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Previous data from spaceflight studies indicate that injured muscle and bone heal slowly and abnormally compared with ground controls, strongly suggesting that ligaments or tendons may not repair optimally as well. Thus the objective of this study was to investigate the biochemical and molecular gene expression of the collagen extracellular matrix in response to medial collateral ligament (MCL) injury repair in hindlimb unloaded (HLU) rodents. Male rats were assigned to 3- and 7-wk treatment groups with three subgroups each: sham control, ambulatory healing (Amb-healing), and HLU-healing groups. Amb- and HLU-healing animals underwent bilateral surgical transection of their MCLs, whereas control animals were subjected to sham surgeries. All surgeries were performed under isoflurane anesthesia. After 3 wk or 7 wk of HLU, rats were euthanized and MCLs were surgically isolated and prepared for molecular or biochemical analyses. Hydroxyproline concentration and hydroxylysylpyridinoline collagen cross-link contents were measured by HPLC and showed a substantial decrement in surgical groups. MCL tissue cellularity, quantified by DNA content, remained significantly elevated in all HLU-healing groups vs. Amb-healing groups. MCL gene expression of collagen type I, collagen type III, collagen type V, fibronectin, decorin, biglycan, lysyl oxidase, matrix metalloproteinase-2, and tissue inhibitor of matrix metalloproteinase-1, measured by real-time quantitative PCR, demonstrated differential expression in the HLU-healing groups compared with Amb-healing groups at both the 3- and 7-wk time points. Together, these data suggest that HLU affects dense fibrous connective tissue wound healing and confirms previous morphological and biomechanical data that HLU inhibits the ligament repair processes.
- hindlimb suspension
- real-time quantitative PCR
the likelihood of an astronaut or cosmonaut receiving an injury that would require repair is highly probable on extended duration spaceflight missions, such as during duty on the International Space Station or on travel to the Moon or Mars. Some astronauts and cosmonauts participate in extravehicular activity, by moving, manipulating, and constructing high-mass hardware and/or participate in other tasks that may increase the probability of being injured. Unfortunately, there is very little spaceflight data from human, nonhuman primate, or rodent experiments on injury repair or trauma associated with the musculoskeletal system. The few pieces of data obtained from studies on rats have indicated that weightlessness may retard the repair of load-bearing bone and skeletal muscle during acute spaceflight exposure (20, 21, 24). Wound repair in the extracellular matrix (ECM) of skeletal muscle has also been shown to be delayed after 14 days of spaceflight (36). Rodent cutaneous tissues subjected to wound repair during spaceflight also showed an impaired ability to heal and then respond to in vivo platelet-derived growth factor (PDGF-BB) and bovine basic fibroblast growth factor (bFGF) supplementations (11).
A common way to simulate the effects of spaceflight on the musculoskeletal system has been with the implementation of the rat hindlimb unloading (HLU) model, used by National Aeronautics and Space Administration (NASA) and investigators worldwide, to study the physiological adaptations associated with acute and chronic microgravity (28, 29). Muscular atrophy and decreased muscle strength are common observations in rats subjected to acute bouts of HLU (3, 17, 33, 35, 37). Our laboratory (35, 38–40, 42) has previously demonstrated that bone and dense fibrous connective tissues subjected to acute bouts (7–28 days) of HLU are significantly compromised in strength, integrity, and biochemical composition because of a reduction in ground reaction forces (GRF). Rodents subjected to 14 and 21 days of HLU had significantly weaker bone-ligament-bone junctions (40) and reduced Achilles tendon ultimate stress and tangential moduli (1). In essence, HLU has deleterious effects on the strength and energy storage capacity of tissues containing ECM collagens.
Ligament tissues are injured more often than any other joint structure (2), and ligament injuries result in a significant disability in ∼100,000 patients per year. Despite current modalities of treatment, 15% of the patients with a medial collateral ligament (MCL) injury develop early signs of arthritis, suggesting an incomplete return to knee stability. Because knee ligaments have a high incidence of injury on Earth, the possibility exists that weakening of the knee structure and surrounding tissues during space travel could predispose astronauts and cosmonauts to potential ligament injuries and probable reloading injuries on return to the Moon, Mars, or Earth after extended-length stays in space.
Obtaining access to subjects that have orthopedic wounds and subjecting them to space travel is virtually impossible because of numerous factors. We have created a ground-based rodent model to study the orthopedic repair of ligaments during reduced GRFs. The biomechanical, morphological, and histological properties of transected rat MCLs subjected to 21 and 49 days of HLU were previously studied by our research group (30). HLU time course healing experiments revealed significant reductions in ligament maximal force, ultimate stress, elastic modulus, and low-load properties that did not improve ligament strength and stiffness to presurgical status over time (30). HLU animals possessed MCLs with abnormally formed scars and uneven fibroblast cell distribution compared with ambulatory ground controls, suggesting that GRFs are important in the micro-organization of the collagen fibrillar network at the ligament scar site (30). More recently, HLU was also shown to inhibit cell proliferation of the corneal epithelium in a mouse model to study wound healing in the eye (25). HLU caused a delay in the onset of corneal wound closure by ∼12 h over a 96-h period (25).
The primary purpose of this study was to elucidate the biochemical and molecular gene expression changes in the healing MCL challenged by acute (3 wk) and extended-duration (7 wk) HLU. We hypothesized that HLU would 1) diminish the healing capabilities of surgically transected knee ligaments to self-repair, 2) compromise the biochemical properties of the ligament ECM, and 3) alter the ECM gene expression profiles of the healing MCLs. The healing characteristics of the rodent MCL tissues was assayed with specific biochemical and gene markers to assess the integrity, maturation, and molecular organization of collagen, the main ECM protein located within ligaments. Biochemical analyses were conducted of 1) hydroxyproline (Hyp), an imino acid marker used as an index of total collagen content measured by HPLC, 2) the mature collagen cross-link hydroxylysylpyridinoline (HP), which improves ECM strength and tissue quality in healing ligaments, also measured by HPLC, and 3) MCL cellularity by DNA measurement to assess the state of cell proliferation in the healing ligament. Real-time quantitative PCR gene markers were used to determine the upregulation or downregulation of specific genes that impact the ECM in healing MCLs as previously determined (15, 18, 19, 32, 34). To assess new collagen synthesis in remodeling ligament, the genes encoding collagen type I (Col1A2), collagen type III (Col3A1), and collagen type V (Col5a1) were assayed. In addition, genes encoding other ECM ligament constituents important to MCL tissue function were measured: lysyl oxidase, the enzyme catalyzing collagen cross-link formation; decorin and biglycan proteoglycans, which are important in the assembly of collagen fibrils during fibrillogenesis; fibronectin, an ECM structural protein expressed during early wound healing; matrix metalloproteinase-2 (MMP-2 or gelatinase-A), a degradative enzyme that aids in the resporption and remodeling of the ECM; and finally tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), which inhibits the activity of specific MMPs during remodeling. Together, the quantitative measurement of the genes mentioned will provide information on the molecular ECM profile of healing MCLs during HLU.
MATERIALS AND METHODS
The Institutional Animal Use and Care Committees at the University of Houston, University of Wisconsin-Madison, and NASA-Ames Research Center approved this study. Specific details of the animal model and design of the experiment have been published previously by our research group (30). Male Sprague-Dawley rats (200 g; Simenson Labs, Gilroy, CA) arrived at NASA-Ames Research Center and were allowed to acclimatize in cages for 7–10 days. Two groups of 30 animals each were divided into 3- and 7-wk wound-healing groups. Each time group contained 10 randomly selected animals that were divided into the following subgroups: sham control, ambulatory healing (Amb-healing), and HLU-healing.
