Is the Nicolai EBOXX E14 the most radical e-bike so far?
First glimpsed over two years ago, the Nicolai EBOXX E14 is finally coming out. And it's still a progressive beast. No derailleurs. Lots of length.
NICOLAI has been building mountain bikes for over 20 years, and for over 10 years electric assisted 'eMTB'. The question "Who needs that?" or the quote "I am still too fit for that," is rarely heard today. Why? Because people who are interested in e-bikes quickly learn that riding an eMTB is still sport. The advantages are obvious: differences in fitness between friends are levelled, the range is increased and ultimately, you can have longer riding time and pleasure.
For this year, Nicolai's eMTB called the EBOXX-2019 is developed and produced precisely for this riding experience. This is a special product because Nicolai is one of the few remaining companies to produce bicycle frames in Germany. In our own factory, all components are CNC-milled from high-quality aircraft aluminium and precisely welded together.
The high-quality welding beads have established themselves as a trademark in the industry. A Nicolai bike can therefore also be recognised without a logo.
Kalle Nicolai says: "It goes without saying that Nicolai only selects very high quality components for the complete bike. Exactly the ones we have tested and also continue to use ourselves. In other areas of performance, we continue today with our so-called Geolution geometry for increased safety, traction, and speed, in all riding situations.
All in all, the bike has an unrivalled and incomparably good riding performance. The reason lies in the sum of all the big small details.
BOSCH CX with KIOX. The small, scratch-resistant colour display is controlled by the operating unit at the left grip. It provides data on riding speed, power, heart rate, battery charge and much more. Magnetic holder to remove and attach the on-board computer.
The 500 Wh battery is integrated into the down tube and can be charged on or off the bike.
GeoMetron is the geometry concept on which all current Nicolai models are based. The concept is based on the theory that a longer wheelbase, a slacker steering angle and a steeper seat angle make the bike faster, smoother, easier to climb and safer.


