. It is currently unclear which of these two pathways actually functions, or is more dominant in vivo, but it is often surmised that maturation of preadipocytes into adipocytes is one of the most important mechanisms in obesity (1, 9, 10, 18, 29, 51, 76).
Conservative treatment of obesity is generally aimed at decreasing the mass of excessive adipose tissues by changing the balance between calorie intake and expenditure, via physical exercise, diet control, or both. Current understanding of cellular mechanotransduction, however, might suggest that there is more to obesity than energy imbalance. Specifically, the responsiveness of cells to their mechanical loading environment was already demonstrated for many cell types (22–24, 36, 78); osteoblasts, for example, produce mineralized bone given a sufficient (dynamic) mechanical stimulation (5, 36, 66). Adipocytes in weight-bearing adipose tissues that are exposed to static large tensional and compressive mechanical strains in vivo (47, 68) might be mechanosensitive and mechanoresponsive as well. Using a combined MRI and finite element modeling approach, Linder-Ganz and colleagues (47, 48) found that, during normal sitting, adipose tissues at the buttocks are subjected to tensile strains that are as high as 30% (averaged across subjects in their study); a lying posture induces tensile strains that are approximately half that magnitude. Hence, given that adipocytes are subjected to substantial tensile strains physiologically at weight-bearing adipose tissues, it is important to determine whether sustained tensile strains are promoting adipogenesis or not, in the context of a sedentary lifestyle, obesity, and adipose-related diseases.
Here we hypothesize that sustained static mechanical loading of preadipocytes can influence the adipogenesis process (i.e., the differentiation response) in these cells. Dynamic loading modes such as cyclic stretching or vibration were generally found to suppress adipocyte differentiation in cell cultures of different developmental stages, e.g., preadipocytes or mesenchymal stem cells that originate from adipose tissue or bone marrow (8, 12, 35, 61–63, 72–74, 77). While the effects of dynamic loading have been extensively investigated (8, 12, 35, 61–63, 72–74, 77), there has been little investigation into the effects of static loading on adipogenesis. To our knowledge, the influence of static loading on differentiation of adipocyte cultures was examined in only two studies so far (27, 32). Specifically, static loads appeared to have a dual effect on adipogenesis, where static stretching applied to mature hypertrophic adipocytes accelerated differentiation (27) but static compression (32) impeded it. Nevertheless, these studies (8, 12, 27, 32, 35, 61–63, 72–74, 77) were limited by the methodology of destructive testing of lipid contents in the cultures, which was done by chemical staining (Oil Red O), Western blotting, and reverse transcription polymerase chain reaction (RT-PCR) (8, 12, 27, 32, 35, 61–63, 72–74, 77). Destructive testing limits the number of time points at which the cells can be studied and compromises the statistical power since cultures cannot be used as their own controls. Most importantly, there was no study so far that looked at the effects of static mechanical loading at the preadipocyte stage, where cells were not yet differentiated, and hence, the question of whether the differentiation process in adipocytes itself could be regulated by mechanical loading needs to be addressed.
In the present work we cultured preadipocytes on elastic substrata and applied sustained static tensile strains to the substrata while inducing differentiation in the cultures. Using our new nondestructive image processing-based method for measuring the numbers and sizes of lipid droplets (LDs) in maturing adipocytes (57), we monitored the lipid production (adipose conversion) in the cultures every 2–3 days for about 3–4 wk. In addition, we examined the effects of mitogen-activated protein kinase kinase/mitogen-activated protein kinase (MEK/MAPK) inhibitor and peroxisome proliferator-activated receptor (PPAR)-γ antagonist on adipogenesis in stretched and nonstretched cultures. Our present findings are relevant to research of obesity and adipose-related diseases as well as to adipose tissue engineering.
Cell culturing.
We cultured preadipocytes as described previously (56, 57). Specifically, mouse embryonic 3T3-L1 cells (American Type Culture Collection) were cultured in a growth medium (GM) consisting of high-glucose Dulbecco's modified Eagle's medium (DMEM, 4.5 mg/ml; Biological Industries, Israel), 10% fetal bovine serum (Biological Industries), 1% l-glutamine (Biological Industries), 0.1% penicillin-streptomycin (Pen-Strep; Sigma, Israel), and 0.5% 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES; Sigma). The maximum confluence allowed for the cultures was ∼80% before passages. Differentiation was induced by changing the GM to a differentiation medium (DM). The DM consisted of the aforementioned GM, supplemented with 5 μg/ml insulin (Sigma), 1 μM dexamethasone (Sigma), and 0.5 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma). Three days after induction of differentiation, the DM was replaced by a supporting medium (SM) consisting of the GM supplemented with 5 μg/ml insulin (Sigma). The SM was changed every 2–3 days.
Cell-stretching apparatus.
We selected to load the preadipocytes in a stretching mode based on our previous work, which showed that tissue compression associated with weight-bearing postures involves coupled, substantial stretching of the cell bodies (47, 48, 68). Accordingly, we built an apparatus for applying equibiaxial and homogeneous tensile strains to cell culture substrata (Fig. 1). A six-well culture plate with an elastic substrate in each well was mounted between the two frames of the apparatus. Rotation of two screws brings the frames closer together and thereby stretches the elastic substrata over Teflon rings positioned underneath the culture plate (Fig. 1C). Four clampers set between the two frames bound the distance between the frames and therefore control the final magnitude of mechanical strain developing in the substrata. This apparatus was carefully designed, in terms of its overall size and material components, to allow it to function in an incubator at 90% humidity, 5% CO2, and 37°C. Accordingly, the dimensions of the apparatus were 20 × 11 × 6 cm. The bottom frame of the apparatus was made of polycarbonate, and the other parts were made of Stainless Steel 303. The apparatus was sterilized using 70% ethanol (Bio Lab) before culturing.
We verified and characterized the homogeneous and biaxial strain state in the substrata (4, 36, 71) as follows. When the elastic substrata were in the undeformed configuration, we photographed three marks that were previously drawn on the substrata. Then, the top frame was lowered to a measured distance from the bottom plate (d) and we photographed the marks again in the deformed configuration. These images were processed by a MATLAB code (MathWorks), and the three Eulerian strain components (Ecc, Err, and Erc) were calculated for each d using the finite strain theory, as detailed previously (5, 20, 45). The two normal strain components, Ecc and Err, are the strains along the circumferential and radial axes, respectively, whereas Erc is the shear strain component. We calculated these stain components for d in a range of 0.83–1.51 cm, which are the minimum and maximum d allowed by the apparatus. The average increment in d was 0.08 cm. We repeated this calibration experiment five times and calculated the mean and standard deviation of each strain component (Fig. 2A). After verification of an equibiaxial strain state (Ecc = Err, Erc ≈ 0) by means of a two-way analysis of variance (ANOVA) for the factors of strain component and d (P < 0.05), the effective tensile strain E was calculated as the average of Ecc and Err (Fig. 2B). We fitted the plot of E versus d with a 2nd-order polynomial function (Pearson correlation coefficient = 0.98): 1 We verified that the substrata for cell culturing in the BioFlex plates are linear elastic by means of uniaxial tensile tests, where we stretched three specimens of the substrata (14 cm long, 2 cm wide) at a rate of 5 mm/min (Instron 5544 model, High Wycombe, UK). The substrata demonstrated a linear elastic behavior up to a strain magnitude of 18%. Additionally, we conducted viscoelastic creep experiments to ensure that strains in the substrata do not increase significantly over time under the sustained loads. For this purpose, we calculated the three Eulerian strain components in the substrata immediately after applying the maximal deformation (E = 12%) and after 24 h, we repeated this experiment five times as well (Fig. 2C). Given that the substantial viscoelastic changes in strain occurred shortly after the load was applied (i.e., within several hours) and were negligible thereafter, we concluded that creep effects could be ignored since changes in the strains, even after 24 h, were within the accuracy of strain measurements (Fig. 2C).
Experimental design.
Using the cell-stretching apparatus described above in Cell-stretching apparatus, we subjected six triplicates of 3T3-L1 cultures to substrate tensile strains of 12%, which is an intermediate value with respect to normal physiological tensile strain distributions in weight-bearing adipose tissues during sitting and lying (47, 48). We deliberately selected to deliver constant, continuous strains (rather than intermittent loading) over the entire culturing period to determine the effects of static loading per se on lipid production in the cultures (that is, without any masking from dynamic loading effects).