Rats in the Amb-healing and HLU-healing groups were subjected to bilateral MCL transection under isoflurane inhalation anesthesia using a non-rebreathing delivery system via nose cone. All animal surgeries were performed in aseptic conditions in a sterile field. Bilateral skin incisions of ∼6–8 mm in length were made on the medial aspect of the knee, exposing the MCL at the femoral-tibial joint line. The MCL transection was made with a no. 11 sterile scalpel blade. The wound area was immediately sutured closed with simple interrupted surgeons’ knots using 4-0 chromic gut (Ethicon, Somerville, NJ) in the subcutaneous fascia and 3-0 Ethilon black monofilament nylon to close the skin. The wounded area was cleaned, and Panalog antibiotic ointment was applied postoperatively as needed. Sham control animals were prepared and surgically manipulated in the same manner; however, no surgical transection was performed on the MCL. Postsurgery analgesic (3 μl) Tylenol-codeine (120 mg/5 ml Acetaminophen, 12 mg/5 ml codeine phosphate) was delivered to each animal's 500-ml water bottle for 72 h. Sham and Amb-healing animals were free to move about in their cages after surgery. Animals subjected to HLU were allowed 24 h of recovery before being suspended.
HLU rats were deprived of GRF to their hindlimbs with the use of the Morey-Holton tail suspension protocol at NASA-Ames Research Center Animal Care Facility as previously published (29). Animals were maintained in a 24°C room, exposed to a 12:12-h light-dark cycle, and given rat chow and water ad libitum. Research staff and veterinarians checked animals two or three times per day for skin wound health, overall well being, water (ad libitum) and food consumption (Purina rat chow), and adaptation to the HLU harness on their tails. The nylon sutures were removed 5 days postoperatively with no apparent stress to the animals. The animals were euthanized after either 3 or 7 wk of unloading. There were 20 MCLs per group, which were distributed and stored at −85°C as follows: 10 ligaments per group were for protein (biochemical) analyses and 10 ligaments per group were submerged in 200 μl of RNALater (Ambion, Austin, TX).
Tissue sample preparation.
For preparation of tissues for DNA measurement, the method of Vailas et al. (41) was used. An individual lyophilized MCL (1.7–3.6 mg) was placed into brown borosilicate glass screw vials with polytetrafluoroethylene-silicone-lined screw caps. Each vial received 1.2 ml of a papain (P-3125; Sigma-Aldrich, St. Louis, MO) solution (1 ml = 28 mg of protein, 33 U of enzyme/mg protein; therefore, 924 U/ml of papain enzyme were used to digest ligament samples). This solution contained 0.001 M cysteine-hydrochloride, 0.01 M EDTA, and 200 μl/10 ml digest of papain suspension (type III; Sigma-Aldrich) in a 0.01 M phosphate buffer (pH 6.5). In addition, the DNA standards (Sigma-Aldrich) were subjected to the same treatment to determine recoveries for DNA. Samples were heated in an oven at 65°C for 48 h. Digests were extracted with chloroform in a chloroform-to-digest volume ratio of 10:1 and centrifuged at 5,000 rpm for 10–15 min. Samples were frozen at −20°C until analyzed. The recoveries for DNA were between 94% and 98% for each assay as reported using similar methodology (9).
Contralateral MCL tissues destined for imino acid (Hyp) and trifunctional peptide (HP cross-link) analyses were hydrolyzed in 1.0 ml of 6 M HCl for 24 h at 110°C using brown borosilicate polytetrafluoroethylene-silicone-lined screw-top vials further sealed with Teflon tape. Individual aliquots (200 μl for Hyp and 700 μl for HP cross-links) of each MCL sample hydrolyzate were aliquoted to 12 × 75-mm borosilicate test tubes, evaporated to dryness (Savant Speedvac Thermo-Fisher, Holbrook, NY), and stored at −80°C until further analyses.
Hyp is a specific index for all phenotypes of collagen, representing 14% of collagen's mass. Amino acid analyses of Hyp was performed with the precolumn derivatizing Edman reagent phenylisothiocyanate (Pierce Chemicals, Rockford, IL) before analysis, according to Martinez et al. (26). Phenylisothiocyanate-derivatized amino acid samples (200 μl of original dried hydrolyzate) were redissolved in 1.0 ml of sample diluent (5 mM sodium phosphate dibasic, pH 7.40 in 5% acetonitrile by volume), filtered through a 0.45-μm filter (GHP Acro-Prep filter plate, Pall Life Sciences, Ann Arbor, MI), eluted isocratically (140 mM sodium acetate-tetraethylammonium-EDTA buffer solution) at a 1.0 ml/min flow rate using a 10 cm × 4.6 mm C18 PICO-TAG column (Waters, Milford, MA) on a Waters model 2695 Alliance HPLC, and monitored on a Waters model 2487 dual wavelength UV/Vis detector at 254 nm. The retention time for Hyp was 2.6 min, the imino acid chromatograms were quantified relative to a known amount of derivatized Hyp (Sigma-Aldrich) standard, and the recovery for the imino acid analyses was 95%.
HP collagen cross-link content determination.
The collagen cross-link analysis (HP) was performed by ion-paired C18 reverse-phase HPLC (Waters 2695 Alliance) method, previously used by our laboratory (26). Aliquots (700 μl) of the dried, hydrolyzed samples were redissolved in 0.5 ml of 1% n-heptafluorobutyric acid (Pierce) and filtered through a 0.45-μm filter (GHP Acro-Prep filter plate; Pall Life Sciences). Elution of the HP cross-link was performed isocratically using 15% acetonitrile (EM Science, Gibbstown, NJ) in 0.01 M n-heptafluorobutyric acid at 1.0 ml/min with a 16-min retention time and was monitored with a Waters 470 fluorescent detector (excitation 290 nm, emission 395 nm). Standardization of our system was performed by applying a commercially available HP standard (Metra Biosystems, Palo Alto, CA). The recovery was determined by applying a pure collagen source (bovine Achilles tendon; Elastin Products, Owensville, MO) and was shown to be 99%. The HP collagen cross-links are expressed as mole HP cross-links per mole collagen.
DNA content measurement.
The estimation of the ligament cell density was achieved by determining the amount of DNA using a modified method of Bonting and Jones (5) and as previously performed by our laboratory (9, 38). A stock solution of 0.1 mg NaDNA (type I; Sigma-Aldrich) per 1 ml distilled H2O was prepared and used as a standard. DNA assay samples were made by diluting 100 μl of the sample extracted with chloroform with 300 μl of 0.1 N NaOH. To this, 400 μl of the indole-HCl (50:50 wt/vol) reagent were added and briefly vortexed. Samples were heated for 20 min at 100°C, cooled to room temperature, and read in triplicate with a Dynatech MR5000 plate reader at 490 nm.
Frozen MCLs were placed into RNase-free 1.5-ml round bottom microcentrifuge tubes (Eppendorf-Brinkman) containing a 4.7-mm stainless steel ball. The isolation of total RNA from ligament was performed with Mixer Mill 300 (Qiagen, Valencia, CA) with two 24-well adaptors and the RNeasy dense fibrous tissue isolation kit (Qiagen) according to the manufacturer's protocol. Total RNA was quantitated by small dilutions and measured by 260-nm optical density on a spectrophotometer (ND-1000, NanoDrop, Wilmington, DE).
Complimentary DNA was synthesized by running 30-μl reverse transcription reactions using SuperScript II RNase H− reverse transcriptase (Invitrogen, Carlsbad, CA) according to the company's instructions with some minor modifications by our laboratory. In the first denaturation reaction, 200 ng of ligament total RNA were added to 1.5 μl of oligo dT(15) primer and diethyl pyrocarbonate water to a total volume of 20.1 μl. Each sample was incubated at 70°C for 10 min in an MJ thermocycler (MJ Research/Bio-Rad, Hercules, CA). After denaturation, a second reaction solution was added containing 6.0 μl of 5× first-strand buffer, 3.0 μl of 0.1 M DTT, 0.6 μl of 200 ng/μl dNTPs, and 0.3 μl of SuperScript II Moloney murine leukemia virus RNase H− reverse transcriptase (50 U/μl) for a total of 9.9 μl per tube. Samples were incubated at 42°C for 50 min followed by 70°C for 15 min. The cDNA samples were stored at −20°C until further quantitative PCR processing.
Real-time quantitative PCR analysis.