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Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient
Xiang Ren1,2,
Fuyou Wang1,
Cheng Chen1,
Xiaoyuan Gong1,
Li Yin1 &
Liu Yang1
Cartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM).
In this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 107 cells/mL; Group B, 1 × 107 cells/mL; and Group C, 0.5 × 107 cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis.
We found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern.
Our results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.
Articular cartilage is a zonal tissue with a depth-dependent gradient of cells and extracellular matrix (ECM). From the articular surface to calcified cartilage, cartilage is usually divided into a superficial zone, a transitional zone, and a deep zone [1]. Although it is only a few millimeters in thickness, articular cartilage provides a nearly frictionless weight-bearing surface in diarthrodial joints and endures life-long extreme loading stress during limb motion. The functional properties of articular cartilage are correlated with its structural variations according to depth [2].
Articular cartilage is aneural, avascular, and alymphatic and has limited self-repair ability [3]. Once cartilage defects are caused by trauma, degenerative diseases, tumors, or other factors, they inevitably lead to progressive joint damage [4]. To date, it is still a substantial challenge for orthopedists to determine how to repair cartilage defects and restore joint function [5]. All treatments only relieve joint pain and delay the course of degeneration [6]. In the end, patients suffering from joint diseases have no choice but to undergo arthroplasty, which still has unresolved problems [7, 8].
Cartilage tissue engineering is a promising approach to repair and regenerate cartilage tissue through a combination of manufacturing technology and life science [9]. Aiming to mimic the articular cartilage in structure and function, the emphasis of recent research has shifted from homogeneous engineered cartilage to zonal engineered cartilage. Klein et al. [10] used chondrocyte subpopulations to produce zonal-specific ECM. Ng et al. [11] fabricated a chondrocyte-seeded agarose construct with distinct material properties in conjoined regions using a novel layered agarose technique. Woodfield et al. [12] achieved gradient distribution of cells and ECM by seeding chondrocytes into a polymer scaffold with a pore size gradient. However, little attention has been paid to the cell density gradient that is present in mammalian articular cartilage [13]. Zonal cartilage has a zonal chondrocyte density [14], different cell-cell communication [15], and different relationships between the cells and their environment [16–18].
In the present study, we hypothesized that the cell density gradient plays an important role in forming the zonal distribution of ECM. Zonal engineered cartilage was fabricated through bioprinting of collagen type II hydrogel constructs with a biomimetic chondrocyte density gradient. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis. The objectives of the study were to offer a new strategy for producing zonal engineered cartilage and to assess the interactions between the total cell density and the cell distribution pattern to construct engineered cartilage.
Chondrocyte isolation and expansion
Chondrocytes were isolated from the knee joints of juvenile New Zealand white rabbits via enzymatic digestion. The animals were provided by the laboratory animal center and were sacrificed with the approval of the Animal Care and Use Committee at the Third Military Medical University. The cartilage was sequentially digested with 0.1 % trypsin in PBS at 37 °C and 5 % CO2 for 30 min, followed by treatment with 0.025 % collagenase II (Sigma, USA) in high-glucose Dulbecco's modified Eagle's medium (DMEM; HyClone, China) supplemented with 10 % fetal bovine serum (FBS; HyClone, China) and 1 % penicillin and streptomycin (P/S; Beyotime, China) at 37 °C and 5 % CO2 overnight. The isolated chondrocytes were expanded in 225 cm2 culture flasks in high-glucose DMEM supplemented with 10 % FBS.
Collagen type II pre-gel preparation
Collagen type II was extracted from swine knees via pepsin treatment and salt precipitation, as previously described [19]. The swine knee joints were obtained from the nearby slaughter market and used with the approval of the suppliers. The collagen type II precipitant was dissolved in 0.15 M hydrochloride acid to obtain pre-gel at a 10 % (wt/vol) final concentration, which was then stored at 4 °C.
Cell-laden construct fabrication
A bioprinter developed by our laboratory based on open-resource systems consists of robotic arms with three-axis motion, a stationary print platform, and a changeable print head. The print head on the X-axis robotic arm consists of piston-driven microscale depositional equipment. The bioprinter is controlled by software written by our laboratory.
Passage 3 chondrocytes were detached from culture flasks using 0.25 % trypsin (HyClone, China). Cell viability was assessed using the trypan blue exclusion test. The cells were counted with a hemocytometer and aliquoted according to different seeding densities. The collagen type II pre-gel was adjusted to pH 7.0 through a dropwise titration of 10 M sodium hydrate. The chondrocytes and collagen type II pre-gel were gently mixed in Eppendorf tubes at temperature below 20 °C.
Our earlier study found that cell distribution patterns could not be well maintained in a printed hydrogel construct as thin as articular cartilage tissue due to zonal fusion caused by the hydrogel's low mechanical strength. In the present study, a computer-aided design (CAD) model of a cylinder with a 6 mm height and a 4 mm radius was created, as shown in Fig. 1(a). The constructs were printed layer by layer, from the bottom to the top, on a sterile platform on a super-clean bench at a temperature below 20 °C, as shown in Fig. 1(b). A 25-gauge needle (0.25 mm inner nozzle diameter) was used. The deposition speed was set to 20 mm/s, and the layer thickness was set at 0.4 mm. Every construct had 15 layers. Three groups were created according to the total cell seeding density in the collagen type II pre-gel: Group A, 2 × 107 cells/mL; Group B, 1 × 107 cells/mL; and Group C, 0.5 × 107 cells/mL. Based on Hunziker's study [14], the total cell density within the articular cartilage of the human medial femoral condyle is 1 × 107 cells/mL. Group B was designed to have a biomimetic total cell density, while Group A was designed to have a higher total cell density, and Group C was designed to have a lower total cell density. Each group included two types of construct: one with a cell density gradient and the other with a homogeneous cell distribution. According to previous research [14], the ratio of chondrocyte densities in the superficial zone (top 10 %) to the transitional zone (middle 10 %) to the deep zone (remaining 80 %) of normal adult human articular cartilage is approximately 3:2:1. As depicted in Fig. 2, the first 11 layers (deep 74 %) of the printed construct were designed to be the deep zone, the middle 2 layers (middle 13 %) were designed to be the middle zone, and the top 2 layers (top 13 %) were designed to be the superficial zone. The construct with the homogeneous cell distribution had a single cell density, which was equal to the total cell density. For the condition of the construct with the cell density gradient, the ratio of three zonal cell densities was 3:2:1, and the ratio of the three zonal volumes was 13 %:13 %:74 %. As the total cell densities were known for each group, the zonal cell densities shown in Table 1 could be obtained by solving a three-variable linear equation.
Images of the CAD model and the collagen type II hydrogel constructs. a Cylinder generated by CAD (4 mm radius and 6 mm height). b Printing on a sterile platform. c Constructs cultured in a 24-well plate. d Construct harvested at 3 weeks
Study design schematic. a The construct with the cell density gradient and the construct with the homogeneous cell distribution were fabricated from bottom to top. b The constructs were cultured in a 24-well plate. c Three cell seeding density groups (2 × 107, 1 × 107, and 0.5 × 107 cell/mL) were created. Constructs cultured in vitro were harvested at 0, 1, 2, and 3 weeks for biochemical assays, a live/dead assay, RT-qPCR and histological analysis
Table 1 Zonal cell densitya in each group
The fabricated constructs were cross-linked in a humidified incubator at 37 °C for 30 min [20] and were then moved into a 24-well plate and cultured in high-glucose DMEM supplemented with 10 % FBS, 1 % P/S, and 2.5 μg/mL amphotericin B (Beyotime, China) at 1.3 mL per well at 37 °C and 5 % CO2, as shown in Fig. 1(c). The culture medium was changed every other day. The constructs were harvested at 0, 1, 2 and 3 weeks, as shown in Fig. 1(d).
Cell viability test
Cell viability was assessed using the Calcein AM (KeyGen Biotechnology Nanjing, China) and propidium iodide (PI, KeyGen Biotechnology Nanjing, China) molecular probes. Live cells were stained with Calcein AM (green), and dead cells were stained with PI (red). The constructs were sectioned and incubated in PBS containing 0.2 μM Calcein AM and 0.6 μM PI at room temperature for 1 h and were then rinsed in PBS. A full-thickness plane was imaged by fluorescent microscopy (Olympus IX71 microscope). The total cell number in each field of view could be quantified by separately counting green and red fluorescent dots. The percentage of live cells was calculated by dividing the number of live cells by the total cell number in 3 random images at 20× magnification.
Gene expression analysis by RT-qPCR
Total RNA was extracted from the constructs using the Cartilage RNAout Kit (TIANDZ, China). The RNA concentrations and purity were measured using a NanoDrop 2000 (Thermo Scientific, USA). Reverse transcription was performed with the QuantiTect Reverse Transcription Kit (Qiagen, USA). Real-time PCR was performed with the QuantiFast SYBR Green Kit (Qiagen, USA). All procedures followed the manufacturers' protocols. The expression levels of the collagen type I (COL1A1), collagen type II (COL2A1) and aggrecan (ACAN) genes were normalized to GAPDH, which was used as an endogenous control gene, according to the 2-△△CT method. The target gene primers were designed as follows: COL1A1 forward, 5-TTGGCAAAGAGGGCGGCAAAGGC-3, and reverse, 5-GAGCACCAGCAGGTCCGTCAGCA-3; COL2A1 forward, 5-ACGCTCAAGTCCCTCAACAACCAG-3, and reverse, 5-GTCTATCCAGTCACCGCTCTTCCA-3; ACAN forward, 5-ACTTCCGCTGGTCAGATGGA-3, and reverse, 5-TCTCGTGCCAGATCATCACC -3; and GAPDH forward, 5-GAAGGTGAAGGTCGGAGTC-3, and reverse, 5-GAAGATGGTGATGGATTTC-3.
Biochemical analysis