The 3T3-L1 preadipocytes were cultured according to the protocol described in Cell culturing. Next, 105 cells were seeded per each well in a six-well BioFlex collagen-coated culture plate (Flexcell). When a confluence of ∼90% was achieved, the culture plate was mounted in the cell-stretching apparatus, and three wells were subjected to stretch, whereas the other three wells were used as nonstretched control cultures. Differentiation was then induced in all the wells as described above in Cell culturing. The cells used for the experiments were always younger than passage 13. We monitored the cultures during periods of 18–28 days (until cultures became too confluent for adequate monitoring). Cultures were inspected almost daily and a normal differentiation process was always observed. Every 2–3 days we digitally photographed the cultures under a phase contrast microscope (Eclipse TS100, Nikon) at an original magnification of ×40 using a field of view (FOV) of 220 × 165 μm2 to optically document the progress of adipose conversion. Photographs were always taken without removing the culture plates from the cell-stretching apparatus; hence stretched cultures remained stretched during the visualization as well. Three different FOVs were considered in each well, and at least five cells were captured in each micrograph. We analyzed the micrographs using our previously developed nondestructive adipocyte-micrograph image processing method. The validation studies of our image processing method for monitoring intracytoplasmic triglycerides, which are specific markers for the adipose conversion (60), were reported in our previous work (57). In brief, Or-Tzadikario et al. (57) compared diameters of 90 randomly selected and automatically measured LDs with manual measurements of the same LDs, obtained using the SigmaScan Pro software (Systat Software), which is commonly employed for measuring entities in micrographs (57). That paper reported that the agreement between the automatic and manual measurements was very good (57), and therefore it was concluded that the image processing-based technique was adequate for monitoring the development of LDs over time. Additionally, examples of our image processing algorithm implemented on Oil Red O- and Nile Red-stained adipocytes (56) versus nonstained adipocyte cultures (Figs. 3 and 4) demonstrate the ability of our image processing technique to correctly identify the locations and sizes of intracytoplasmic lipid droplets. Oil Red O specifically stains intracytoplasmic triglycerides by dissolving in the lipoid substance (60), and Nile Red detects intracellular LDs under fluorescence microscopy by fluorescing in the presence of triglycerides (26). Hence, these comparisons provide additional visual verification of our presently used image processing-based nondestructive technique. In the image processing algorithm, each micrograph was converted into a binary image, in which only LDs were in white and everything else was blackened (Fig. 4). Then, we manually marked cells in each micrograph (5 cells per each micrograph). Our MATLAB code (provided in full in the Appendix of Ref. 57) automatically calculated the percentage area covered by LDs, the number and diameter of LDs, and the number of differentiating cells in a FOV. Subsequently, we calculated the ratio of each of these adipogenesis parameters in the stretched cultures over the nonstretched cultures in the same six-well plates. The above experiment was repeated six times, and descriptive statistics of all the aforementioned parameters (means and standard deviations) were calculated for the entire data set from the six trials. Finally, we compared each property ratio to unity (unity indicates that stretching had no effect on adipogenesis) using ANOVA for the factor of time postinduction of differentiation. P < 0.05 was considered statistically significant.
Measurements of triglyceride content and leptin expression.
We conducted Oil Red O staining studies to compare between lipid contents in stretched versus nonstretched cultures at day 17 postinduction of differentiation. The staining protocol was previously described (60). In brief, a working solution of Oil Red O was prepared by dissolving 4.2 g of Oil Red O (Sigma) in 1,200 ml absolute isopropanol. Subsequently, we added 900 ml of double-distilled water (DDW) to the solution and left it overnight at 4°C. Triplicates of stretched and nonstretched cultures were fixed for 10 min with 4% paraformaldehyde, then washed with phosphate-buffered saline, stained for 1 h by complete immersion in a working solution of Oil Red O and carefully rinsed with DDW. The stained cultures were then digitally photographed. These micrographs were analyzed by a red color threshold: each red pixel in the red-blue-green micrograph was converted into a white pixel and everything else was blackened. Furthermore, 1 ml of isopropanol was added to the stained cultures, and the absorbance of the extracted dye was immediately monitored spectrophotometrically at 510 nm (SpectraMax 340PC384, Molecular Devices). The Oil Red O absorbance between the stretched and nonstretched cultures was compared using a nonpaired t-test.
We further conducted alkaline phosphatase (ALP) histochemistry staining studies since ALP activity and fat storage were found to be tightly linked during 3T3-L1 differentiation (2). Specifically, triplicates of nonstretched versus stretched cultures were fixed in immersion of 0.3% paraformaldehyde for 1 h. Diazonium salt solution was prepared by dissolving 25 mg of Fast Blue RR salt in 48 ml DDW. Naphthol AS-MX phosphate alkaline (2 ml) was then added to the solution. The reagent mixture was incubated with the fixed culture cells for 30 min in room temperature. Cultures were then rinsed thoroughly in DDW.
In addition, we examined the extent of leptin expression, being one of the most abundant adipocytokines (55), in the stretched versus the nonstretched cultures. For this purpose, we used a Mouse Leptin ELISA kit (ab100718; Abcam), which is an in vitro enzyme-linked immunosorbent assay for quantitative measurement of mouse leptin in culture supernatants. The media in triplicates of stretched and nonstretched cultures were sampled at days 4 and 17 postinduction of differentiation, and the amount of leptin was measured using the ELISA kit according to the manufacturer's instructions. We compared the concentration of the expressed leptin between the stretched and nonstretched cultures using a nonpaired t-test.
Examining the roles of PPARγ and MEK signaling.
We examined the effects of PPARγ antagonist and MEK inhibitor on the adipogenesis in the stretched and nonstretched cultures, as follows. Six triplicates of 3T3-L1 preadipocyte cultures were grown on elastic substrata as described above in Cell culturing. When a confluence of ∼90% was achieved, differentiation was induced in all the wells (again as described in Cell culturing). The substrata in three triplicates were then subjected to 12% strain. A selective and irreversible PPARγ antagonist GW9662 (Calbiochem) or MEK/MAPK inhibitor (PD98059; Calbiochem) was added to the DM in one triplicate of the stretched preadipocyte cultures and in one triplicate of the nonstretched preadipocyte cultures at a concentration of 10 μM and 50 μM, respectively; these concentrations were previously shown to be highly specific to the PPARγ transcription factor and the MEK/MAPK pathway, respectively, in 3T3-L1 cells (3, 14, 15, 31, 38, 42, 43, 58). Control cultures received dimethyl sulfoxide as a vehicle. The DM was changed every 2–3 days. Given that in our preliminary experiments we observed stretched-induced accelerated lipid production at day 17 postinduction of differentiation (Fig. 5), we digitally photographed and analyzed the cultures at that time point. The micrographs were processed as described above in Experimental design, and the following adipogenesis parameters were calculated: 1) percentage lipid area per FOV, 2) mean diameter of LDs per cell, 3) mean number of LDs per cell, and 4) mean number of differentiated cells per FOV. The data were analyzed by means of the Mann-Whitney statistical test using SPSS software (version 17, IBM). Differences were considered to be significant when P < 0.05.
Example comparisons between micrographs of stretched and nonstretched cultures at different experimental time points are provided in Fig. 4 (processed images are shown at the top right frames). The progress of adipose conversion was well observed in both the stretched and nonstretched cultures, but LDs in the stretched cultures (Fig. 4, right) became substantially greater than in the nonstretched cultures (Fig. 4, left) at corresponding time points after ∼10 experimental days. Consequently, larger cells were typically observed in the stretched cultures (Fig. 4). These example micrographs are visual evidence for the accelerated lipid production in the stretched adipocyte cultures starting from the second week of the experiments.
Quantitative analyses of our experimental data are provided in Fig. 5. The ratio of %-lipid area per FOV in the stretched over nonstretched cultures was significantly greater than unity (P < 0.05) and reached a value of ∼1.8 on average for the period between 8 days after induction of differentiation plus stretching and the end of experiments. The superior coverage of the FOV by LDs in the stretched cultures over that period was due to significantly greater sizes of LDs with respect to nonstretched cultures starting from approximately day 10 postinduction of differentiation and stretching (P < 0.05; Fig. 5B), as well as due to the existence of significantly more LDs per cell between days 10 and 17 (P < 0.05; Fig. 5C). Specifically, on the last week of experiments (i.e., starting at about day 21), LD diameters in adipocytes belonging to the stretched cultures were at least ∼1.4 times greater than LDs in the nonstretched cultures (Fig. 5B). At day 28 postinduction of differentiation, LD diameters in the stretched cultures were ∼1.8 times greater than in the nonstretched cultures. The peak value of the ratio of mean number of LDs per cell in stretched over control cultures, being ∼1.8, occurred approximately at day 15 postinduction of differentiation and stretching (Fig. 5C). Interestingly, the statically stretched cells also differentiated significantly faster than the cells in the nonstretched cultures within the first ∼10 days of the experiments (Fig. 5D, P < 0.05). For example, on experimental days 8 and 10 there were on average ∼2.1 times and ∼1.8 times greater numbers of differentiating cells (i.e., cells which contained identifiable LDs) per FOV in the stretched cultures than in the nonstretched cultures, respectively (P < 0.05; Fig. 5D). However, at the portion of the time course starting around experimental day 13 and lasting until experiments terminated, cells in the stretched and nonstretched cultures differentiated at a statistically indistinguishable rate (Fig. 5D).
Measurements of triglyceride content and leptin expression.
Micrographs of nonstretched versus stretched cultures stained with Oil Red O, 17 days postinduction of differentiation, and the corresponding processed micrographs presenting only the red pixels are provided in Fig. 6. These processed images (Fig. 6, right) provide additional visual verification of our image processing-based method as a tool for identifying intracytoplasmic triglycerides. In addition, the accelerated adipogenesis in the stretched cultures can clearly be observed (Fig. 6). Consistent with our results that were obtained using the image processing algorithm, the Oil Red O absorbance measured in the stretched cultures (0.28 ± 0.02 optical density) was significantly greater than in the nonstretched cultures (0.23 ± 0.05 optical density) (Fig. 6C) (P < 0.05).