For a specific gene of interest, 5′ Taqman hydrolysis quantitative PCR assays were performed by an MX-4000 real-time quantitative PCR machine (Stratagene, La Jolla, CA). For each quantitative PCR assay, the sequences for forward and reverse oligonucleotide primers (20–24 nt), fluorescent reporter probes (20–25 nt) containing a 5′-FAM, -HEX, or -Cy5 fluorescent reporters, and a 3′-BHQ 1–3 quencher (Biosource International, Camarillo, CA) were generated with Beacon designer 2 Taqman multiplexing software (Premier Biosoft, Palo Alto, CA). The genes of interest and sequences are listed in Table 1. Standards were generated with synthetic DNA amplicon oligonucleotides (Biosource International) spanning the entire quantitative PCR amplification (75–150 nt) region. A standard curve methodology was performed to quantitate the specific gene targets of interest. Specific standard aliquots (108 to 101 copies of cDNA) were used to generate a linear curve (>R2 = 0.995) with a quantitative PCR efficiency (slope) of −3.3 to determine the cDNA copy number of triplicate replicates. Each quantitative PCR reaction consisted of 12 min at 95°C to activate the HotStar Taq polymerase (Qiagen) followed by 40 cycles of two-step thermocycling (45 s at 94°C and 12 s at 60°C). The cDNA copy number of the target gene was normalized to the copy number of cyclophilin A, which remained constant independent of treatment protocol. Quantitative PCR gene expression measurements were converted to mean multiples of changes compared with 3-wk sham animals. Multiples of changes from 0 to 1 and 0 to −1 are nonlinear. Therefore, the multiples of changes of mean gene expression were log10 transformed before statistical analysis was performed.
To evaluate the differences among groups for each dependent variable, a two-way ANOVA was applied. When the F tests were significant, Duncan's post hoc tests and least square means used to further identify differences between group pairs. Statistical significance level was set at P < 0.05.
All animals tolerated the anesthesia, MCL surgical transections, recovery, and HLU well. The initial body weights were not significantly different at the beginning of the time course study (Table 2). As with the majority of rodent HLU protocols, the HLU animals failed to grow at the rate of the sham and Amb-healing groups within the first 7 days, as previously shown in rat growth charts from an earlier publication by our research group (30). HLU-healing body weights at the end of each study were significantly smaller than age-matched sham and Amb-healing groups (P < 0.05).
Extracellular Matrix Biochemistry
There was a significant (15–25%) decrease (P < 0.05) in Hyp concentration (Fig. 1), an index of total collagen, in both the 3-wk Amb-healing and HLU-healing groups compared with the 3-wk sham group. All 7-wk groups showed recovery to sham control levels. The HP collagen cross-link content (Fig. 2), a physiological marker of collagen maturation, was significantly (25–30%) smaller (P < 0.05) in the 3- and 7-wk Amb- and HLU-healing groups. The content of HP cross-links was significantly greater (P < 0.05) in the 7-wk sham group than in the 3-wk group. HLU and wound repair in the MCL did not affect the accumulation of HP cross-links compared with Amb-healing in either the 3- or 7-wk groups.
We used tissue DNA concentration to measure tissue cellularity. The DNA concentrations (Fig. 3) in the 7-wk HLU-healing group was significantly greater (P < 0.05) than in the 3-wk sham control group and all other groups. The tissue cellularity was smaller (30–60%) in 7-wk sham and Amb-healing groups. The tissue cellularity in the 7-wk HLU-healing group remained significantly elevated compared with that shown in sham and Amb-healing age-matched controls (P < 0.05).
MCL Tissue Gene Expression Levels
Changes in collagen types I, III, and V gene expression are shown in Fig. 4, and the specific numerical values and changes (in fold) of other ECM markers are shown in Table 3. The HLU-healing group demonstrated impairment in the time course of collagen gene expression events during wound repair remodeling compared with that shown in the Amb-healing group. In Fig. 4A, collagen type I (Col1a2) gene expression levels remained constant during the first 3 wk of wound healing. However, at 7 wk of wound healing, the HLU-healing group had significantly depressed collagen type I gene expression levels compared with sham and Amb-healing groups (P < 0.05). Collagen type III (Col3a1) and collagen type V (Col5a1) gene expression levels were not significantly different after 3 wk of wound healing (P > 0.05). The Amb-healing group had significantly elevated levels of Col3a1 after 7 wk of healing (Fig. 4B), whereas the HLU-healing group remained significantly lower (P < 0.05). Likewise, HLU-healing group Col5a1 gene expression levels (Fig. 4C) remained significantly lower than levels in Amb-healing controls (P < 0.05).
Other ECM genes that were either upregulated or downregulated during wound healing are depicted in Table 3. All mean (in fold) changes were compared with the 3-wk sham control group, which was set at 1.0 (data not shown). Fibronectin gene expression is ∼2.6-fold elevated after 3 wk of healing compared with the sham control group; however, significant reductions in fibronectin expression are shown at 7 wk in the sham control and Amb-healing groups, although levels remained elevated in the HLU-healing group. Decorin, a chondroitin/dermatan sulfate proteoglycan associated with collagen types I, III, and V was significantly downregulated in the 3-wk HLU-healing group and stayed elevated compared with sham control levels after 7 wk of wound healing. Decorin gene expression levels are virtually absent in the 7 wk Amb-healing group. Similarly, biglycan, a small cell surface or pericellular proteoglycan with similar properties to decorin, shows similar decrements at 3 wk in the HLU-healing group and remains significantly elevated compared with the 7 wk Amb-healing group (P < 0.05). Results for lysyl oxidase, the gene that codes for the enzyme that catalyzes collagen cross-link formation, are also shown in Table 3. Lysyl oxidase is reduced in the 3-wk Amb-healing and HLU-healing groups (P < 0.10), which parallels the collagen cross-link content in Fig. 2. However, HLU-healing does not alter the expression of lysyl oxidase vs. the Amb-healing controls. As shown in Table 3, MMP-2 (gelatinase A) and TIMP-1 were normally elevated during normal and pathological ECM remodeling. MMP-2 activity was altered in the HLU-healing group at 7 wk compared with the Amb-healing group (P < 0.05). No changes in MMP-2 gene expression from sham controls were apparent at 3 wk (Table 3). TIMP-1 expression at 3 wk was significantly reduced in the (3 wk) HLU-healing group (P < 0.05) compared with the Amb-healing group; however, TIMP-1 expression remained greater in the HLU-healing group at 7 wk.
Data from this study support our hypotheses that HLU diminishes the healing of dense fibrous connective tissues and degrades the repair processes. This is evidenced by changes in collagen ECM, changes in tissue cellularity, and altered gene expression of ECM components involved in ligament wound repair processes. The biochemical and molecular analyses confirm the biomechanical measurements from our previous study showing significantly reduced maximal force, ultimate stress, and elastic modulus in HLU-healing animals after 3 wk of MCL healing, which was not improved with an additional 4 wk of ligament healing (30). HLU-healing animals at 3 and 7 wk also showed altered cellular and ECM microarchitectures at the scar site compared with results shown in Amb-healing animals, suggesting that GRFs are necessary for proper collagen fiber alignment and orientation (30).
Extracellular protein and molecular gene markers characterizing healing ligaments in rats, rabbits, and humans have been well documented in reviews on injury and repair of ligaments and tendons (13, 14, 18, 31, 44, 45). Results from the Amb-healing groups in our study are consistent with the well-characterized processes of wound healing previously described for the time points used in our study (13, 14). The MCL heals through three processes, including hemorrhage with inflammation, followed by ECM and cellular proliferation, and concluding with active remodeling and maturation of the collagen ECM (14). Evidence provided in this study suggests that genes encoding the three main collagen phenotypes (Col1a2, Col3a1, and Col5a1) within ligaments are differentially expressed in HLU-healing animals compared with Amb-healing animals at 3 and 7 wk, thus emphasizing the importance of mechanical stress on ligament ECM gene regulation during wound healing.
ECM Collagen and Maturation