After harvesting, the constructs were weighed and then digested for 24 h in 1 mL of 0.1 M PBS containing 2.5 U of papain (Beyotime, China), 5 mM cysteine-HCl and 5 mM Na2EDTA at 60 °C. The cell number was assessed based on the DNA content using Hoechst 33258 dye (KeyGen Biotechnology Nanjing, China), as previously described [21]. Hoechst 33258 is a fluorescent nucleic acid stain used to quantitate double-stranded DNA in solution. The fluorescence of the samples was converted to cell numbers using a cell-number standard curve created previously. The glycosaminoglycan (GAG) content was determined using the 1, 9-dimethylmethylene blue (DMMB; Jianglaibio, China) dye-binding assay and a shark cartilage chondroitin sulfate reference sample (C4348; Sigma, USA). The GAG content was normalized to the cell number to assess the single-cell biosynthetic activity.
Histological analysis
The constructs were fixed in 4 % paraformaldehyde, dehydrated in a sequential ethanol series and embedded in paraffin. Sections (5 μm) were cut, deparaffinized and stained with hematoxylin-eosin (H&E) for cell distribution assessment and with Alcian blue for sulfated GAG assessment. For immunohistochemistry, rabbit polyclonal antibody Anti-Collagen I (bs-0578R; Bioss, China), Anti-Collagen II (bs-11929R; Bioss, China) and Anti-PRG4 (bs-1175R; Bioss, China) primary antibodies were used to detect collagen type I, collagen type II, and PRG4, respectively, synthesized by the rabbit chondrocytes. The primary antibodies were recognized using the Biotin-Streptavidin HRP Detection System (SP-9000; ZSGB-BIO, China) and a DAB kit (ZLI-9017; ZSGB-BIO, China). All procedures followed the manufacturers' protocols.
Statistical evaluation was performed using GraphPad Prism software (version 6.02). The data are presented as the mean ± standard deviation (SD) and were analyzed using analysis of variance (ANOVA) followed by Tukey's honestly significant difference post hoc tests. Two-way ANOVA was used to reveal the differences in the total cell number, cell viability, GAG content and GAG/cell numbers among groups. One-way ANOVA was used to analyze the gene expression levels. In this study, p values less than 0.05 were considered statistically significant.
Construct characterization
The constructs were successfully fabricated and had uniform geometrical dimensions, with an average volume of 0.3 ± 0.05 mL. The physiochemical properties of the 10 % (wt/vol) collagen type II pre-gel adequately matched the requirements for hydrogel biofabrication [22]. As shown in Fig. 3, the results of the H&E staining showed that both the homogeneous and the gradient cell distribution patterns were effectively established and were maintained throughout the whole culture period.
Representative images of H&E staining of the construct with the homogeneous cell distribution and the construct with the cell density gradient (scale bars, 200 μm)
Cell viability
The trypan blue exclusion test showed that 98 ± 1 % of the chondrocytes that had detached from the culture flasks were alive. To assess the damaging effect of printing on cell viability, cell viability tests were performed on the first day after fabrication, as shown in Fig. 4(a). The average live cell percentage was 93 ± 3 %. No significant difference was observed between the two types of construct or among the different groups. As shown in Fig. 4(b), a slight decrease in the total cell number was observed in all groups during culture, but the difference was not statistically significant.
Cell viability after fabrication and the total cell number in the constructs. a The live cells were stained with Calcein AM (green dots), and the dead cells were stained with PI (red dots). The images in a, b, and c represent the construct with the homogeneous cell distribution in Group B on the first day after biofabrication; the images in d, e, and f represent the superficial zone of the construct with the cell density gradient in Group B after 1 week of in vitro culture; the images in g, h, and i represent the middle zone of the construct with the cell density gradient in Group B after 2 weeks of in vitro culture; and the images in j, k, and l represent the superficial zone of the construct with the cell density gradient in Group A after 3 weeks of in vitro culture (scale bars: 100 μm). b Total cell numbers in constructs in Groups A, B, and C after 3 weeks of in vitro culture
To evaluate the phenotypic alterations of the chondrocytes in the collagen type II hydrogel constructs, the relative expression levels of COL1A1, COL2A1 and ACAN compared to GAPDH were determined by real-time PCR, as shown in Fig. 5. During the 3-week in vitro culture, COL1A1 expression remained low, and it was down-regulated throughout the culture period. However, the expression levels of both COL2A1 and ACAN were significantly increased (p < 0.05).
RT-qPCR analysis of the COL1A1, COL2A1, and ACAN expression levels in the construct with the cell density gradient and the construct with the homogeneous cell distribution in Groups A, B, and C after 3 weeks of in vitro culture. COL1A1: collagen type I; COL2A1: collagen type II; ACAN: aggrecan. The * indicates significance (p < 0.05). The error bars show the SD (n = 3)
GAG content quantification
The total GAG contents in the constructs are shown in Fig. 6(a). During the first week of culture, the GAG content was positively correlated with the total cell density. Group A had the highest GAG content, and Group C had the lowest. During the second and third week, only Group C had a lower GAG content compared to Group A or B (p < 0.05). The differences between Group A and B were not significant.
GAG content of the constructs and GAG content normalized to the cell number. a GAG content of the constructs in Groups A, B, and C after 3 weeks of in vitro culture. b GAG content normalized to the cell number. The * indicates significance (p < 0.05). The ^ indicates a significant difference between the gradient construct in Group A and the gradient construct in Group B, the homogeneous construct in Group B, and the gradient construct in Group C (p < 0.05). The # indicates a significant difference between Group A and Groups B and C (p < 0.05). The error bars show the SD (n = 3)
To assess the average single-cell GAG production, the total GAG content was normalized to the cell number, as shown in Fig. 6(b). In the first week, no significant differences were observed among the three groups. In the last 2 weeks, the average single-cell GAG production in Group A was the lowest among the three groups. Because there was no significant decrease in the cell number, several of the cells in Group A might have entered the static state due to the limited nutrient supply. Group B had the highest average single-cell GAG production. Interestingly, there were significant differences between the construct with the cell density gradient in Group B and the constructs in Group A and C, although there was no significant difference within Group B. This result might be due to a synergistic effect of the total cell density and the cell distribution pattern. At 3 weeks, a significant difference was observed in the constructs with the homogeneous cell distribution between Group B and Group C, which might have been due to an effect of the total cell density.
Alcian blue staining and immunohistochemical analysis of collagen types I and II and PRG4
To assess the ECM distribution in the constructs, Alcian blue staining for GAG and immunohistochemical analysis of collagen types I and II and PRG4 were performed. As shown in Fig. 7, the positively stained cells were brown, the negatively stained cells exhibited blue nuclei, and GAG in the matrix was also stained blue. Nearly all of the chondrocytes in the constructs stained positively for collagen type II, many stained positively for PRG4, and a few stained positively for collagen type I. In the constructs with the cell density gradient, the collagen type II, PRG4 and GAG contents were concentrated in the superficial zone and decreased with depth.
Zonal distribution of ECM in the constructs with the cell density gradient in Group A after 2 weeks of in vitro culture. The images in a, b, c, and d represent the superficial zone; the images in e, f, g, and h represent the middle zone; and the images in i, j, k, and l represent the deep zone. Images of collagen type I immunohistochemical (IHC) staining (a, e, and i); collagen type II IHC staining (b, f, and j); PRG4 IHC staining (c, g, and k) (scale bars: 100 μm). Images of Alcian blue staining (d, h, and l). The white arrows indicate positively stained cells, and the black arrows indicate negatively stained cells (scale bars: 200 μm)
The ultimate goal of tissue engineering is to generate tissues or organs in vitro to repair and reconstruct impaired body function. As cartilage is aneural, avascular and alymphatic, it was predicted that cartilage would be one of the first tissues to be successfully replicated in the laboratory. However, this assumption proved to be incorrect. Engineered cartilage is fragile in vivo and has difficulties in integrating with the surrounding tissues [23]. One of the main defects of engineered cartilage is its lack of zonal properties compared to natural cartilage [24].
Chondrocytes are the sole resident cell type in articular cartilage [25]. A chondrocyte density gradient is one of the zonal properties of mammalian articular cartilage [13]. From the superficial zone to the deep zone in the human femoral condyle, the ratio of the zonal chondrocyte densities is approximately 3:2:1 [14]. In the present study, we printed collagen type II hydrogel constructs with a biomimetic chondrocyte density gradient and focused on the biological effects of the cell density gradient and the total cell density. To compare the two different cell distribution patterns at a specific total cell density, two types of construct were designed: one with a cell density gradient and the other with a homogeneous cell distribution. To evaluate the interactive effects of the total cell density and the cell distribution patterns, three groups were created according to the total cell seeding density. The results of the study demonstrated that total ECM production was positively correlated with the total cell density in the early culture stage, that both the cell distribution and the total cell density had effects on the cells' biosynthetic ability, and that the cell density gradient distribution resulted in a gradient ECM distribution.