Representative micrographs of nonstretched versus stretched cultures stained for ALP activity are provided in Fig. 7, A and B, respectively. Differentiated adipocytes and the accumulated LDs can be observed in dark gray and white, respectively. Stained cells indicated activity of ALP. Consistent with the Oil Red O staining results, we visually observed greater ALP activity in the stretched, compared with the nonstretched cultures, which further supports our findings that adipogenesis is accelerated in the stretched cultures.
Leptin concentrations measured at days 4 and 17 postinduction of differentiation in the stretched versus the nonstretched cultures are presented in Fig. 8. At day 4 postinduction of differentiation, the leptin concentration in the stretched cultures was 1.23 times higher than in the nonstretched cultures, but this difference was not statistically significant (Fig. 8). Nonetheless, at day 17 postinduction of differentiation, the leptin concentration in the stretched cultures was significantly higher than that in the nonstretched cultures, being 3.73 ± 0.32 versus 3.38 ± 0.28 pg/ml (P < 0.05) (Fig. 8). Given that leptin expression was found to be positively associated with the sizes of adipocytes (67), and the latter positively correlates with the differentiation stage of the adipocytes (65), these findings provide additional evidence that the lipid production process was enhanced in the stretched cultures.
Examining the roles of PPARγ and MEK signaling.
The adipogenesis parameters calculated in stretched and nonstretched cultures with and without MEK inhibitor are presented in Fig. 9. When comparing between stretched cultures treated with the MEK inhibitor and stretched cultures that did not receive this treatment, we found that the %-lipid area per FOV was statistically indistinguishable (Fig. 9A). Likewise, when comparing nonstretched cultures treated with the MEK inhibitor to nonstretched cultures without treatment, %-lipid area per FOV was indistinguishable (Fig. 9A). Differences in %-lipid area per FOV came out as significant (Fig. 9A; 16.34 ± 4% vs. 10.2 ± 1.4%; P < 0.05) when comparing stretched versus nonstretched cultures that were not treated with the MEK inhibitor (which is consistent with our previous results), but when comparing stretched cultures that received the MEK inhibitor treatment to nonstretched cultures that did not receive the treatment, there was no statistically significant difference again. In this latter case, even though LDs were significantly smaller in the stretched cultures with MEK inhibitor (Fig. 9B; 5.51 ± 1.1 μm) compared with the nonstretched cultures without the MEK inhibitor (Fig. 9B; 6.58 ± 1.67 μm) (P < 0.05), significantly more differentiated cells per FOV (Fig. 9D; 24.39 ± 8.89 vs. 14.98 ± 4.97; P < 0.05) produced approximately the same amount of lipids in the stretched cultures with MEK inhibitor and in the nonstretched cultures without the MEK inhibitor. In other words, inhibition of the MEK/MAPK signaling pathway reduced the extent of adipogenesis in the stretched culture group, bringing it to the baseline level of the nonstretched group without the MEK inhibitor.
The adipogenesis parameters calculated in stretched and nonstretched cultures with and without PPARγ antagonist are presented in Fig. 10. Adding PPARγ antagonist to the nonstretched cultures reduced the %-lipid area per FOV from 10.2 ± 3% to 7.6 ± 3.5% (Fig. 10A), and in the stretched cultures, the PPARγ antagonist reduced the %-lipid area per FOV from 16.34 ± 4% to 14.4 ± 2% (Fig. 10A), but both decreases were not statistically significant. Even though LDs were significantly smaller in the stretched cultures with the PPARγ antagonist compared with the stretched cultures without the PPARγ antagonist (Fig. 10B; 6.8 ± 1.5 vs. 8.26 ± 2.35 μm, respectively; P < 0.05), greater numbers of LDs per cell (Fig. 10C; 15 ± 10.7 vs. 12 ± 7.4) and greater numbers of differentiated cells per FOV (Fig. 10D; 16.15 ± 6.4 vs. 14.7 ± 3.2) both contributed to the similar coverage of the FOVs by lipids. Most importantly, the %-lipid area per FOV in the stretched cultures, either with or without the PPARγ antagonist, was significantly greater than in the nonstretched cultures without the PPARγ antagonist (Fig. 10A; 10.21 ± 3%) (P < 0.05). In the case of the stretched cultures without the PPARγ antagonist, this latter observation was manifested by significantly greater LDs (Fig. 10B; 8.26 ± 2.3 vs. 6.58 ± 1.66 μm) (P < 0.05). In the case of the stretched culture with the PPARγ antagonist, the superior coverage of the FOV by lipids, compared with the nonstretched cultures without PPARγ antagonist, was attributed to 1) greater sizes of LDs (Fig. 10B; 6.8 ± 1.6 vs. 6.58 ± 1.66 μm); 2) greater numbers of LDs per cell (Fig. 10C; 15 ± 10.8 vs. 11.6 ± 7); and 3) more differentiated cells per FOV (Fig. 10D; 16.15 ± 6.14 vs. 14.9 ± 5). In other words, the PPARγ antagonist did not suppress the accelerated lipid production in the stretched cultures.
The present study provides clear evidence that static stretching stimulates adipocyte cultures to produce lipids at a faster rate. Our results show that both the larger numbers and greater sizes of LDs contributed to the consistently superior coverage of the FOV by lipids in the stretched cultures, starting at approximately day 10 postinduction of differentiation and stretching. These experimental data hence support our hypothesis presented in the Introduction that adipocytes are mechanosensitive and mechanoresponsive and that the adipose conversion process is influenced by mechanical stimulation.
Adding PPARγ antagonist reduced the %-lipid area per FOV in both the stretched and nonstretched cultures, which is consistent with knowledge regarding the regulation of adipogenesis by PPARγ (21, 50, 75). However, in the present study, this antagonist did not suppress the mechanoinduced accelerated lipid production observed in the stretched cultures. It is recognized that the PPARγ transcription factor is involved in the initial phase of cell differentiation, but once cells are on the adipocyte-phenotype pathway, the functionality of PPARγ is less or not at all influential. Thus, a downstream effector should play a role in this process, and hence we examined the MEK/MAPK pathway and we did find that inhibiting this pathway had a significant influence on suppressing the accelerated adipogenesis in stretched cultures. Indeed, the MAPK signaling pathway was previously found to be mechanically activated in various cell types (e.g., vascular endothelial cells, human fibroblasts, and mesenchymal stem cells) and in response to different mechanical loading regimes (e.g., shear stresses, tensile loading, and static or dynamic hydrostatic pressures) (37, 41, 49, 54). Importantly, consistent with our present results, Tanabe and colleagues (72) also found that the extracellular signal-regulated kinases (ERK) system was mechanically activated in 3T3-L1 preadipocytes (considering that ERK is activated by the MEK). Nevertheless, in the study of Tanabe and colleagues (72), activating the ERK/MAPK pathway resulted in inhibition of adipogenesis in the stretched cultures, which is the opposite type of effect reported here. Indeed, the ERK/MAPK was found to have both inhibitory (17, 33, 39, 64) and stimulatory (7, 40, 52, 58, 79) effects on adipose conversion. Considering that Tanabe and colleagues used a different mechanical loading regime (their cells were cyclically loaded to 120% or 130% of their undeformed length at a frequency of 1 Hz for 15 or 45 h), and given that stimulation of the ERK/MAPK pathway might have opposing effects in the process of adipogenesis depending on the time of activation during the differentiation process (57, 71), either inhibition or acceleration of the adipogenesis could be expected. In a recent paper, Hara and colleagues (27) examined the effects of static stretching of up to 120% in mature 3T3-L1 adipocytes that were exposed to the stretch for 72 h. Rho-Rho kinase was found to be activated and contribute to the accelerated adipogenesis in the stretched adipocytes. The different pathways that were found to be involved in our present study and in the Hara et al. study could be a result of different durations of stretching (72 h vs. 17 days), different developmental stages of the stretched cells (mature adipocytes vs. preadipocytes), and different loading modes (20% static uniaxial strain vs. 12% equibiaxial strain). Interestingly, even though two different signaling pathways were found to be activated by mechanical stretching (MEK/MAPK in our study, and Rho-Rho kinase in the Hara study), both pathways show the same influence of accelerating the adipogenesis in stretched cultures.
There is a debate in the literature regarding the role of the ERK/MEK pathway in insulin signaling, as there are studies which report that activation of ERK/MEK stimulates insulin signaling (28) but others reported lack of influence (44) or an inhibitory effect (6, 19). Specifically, the accelerated lipid production observed in the stretched cultures could be an outcome of enhanced insulin signaling by the MEK pathway, as suggested by Harmon and colleagues (28), who showed that MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes. On the other hand, this phenomenon might be driven by other mechanotransduction pathways as well, such as mechanosensitive channels, in which gating is initiated in response to mechanical stimuli, resulting in catalyzed transport of glucose across the plasma membrane (34, 58). In this case, insulin signaling could be unaffected, as suggested by Lazar and colleagues (44), who found that MEK inhibition does not block stimulation of glucose utilization by insulin. The work of Fujishiro and colleagues (19) suggests that insulin signaling could even be inhibited when the ERK pathway is activated, as they found that ERK markedly suppresses the expressions of insulin receptor (IR) and its major substrates (IRS proteins) as well as that of the glucose transporter GLUT4. Likewise, Bost et al. (6) concluded that the IRS-1 protein is phosphorylated by the ERK pathway and that this serine phosphorylation exerts an inhibitory effect on the insulin signaling. Now that the present study points to involvement of the MEK pathway in accelerated adipogenesis in stretched adipocytes, future research should focus on the interactions between MEK and insulin signaling in such stretched cells.