A significant reduction in the imino acid Hyp, a marker of total collagen in connective tissues, in both of the 3-wk surgically wounded groups (Amb-healing and HLU-healing) is in agreement with rat and rabbit time-course wound healing models of ligaments and tendons (6, 16, 31). Postsurgical tendon gap transection in rats showed little change in Hyp after 30 days (6). However, tendon ultimate stress increased to 70% of controls and the HP collagen cross-link doubled in content, suggesting that an improvement in tendon strength following wound healing can occur without an increase in total collagen (6). Similarly, Achilles tendon repair in rabbits was studied 15 days after surgery and showed that healing tendons recovered 80% of the collagen and 60% of the HP collagen cross-links compared with normal tendons (31). The mature HP cross-links have also been reported to never obtain presurgical levels 1 yr postinjury after subsequent surgical repair (18), which may, in part, contribute to the repaired tissue's decrement in postsurgery strength. In our study, a decrement in total collagen and HP cross-link contents after MCL surgery was demonstrated in both surgical wound healing groups. Furthermore, HLU did not improve or reduce collagen concentration or maturation compared with ambulating animals. An additional 4 wk of healing showed no differences in collagen content or maturation vs. sham controls, suggesting that other ECM factors, such as the organization of the collagen fibers or proportion of specific collagen phenotypes comprising the ECM, may contribute to our previously reported strength decrements in both the 3- and 7-wk groups (30).
Ligament repair during early wound healing is well characterized by a disorganized, hypercellular repair area at the scar site with a large number of inflammatory cells and migrating fibroblasts (14, 23). Matrix flaws and a greater degree of cellularity are indicative of a delay in the wound repair process (14, 23). In our study, ligament cellularity was measured via DNA content from papain-digested sham and surgically transected ligaments. HLU-healing animals had significantly elevated levels of cellularity 3 wk postsurgery, and the levels remained elevated at 7 wk postsurgery. The tissue cellularity data were confirmed by hematoxylin and eosin histology from MCL scar site tissue sections, as previously reported by our research group (30), suggesting that ambulatory animals have improved collagen bundle formation (microarchitecture) and reduced tissue cellularity, whereas HLU-healing groups maintain discontinuities and voids in the scar tissue with an elevated number of cells (30). It remains to be elucidated whether the hypercellularity in the HLU-healing ligaments at both time points are because of a greater number of inflammatory cells or fibroblastic cells or a combination of both cell types.
Differential Expression Extracellular Matrix Genes During Ligament Repair
New molecular methodology for dense fibrous connective tissues, which contain a substantially reduced number of fibroblasts compared with other tissues, has allowed researchers to measure the differential expression of key genes involved in the ligament repair process from attomole quantities of reverse-transcribed total RNA. Early studies investigating gene quantitation and ligament repair in rabbits by Hart and colleagues (8, 32, 34) have provided seminal data on the differential expression of genes encoding structural proteins and enzymes during wound repair. On the basis of previous literature (32, 34), key regulatory genes (collagens type I, III, V, fibronectin, lysyl oxidase, decorin, biglycan, MMP-2, and TIMP-1), involved in the production and remodeling of the ligament ECM were measured from sham and surgically repaired MCLs. Supporting our hypothesis that HLU delays ligament wound healing, the expressions of collagen types I (Col1a2), III (Col3a1), and V (Col5a1) in the 7-wk HLU-healing group were significantly reduced compared with that shown in Amb-healing animals. Interestingly, there was no significant difference in collagen gene expression at the 3-wk time point. The reduced gene expression of collagen types I, III, and V at the 7-wk time point in the HLU-healing group could indicate that the expression of newly synthesized collagen may be reduced during HLU even though the total collagen (new + old collagen protein) showed no change compared with the Amb-healing groups. The expression of type III collagen is usually greater than the expression of type I collagen during early wound healing. However, with time, further tissue remodeling proceeds and type I collagen becomes the predominant fibrillar collagen while simultaneously improving dense fibrous connective tissue strength (14). Our results indicate that HLU differentially suppresses collagen types I, III, and V expression at 7 wk, thereby influencing the remodeling phase of ligament wound healing.
In addition, fibronectin, a key protein that aids in the synthesis of provisional granulation tissue during early wound repair, is expressed at greater levels in the 7-wk HLU-healing group. Similarly, decorin and biglycan, proteoglycans involved in the purported inhibition and/or regulation of collagen fibril and fibrin assembly (10, 12, 43), demonstrated a significantly greater expression in the 7-wk HLU-group compared with the Amb-healing group, suggesting that normal fibrillogenesis in the HLU-healing animals may be compromised. The importance of decorin gene and protein expression in ligament healing needs further study.
MMP-2 (gelatinase A) and TIMP-1 gene expression levels were measured to determine whether the collagen remodeling process during wound healing and HLU was altered at different time points. MMP-2 activity is well known to digest basement membrane (type IV collagen) and type I collagen more efficiently than other collagenases (7, 22). TIMP-1 inhibits collagen degradation by inhibiting MMP activity, and its greater level of expression during wound healing in ligament suggests that TIMP-1 may regulate collagen resorption by MMPs. In our study, a smaller expression of MMP-2 and a greater TIMP-1 expression level in the 7-wk HLU-healing group suggest that ECM turnover and remodeling may be inhibited by HLU. It is unknown whether HLU has a direct effect on normal MMP activity during tissue remodeling. Future studies dedicated to measuring the delicate balance of MMP and TIMP enzyme activity through zymography in dense fibrous connective tissue during the combination of wound healing and unloading could confirm protein turnover and remodeling activity. Finally, the gene encoding lysyl oxidase, a copper-dependent metalloenzyme that catalyzes the first steps in collagen cross-link formation (reducible cross-link formation), showed a trend toward less expression at 3 wk in the HLU-healing group. HP cross-link formation is depressed in both of the 3-wk surgical groups; however, it remains uncertain whether labile reducible collagen cross-link formation confers the same strength within the healing tissue compared with mature stable collagen cross-links and/or whether is there a direct correlation between lysyl oxidase gene expression and collagen cross-link formation during wound healing.
This study has limitations that should be addressed in future investigations. Immunohistochemical localization of proteins in and near the scar area would help in ascertaining the protein expression of the specific genes that were measured using real-time quantitative PCR. In addition, the use of in vivo collagen formation markers, such as the conversion of [3H]proline to [3H]Hyp to measure new collagen secretion (specific activity), as well as gel zymography to determine ECM resorption, could provide further evidence of potential dense fibrous connective tissue turnover rates during wound healing and HLU.
HLU is a model that mimics some of the physiological aspects of spaceflight; in our experiments, the model was used to decrease the GRFs during ligament wound healing to simulate potential orthopedic healing situations that may occur during long-term space travel. Using the HLU model, other investigators have suggested that wound healing is also depressed in other tissues with an adequate blood supply, such as in corneas (25). We postulate that a decreased blood flow to the hindlimbs could exacerbate the wound healing process during HLU as previously demonstrated in rat HLU studies measuring vascular changes (4, 27).
In summary, our data support the concept that HLU depresses normal ligament wound healing processes and produces inferior ECM material at the wound transection site. Biomechanical, biochemical, and molecular gene expression data show a diminished wound healing response of the MCL when deprived of GRFs.
This research was supported by National Aeronautics and Space Administration Grant NAG9-1152 (D. A. Martinez, A. C. Vailas, R. Vanderby, and R. E. Grindeland).
The authors thank the following individuals at the NASA-Ames Research Center who contributed to animal care: Tom Wang, Fang Yuan, Megan Moran, Lisa Baer, Cyra Fung, Guilia Gurun, and David Garcia. We also express appreciation to the members of the Connective Tissue Physiology Laboratory who helped in the preparation of the rat MCL biochemical and gene expression assays: Dr. Antonios Kyparos, Dr. Laura Gutiérrez, Kavita Davé-Coombs, Andrea Creath, Nancy Garcia, John Fink, Hai-Yen Hoang, Phoung Le, and Andrew Lust.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society