A primary goal of biofabrication is to achieve high cell viability in constructs [26]. When cells are deposited within biomaterials using a bioprinter, they are exposed to shear stress, and damage may occur [27]. Attention should be paid to both the rheological parameters (e.g., viscosity) of the printing material and the printing parameters (e.g., dispensing pressure and nozzle size) [28, 29]. In our study, all chondrocytes were encapsulated in a 10 % (wt/vol) collagen type II pre-gel. Thus, the viscosity of the mixture of chondrocytes and pre-gel depended on the cell density. With the same printing parameters, the superficial zone of the construct with the cell density gradient in Group A had the highest risk of cell damage because it had the highest cell density. Nevertheless, the cell viability test showed that 93 ± 3 % of the chondrocytes were alive after printing. No significant difference in cell viability was observed between the two types of constructs or among the three groups, which was consistent with previous studies [22]. The cell viability was also determined by quantifying the total cell number. As shown in Fig. 4(b), there were no significant changes in the total cell numbers in any construct throughout the culture period. Cell apoptosis might account for the slight decrease.
As a basic ingredient in tissue engineering, scaffolds should provide a platform that directs cell adhesion, proliferation, and ECM synthesis. Designing a biomimetic scaffold to accommodate cells in a suitable milieu is one of the most important strategies in tissue engineering [30]. Hydrogels possess many characteristics of a natural ECM and are widely used for three-dimensional cell culture [31, 32]. In articular cartilage, chondrocytes dwell in the natural collagen type II hydrogel networks, the interstitial voids of which are filled with a large amount of fluid components and some GAG [1]. Based on the biomimetic component idea, we chose collagen type II hydrogel to construct engineered cartilage. The results of the RT-qPCR and immunohistochemical analyses of collagen types I and II showed that chondrocytes cultured in collagen type II hydrogel retained their chondrogenic phenotype. Meanwhile, many studies have demonstrated that collagen type II has the ability to retain the biological functions of chondrocytes and is appropriate for engineering cartilage [33–35].
Cells have a core role in tissue engineering systems. The main function of cells is to synthesize and maintain ECM. Once cells settle down on scaffolds, they sense the environment and react accordingly. Both the cell seeding density and the cell distribution pattern are important parameters when designing a tissue engineering construct. It has been demonstrated that the cell seeding density is a positively correlated with ECM production [36, 37] and that an anisotropic distribution of cells leads to an anisotropic distribution of ECM [12]. However, few studies have investigated these two parameters simultaneously and quantitatively. In the present study, we evaluated the two factors in one system by comparing two cell distribution patterns at three total cell densities. The results showed that the constructs in Group B (1 × 107 cells/mL total cell density), and particularly the construct with the cell density gradient had two advantages, namely, improved total GAG production and single-cell biosynthetic ability, over the constructs in the other two groups at 2 weeks of in vitro culture. The results showed the synergistic effects of the cell distribution pattern and the total cell density. Meanwhile, the constructs with the cell density gradient had the advantage of zonal ECM properties similar to those of natural cartilage compared to the constructs with the homogeneous cell distribution.
In previous studies [12], a cell gradient distribution was achieved using a scaffold with a pore size gradient, in which the cells were randomly settled on the surface of the polymer fibers. The zonal cell density and the total cell number in the scaffold could not be quantified. Furthermore, the cell distribution pattern in the scaffold was not stable during culture. In addition, different pore sizes may have different biological effects on cells [38]. In our study, chondrocytes and collagen type II pre-gel were mixed to produce the required cell density. The zonal proportions and cell density gradient in articular cartilage were well duplicated in the construct by bioprinting. The cell distribution patterns remained stable throughout the culture period. The results of the immunochemistry and Alcian blue staining demonstrated that the new ECM had a gradient distribution in the constructs with the cell density gradient. This observation was more meaningful for PRG4, which is a specific protein of the superficial zone that is responsible for surface lubrication [24, 39].
Our study provided a new strategy to construct zonal engineered cartilage. Compared to traditional scaffold fabrication methods, hydrogel bioprinting has the advantages of being able to fabricate constructs with an arbitrary geometry on the macro-scale, particularly for constructs with a curved surface, and to accurately control the cell distribution in constructs on the micro-scale. Zonal engineered cartilage was fabricated by bioprinting a construct with a cell density gradient. This technique is easy to perform, and there are no difficulties in isolating and expanding the chondrocyte subpopulations. More importantly, by optimizing the total cell density and the cell distribution pattern, the chondrocytes can be induced to exhibit maximum biosynthetic ability. Researchers may no longer need to wait for long periods to allow chondrocytes to expand to achieve maximum ECM deposition in a scaffold. In addition, the study showed that structurally biomimetic engineered cartilage could be constructed by hydrogel biofabrication. The constructs with a cell density gradient could be a good platform for investigating cell-cell communication and the relationships between cells and their environments.
The study also has certain limitations. First, articular cartilage is a weight-bearing tissue. Mechanical stimuli play an important role in mediating chondrocytes' biological functions [18]. Zonal chondrocytes sense the zonal mechanical environment before producing a specific ECM. In this study, however, the results were only observed in static culture. Further studies should be performed to observe more differences between the two types of constructs in an appropriate mechanical environment (e.g., via culture in a bioreactor). Second, constructs with a biomimetic cell density gradient should be evaluated in vivo to observe whether these constructs are able to better integrate with the surrounding tissues and produce a satisfactory clinical outcome. Furthermore, we did not pay much attention to the effects of the total cell density and the cell distribution pattern on the subpopulation phenotype. In future studies, effort should be devoted to retaining and/or creating a subpopulation phenotype [40]. It would also make sense to fabricate engineered cartilage with a biomimetic distribution of zonal marker proteins.
In this study, we printed collagen type II hydrogel constructs with a biomimetic chondrocyte density gradient and focused on the biological effects of the cell distribution and the total cell density. The results from the in vitro static culture showed that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern. The results suggested that zonal engineered cartilage could be constructed by bioprinting a collagen type II hydrogel construct with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to obtain synergistic biological effects.
ACAN, aggrecan; ANOVA, analysis of variance; CAD, computer-aided design; COL1A1, collagen type I; COL2A1, collagen type II; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; FBS, fetal bovine serum; GAG, glycosaminoglycan; H&E, hematoxylin-eosin; P/S, penicillin and streptomycin; PI, propidium iodide; SD, standard deviation
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Barnes CP, Pemble CW, Brand DD, Simpson DG, Bowlin GL. Cross-linking electrospun type II collagen tissue engineering scaffolds with carbodiimide in ethanol. Tissue Eng. 2007;13:1593–605.
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Malda J, Visser J, Melchels FP, Juengst T, Hennink WE, Dhert WJA, Groll J, Hutmacher DW. 25th Anniversary Article: engineering hydrogels for biofabrication. Adv Mater. 2013;25:5011–28.
Steinert AF, Ghivizzani SC, Rethwilm A, Tuan RS, Evans CH, Noth U. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res Ther. 2007;9:213.
Klein TJ, Malda J, Sah RL, Hutmacher DW. Tissue engineering of articular cartilage with biomimetic zones. Tissue Eng Part B Rev. 2009;15:143–57.
Archer CW, Francis-West P. The chondrocyte. Int J Biochem Cell Biol. 2003;35:401–4.
Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat Biotechnol. 2014;32:773–85.
Ferris CJ, Gilmore KG, Wallace GG, Panhuis MIH. Biofabrication: an overview of the approaches used for printing of living cells. Appl Microbiol Biotechnol. 2013;97:4243–58.
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Nair K, Gandhi M, Khalil S, Yan KC, Marcolongo M, Barbee K, Sun W. Characterization of cell viability during bioprinting processes. Biotechnol J. 2009;4:1168–77.
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We are grateful to the laboratory assistants in our center for all they've done for collecting data and technical supports.
This work was supported by grants from the National Natural Science Foundation of China (No. 31130021), the PLA Research Foundation (No. SWH2013JS08), and the International Cooperation Foundation of Chongqing Science and Technology Commission (No. CSTC2014GJHZ110003).
There is a willingness to share primary data related to the research on request to the Center for Joint Surgery, Southwest Hospital, the Third Military Medical University, Chongqing 400038, P.R. China. Please see: http://www.jointsurgery.com.cn/.
XR, FW, CC, XG, LYin and LYang made substantial contributions to the study conception and design and to the acquisition and statistical analysis of the data in the study. All authors read and approved the final manuscript.
The animals were sacrificed with the approval of the Animal Care and Use Committee at the Third Military Medical University. All procedures complied with the Basel Declaration and its later amendments or comparable ethical standards.
Center for Joint Surgery, Southwest Hospital, the Third Military Medical University, Chongqing, 400038, People's Republic of China
Xiang Ren, Fuyou Wang, Cheng Chen, Xiaoyuan Gong, Li Yin & Liu Yang
Orthopedic Department, 452nd Hospital Chinese PLA, Chengdu, 610021, Sichuan, People's Republic of China
Xiang Ren
Fuyou Wang
Cheng Chen
Xiaoyuan Gong
Li Yin
Correspondence to Liu Yang.
Ren, X., Wang, F., Chen, C. et al. Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient. BMC Musculoskelet Disord 17, 301 (2016). https://doi.org/10.1186/s12891-016-1130-8
Chondrocyte
Collagen type II
Cell density gradient
Total cell density