The understanding that adipocytes respond to mechanical loading and thus the latter can be used to regulate their differentiation response and control their lipid production is consistent with knowledge regarding mechanotransduction in other cell types, including cardiomyocytes, vascular endothelial cells, smooth muscle cells, skeletal muscle cells, and osteoblasts, which are all responsive to mechanical loads (22–24, 36, 78). Moreover, given that adipocytes originate from the same progenitor cells as do osteoblasts and myocytes, that is, they all normally mature from mesenchymal stem cells (11), it is only reasonable to expect that mechanical signals would influence adipocytes as well. Our present findings clearly indicate that a static (sustained) stretch accelerates lipid production in adipocytes, which could theoretically correspond, for example, to the increased adipose mass in the buttocks of obese individuals, where soft tissue stretching during weight bearing is substantial (13, 48, 70). Contrarily, cyclic stretches delivered at physiological frequencies appear to inhibit lipid production, which is rather intuitive given that physical exercise would generally involve cyclic loading of soft tissues, and since exercise is associated with a decrease in fat tissue mass over time. Specifically, there are numerous reports regarding the inhibiting effects that cyclic stretches induce, in which the differentiation was examined using Oil Red O staining. Additionally, RT-PCR, Western blotting, and immunofluorescence analyses were conducted for identifying gene and protein expressions which are triggered by such cyclic mechanostimuli (8, 12, 35, 61–63, 72–74, 77). Tanabe and colleagues (72, 73) found that cyclic stretching of 3T3-L1 preadipocytes to 120% or 130% of their undeformed length, at a frequency of 1 Hz, for 15 or 45 h, inhibits differentiation. The inhibition of the adipogenesis was found to be mediated by activation of the ERK/MAPK pathway. Later on, Turner and colleagues (77), who exposed human umbilical cord perivascular cells to equibiaxial cyclic stretch (10% half-sine waves at a frequency of 0.5 Hz for 24 or 60 h) observed inhibition of adipogenesis, however, through the transforming growth factor-β1/Smad signaling pathway. David and colleagues (12) assessed the differentiation of stretched versus nonstretched bovine mesenchymal stem cells as well as of cells from a C3H10T1/2 pluripotent cell line, and they also found that 300 cycles of elongation per day, to a magnitude of 4,000 μϵ at a frequency of 1 Hz (over a 2-wk period) inhibited differentiation into adipocytes. Sen and colleagues (8, 61, 62) again documented inhibited differentiation to the adipocyte phenotype in the C3H10T1/2 cell line as well as in marrow-derived mesenchymal stem cells, where cells were cyclically stretched to 2% strain at a rate of 10 cycles per minute, up to 3,600 total cycles (over 5 days). The inhibition of adipogenesis in these studies was reported to occur due to activation of the β-catenin pathway. In a later study, Sen and colleagues (63) delivered the following mechanical loading regimes to C3H10T1/2 cells: 1) low-intensity vibration, which they defined as strains below 0.001% applied at a frequency of 90 Hz; as well as 2) high-magnitude cyclic strains (2%, 0.17 Hz). They observed that two 20-min sessions, 1 or 3 h apart, of either low-intensity vibration or high-magnitude strains, suppressed adipogenesis via activating the β-catenin; adding more loading sessions further increased the suppressive effect. Likewise, Tirkkonen et al. (74) used vibrations to mechanically load human adipose tissue stromal cells. A 14-day period of 3 g peak acceleration at a frequency of 100 Hz, which was applied for 3 h per each experimental day, was similarly found to inhibit differentiation into adipocytes. Consistently, Huang et al. (35) delivered equibiaxial peak strains of 10% to rodent stromal cells at a 0.5 Hz frequency for 48 h and observed inhibition of adipocyte differentiation, but when the peak strain was lowered to 2%, the suppressive effect was significantly milder, or even nonexistent when the peak strain was reduced to the 0.5% level. To summarize, different signaling pathways were found to be mechanically activated following cyclic stretching of adipocytes, preadipocytes, or progenitor cell cultures, and the fact that multiple pathways were identified could be due to the differences in experimental protocols and cell types. Combining our present results that concern static stretching with the aforementioned studies, which all investigated the effect of cyclic stretching (8, 12, 35, 61–63, 72–74, 77), we can suggest that sustained static stretching delivered to adipocytes (within a certain physiological range) can stimulate them to produce lipids in vitro, whereas dynamic (cyclic) stretching is able to inhibit lipid production. Such a potential dual-mechanotransduction response is known to exist in other cell types as well, e.g., in osteoblasts whose activity is stimulated by cyclic loading but depressed under static loading (5, 37), being the opposite of the type of response suggested herein.
Our present results may be the foundation of additional work that should be focused on monitoring the adipose conversion in more physiological conditions, for example, using three-dimensional tissue-engineered adipose constructs exposed to different levels of stretch or to compound loading (e.g., hydrostatic pressures and tensile strains applied simultaneously). The effects of intermittent loading can be studied as well, to determine whether occasional relief periods in the stretching are able to counterbalance the accelerated lipid production. Additionally, it may be worthwhile to study how the adipose conversion process is influenced by different concentrations of insulin in the differentiation medium (57) (e.g., in the context of examining interactions between MEK and insulin signaling) or by different levels of glucose (e.g., in the context of a calorie-rich diet), and these biochemical modifications can in turn be studied in combination with mechanical loading. Such studies may eventually open new research paths, driven by mechanotransduction, to explore mechanisms in obesity and related conditions.
This research work is being supported by a grant from the Ministry of Science and Technology, Israel and the Ministry of Research, Taiwan (A. Gefen), and also, partially, by a grant from the Ela Kodesz Institute for Cardiac Physical Sciences and Engineering (A. Gefen).
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: N.S. and A.G. conception and design of research; N.S., R.G., O.S.-Y., U.Z., and D.B. performed experiments; N.S., D.B., and A.G. analyzed data; N.S., D.B., and A.G. interpreted results of experiments; N.S. and A.G. prepared figures; N.S. and A.G. drafted manuscript; N.S., D.B., and A.G. edited and revised manuscript; N.S., R.G., O.S.-Y., U.Z., D.B., and A.G. approved final version of manuscript.
The authors are thankful to Maya Baranes, Ayelet Levy, and Sarit Enzer for technical assistance in running the experiments. Shira Or-Tzadikario is also thanked for the technical help with the cell culturing.
- Copyright © 2012 the American Physiological Society
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Rumors of Microsoft Office on Metro Tablets? It better be a fact
Microsoft gave users and developers many things to think about at the BUILD conference in California this last week. One that is getting a lot of traction is the idea that Microsoft will be developing a Metro style, touch...Read More

subset: commoncrawl


text:
News | Newsbriefs | Events | Sports | Schools | People | Police | Legals | Bulletin Board | Letters | Progress | Readers Choice
Minnesota Municipal Power Agency recently installed a solar array at North High School as part of the Hometown Solar Grant program.
submitted photo
The Washington County Board of Commissioners received a Pollinator Community Award in early September for its Pollinator Friendly Resolution passed in February 2016.
Washington County commissioners are striving to find a happy medium – preserve the treasure that is the Historic Courthouse, while making the building available for community events and touring visitors.
Ramsey County Parks and Recreation, in partnership with the Metro Bowhunters Resource Base, will again conduct controlled archery deer hunts this fall throughout the county parks system.
Ramsey County voters have several options for voting in the presidential election prior to Election Day Nov. 8, either by mail or in person, between Sept. 23 and Nov. 7.
Absentee voting for the statewide general election began Sept. 23 and continues through Monday, Nov. 7.
Trail lights on three routes will turn on at dusk to invite night hikers to the Lake Elmo Park Reserve.
Guests are invited to experience Washington County's Lake Elmo Park Reserve's lighted cross-country ski trails for night trail running or hiking. The lights started being used Sept. 2.
The new 106,654-square-foot senior living community will offer a variety of living solutions for older adults, and will feature community rooms and underground parking in addition to a movie theater, fitness center and hair salon.
Arbor Glen Senior Living, a new $16 million senior living community located in Lake Elmo has broken ground.
Washington County will work with Lake Elmo to create a sidewalk crossing at the railroad tracks on County State Aid Highway 17 as part of the improvement and replacement of the infrastructure in the city's downtown.
During the 2016 general elections, approximately 1,000 election judges will be needed to staff polling locations throughout Washington County.