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The Ramo Buchon, a beautifully crafted bouquet, is more than just a bundle of flowers – it’s a symbol of timeless love, an elegant surprise that can leave a lasting impression on your loved one. In this article, we’ll explore the enchanting world of the Ramo Buchón, its history, significance, and how it can be the perfect expression of your deepest emotions.
The Art of Gifting: What is a Ramo Buchon?
A Ramo Buchon, often referred to as a “Hatbox Bouquet,” is a unique and artistic arrangement of fresh flowers presented in a round or cylindrical box. It’s a striking departure from traditional bouquets, as it combines the elegance of a floral display with the practicality of a keepsake box. The arrangement is characterized by its exquisite symmetry, with carefully selected blooms and foliage.
The History and Significance
The tradition of giving flowers dates back centuries, with each flower bearing its own symbolism. However, the Ramo Buchón is a relatively recent addition to the world of floral arrangements. Originating in France, it was initially created as a stylish and contemporary alternative to traditional bouquets. Its design was meant to capture the essence of romance and luxury, making it a perfect gift for special occasions.
The term “Buchón” itself has its origins in the Spanish language, meaning “pigeon.” The association between this bird and the arrangement is quite poetic. Just as pigeons are known for their loyalty and lifelong partnerships, the Ramo Buchón signifies enduring love and commitment.
The Perfect Surprise
A Ramo Buchón is the embodiment of elegance and surprise, making it an ideal choice for special moments. Here are some occasions where this exquisite arrangement can leave a lasting impact:
Celebrate the enduring love you share with your partner by surprising them with a Ramo Buchón. Its elegant presentation and carefully chosen blooms can express your deep affection.
2. Valentine’s Day
What better way to declare your love on Valentine’s Day than with a gift that symbolizes lifelong devotion? A Ramo Buchón is sure to make your loved one’s heart skip a beat.
Make your loved one’s birthday extraordinary with a gift that stands out. The artistic arrangement of a Ramo Buchón is a wonderful surprise that will be cherished.
Mark this significant milestone in your loved one’s life with an elegant Ramo Buchón. It’s a unique gift that expresses your pride and best wishes for their future.
5. Apology and Reconciliation
When words are not enough, a heartfelt Ramo Buchón can speak volumes. It’s a thoughtful gesture to mend relationships and express your sincere apologies.
The Elements of a Ramo Buchon
A typical Ramo Buchón consists of carefully chosen flowers and foliage, arranged in a circular or cylindrical box. Each element is selected for its unique characteristics, colors, and fragrance. Here are some key elements that make a Ramo Buchón truly special:
1. Premium Flowers
The heart of a Ramo Buchón is its premium flowers. These are often exquisite roses, lilies, orchids, or other luxurious blooms. Their beauty and fragrance are enhanced by their unique arrangement.
2. Artistic Presentation
The presentation is crucial to the impact of a Ramo Buchón. The flowers are often neatly arranged in a circular or cylindrical box with meticulous attention to detail.
3. Keepsake Box
The box itself serves a dual purpose. It not only showcases the flowers but also provides a keepsake for your loved one to treasure long after the flowers have faded.
You can make the Ramo Buchón even more special by personalizing it with a heartfelt message or the recipient’s name. This touch of personalization adds a deep emotional connection to the gift.
Why Choose a Ramo Buchon?
The Ramo Buchón is more than just a bouquet – it’s an experience. Choosing a Ramo Buchón as a gift offers several unique advantages:
1. Lasting Impressions
The elegant presentation and exquisite flowers will leave a lasting impression on the recipient, making it a memorable gift.
The Ramo Buchón’s association with pigeons and lifelong partnerships adds depth and meaning to your gift, conveying your love and commitment.
It’s suitable for a wide range of occasions, from romantic gestures to expressions of gratitude or apologies.
4. Keepsake Value
The keepsake box can be used to store mementos or as a decorative piece, ensuring your gift continues to be a cherished part of their life.
In the world of gift-giving, the Ramo Buchón stands out as a symbol of timeless love and elegance. Its unique design, premium flowers, and the deep symbolism it carries make it a perfect choice for expressing your deepest emotions on special occasions. Surprise your loved one with a Ramo Buchón and create a moment that they will treasure forever.