redpajama_set_name: RedPajamaCommonCrawl


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David Scott Dibble for Senate District 61
Scott Dibble is running for re-election to SD 61. He currently represents SD 61, which includes parts of South, Southwest, and downtown Minneapolis. Dibble was first elected to the Senate in November 2002. Prior to the senate, he served one term in the Minnesota House of Representatives, becoming the third openly gay legislator to serve in the Minnesota Legislature. As a state senator Dibble has been influential on matters of human rights, transportation and transit, energy efficiency, the environment, housing and economic development.
On reforming Minnesota's transportation system:
"Transportation now creates more green house gases in Minnesota than electricity generation. I'm proud of my role as past chair of the Transportation Committee in delivering bills that provide the highest ever levels of funding for transit, biking and walking, and connecting those investments to climate, land use and equity – ensuring that we serve those who most need mobility and access to destinations. I will continue to push for this. I supported an initiative to first deploy electric buses in the communities most impacted by transportation pollution. Additionally the Minnesota Green New Deal would require that we eliminate fossil fuels from our transportation system and focus economic opportunity and benefits to those who have been traditionally left out."
Visit the campaign website to support Scott Dibble: https://www.scottdibble.com


Server monitoring is the best way to prevent catastrophic data failure on a business wide scale. Our IT support London team is able to remotely view the condition of your server, 24 hours a day, 365 days a year. Using our knowledge both of the normal power and data load your business runs through the server, and the capacities of the server itself, we'll be able to spot any problems long before they become serious, access the server either remotely or physically, and solve the issue.
Remote access or hands on repair?
Depending on the condition of your server, we may choose to go in remotely and change its settings; or we may need to attend your premises physically and inspect the hardware in more detail. You don't need to worry about a thing: if there's a serious problem, we will migrate your data safely to a temporary server while we fix or replace the item in question.
Out of date software is one of the most common causes of erratic server behaviour. We don't only monitor your hardware to ensure that it's physically performing as it should: we also keep an eye on the licenses and known upgrades for the software you run on it. With a complete server monitoring package from our IT support London team, you'll never have to worry about keeping track of all your licenses and upgrades again. Let us take the worry out of running an IT department: leaving you free to concentrate on the things you do best.
Our server monitoring software allows us to perform nearly all monitoring, diagnosis and repair without physical access to your machine. We can instantly call up server status reports, and view the data load on your network in real time. We can look at predictions for the power and memory space your disk will use in the future, and access historical reports to examine the past performance of the unit. We can see every event that happens in the server. If necessary we can isolate it, transfer the data load to a different machine or go inside to perform a fix.
No problem. Our IT support London team can monitor, assess and fix multiple servers, either in an installation designed by us or in a historical installation. We can also assess the current efficiency of your server setup, recommend changes and consult on the design of a new network. Talk to us today about server monitoring and design: and start tomorrow with a more streamlined and power-efficient network.
Power outages are a frequent cause of service interruption – usually, they happen when one server is asked to perform too many power-intensive tasks at once. Our IT support London team can monitor the power used by all of your servers, and remotely activate switches to keep every task running at the optimum speed.
Server monitoring – What options?


Since I started blogging back in October 2011 I have been so lucky to get in contact with a large number of economists others with interest in monetary policy issues. That has been very rewarding and I want to thank everybody that are reading and commenting on my blog. You make it more inspiring for me to blog.
However, I would like to get in contact with even more 'monetary nerds' around the world and I would also love to help facilitate contacts between others with similar interests.
I am therefore setting up a Global Monetary Policy Network (GMPN). GMPN is not a club or an association, but more like a database of 'monetary nerds' around the world.
I therefore welcome all of you to drop me a mail about your own interests in monetary policy, monetary theory and monetary history. Let me know about your background and why you are interested in monetary issues. So are you interested in monetary policy or monetary history – or financial and banking regulation for that matter?
I will not make any of the information public, but I hope to be able to bring 'monetary nerds' from around the world together.
So if you for example are a PhD student from Sweden who would like to research mobile banking in Africa I might be able to bring you in contact with an Kenyan economics professor or are you a central banker in Europe that would like to learn about banking resolution in New Zealand then I might have a contact for you.
You might also be a journalist who is interested in getting into contact with monetary experts around the world.
So in that sense I see GMPN as a possibility to build a database of interesting people. There are no conditions for joining the network and it is obviously for free. Economics professor, students, journalists and policy makers are all very welcome.
Finally it is certainly no condition that you are a Market Monetarist or think NGDP targeting is a great idea. The only condition is that you have an interest in monetary issues and is willing to share some information about yourself with me.
For those of you I already know I also welcome you to drop me a mail – so I can update my "data" on you and your interests.