Ramsey County Master Gardeners are offering two information sessions to help community members interested in becoming master gardeners learn more about the program and how to apply. Master gardeners and interns will be on hand to answer question
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text:
If you've ever seen the musical Oliver!, chances are you didn't walk out of the theater thinking that the main character was a lazy, entitled asshole who didn't need another helping of gruel as much as he needed a swift kick in the shorts. Well, apparently, there is one person in America who thought that, and he's currently fucking running for fucking President. During an appearance at Harvard's Kennedy School of Government, GOP candidate Newt Gingrich called child labor laws "stupid," arguing, "it is tragic what we do in the poorest of neighborhoods, entrapping children in… child laws, which are truly stupid."
But wait! Maybe that just seems horrible out of context. Maybe he said "entrapping children in child laws, which are truly stupid… if the children in question appeared in Baby Geniuses, because damn, that was a stupid movie." Or maybe he followed up his statement by tugging on a cord, releasing a giant shower of Smartees from a trap door, and gleefully tossing them to the audience.
However, it seems like none of these things happened, and Newt does, in fact, believe that child labor laws are stupid:
Most of these schools ought to get rid of the unionized janitors, have one master janitor and pay local students to take care of the school. The kids would actually do work, they would have cash, they would have pride in the schools, they'd begin the process of rising… you're going to see from me extraordinarily radical proposals to fundamentally change the culture of poverty in America.
So correct me if I'm wrong, but it sounds like Gingrich is proposing that poor children — presumably under the age of fourteen, which is the minimum age for minors to work under the Fair Labor Standards Act — get off their poor, tiny asses and start scrubbing urinals for a few bucks an hour, or perhaps run for district superintendent. Clearly, this man has read the Wayside School series enough times to think that it serves as an appropriate model for public education; of course, it does not, and the Industrial Revolution similarly does not serve as a template for modern-day child labor laws.


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I was talking with a friend of mine last Friday and he asked me what I still wanted to do with my career. As I talked through my goals and dreams, I realized that they came together in a sort of bucket list for work experiences.
I like the idea of a work-related bucket list because it takes the pressure off reaching ONE outcome or level or title or position. When we look back at our careers, we remember the great experiences - the awesome project, the team wins, the day you invented a new idea, your first big presentation.
Perhaps career success is not about achieving a particular place, but in checking off as many bucket list items as you can. Career happiness and fulfillment is a function of doing many of the things on your list, not particular things.
Imagine the positive momentum you would create by actively working both your life and career bucket lists? Having ONE goal or just a couple does not serve us well because we don't know what we don't know and we cannot predict what the future will hold. Defining our success or happiness based on a particular outcome is unhelpful. (butterfly affect comes to mind again)
What might be on your career bucket list? Get your week started off with a bang by taking a few moments to start your list and plot how to start ticking away at the experiences that most interest and intrigue you. And for extra credit, make an unreasonable request to help something happen this week.


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The South American side kick-off their Group B campaign again Ange Postecoglou's unfancied team at Cuiaba's Arena Pantanal on June 13 before facing Spain and the Netherlands.
And despite the Socceroos having been written off as likely whipping boys, Sampaoli has claimed to be wary of a match his players cannot afford to lose if they are to progress to the knockout stages.
"Always the first opponent is the most complicated," he said.
"In a tournament where you need to quickly (gain points), there will be nervousness, beyond what Australia can offer.
"That will be the hardest game … anxiety becomes secondary when you have so much work to do. (This is) no time to think about how many days are left to face Australia or who are the players going out. We must continue to strengthen the group."
While Brazil and Argentina are favourites to win on the 'home soil' of South America, Sampaoli's talented side are also being talked up as potential dark horses, with the weight of domestic expectations hanging over his players.
"The enthusiasm of the people requires us to prepare in the best way," he said.
"(We will) try … all through the preparation for the players (to) arrive in the best way to confront many rivals (with different qualities). Beyond the position (the) team will finish in our group, it is important that Chile does not lose shape against such prestigious opponents.
"My responsibility is to prepare the squad to face a very competitive group. I must try (to make sure) that players who came in (at) different point of preparation are (at their) best.
"Our priority is to work, (the intensity of) which is negotiable. Planning is scheduled in a certain way, but here athletes have to (combine) the mental with the physical (and) with the football. So, that's what we are (at)."
For more sports news visit allSports.com.gh
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Nov 27, 1990
|Filing date
|Dec 28, 1988
|Priority date
|Dec 28, 1988
|Fee status
|Also published as
|Publication number
|Russell W. Holbrook
|Original Assignee
|Pitney Bowes Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (16), Referenced by (61), Classifications (11), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention relates to a front end feeder for a mail handling machine, and in particular for a machine for high speed processing of mixed mail.
State of the art mailing machines can perform such automatic functions as handling mail of different sizes and thicknesses, envelope sealing, mail weighing, mail stamping, and mail sorting. The typical processing sequence starts at the front end of the machine where the mail is stacked. The stacked mail is then registered against a reference wall of the machine and the next step in the process is to feed the mail to a singulator to remove individual mail pieces from the bottom of the stack and thereafter process those individual mail pieces in serial fashion through the various modules of the machine.
Special problems arise when the mail to be handled is mixed mail, meaning envelopes containing inserts that have their flaps sealed, or closed but unsealed, or open. The problems intensify when an added requirement is the ability to process envelopes of varying sizes, for example from No. 6 to No. 15, and of varying thickness, say from thin air mail with a single insert up to three-quarters of an inch. Further problems arise when an additional added requirement is high-speed processing, up to four per second. To our knowledge, there exists no mail handling machine capable of high speed processing of mixed mail of varying size and thickness.
An object of the invention is a front end feeder for high-speed processing of mixed mail.
Another object of the invention is a front end feeder capable of delivering mail pieces to a singulator at the rate of up to four per second.
Still another object of the invention is a front end feeder capable of properly feeding mixed mail to a downstream singulator.
A further object of the invention is a front end feeder capable of properly feeding envelopes having a wide range of sizes and thicknesses to downstream modules for further processing.
These and other objects and advantages as will be apparent hereinafter are achieved with a front end feeder comprising a hopper region for receiving a stack of horizontally oriented mail and including a bottom or deck surface and an upstanding wall serving as a registration surface against which the flap edge of the envelopes is to be made to bear.
In accordance with one broad aspect of the invention, the hopper region is provided with means to deliver the mail pieces pre-shingled to the downstream module. A feature of this aspect of the invention is the provision of means for fluffing the mail to enable the mail to slide more easily over one another.
In accordance with another broad aspect of the invention, means are provided for continually urging the mail pieces while in the hopper region downstream as well as toward the registration wall. A feature of this aspect of the invention is compound slanting of the mail deck in the hopper region.
In accordance with a further broad aspect of the invention, the registration wall is movable, and means are provided for the registration wall to tamp with varying force the adjacent edges of flapped mail pieces.
These and other features and advantages will become clearer from the detailed description given below of one embodiment of a front end feeder of the invention, taken in conjunction with the accompanying drawings wherein:
FIG. 1 is a schematic side view of part of a mail handling machine employing one form of front end feeder in accordance with the invention;
FIG. 2 is a more detailed side view of the front end feeder illustrated in FIG. 1;
FIGS. 3 and 4 are perspective views of part of the feeder of FIG. 1 illustrating action of the tamper subsystem;
FIG. 5 is a schematic side view of the tamper and nudger subsystems used in the feeder of FIG. 1;
FIG. 6 is a perspective view of the feeder of FIG. 3 illustrating operation with multiple flapped envelopes;
FIGS. 7-9 are top schematic views illustrating the envelope driving and nudging actions of the feeder of FIG. 1;
FIGS. 10-12 illustrate the shingling action of the front end feeder of the invention;
FIG. 13 is a rear perspective view of the tamper subassembly used in the feeder of FIG. 1;
FIGS. 14 and 15 are exploded and perspective views, respectively, of a composite roller for use in the feeder of the invention;
FIGS. 16-18 are side views illustrating the shingling action of the feeder of the invention;
FIGS. 19 and 20 are top views, in different positions, of the composite rollers for use in the feeder of the invention;
FIGS. 21-24 are schematic side views illustrating the fluffing action of the composite rollers;
FIG. 25 is top view of the front end feeder of the invention with part of the deck removed showing the synchronized driving of the nudger and tamper subsystems;
FIGS. 26 and 27 are perspective and top views, respectively, of the guiding structure for the envelope flaps just downstream of the tamper subsystem.
FIG. 1 illustrates schematically the front end of a mailing machine 5 comprising a hopper 10 holding a stack 11 of registered mail in a horizontal position on a deck 12. A forward-drive mechanism 6 mounted below the deck 12 moves the stack downstream (to the right in FIG. 1) toward the singulator module, designated 15. Following singulation, the unsealed mail has the profile of its flap generated, and information based on the profile is fed via a computer to a moistener which wets the flap glue line which is then sealed. This occur at the stations indicated generally at 16 in FIG. 1.
One of the features of the feeder of the invention is the guideless hopper. Unlike other mailing machines, there are no rear props or side guides in front that the operator must adjust to hold the stack in place. By eliminating the need for such guides, the feeder of the invention can truly be a mixed mail feeder, i.e. capable of handling mail of varying thickness and varying size, both flapped and unflapped.