domain: design


text:
Rocco Famia was shot three times as he prepared to go to work during the early morning hours of August 26, 1927. The shots were fired from outside through a window and into the kitchen of his Lytle St., Hazelwood home.
Reports linked the murder to tensions between bootleggers, a Prohibition-spurred trade that was dominated by Italian immigrants and that became particularly violent in the five-year period beginning in 1927.
Seven men, all recent Italian immigrants with extensive criminal records, were arrested in Clairton that same day.
Famia’s wife, Sophia, testified before the grand jury that her husband was murdered by Vincenzo Ciccia and Oreste Delforte as a threat to her not to testify against the them for a different crime.
Sophia Famia provided that same evidence at trial. Ciccia and Delforte offered an alibi defense, including testimony from multiple witnesses who could place them in Clairton, more than ten miles to the south, at the time of Famia’s killing.
The two men were convicted on December 17, 1927, after a week-long trial. The jury recommended the death penalty.
A motion for a new trial was argued on December 27, 1927. Though such a motion was a routine occurrence, a formality between the conviction and the formal imposition of a death sentence, it was granted on December 30, after a three-judge panel determined the evidence did not support their convictions.
At retrial, the state argued the same dubious case, centering on the testimony of Sophia Famia. The defendants offered the same alibi defense. Ciccia and Delforte were acquitted on March 3, 1928. No other arrests were made for the killing of Rocco Famia.
After being released from jail, the two men raised families and ran small businesses. Oreste Delforte died in Canandaigua, NY, in April 1966. Vincenzo Ciccia died in Pittsburgh on December 6, 1973.

domain: news


text:
Jonathan Green 10318 Bb 5 Pound Orig Grass Seedby Jonathan Green
Black beauty, 5 pounds, original grass seed mixture, tall fescue plant roots deeper than other grasses & is ideal for stressful growing situations caused by hot sun, deep shade & poor soils, thrives in clay or sandy soils, is more drought, heat & cold tolerant, will improve an existing lawn or establish a great new lawn.
- Product Class
- Full Sun Grass Seed
- Part Number
- Related Departments
- Lawn, Garden & Outdoor ToolsLawn CareGrass Seed Assorted
- Shipping Weight
- 5.52 pounds
- Domestic Shipping
- Item can be shipped anywhere in the US including APO/FPO addresses and territories.
- International Shipping
- Item can be shipped anywhere in Canada including the territories.