In today's world it's not always easy, or even possible, to find the perfect house with the ideal number of rooms, bathrooms, or living space. Sometimes the potential your home holds isn't obvious. Little changes can have a tremendous impact on opening up new avenues that will give you the bonus of more space with the benefit of avoiding uprooting the family. Sphere can help you unlock your properties potential.
Sphere can help you with all your construction projects large or small.
Our workforce specialise in all types of structural work including basement, garage and loft conversions, extensions, roof repairs, kitchen and bathroom installations. Using only the best quality materials and products available we will take control and oversee the entire project through to completion, including any lighting, plumbing and electrical needs and damp proofing.
We try to design and construct in an environmentally friendly manner by the use of sustainable and eco-friendly materials. When we plan an extension we would attempt to maximise solar gain by orientating the majority of habitable room windows in a predominantly southerly elevation or by the use of sun tunnels. This will of course reduce the dependence upon supplementary heating and lighting and reduce heating and lighting expenditure.


Last week, a biased but fairly thorough analysis revealed that Massachusetts is the best state in the nation. Even if the weather can get a little miserable, the Bay State tops (or is at least close to topping) the charts in education, health, and wealth, and recently pushed California out of its number one spot as America's most energy-efficient state.
And it looks like it's going to keep that energy-efficient crown for a while. Starting in 2014, large institutions including hotels and restaurants are going to be banned from throwing away their leftover food. Instead, the food waste will go to composting sites and specially-designed treatment plants to convert the scraps into energy, heat, and fertilizer.
The specially-designed plants use bacteria to break down the leftovers into methane, which is then burned like natural gas to make electricity. This kind of system is already in place in parts of Europe, and state officials hope to expand it to home and small restaurant waste, too.
Expect the rest of the country to catch up to the Best State in a couple of decades.


text:
Q: Get Spreadsheets from a folder and modify them I have a folder called "Adform" in my Drive and I want to take all the spreadsheets from there and modify them by adding new columns, change headers.
At this point I managed to get the files name, one by one, but I don't know how to use SpreadSheetApp to modify those Spreadsheets.
var folders = DriveApp.getFoldersByName('Adform');
    while (folders.hasNext()) {
      var files = folders.next().getFiles();
      while (files.hasNext()) {
        var file = files.next();
        Logger.log(file.getName());

    }
  }

A: How to loop through all the Spreadsheets in a Drive folder
You are almost there. The best way in my opinion is to check if the mime type of the file is application/vnd.google-apps.spreadsheet, and if so, get the id. Then pass this id to another function that will modify the Spreadsheet in any way that you want.
function myFunction() {
  var folders = DriveApp.getFoldersByName('Adform');
    while (folders.hasNext()) {
      var files = folders.next().getFiles();
      while (files.hasNext()) {
        var file = files.next();
        // Check if file is a spreadsheet
        if (file.getMimeType() === "application/vnd.google-apps.spreadsheet") {
          // Call the function to modify the spreadsheet with the file id
          modifySpreadhsheet(file.getId())
        }
    }
  }
}

function modifySpreadsheet(id) {
  // Open the file with the id
  var file = SpreadsheetApp.openById(id);
  // Get all the sheets
  var sheets = file.getSheets()
  // Get the first sheet
  var firstSheet = sheets[0]
  // Get the range that contains all the data in that sheet
  var range = firstSheet.getDataRange();
  // Get all the values in that range
  var values = range.getValues();

  Logger.log(values)
}

This sample function will just get the first sheet of the spreadsheet and then Log all the data within that spreadsheet to the logger.
Then you can use functions like setValues(values) to modify the content as needed.
References

*

*getMimeType()

*getId()

*openById(id)

*getSheets()

*getDataRange()

*getValues()

redpajama_set_name: RedPajamaStackExchange


text:
PSA and Renault hits government plans to reverse diesel policy
Article by Christian A., on December 5, 2014
A plan by France to get rid of its policies encouraging diesel cars has met criticisms from the country's own carmakers -- PSA/Peugeot-Citroen and Renault. French Prime Minister Manuel Valls has announced that the government will get rid of policies that have "favored the diesel engine" and will raise excise tax on diesel fuel as well as remove incentives for diesel car purchases.
PSA remarked that a change in the policy will hurt its competitiveness as it has been considered a leader in diesel technology. A spokeswoman for PSA told Automotive News Europe that diesel is a key technology to cut greenhouse gas and to fight against climate change.
She noted that the increase in excise tax on diesel will place France at a disadvantage compared with Germany. For decades, France has encouraged consumers to select diesel cars by imposing lower fuel and purchase taxes.
But that policy is bound to be changed soon, no thanks to green campaigners who claim that diesel emissions damage air quality in busy cities and cause health issues. Both PSA and Renault remarked that any tax hike on diesel fuel will not improve air quality.
A spokesman for Renault remarked that the tax does not "differentiate between modern and older diesel engines, and thus has no real environmental impact."
The carmaker has noted that said planned fuel tax hikes will raise the price of diesel by 4 cents, and by 2 cents for gasoline. It is estimated that around 80 percent of motorists in France use diesel-powered cars.
Topics: psa, renault, france, diesel, engine


He's always made the entire experience very enjoyable.
I would recommend their Real Estate services to anyone.
I have used Garrett @ Brentview Realty to both BUY and SELL a house. Both were great experiences! Garrett is very professional, yet personable and I would recommend their Real Estate services to anyone.


As Reddit looks to build out its performance advertising business, the social site has begun offering cost-per-click bid types.
Though CPC ads are table stakes, Reddit's vice-president of brand partnerships, Zubair Jandali, said this is a major step forward as the site aims to round out its ad offerings.
"This is a point of inflection that helps get us to parity in the market and will change the way advertisers think about our capabilities," said Jandali.
According to the IAB, 61% of half-year 2018 internet advertising revenue was priced on a performance basis. Zubair told The Drum that offering CPC ads will open the site up to a "new class of advertisers".
"Reddit serves as a release valve for marketers looking for an alternative to Facebook, Google and Amazon, and we're excited about that simply because if they've found efficiency on those platforms, they'll likely find efficiency with ours, but with new users and a different environment," said Zubair.


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CABIN FEVER [2016] (REVIEW)
Dir. Travis Z
As far as the 2000's go the film industry will hardly denote any horror film in that decade as a classic, but one thing is undeniable, it has its fair share of fun. Of these fun films, along with FINAL DESTINATION and WRONG TURN is a small cabin in the woods thriller from Eli Roth called CABIN FEVER. Five youths out in the woods succumb to a flesh-eating virus and subsequently, all die under nasty circumstances. A low budget flick that quickly garnered cult status amongst a generation of teens and one that's tried to keep the series going to dire effect. Ti West failed with the first sequel and PATIENT ZERO was a sub par attempt to recapture the gore of the original. So what next? 13 years later, and here comes the remake.
Produced by Eli Roth and directed by Travis Zariwny (INTRUDERS, SCAVENGERS) CABIN FEVER is less of a remake and more a case of flogging a dead horse. If after 3 attempts you can't move the series any way forward, it's time to start a new franchise. Zariwny refuses to add anything to CABIN FEVER other than bigger production values, if anything, this is the film Eli Roth wanted to make if he had the budget. Better sound quality, a clean visual aesthetic and model stars all make this a polished version of the original, and if this was the first edition it would be a killer, unfortunately it loses the charm of the original and instead is just a shadow of its former self.
There's no point going into the plot or cast as this is just a rereading of the original script and while the deaths are a little more graphic, there's nothing to entice fans to this entry and newbies would be better placed to watch the original for its grubby charm.
It would be interesting to see a truly great sequel but by rehashing the same material, it's hardly likely that this will get the chance and maybe a complete rewrite and revision will pop up in another 10 years. Here's hoping not but we've seen rarer things happen. CABIN FEVER is not the remake anyone wanted but you could do a lot worse.