FIG. 2 is a more detailed side view of the hopper region 10. It includes a deck 12 which is supported in a fixed position from below. An extension piece 20 is fixed at its left side and terminates in a tilted back wall 21. On the rear is mounted a registration side wall 22, comprising a lower vertical part 23 and an angled backward upper vertical part 24. The drive means are not shown in this view. The dashed vertical line 25 roughly demarcates the hopper region 10 from the downstream singulator 15 (not shown in this view). The deck 27 at the singulator is horizontal, i.e., level when viewed from the front (though it can be slanted downward toward the rear wall), but the deck 12 in the hopper region is angled upward by an angle of about 4�-6�, preferably 5�. In FIG. 2, the dashed line 91 is an extension of the deck surface 12, and the angle designated by 90, between line 91 and the horizontal line 27, is about 5�. The guide 28 is located approximately at the transition between decks 12 and 27.
In accordance with this feature of the invention, gravity is used to keep a stack of up to 9 inches high upright in the hopper without guides. By tilting the entire mail deck up to the singulator five degrees up toward the downstream direction of the machine, the stack of envelopes will tend to lean against the back wall 21. To shift the center of gravity of the stack even further back, the back wall in the hopper area is oriented about 100�-110�, preferably 105 degrees, from the surface 12 of the deck, the angle being designated by reference numeral 7. That means that the stack is actually leaning by about 20 degrees from upright. This is more than enough to compensate for the tilt of high stacks from the cumulative effect of all the extra thicknesses of the flaps and thus eliminates the need for a front guide for the stack. The need for a side guide (opposite to the wall 22) is eliminated by a similar use of gravity and by a nudger drive mechanism explained below. By slanting the deck sideways, about 6�, the mail stack is leaned toward the registration wall 22. The lean of the stack toward the back is also enhanced by stripping mail out from under the stack. As the bottom inch of the stack moves into the singulation nip the stack is no longer evenly supported, and it tends to fall upstream or off the left end of the hopper deck 12. This is illustrated in FIGS. 10-12, which is further explained below.
For the machine illustrated in FIG. 2, the top of the rear wall 21 to the deck is only about 4 inches. To accommodate 9 inches of stack height, a rear wall extension (not shown) is provided that pulls up to support a nine inch stack height.
Another feature of the invention is the means by which the mixed mail is properly oriented within the machine. Mail orientation is accomplished using both novel tamping and nudging registration subsystems. The purpose is to get each mail piece in the proper orientation so that as it passes through the rest of the machine it is not skewed and the indicia is printed entirely on the upper right hand corner of the mail piece as is conventional. The object of the tamping subsystem is to register all mail pieces in the stack along the same line whether they are flapped or unflapped. Flapped mail-pieces end up with the inside of the flap pushed up against the inside edge of the desk and unflapped mail-pieces are pushed against a restraint positioned against the inner edge of the deck. The restraint is either the side wall 22 of the tamper or a flap of a subsequent mail piece being held against the inner edge of the deck by the tamper.
To understand this better, reference is made to FIGS. 3 and 4 of the drawings, which is a perspective view of the feeder of the invention, but with the rear wall 21 omitted for clarity. In these figures, the space for the singulator 15 is shown at the right, with its deck 27. Numeral 32 references the forward belt drive in the singulator. Numeral 28 references a barrier plate whose function is to limit the height of the overlapped or shingled mail entering the singulator module. The angle between the decks 12 and 27 is not shown for clarity. The singulator includes a side registration wall 29 forming with the back edge of the deck 27 a slot 30 for passage downstream of the flap of a flapped envelope. An object of the feeder in the hopper region is to introduce shingled mail into the singulator.
The mail to be processed is placed on the deck 12 of the feeder. If it is open flapped mail, the side wall 23, 24 is moved apart from the rear edge 31 of the deck to form an open slot 35, which is aligned with the slot 30 in the singulator. As shown in FIG. 3, the envelopes are placed face down with their overlapped flaps extending downward in the slot 35. If the mail is unflapped, that is, with closed flap, sealed or unsealed, the mail is stacked flap down with the flap fold edge 36 adjacent the side wall 23, 24. In this case, the latter has been moved inward to close the slot 35.
The tamper mechanism is incorporated behind the wall 23, 24 and functions when there are envelope flaps in the slot 35. As will be explained below, the wall 23, 24 is movable and can be caused to exert a varying force on the flaps in the slot 35. The force is maintained high in between feed cycles to define and maintain registration along the letter deck edge 31, and the applied force is relieved to allow free movement of mail when downstream movement is required. This is achieved by causing the tamper or registration wall to push on whatever flaps are between it and the inside edge 31 of the deck 12.
FIG. 13 is a schematic view of the tamper mechanism, seen from the back of the side wall 23, 24. The deck 12, as mentioned, is fixed. The side wall 23, 24 is movable relative to the deck 12, being mounted on linear slides 38. Inside the wall is mounted a dashpot 40 connected to a pushrod 43 mounted in a linear bearing 39 (FIG. 5) supported at 42, the pushrod 43 acting as a cam follower which engages a face cam 45. The dashpot 40 contains a light spring to urge the pushrod 43 against the cam face 45. The latter in turn is mounted on a shaft 46 driven or rotated by a motor 47 mounted beneath the deck 12. As later described, the shaft 46 is part of the forward drive mechanism in the hopper region. A tension spring 49 anchored to a base support post 48 at its right end and to the movable wall 23 at its left end functions to provide a maximum biasing force tending to pull the wall 23 against the deck edge 31.
To feed open flap envelopes the user moves the hopper wall back to create a gap 35 (FIG. 6) for flaps, then loads the hopper region 10. The hopper wall 23, 24 then moves the stack toward the machine front so that the inside of the flap on the bottom most envelope is registered against the rear edge 31 of the letter deck 12 ready to be processed. However, due to the high force required to register the flapped stack, the flaps tend to become pinched between the rear edge 31 of the letter deck and the hopper wall 23. To alleviate this situation, the hopper wall is synchronously coupled to the motor driven face cam 45 through the air dashpot 40. The dashpot 40 is adjusted so that the force the wall 23 transmits to the mail stack varies from approximately a small value of about 3 ounces to a larger value of about 24 ounces. During the downstream feed cycle (explained below) the force drops to allow free movement of the envelopes in the hopper. Between feed cycles the force rises to approximately 24 ounces to tamp and register the bottommost envelope preparing it for processing. An advantage of the dashpot-cam configuration is that a force rather than a displacement is applied to the wall 23 regardless of the wall's linear position. The dashpot is adjusted so the balance of forces on the wall is such that it appears motionless at all times.
The face cam provides, essentially, two extreme positions at opposite sides along its circumference and a gradual taper between the two extreme positions. In one, the minor lobe position, the pushrod 43 tends to be moved furthest to the right in FIG. 5, providing the heavy tamping force, and in the opposite extreme position, the major lobe, the pushrod 43 is moved furthest to the left in FIG. 5 providing the light force relief position. The rotation of the cam 45 is synchronized with the rotation of the nudger so that when the nudger is moving the envelopes downstream, the light force is applied, whereas when the nudger is fluffing the mail stack, explained below, the heavy force is applied.
Suitable sensors can be provided, if desired, to activate mechanisms to disengage the tamper from the cam 45 when no open flap is detected, in which case the spring 49 will move the tamper housing to close the gap 35. Alternatively, when a flap is detected, then the tamper is activated to function as described above. However, an advantage of the prepared system as described above is that no additional sensors are required, and, even though no flaps are present and the wall is pulsating, it does not interfere with the machines normal operation and is not objectionable.
The side wall 23, 24 moves sufficiently to form a slot 35 to accommodate the thickness of many flaps (up to 0.75 inch) between the wall 23, 24 and the registration edge 31 of the deck. As further illustrated in FIG. 6, the upper side wall part 24 is angled backwards about 15-19 degrees, preferably about 17 degrees, with respect to the lower portion 23. This is to accommodate the thicknesses of many flaps and to keep the right, non-flapped, edges of the envelopes in substantial alignment.
The mechanism for moving the mail downstream in accordance with another feature of the invention comprises a nudging subsystem. This drive moves mail in the mail hopper in two directions; downstream in the direction of mail flow through the machine, and toward the registration wall. In addition, as explained below, the stack is also moved upwardly in a fluffing action. Being able to feed the bottom item in a vertical stack allows a mailing machine or like paper handling device to be easy to load and to occupy a minimum of table space. This fluffing feature permits bottom feeding, which also has the advantage of being less sensitive to stack height within a reasonable range.
Another feature of this aspect of the invention is the shingling of a vertical stack of mail in preparation for singulation. Shingling helps reduce the drag forces on the lowermost item in the stack while it is being singulated.
The forward drive of the invention, in a preferred embodiment, uses a plurality of composite rollers 50 of the construction shown in FIG. 14. Each roller 50 consists of a wide core or center element 51 having a circumference 52 which is concentric with its trilobular hole 53. On this circumferential surface is elastically mounted a frictional tire 54. Located eccentric to the trilobular hole ar two cantilever shaft portions 55, one shown in FIG. 14 extending to the left, and the other extending to the right and not visible in FIG. 14. Thin rollers 56 and 57 with low friction surfaces are mounted on these shaft portions 55 and are retained by means of, for example, snap latches 58 and 59. One thin roller is positioned on each side of the wide center portion. As will be noted, the shaft portions 55 are eccentric with respect to the hole 53, and are positioned such that the thin outer roller portions are offset by about 180�. See also FIG. 19. The result is that each of the outer roller portions 56, 57 extend beyond the circumference 52 of the center roller portion 51 over a small arc of about 45�. The reason for allowing the thin roller to extend beyond the outside diameter of the center roller portion is explained below.