domain: agronomy


text:
Weitere Kapitel dieses Buchs durch Wischen aufrufen
Design science and natural science are complementary research paradigms in the management disciplines. Fundamentally the task of management is to develop, articulate, and achieve organizational goals and purposes. Design science research addresses that task by creating novel and effective artifacts that are demonstrated to improve managers’ capability to change “existing situations into preferred ones” (Simon (1996), p. 130). Natural science research addresses it by developing theories that provide deep, principled explanations of phenomena, justified by rigorous empirical evidence that managers can use to guide their actions. Designed artifacts have no special dispensation from the laws of nature; however, business organizations and the environments in which they operate are social constructions (Searle, J. R. (2006) Social ontology: some basic principles, Anthropological Theory 6 (1), pp. 12–29). They are themselves artifacts designed to achieve human goals, purposes, and intentions, influenced by and operating within the context of emergent and intentional human behavior. Furthermore, natural science explanations of how or why an artifact works or does not work may lag years behind the application of the artifact. If academic research is to make significant contributions to management practice it must utilize the results from each paradigm to guide the other. There is evidence that this integration is beginning to take place in several management disciplines including information systems and organizational science. This paper summarizes and assesses this emerging work.
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- Design Science in the Management Disciplines
Salvatore T. March
Timothy J. Vogus
- Springer US
- Chapter 14
Neuer Inhalt/© ITandMEDIA

domain: literature


text:
I love me some quiche. Something about cheesy eggs with a flaky crust just does it for me. And it’s super easy to make. I was feeling a little under the weather today, but wanted something warm and comforting for dinner, so I spent 10 minutes in the kitchen preparing this and man am I glad I did! Not sure if this is weird, but I always make quiche for dinner, not breakfast. Anyone else?…Bueller?…Bueller? (80s movie reference for those of you that don’t know.)
..mmmm, ohhh yeahhhh. And I totally forgot to take a picture of a nice cheesy slice because I gobbled it down as soon as it hit the plate…and then went back for seconds. The sausage in this was so flavorful and added a nice kick.
I’ll be living off of this for the next day or two since, like some other baked dishes – including homemade mac and cheese – I think quiche is even better reheated.
What are your favorite quiche fillings? Are there any meals you like better the next day?

domain: food


text:
They say it takes diverse kinds of people to make the world go round — and the same principle is applicable to literature as well. As a student, whether you are reading fantasy, drama, comedy or tragedy, you are going to read about different types of characters that keep the story moving and also keep you intrigued.
Now you may already know about the protagonist and antagonist as two distinct types of characters in literature. However, the types of characters in fiction goes well beyond these two categories.
That is why we have presented this elaborate guide on different character types featured in almost every literary work. Before we delve into the types, you must know that there are two main ways to classify them: based on their role, and based on quality.
Types of character based on their roles
Characters based on a role refers to the part that one portrays in a work of fiction. In this specific type, the most significant character is the protagonist. This indicates that all other characters are developed according to their relationship to the protagonist. Basically, these types elaborate on how characters interact with each other and influence one another.
Protagonist or the central character
This type of character hardly needs any special introduction, as most of us can identify them easily. The protagonist is the star of the story. Most of the action within the story revolves around them. Some popular examples of the protagonist from the world of fiction would be Sherlock Holmes, Hercule Poirot, Robert Langdon, Frodo Baggins, etc.
Every single work of fiction must come with a protagonist. This type of character is vital because other types of characters in a story are defined in relation to the protagonist.
In this case, you must also remember that a work of fiction may not revolve around one single protagonist. There may be more than one protagonist to a story as well. C.S.Lewis’s popular novel series, Chronicles of Narnia, is a great example of a narrative with multiple protagonists. Another popular example would be Enid Blyton’s Famous Five.
In stories narrated in the first-person point of view, the protagonist is usually the narrator, but there are exceptions as well. The narrator can also be someone close to the central character (like Nick Carraway in The Great Gatsby).
The Antagonist Or The Villain
This type of character stands in direct contrast with the protagonists. Simply put, this character stands against everything that the protagonist supports. They are as much integral to a story as the protagonists because they have the crucial role of anti-hero to play in the conflicts, that arise within a story. Lord Voldemort, from J.K.Rowling’s iconic Harry Potter series, serves as the finest specimen of an antagonist. As said without a villain, the best heroes wouldn’t have risen. So for any story to get that shine of hero , a proper vitiated antagonist or the Villain character is imperative.
Even though most of the time, the antagonists display evil traits, but there are prominent exceptions in this case.
Various literary pieces have different depictions of antagonists in terms of anatgonist’s
- Mental capability
- Ability to portray the character traits to the maximum evil trough
- Ability to maintain moral dis-synchrony with the readers, viewers etc.
- Ability to match with the character arc of the story, that the presenter wants to create.
- Ability to make an impact regarding the fineness, verbose, delivery of messages.
Some literary pieces portray their protagonist as an anti-hero who lacks the typical heroic attributes. William Shakespeare’s Macbeth is a perfect example of an anti-hero. Similarly, some antagonists have been portrayed as morally ambiguous characters, the classic example of which would be Severus Snape from the Harry Potter series.
Another important thing to remember about the antagonists is that they may not always be a person, and can also be a situation or a condition (like depression or anxiety). An example of this would the self-obsession and vanity of Dorian Gray, which ultimately causes his downfall.
Some of the prominent antagonists in English literature are Iago, Professor Jim Moriarty, Mrs. Danvers, Dr Frankenstein etc.
Most literary works have a protagonist and a deuteragonist (or group of deuteragonists). This character is not exactly in the spotlight, but he/she plays a pivotal role in the story.
These characters are often found in the company of the protagonist — giving advice, standing by them when in need, and generally lending a helping hand.
Their presence and close connection with the protagonist give the story warmth and heart. Introducing these characters indicates that it is not just about the protagonist’s journey, however, about the friends, they make along the way. Of course, not all deuteragonist is categorised as friends; some are arch-enemies. But even these less-friendly deuteragonists still add a certain depth to a story.
Moreover certain axioms for charecters to play a proper deuteragonist are
- They should not superimpose the protagonist in the story
- The actionable part must involve the protagonist more than the deuteragonist
- Deuterganosit’s role is to supplement and suffice the protagonist’s role in the story.
Some prominent examples of deuteragonists are Jane Bennet, Dr. Watson, Mercutio, Samwise Gamgee, Ron and Hermione, etc.
This type of character is a little tricky to pin down. Since many stories focus so much on the protagonist’s love interest that other relationships get left out in the cold. However, the connection shared between the confidant and the protagonist still has a profound impact on the readers.
Confidants are often closest friends, but they may also be a potential love interest or even a mentor. The protagonists shares their emotions and thoughts with this individual, even the things they’re reluctant to share with anyone else. However, the confidant can also be an individual that the protagonist turns to, not because they want to, but because they believe they have no other option.
Some brilliant examples of confidante would be Horatio, Friar Laurence, Mrs Lovett, Albus Dumbledore, Hannibal Lecter, etc.
These types of characters in the literature appear in and out of a narrative. You may barely find them in one or two scenes from the literary piece.
Having said that, a well-rounded story still needs a few tertiaries to take it forward. In fact, you may find these characters in your day-to-day life as well. Like a baker from your favourite bakery, or the random guy you sit next to in a bus.
Some specimen of the tertiary characters in literature are Madame Stahl in Anna Karenina, Radagast in The Lord of the Rings, Parvati and Padma Patil in Harry Potter, Fabrizio and Calo in The Godfather, etc.
A foil character is someone whose values and personality essentially clash with the protagonist’s. This clash demonstrates the central character’s defining attributes, offering the readers a clear idea of who they truly are.
The foil’s exact relationship to the protagonist relies on the differences between them. For instance, if the protagonist is introverted, their foil might be super extroverted, but that wouldn’t necessarily lead the two of them to become friends. However, if the central character is kind and their foil tends to be self-serving, they’re probably not going to get along.
Lydia Bennet, Draco Malfoy, Paris, Circe, are some of the famous examples of foil characters in literature.
Types of characters based on quality
Character quality denotes what kind of character someone is. This doesn’t indicate their temperament, like being helpful or mean, but instead their nature within the story, like being dynamic or static.
These types happen to focus on narrative purpose in a story. For instance, a dynamic figure presents a compelling arc for readers to follow, and a symbolic one represents some underlying moral or theme.
This one’s quite self-explanatory. A dynamic character is one who evolves over the course of a narrative. They often evolve to become better or wiser. The character may change positively and negatively depending on the need of the story.
Some of the most common examples of dynamic characters are Don Quixote, Elizabeth Bennet, Neville Longbottom, Han Solo, and Walter White.
Static or flat characters
This types of characters are the exact opposite to their dynamic counterparts. These static figures often tend to be unlikable, like Cinderella’s stepsisters or the Dursleys from Harry Potter— their ignorance or mistreatment towards the protagonist makes them people that the readers “love to hate,” and amplifies sympathy for the protagonist.
Some other valid examples of static characters are Miss Havisham, Mr Collins, Sherlock Holmes (a rare static protagonist), etc.
A symbolic character is incorporated to represent something more important than themselves. This type of character must also be utilised sparingly, or at least subtly, so the reader doesn’t feel like the symbolism is too heavy. As an outcome, the true nature of a symbolic character may only be fully realised at the very end of a story.
Some pertinent examples of symbolic characters are Aslan (symbolises God/Jesus in The Chronicles of Narnia), Gregor Samsa (symbolises the difficulty of change/being different in The Metamorphosis), Jonas (symbolises hope in The Giver), etc.
Stock characters are those familiar figures that feature in the stories time and again. Even though it’s best not to overuse them, but they can really help make readers feel “at home” in your story.
The idea is to not just rely on their archetypal features. Take Albus Dumbledore, for example. He might seem like a “stock” mentor in his appearance and sage manner. However, his wisecracks and weaknesses highlighted later in the series reveal that he's a fully-fledged character in his own right.
Wrapping it up,
Not every literary work will include every type of character. But the more learn about the different character types, the easier it will be to evaluate them while working on an academic task.
Different types of characters are simplified by our experts
While working on a literature assignment, you may have trouble analysing the characters within a literary piece of work. But that doesn’t mean you should spend sleepless nights trying to put together the task. This is when you should approach the experts on MyAssignmenthelp.
Whether it’s foil literary definition or flat character definition, our experts will evaluate the character in most appropriate way possible. They are perfectly aware of each of the different types of characters that exist in the realm of literature.
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domain: artistic