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One of History's Foremost Anti-Slavery Organizers Is Often Left Out of the Black History Month Story
By PAUL ORTIZ | TIME
Today, the Reverend Dr. Henry Highland Garnet is the most famous African American you never learned about during Black History Month. In the 19th century however, Garnet, who lived from 1815 to 1882, was recognized as one of the foremost anti-slavery organizers in the world. He was the founding president of the American and Foreign Anti-Slavery Society, an organization that waged international campaigns against human trafficking and Cuban patriot José Martí called him America's "Moses."
And, though he is often left out of the popular story of abolition, Rev. Garnet's work of building bridges of solidarity offers a unique perspective on what it means for the U.S. to value human rights in an international context in the 21st century.
Born a slave in Maryland, Garnet was carried by his parents to freedom in the North at the age of 9. As a teenager, Garnet attended Noyes Academy in New Hampshire, an integrated institution of higher learning founded by anti-slavery advocates. Suffering from an ailment that would eventually cost him his leg, the enterprising student discovered one day that area white farmers were plotting to destroy the school. Garnet's biographer noted that he "spent most of the day in casting bullets in anticipation of the attack, and when the whites finally came he replied to their fire with a double-barreled shot-gun, blazing from his window, and soon drove the cowards away." Though the mob eventually destroyed the school, Garnet's covering fire allowed his fellow students to escape under the cover of darkness.
Rev. Garnet's speeches on slavery and liberation captivated audiences. At the National Negro Convention held in Buffalo in 1843, the 27-year-old minister came forward with an audacious plan to end slavery, urging an armed uprising of the slaves. "If you would be free in this generation, here is your only hope," he said. "However much you and all of us may desire it, there is not much hope of redemption without the shedding of blood. If you must bleed, let it all come at once—rather die freemen, than live to be slaves."
Garnet came to this radical conclusion because he believed that it was the only way to stop the United States from spreading slavery via warfare across the continent: "The Pharaohs are on both sides of the blood red waters!" Rev. Garnet lectured. "You cannot move en masse, to the dominions of the British Queen—nor can you pass through Florida and overrun Texas, and at last find peace in Mexico. The propagators of American slavery are spending their blood and treasure, that they may plant the black flag in the heart of Mexico and riot in the halls of the Montezumas."
Garnet interpreted the contested U.S. invasion of Mexico that launched the Mexican-American War in 1846 as a diabolical plan to re-plant slavery's banner in a republic that had effectively abolished chattel bondage. In contrast to those politicians who preached the hatred of Mexicans, Garnet reminded Americans that fugitive slaves regularly found sanctuary in Mexico. Garnet lauded the Mexican people as "liberty-loving brethren," and "ultra-abolitionists." Garnet's understanding of the role that Mexican and Latin American abolitionists played in the global struggle against slavery informed his later human-rights crusades.
Full article at TIME
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text:
Sample Essay on Aristotelian Analysis of Pastoral Elegies- Lycidas and Adonais
Monday, 05 March 2018 / Published in Literature Review
A pastoral elegy is a type of poem whose meaning and significance is centered on death and the heavenly rural life. This type of poem is often written in an expressive way and in a somber tone to express disappointment in the demise of a loved one. The type of life expressed in pastoral elegies is that in a modest rural setting. On numerous occasions, shepherd and farmers always form subject of these poems. In addition, affection and death are popular themes in these poems. The main objective of this paper is to compare Milton's Lycidas to Shelley's Adonais especially on the way these poets use conventions of pastoral elegy. The paper will also provide a critical view of Aristotelian analysis of these pastoral elegies.
Comparison of Milton's Lycidas and Shelley's Adonais
Both poems were composed as pastoral elegies. Shelley's Adonais was composed upon the demise of John Keats. Just as many admirers that Keats had, Shelley held the belief that Keats died as a result of cruel and depressing reviews of his poetry. Despite its false nature, it was widely held that Keats suffered rapture in his lungs as a result of anger from unwanted criticism. Milton's Lycidas is also another of the same kind in memory of his friend Edward King, a talented youth with abundant intellect, who in the view of Milton, died before his time.
The titles of both poems are derived from past activities and myths of the Greeks. Milton uses the title Lycidas not just to moan his friend but also to obtain some poetic advantages. The designation Lycidas a Greek name occurred in numerous classical sources. This is aimed at providing a perfect comparison between his dead friend and a Greek singer, who was talented but, was afraid of losing to his competitor. Shelley also uses the title Adonais as a way of bringing in older traditions to make emotional complications appear simpler. The Greek legend Adonis is loved by both two women but this love makes him decide to live on earth. He is however attacked and killed by a bear. It is said that every year Greek women commemorate by mourning the death of the young prince and later rejoice on his resurrection. This myth forms the foundation of this poem since Shelley believes that just like the prince Keats, she will be immortal. This is because just as Adonis, Keats died at a younger age.
Both pastoral elegies begin with a feeling of dejection but end in optimism. In Lycidas, Milton begins by stating the reasons why he has to write his poem. When he says, "Bitter constraints and sad occasions dear…for Lycidas is dead." This is an indication that the poet feels dejected and in sorrowful. He even calls upon everyone to mourn the death of an intelligent man who died before his time. The mourning is for a fellow poet who shall not go without mourning in singing. The sorrowful and mournful tone is also depicted in Shelley's Adonias. "I weep for Adonias- he is dead." The beginning of the poem shows how the overall feeling that the poet has as he begins to write. These poems however end in optimism. In Lycidas, the poet ends by stating some form of Christian consolation. This is when he requires mourners to weep no more because Lycidas will rise to heaven where he will listen to nuptial songs. His is a deity who should not be mourned but celebrated for he will do good things to others. In Shelley's Adonais, the poet realizes that Keats takes party in the eternal which means he does not die. This realization helps in the eventual happiness and the change in the view from sadness to optimism. The personal immortalizes Keats in numerous ways. "He is made one with nature" the persona says. The poet even dares those still in mourning to join in celebrating Keats newfound immortality. Those who do not quit mourning are in danger of finding themselves in a tomb. Shelley even accepts the demise of Keats because he has become a light that shines an inspiration to the world. He will continue shinning, if like Adonis, he is not forgotten.
Other than using the traditional setting to bring out the intended message, Milton and Shelley direct the blame of the poets' deaths on different people and entities. Shelley lays the blame on literary criticism that was published prior to Keats'. He ridicules the weaknesses of the critics as compared to those of Keats. Shelley appears to be unaware that tuberculosis was the cause of her friend's death. In Lycidas, the poet wonders why the nymphs were unable to save his fellow poet from drowning. It is important to note that although he is aware that the nymphs are not beings that can save one from drowning, Milton still looks for an entity that he can accuse of having brought death to his friends. As the poem progresses Milton notices that if even the muse calliope was unable to save his friend then it was his fate to die.
The two poems also use the outwitting form of pastoral elegy in which a shepherd of natures is lamenting the demise of another. From a literal perspective, the main role of a pastoral elegy is to bring in a reflexive point of view in its use of ancient traditions to simplify complicated phenomena. The poet uses Adonis, his two lovers Aphrodite and Persephone, although in the elegy Aphrodite (Urania) is depicted as Adonis' mother probably because Keats never had a lover. This myth is used to derive the intended meaning of the poem, especially in lamenting the poet's death and the eventual joy upon the discovery on the immortality of Keats. In Lycidas, the persona recalls how he and a person named Lycidas had been good shepherds, both poets. However Lycidas dies and this is saddening to the persona. After lamenting his friend's death, despite encouragement from Apollo, it is the speech from St. Peter about unworthy poets that lead him into apologizing on behalf of all the other shepherds. He then asks the valley to lay flowers on King's coffin despite knowing that he drowned and his body lies at the sea bed. The realization that Lycidas is not dead but is reborn in heaven where the heavenly saints provide him with entertainment is a relief to Milton. Pastoral elegy applied in these poems provides the reader with an understanding of the process of grief which begins with a feeling of dejection and disappointment characterized by mourning as the poems progress the poet realizes that it is pointless to mourn as the dead are alive in heaven watching over the living.
Aristotelian Analysis of Pastoral Elegies
For an elegy to qualify as a tragedy in Aristotelian perspective, it must be an imitation of an action that is serious, complete and of a certain magnitude in language, in form of action in its endeavors to realize its katharsis (cleansing) of tragic emotions and fear. From an Aristotelian perspective, these poems qualify as tragedies. Both poems have tragic heroes of great philosophical and literary relevance. Lycidas derives its story from that of Lycidas a great poet and singer who died before his time. Just like Lycidas, his friend King was a great poet who died before he could acquire fame from his poetic work. When he says, "Lycidas is dead, dead ere his prime." This is also the case Shelley's Adonais. The poet borrows from the Greek mythology of Adonis son of Zeus who dies when on earth after an attack by a boar. The fear of death leads to numerous dramatic performances which from Aristotelian perspective is a characteristic of a tragedy. There is however a turn of events when the protagonists in these poems emerge as winners despite their physical death.
The plots of the poems appear as whole since they have a start, middle and a completion. Both poems begin with the death. In Lycidas, the poem begins with the information on the death of Lycidas who dies before his time. It is a poem in memory of a fellow poet. In Shelley's Adonais, the persona begins by calling people to mourn the death of a good friend Adonais. In the middle, there poems are characterized by mourning and blames on who caused the deaths of the good friends. In Lycidas, the persona questions the nymphs and muses on their role in Lycidas' death. He also desires to know the one who was on watch when Lycidas died. In Shelley's Adonais, the blame is on the critics whose objections led to the death of Adonais. The persona beckons mourners and recognizes that even nature mourns Adonais as the wind and oceans stop to pay tribute to the fallen hero. The climax is when there is an unexpected alteration in the mood of the poem. In Lycidas, the persona comes to a realization that his acquaintance is not dead but is in heaven. He tells other shepherds that Lycidas is in a place where he "hears the unexpressive nuptial song." The shepherds should weep no more. The climax and end of Shelley's Adonais is when there is a sudden change in the tone of the poem when the persona realizes that his friend is in heaven safe from his enemies. In the end the persona concedes to the passing of his friend with the hope that Adonais' light continues to guide her for the rest of her life.
Aristotelian of the pastoral elegies by Milton and Shelley as tragedies provides an essential way through which these poems can be understood. The introduction of tragic heroes such as Lycidas and Adonis provides a proper understanding to the poem since it enables the reader to find a perfect relationship between these heroes and the person for whom the poem was written. Milton wrote the poem Lycidas in reminiscence of his friend Edward King. Shelley wrote his in memory of his friend John Keats. Just like King, Lycidas died at an early age before he could enjoy the fruits of his success. The emotional language used in these poems suggests that the poet was a close friend to the deceased friend. The tragic death of Adonis son of Zeus for instance brings out sorrow and despair from his mother who calls upon death to take her away just as it took her son. The climax characterized by a sudden change in the tone of the poems helps in recognizing that the tragic hero, though dead, emerges as the victor at the end.
Percy Shelley's Adonais and John Milton's Lycidas are poems written in memory of their friends and fellow poets John Keats and Edward King respectively. The two poems fall in the classification of pastoral elegies. To be precise, these poems are elegies since they were written to mourn the death of someone. The widespread depiction of the shepherd subjects and descriptions makes the poems pastoral. These poems can also qualify as tragedies from an Aristotelian perspective. This is because they contain imitations of actions with serious consequences. The presence of tragic heroes drawn from ancient Greece, Lycidas and Adonis, create a point of comparison and understanding of the essence of the deceased friends to the poets. These heroes though defeated at the beginning emerge victories at the end. The plot of the poems are single and complex since they contain reversals, news that was at first sad is revealed as good in the end, and recognition where there is a change from ignorance to awareness.
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Looking at the dad figure..... Ehhhh.... He casually told a toddler that his adopted mother had "complications". Me thinks our boys may have at the very least suffered emotional neglect. (I'm gonna say Tiger-Dad was better "dad")
Claudus was definitely not an easily likeable character. Vast contrast to the original series Claudus.
Mark M, Sep 8, 2014
Daremonger Thunderian Legend
That's true. Both Clauduses are different.
Daremonger, Sep 8, 2014
As a leader and lord of the Thundercats the OS Claudus was definitely the superior leader.
Most definitely.
I have mentioned before the comparisons to Avatar. I honestly think they had a longer story that would span at least two or three seasons. But I think the way they tried to make the long story arc yet trying to still make it episodic like the OS was a bad idea. I really think they should have just stuck to one idea and tried to successfully pull it off.
Mark M, Sep 10, 2014
The NS did try to do too much, which proved to be its downfall.
Daremonger, Sep 10, 2014
In some ways it reminds me of some of those Marvel and DC animated movies. To childish to appeal to most adults and to grown up and dark to appeal to the vast majority of children.
I'm not a fan of the old TC series, I was more into he-man, gijoe and tmnt.
Regarding the new series, I ADORE it, I mean, I never thought that i'll in love to a new cartoon, until now, I was more than happy with B:TAS and TMNT (2K3 series).
I give an 8 or 9 to this cartoon. My only complaint? It's a shame we didn't get another season.
TMNachoT, Dec 26, 2014
I like the 2011 series but it is very frustrating when you know they could have done so much better.
Bengali Rampager
Mark M said: ↑
Exactly. I mean the original intro as most people know is EPIC, the 2011 series didn't even have one! The power stones thing was a failure as well, totally taking away from the eye of thundera's uniqueness. I can go on and on. I didn't even get it. Where did mummra get the "animals" again? *sigh*
Bengali, Jan 1, 2015
There was good few things I didn't get and see the point in.
I really agree with what you say about the Eye of Thundera. They really did rob it of its mystical mystery etc.
My biggest problems are the lack of and inconsistency of the characters and the designs.
and the designs.
I don't think the designs of the main ThunderCats were THAT bad. Panthro and Tygra should have kept their classic colors though for sure. I really hated that Mumm-Ra had wings instead of the red torn cape. Oh yea and Tygra having a gun was soooo un-thundercats like. Just blasphemy.
I didn't think Lion-O's design was that heroic. I didn't like the bat wings either. Well Tygra still used his whip so I didn't think using the gun was so bad. They really should have kept the classic colours. Still at least they didn't go all X-men with matching uniforms lol.
I still really hated them not having technology.
LioConvoy said: ↑
Thundertank, Cat's Lair, the armory, etc. They've ALWAYS had guns.
It makes more sense for them to have more than just one weapon. If you were going into a battle would really just want going in with a whip, or a staff? I would take the whip into battle if I had a couple hand guns, a rifle and maybe a couple knifes with me as well.
Having big laser weapons that look like huge battle canons is different than Tygra carrying around a pistol that actually looks like a real hand gun. It was unnessasary. They just tried to make it more modern and failed.
I just picked up the first season on DVD, after watching the first few episodes, I really have to say that it was all over the place from the very beginning.
The first episode implies that there was an ancient battle with the cats vs mummra/lizards but then in 'legacy' we see that the only "battle" that happend was a rebellion somewhere in space.
Mongor Laser Beast TC.org Staff
Just to share my thoughts...
I am not a fan of the 2011 series. I understand that the creators were trying to pay homage to the original show while creating something that was both different and fresh. Unfortunately, their failure was in the execution. For me, many of the elements that made the original series popular were lost. Some fans that I have spoken to have said that the new series is better because there is more continuity between episodes, but shows like South Park and The Simpsons have shown us that animated programs do not need continuity to be successful and interesting. When the new series was advertised, I was excited to see what was it was going to be like, but I was so disappointed as each new episode premiered. I felt the same way when Rob Zombie announced he was remaking John Carpenter's Halloween (my favorite movie). Just like the 2011 series, Zombie had the potential to rewrite the story of Michael Myers and bring new life into the franchise, but his execution was terrible leading to one of the worst remakes, in my opinion. In any case, I hoped that the story in the 2011 series would conclude because I wanted to write and publish an article comparing the two series. Since my PhD dissertation was about adaptations (and remakes to a lesser extent), I feel that an article on the two shows would be something interesting for Tcats fans to read, but I don't feel I could write a justifiable article when the 2011 series finished with too many loose ends.
Mongor, Jan 5, 2015
Bengali, Mongor, I agree with what you are saying. As I have said over all I still think they had the wrong creative team working on the 2011 series. They lost a lot of what really made the original series appeal to its fans.
The first few episodes of the 2011 series are not very good and when a show starts so poor it is hard to keep a fans interested for the next episode. Granted it got a lot better, but the damage was done.
adssse Thunderian Legend
I actually really enjoyed the 2011 series. It is quite different from the original and I guess if what you were looking for was a tweaked/updated version of the classic than I can see the disappointment. I look at them both in their own light (somewhat like the Wildstorm comics etc) and enjoy them. I seem to recall many liking the 2011 series, especially the first couple of episodes. It seems like there was no push behind the series though by WB. Unfortunately they don't appear too invested in the Thundercats property. Hopefully that will change for the anniversary.
adssse, Jan 5, 2015
adssse said: ↑
Upon a second watch without comparing it to the OS I really did enjoy it even with it's faults. If the writing and creative staff had just been a bit more coordinated they really could have produced an amazing show.
Theres really nothing I like about the new series. Everytime I see kit and kat with tales my stomach turns. Panthro comes off as borderline schizophrenic. As a group the ThunderCats seem like angry teenagers. Its just an awful mess. Its so irritating how they didn't put that little mark under their eyes which was such a trademark of the classic...they tried to put it on Leo but off to the side a little..grrr. Oh yea and who was Leo's Avatar girlfriend in 'Legacy'?