In the preferred embodiment shown in FIG. 15, pairs of these composite assemblies 50 are mounted on trilobe shafts 60 so as to establish an in-phase relationship between the roller pair such that the distance between the thin rollers 56, 57 appearing at, for instance, the 6 o'clock position, as shown in FIG. 15, remains constant as the trilobe shaft 60 is rotated. The trilobe shafting also allows the rollers to be rotatingly driven in this established orientation. FIG. 15 also shows the thin rollers 56, 57 extending beyond the circumference 52 of the core element 51 only over a short arc equal to about 90 degrees each. For the remainder of the 180� of the circumference, the core roller 51 extends beyond the thin rollers 56, 57.
FIGS. 16-18 show a side view of three of the assemblies of FIG. 15 located with respect to the horizontal deck 12 of a feeding device and supporting a stack of mail 11 on the frictional tire surface 54 of each roller assembly 50. In the position shown in FIG. 16, the rotational drive supplied to the shafts 60 will move the stack in the direction shown by the arrow. The distance permitted between the shaft assemblies is related to the amount that the frictional tire is exposed above the horizontal deck. The distance between the shafts must be such that any envelope spanning the roller assemblies must be raised high enough by the frictional tire so that its sagging portion does not drape significantly on the deck. As one example, not to be deemed limiting, a 3.5 inch shaft center to center distance and a 0.2 inch tire to deck exposure can be used.
FIG. 17 shows a similar view except the shafts have rotated clockwise (CW) about 45�, and the stack is now supported on the thin, eccentrically mounted rollers 56. In this position, the lowest envelope in the stack is mainly subjected to the frictional force of the stack on top of it. The rollers 56, 57 below offer little frictional drag. In order that there be little or no contact of the envelope with the frictional tire 54 in this roller assembly position, the eccentrically mounted rollers 56, 57 must extend above the tire surface. In the preferred embodiment, they extend approximately 0.10 inches above the tire 54.
In rotating the roller assemblies in a clockwise direction from their position in FIG. 16 to that of FIG. 17, the stack will experience an acceleration in the vertical direction in being displaced from the tire radius to the eccentric roller radius. As the roller assembly continues on in a clockwise direction, shown in FIG. 18, the eccentric rollers arrive at a position where the stack 11 once again is beginning to rest on the tire 54. During this raising and lowering of the stack as the composite assemblies are rotated, a mild to vigorous tossing or fluffing of the stack 11 is effected depending on the rotational speed and the roller assembly geometry. This fluffing of the stack 11 contributes to enabling it to be advanced in a shingled fashion to a singulating device as shown in FIG. 18.
The forward drive system in the preferred embodiment comprises three axial assemblies of two, two, and three composite rollers 50, respectively, as shown in FIGS. 7-9. The shafts 60 of all three assemblies are essentially parallel, but are angled toward the registration wall 23, the angle indicated by 61 being about 10� to 16�, preferably about 13�. The shafts 60 are ganged together and driven by a common motor drive via a pulley 63, mounted under the deck 12, at the same rpm. See also FIG. 5. The same motor also belt drives the shaft 46 which rotates the cam 45. As shown in FIG. 5, the thin rollers in one position extend above the deck activating the stack above.
FIGS. 19 and 20 are top views of the composite rollers, taken after 90� rotation, showing more clearly how in one position, one thin outer roller 56 will protrude to one side while the other thin outer roller 57 will protrude to the other side, and after 180� of rotation later, the other thin roller 57 will protrude, whereas at the 90� and 270� positions (FIG. 20), the center roller 51 protrudes. The effect on a letter 11 of the stack is shown in FIGS. 21-24, showing 270� of rotation of the rollers. As mentioned above, the rim of the core element 51 is of rubber with high coefficient of friction and is relatively wide, whereas the thin rollers 56, 57 on opposite sides may be constructed of plastic with a low coefficient of friction. Thus, when the core element protrudes (FIGS. 21 and 23), the envelopes are driven in the direction of rotation of rollers, downstream or forward, as well as toward the side wall 23 due to the angled position of the rollers (FIGS. 7-9), as shown by the arrow 64 in FIG. 7. In the rotated positions of FIGS. 22 and 24, where the thin outer rollers predominate, the action is mostly vertical to fluff up the stack to reduce frictional forces between the envelopes. This combined forward and fluffing action causes the stack to begin shingling as illustrated in FIG. 18. In addition, the backward tilt of the deck illustrated in FIGS. 10-12 also causes the stack to tilt backward as shown, which is important in reducing the weight of the stack on the lowermost envelopes and makes it easier for the singulator 15 to separate individual mail pieces.
FIG. 10 shows a stack 11 of mail being deposited at the rear in the hopper section against wall 21 before activation of the drive. FIG. 11 shows how activation of the drive typically causes a section 11' of the stack to be separated and driven forward. While the initial forward motion would tend to carry the whole stack forward, the fluffing rollers and the inclined deck tend to cause the upper part of the stack to tilt and fall backward against the rear wall 21, while a handful of envelopes 11' are driven forward. The continued driving and fluffing action causes the initial handful 11' to become shingled 11" and thus pass in that condition under the barrier 28 and is driven forward into the singulator 15 by the belt drive 32. The continued forward drive then causes a second section 11'" to becomes separated from the stack 11 and undergo the same shingling action as the first section 11', and this continues until the hopper becomes depleted of envelopes.
To optimize the above-described action, we have found it desirable to adjust the relative phase of the fluffing rollers in the three axis drive. By "phase" is meant the orientation of the outer fluffing roller 56, 57 on one roller to that on another roller. "In phase" means that, viewed from the front, they are aligned. In particular, it is preferred that the rollers 50 (FIG. 9) on each shaft 60 are all in phase with one another; and the rollers 50 in all three of the assemblies are also in phase with one another.
Another feature that contributes to the pre-shingling action desired is a selection of frictional coefficients for the main center or drive roller 51 for the three roller assemblies. In particular, we prefer that a material be chosen for the drive tire 54 for the three-roller assembly in the extreme upstream position which has the highest coefficient, for the middle two-roller assembly the lowest coefficient, and for the extreme downstream assembly a higher coefficient. This is because the principal advancing forces will be provided by the end roller assemblies. The higher coefficient is especially important for the upstream assembly because of the greater stack weight. Various types of rubber tires with different frictional coefficients are well-known and are available for this purpose.
FIGS. 7-9 also show the profile of the cam face 45 relative to the follower 43. In the position shown in FIG. 7, the follower 43 is on the minor lobe of the cam face and the heavy force is being applied by spring 49 for tamping the flapped envelopes shown at 80. In the view of FIG. 8, 180� of rotation later, the major lobe of the cam face 45 has applied a reverse force to the wall 23 so that a light force now exists, which allows an envelope 80 to be advanced. FIG. 9 shows 180� of rotation later a return to the condition of FIG. 7.
FIG. 25 illustrates a preferred embodiment for driving the cam 45 and roller assemblies. A motor 47 belt-drives 81 shaft 46 to which the cam 45 is attached. The shaft 46 in turn belt-drives 82 the adjacent roller shaft 60, which in turn belt-drives 83, 84 the end roller shafts 60. All the shafts of the drive are supported for rotation by end mounts 85, 86.
It is preferred that the rollers 50 be driven such that the surface speed of the frictional tire 54 is in the range of about 24-32 inches per second (ips). We have found that, for the preferred machine described above intended to handle mixed mail at the rate of up to about four per second, if the surface speed is substantially greater than 32 ips, then excessive vibration of the stack occurs that actually reduces the throughput. On the other hand, when the surface speed falls below about 24 ips, then the mail pieces are not fluffing properly and producing the desired shingling profile. In the range indicated, we prefer the valve of 28 ips as optimum.
As mentioned above, the envelopes are driven forward as well as toward the registration side wall 23, 24. This action is assisted by a tilting of the deck 12 about 4�-8�, preferably 5�, downward toward the side wall, indicated by 66 in FIG. 6 with the dash-dot line 66, being horizontal. FIG. 6 also shows, somewhat schematically, a stack of envelopes 11 whose flaps 67 extend into the slot 35 adjacent the deck edge 31. It is important that the stack 11 as it shingles continues to maintain the envelope flaps 67 in the slot. A further feature of the invention is structure downstream of the tamper wall 23, 24 but before the singulator 15 which is configured to guide the envelope flaps as they shingle down into the slot 35 and into the slot 30 in the singulator module. This structure consists of a vertical wall portion 70 located adjacent the tamper wall 24 and comprising a first surface 71 which slopes downstream, downward and toward the machine front, which intersects a second surface 72 which slopes downward and downstream, merging finally with a nearby vertical major surface 73. Preferably, the surface 72 forms an angle of about 30 degrees-40 degrees, with 35 degrees being preferred, with a vertical plane. This angle substantially matches the angle formed by the leading edges of the ideal shingled stack of mail. It also matches the angle at which the bent lower part 28' of the guide 28 extends. The surface 71 is adjusted to guide the flapped mail stack downstream toward the singulation area without causing any restriction or binding. A preferred angle for that surface is about 107 degrees with respect to the deck, and can vary about 5 degrees either way. See also FIGS. 26 and 27. As shown in FIG. 27, the major surface 73 is angled backwards by a small angle of about 1 degrees to 4 degrees, preferably about 2 degrees. In the figure, line 74 parallels the registration edge 31, and the angle indicated by numeral 75 represents about 2�. The surface 71, is angled indicated by reference numeral 76, preferably between about 35-39 degrees, preferably about 37 degrees, backward with respect to the surface 73. These angular ranges have proven desirable in this particular machine embodiment dealing with No. 5 to No. 15 envelopes with thicknesses up to three-quarters inches.
The operation of the system is based on on-demand feeding, with upstream actions and movements conditioned on the downstream envelope having completed its processing. Assuming this has been done, the nudger tamper subsystem, i.e., the forward drive, is activated whenever there is mail in the hopper covering a hopper sensor (not shown). This is a reflective optical sensor which looks through the hopper deck. Preferably three reflective sensors are provided of which the covering of any one will activate the subsystem. Two are is located in the open area of the hopper and the other is located in the nip area of the singulation module. This ensures that the machine will continue to function while there is any mail piece waiting to be processed.
Each of the features shown and described herein, including the flap edge tamper subsystem, the angled nudger drive subsystem which drives the envelopes downstream as well as toward the side wall, the fluffing action of the drive wheels which together with the angled deck and back support provide the desired shingling action, are believed to be novel in themselves in the preferred environment of a high speed, mixed mail handling machine, and are also considered significant parts and contributors to the high performance of the overall front end feeder combination. Thus, under certain conditions, some features of the present invention may be omitted, or used alone, or used with some but not all of the disclosed features. And the present invention is intended to include individual features of the overall system disclosed herein, as well as combinations of some of the disclosed features without other disclosed features, as well as the overall combination.
The principles of operation described above for these novel subsystems, while considered especially applicable in the environment of a mixed mail handling machine, are also considered applicable to the feeding of other articles from stacks, such as sheets of paper.
Moreover, many of the details given above for the preferred embodiment intended to handle a specific range of envelope sizes and thicknesses are not critical and can obviously be replaced by equivalent means. For instance, the shaft belt drives can be substituted by gearing, and the face cam by any other structure which intermittently forces back the push rod. Alternatively, since these state-of-the-art mail handling machines are frequently controlled by a computer, such as a microcontroller, it is also possible to substitute a solenoid which is pulsed in synchronism with the nudger-fluffer subsystems such that the tamper force is reduced during the envelope driving phase and increased during the stack fluffing phase, or a cam and spring system. Still further, other constructions of the fluffing rollers can be substituted, so long as each roller includes a protruding high friction drive part over part of the circumference and a protruding low friction fluffing part over another part of the circumference. Also the phase relationships of the fluffing and drive parts may be different than as described for different kinds of articles.
While the invention has been described and illustrated in connection with preferred embodiments, many variations and modifications as will be evident to those skilled in this art may be made therein without departing from the spirit of the invention, and the invention as set forth in the appended claims is thus not to be limited to the precise details of construction set forth above as such variations and modifications are intended to be included within the scope of the appended claims.
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|U.S. Classification
|271/2, 271/146, 414/795.7, 414/797.7
|International Classification
|Cooperative Classification
|European Classification
|Dec 28, 1988
Owner name: PITNEY BOWES INC., A CORP. OF DE., CONNECTICUT
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:HOLBROOK, RUSSELL W.;REEL/FRAME:005011/0970
Effective date: 19881219
|May 20, 1994
|Fee payment
Year of fee payment: 4
|May 20, 1998
|Fee payment
Year of fee payment: 8
|May 21, 2002
|Fee payment
Year of fee payment: 12


text:
ACParadise Network: American Cosplay Paradise American Cosplay Experience American Cosplay Snapshots
Cosplayer Ana Aesthetic > Costume of Leeloo (Fifth Element, The)
- Most Recent Photo
- 10-06-2011
- Series
- Fifth Element, The
- Character
- Leeloo
- Special Variation:
- Rubber Suspenders
- Year Completed:
- Construction Difficulty:
About this Costume
- Construction Details:
The wig is a human hair wig dyed and styled by Diana Shepherd. The costume was constructed primarily by myself. The shirt a rather old and baggy sweater I found at Goodwill. The pants made from scratch with a ribbed lyvra, trimmed in black pleather stripping. Belt purchased from Target. Shoes borrowed from my roomie. The suspenders were the most challenging part, taking several days to construct. I used JiaJem's tutorial for the basis, but opted to go with a different Smooth-On product rather than Dragon Skin. Using her pattern as reference, I drafted my own based on my measurements, and used all the same materials Jia suggested to construct the "mold." I'm really proud of this, because I made the suspenders (for the most part) on my own, with a lot of supervision from a friend. This is one of my favorite costumes to wear, ever, and people always comment about how uncomfortable it looks. It's not. It's awesome. And always great to wear in a Georgia summer!
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Analysts link Indian Buddhist temple bombings to Myanmar's ethnic clashes
Some analysts believe a wave of bomb blasts which exploded at one of Buddhism's holiest sites in India could be linked with ethnic clashes between Buddhists and Rohingya Muslims in Myanmar.
The bombs planted across the Maha Bodhi temple in India's Bihar state went off early on Sunday, injuring two monks.
It is the first ever terrorist attack to be launched on Buddhists in India.
Expert analyst Dr Acharya Arabinda says the attack was anticipated after New Delhi police warned state officials that the Maha Bodhi Temple was under threat.
"In October 2012, the New Delhi police arrested some members of the Indian Mujahideen," Dr Arabinda said.
"One of the targets was this temple."
Dr Arabinda believes the attack is clearly linked to ethnic clashes between Rohingya Muslims and Buddhists in Myanmar.
"Lots of Indian Muslims have come to Bangladesh," he said.
"They have been living there for more than 20 years now, and a few of the Indian Muslims have joined terrorist groups."
Kingshuk Nag, editor of the Times of India, says there are several links to the bombings but it is too early to tell who is responsible.
"Somebody has suggested that it's linked to the Rohingyas, therefore this was seen as some sort of a retaliatory effect," Mr Nag said.
"Others have also talked about the Indian Mujahideen group of Muslim terrorists.
"Another line of thinking is that it could be the handiwork of the Hindu fundamentalist groups."
Dr Arabinda has criticised the Indian government and police officials for failing to prevent the blasts.
He believes the government and the police should work together more effectively to prevent any future attacks.
Mr Abhayanand, director general of police in Bihar, says they are not able to confirm who is to blame for the attack.
Topics: terrorism, unrest-conflict-and-war, islam, buddhism, religion-and-beliefs, india, asia, burma
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They're really funky, with a crazy, let-loose style. They're a small band out of Olympia, Wash. I think if it were guys singing, I wouldn't listen to it, because their songs are really kinda sex-nasty, but since it's girls, it makes it kinda fun.
* Robosapien, self-titled
They used to be out of San Francisco, but now they're in Chicago. They're incredibly nice - they stayed at my house (when they came through town) and their music is just good, fun dance music.
* Sister Spit, "Greatest Spits"
This is basically a van full of folks who travel around the country doing spoken word. They were here a few years ago. I really love spoken word, and this is a compilation of artists doing spoken word, so you can't go wrong with that.
* Jurassic Five, "Quality Control"
Hip-hop old-style. They've got smart lyrics and really good beats.
* Mason Jennings
He's been around a long time and I only found out about him a couple of years ago. He plays acoustic-style music that I feel like anybody would like - people who like country would like him and people who like rock would like him too. It makes you feel good to listen to his songs.
Jeff Buckley, "Sketches for My Sweetheart the Drunk"
Disc 1 ... He has an amazing voice range mixed with purely beautiful music and an awesome variety in general.
* Built to Spill, "Keep It Like a Secret"
Oh, Built to Spill.
* Jayhawks, "Sound of Lies"
They consistently produce awesome albums and nobody can keep them down.
* Chris Pureka, "Driving North"
I saw her at Ladyfest South and haven't stopped listening to her album since. She's a great musician and a really nice person.
* Girlyman, "Remember Who I Am"
They're on Daemon as well and they blend folk, rock and sweetness in a way that makes you want to hear more ... all of the time. ...
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Looking for something to do this weekend?
The table is set in Baltimore and Washington counties and Baltimore City for restaurant week.
Life is a peach at festivals celebrating the stone fruit in Westminster, Leitersburg and Sudlersville.
Seafood festivals take place in Havre de Grace and Cambridge.
The Chesapeake Bay Maritime Museum in St. Michaels hosts Watermen Appreciation Day.
The atmosphere gets smoky at the Maryland State BBQ Bash in Bel Air.
Fantasy fun reigns at the Pirates and Wenches festival in Rock Hall and at the Otakon Convention in Baltimore.
To learn more log on to visitmaryland.org, follow them on twitter and Facebook at TravelMD or call 866 – MD Welcome and Discover your Maryland; land of food and fun.