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A proposal has been prepared by the oil services company Wood Group and two UK's unions in a bid to end a dispute over cuts in pay and allowances for offshore workers on Shell's North Sea platforms.
Following the 24-hour and 48-hour strike actions on Shell's platforms in late July and early August, further action was postponed for talks to be held between Wood Group and the two UK unions, RMT and Unite. According to the unions, the workers faced losing up to 30% in pay and allowances.
Unite and the RMT union have had five meetings with Wood Group since the unions decided to suspend their industrial action on 12 August for further negotiations.
Unite reported on Tuesday it will ballot its members on a proposal that could end an industrial dispute that led to the first North Sea strike in a generation.
Unite regional officer John Boland said: "Throughout this dispute, our members have remained united and strong in defense of their pay and conditions, and we have always made clear that we were open to meaningful negotiations.
"The proposal we are putting to members will still involve a large degree of sacrifice on their part. Wood Group has made concessions on key areas of competency and flexibility payments that will reduce the impact of the cuts they originally put forward.
Posted on September 7, 2016 with tags North Sea, RMT, Strike, UK, Unite, Wood Group.


The Isreali enemy has pursued the construction of a cement wall in Adeisseh amidst heightened Lebanese army mobilization, and under the supervision of the international peace keeping forces in the region, NNA field reporter said on Friday.
At noon on Friday, MP Kassem Hashem toured Adeisseh and inspected the Israeli breach in said region.
Lebanon will face this Israeli breach, whether at the political level or otherwise, he said, stressing the importance of the tripartite army-people-resistance defense formula to force the enemy to stop its violations."
The United Nations is responsible for this Israeli aggression, he said, calling for "an end to the breaches of the enemy, which is always thirsty for our land and waters."


Years of extensive research in the field of speech processing for compression and recognition in the last five decades, resulted in a severe competition among the various methods and paradigms introduced. In this paper we include the different representations of speech in the time-frequency and time-scale domains for the purpose of compression and recognition. The examination of these representations in a variety of related work is accomplished. In particular, we emphasize methods related to Fourier analysis paradigms and wavelet based ones along with the advantages and disadvantages of both approaches.
In real-field applications, the correct determination of voice segments highly improves the overall system accuracy and minimises the total computation time. This paper presents reliable measures of speech compression by detcting the end points of the speech signals prior to compressing them. The two different compession schemes used are the Global threshold and the Level- Dependent threshold techniques. The performance of the proposed method is tested wirh the Signal to Noise Ratios, Peak Signal to Noise Ratios and Normalized Root Mean Square Error parameter measures.
This paper studies the effect of different compression constraints and schemes presented in a new and flexible paradigm to achieve high compression ratios and acceptable signal to noise ratios of Arabic speech signals. Compression parameters are computed for variable frame sizes of a level 5 to 7 Discrete Wavelet Transform (DWT) representation of the signals for different analyzing mother wavelet functions. Results are obtained and compared for Global threshold and level dependent threshold techniques. The results obtained also include comparisons with Signal to Noise Ratios, Peak Signal to Noise Ratios and Normalized Root Mean Square Error.


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Site Name: Simmons Park
Location: Apollo Beach (Bahia Beach), FL
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Dangerous Urban Chicken Craze Has Legs
What's next -- clean air?
By Sara K. Smith	• Published at 9:16 pm on August 18, 2009
After all the factory farming industry has done for us, it's shameful that people can't show a little more gratitude. Thanks to massive industrial complexes where cows, chickens, and pigs spend their artificially short lives in dire confinement, ankle deep in their own poo and kept alive on a diet of corn syrup and trash, we have regular access to affordable meats and eggs that are very nearly edible most of the time!
And how do consumers say thank you to these brave souls? They start growing their own chickens. And eggs. In their own backyards.
A number of American cities have already jumped on the "urban chicken" movement, with residents turning their once-scenic and fragrant yards into pestilent dens of stinking guano. Such unreconstructed hippies love to play the modern homesteader, gathering eggs every morning and baling out their coops on weekends.
The latest such Farmer Joes: two women, Gay-Ellen Stulp and Stephany Miskunas, who are trying to get backyard chickens legalized in their Lafayette, Indiana neighborhood.
It's been a blast," Stulp said of her quest. "I can't believe the discussions I've gotten as I go around with my petition.
"It's a little hobby. They are pets. I guess I'm now part of the chicken underground."
Sure, they're pets to you, lady! But to the children of men and women who run and work at the massive egg farms that produce cheap, brittle-shelled, anemic, watery eggs for the rest of us, these chickens are nothing less than a threat to their very way of life.
Plus, if there's one thing this healthcare debate has taught us, it's how terrifically un-American it is to introduce cheap not-for-profit competitors to large, profit-driven companies that have employees and taxes to pay. In other words, the private coop is just one slippery slope away from the private co-op.
So let's hope this dangerous "urban chicken" trend doesn't spread any further. Because if it does, the next thing you know people will be growing their own vegetables to go with their eggs.
And that way lies socialism.
Sara K. Smith can butcher a chicken but she still doesn't know what body part the McNugget comes from. She writes for NBC and Wonkette.


Have an auditory or visual leaner? A student who needs more hand-holding? No problem!
This eCourse supplement consists of online videos for each lesson. The eCourse is offered on a subscription basis (see below).
Students are able to submit questions to Katherine online as they complete the eCourse. View sample lessons.
About the eCourse: Have an auditory or visual leaner? A student who needs more hand-holding? No problem! We now offer an eCourse supplement. The eCourse consists of video lessons that teach the same concepts as in the textbook, but in a different way, walking students through step-by-step using slides and often using different examples. Students using the eCourse still need the textbook, but the eCourse will cut down on the amount of reading necessary. The videos range in length–some are 2 minutes; others 12. The average for the two chapters in eCourse 1 we sampled was approximately 6.3 minutes.
Note: You still need to purchase the Book 2 Textbook and Student Workbook/Teacher Guide. The eCourse is an optional supplement to the program.


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DC Could Soon Be Ending the Snyderverse for Good
The DCEU is in flux as co-leads Gunn and Safran prepare to present their new plan.
byCarson Burton
Posted Dec. 8, 2022, 10:10 a.m.
Buckle up, DC fans; it may be the end of the Snyderverse as we know it.
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DC Studios has been in the middle of a massive shakeup for months now as new leadership at Warner Bros looks to change the future for DC content. According to the Hollywood Reporter, the studio may be undergoing yet another drastic change: the full on shuttering of the Snyderverse
Next week, new heads of studio James Gunn and Peter Safran will lay out their years-long plan to head of Warner Bros Discovery David Zaslav. Their plan, which is currently still in flux, will serve as the blueprint for what DC will become, possibly introducing a completely new vision for the franchise. What does that new vision look like? We don't really know, but it appears it will not include Wonder Woman 3. Reportedly, the film will not move forward under Gunn and Safran's DC.
The Wonder Woman move indicates the possibility of a full-on closure of Zack Snyder's Snyderverse. That means that DC could be without all the heroes Snyder cast in his Justice League film. No more Man of Steel 2 with Henry Cavill, and no more Aquaman with Jason Momoa.
It's already known that both Cavill's Superman and Momoa's Aquaman will make cameos in the Flash movie, set to hit theaters on June 16 of 2023. Cavill is said to have shot his cameo back in September, but the Hollywood Reporter claims that there is a debate inside the studio as whether or not to keep the cameo; if its inclusion promises something the studio would have no plans on delivering, it won't be seen by audiences.
The decision is already a bit precarious as Cavill recently made his triumphant return to screens as Superman in Black Adam back in October. Cavill himself even confirmed that he would indeed be back as Superman. At the time, it was guaranteed that he would be returning for a sequel; now, it's reportedly up in the air.
While the plans are still very much in the air, any number of scenarios are reportedly in play, including Momoa possibly returning as a different DC character entirely. Meanwhile, Black Adam 2 may not happen after its lackluster theatrical showing.
Either way, the Snyderverse has been the backbone of DC for years now, and Snyder has an explosivlely supportive fanbase behind him. Thanks to the outpouring of love for Snyder and his creative vision, Warner allowed the four-hour long Snyder Cut to hit HBO Max. That was under different leadership, though.
What happens to the Snyderverse now that Zaslav is at the helm of Warner? How does Snyder tie into Gunn and Safran's master plan? Will we ever see these heroes again? Right now, it's anyone's guess.
Carson Burton is a freelance news writer for IGN. You can follow him on Twitter at @carsonsburton.