text:
People who live in Okinawa on the Southern tip of Japan have the longest life spans in the world; a unique diet could be the secret to their longevity.
An Okinawan's average life span is about 110 years, genetics have been linked to their good health but diet also plays an important role, nutrition-and-you.com reported.
The oldest man ever recorded, Jiroemon Kimura who died recently at the age of116, was from the impressively healthy region. The oldest person alive now, Misao Okawa, 115, is also from the same area of southern Japan, The Guardian reported.
"The Okinawans have a low risk of arteriosclerosis and stomach cancer, a very low risk of hormone-dependent cancers, such as breast and prostate cancer," Dr Craig Willcox, who wrote a book on the Okinawa diet, said. "They eat three servings of fish a week, on average ... plenty of whole grains, vegetables and soy products too, more tofu and more konbu seaweed than anyone else in the world, as well as squid and octopus, which are rich in taurine - that could lower cholesterol and high blood pressure."
The "longevity diet" in the Okinawa region is pescetarian, it excludes all land animals and animal products but allows seafood. Other staples are soy, and low calorie vegetables, according to nutrition-and-you.
Okinawans eat an extremely low calorie diet, averaging at only one calorie per gram of food. The diet is 20 percent lower in calories than the Japanese overall average. They also eat only 25% of the sugar and 75% of the grains as the average Japanese.
The diet consists of mostly green, orange, and yellow (GOY) vegetable which are packed full of cancer-fighting antioxidants, not to mention a ton of vitamins.
Foods are sorted into four calorie-based categories: featherweight foods, lightweight, middleweights, and heavyweights.
The foods the late Kimura swore by were porridge, miso soup and vegetables. He followed a mantra of "eat light to live long," according to The Guardian.
Oily Fish Lowers Risk Of Breast Cancer
Consumption Of Nuts Related To Lower Prostate Cancer Rate

domain: biology


text:
SC Side Event to the Bonn Climate Change Conference, 8 May 2017
Title: Implementing the Paris Agreement – Perspectives from developing countries
Date: 8 May 2017, 15:00-16:30
Venue: Bonn Climate Change Conference
Organizers: The South Centre and the Third World Network
Description: Speakers from developing countries will share their views on how the Paris Agreement should be implemented and the challenges and opportunities it poses
Walter Schuldt was not able to attend.
This article was tagged: Climate Change, Climate Financing, Conferences of the Parties (COP), Environmentally Sound Technology, Equity Principle, Group of 77 and China (G-77 and China), Technology Transfer, United Nations Framework Convention on Climate Change (UNFCCC)

domain: politics


Er: YAG crystal has an abundant energy degree structure as well as excellent laser efficiency. It can stably give off lasers with wavelengths of 1.64 μm, 1.78 μm, and 2.94 μm, allowing high-concentration ion doping, as well as the power degree life time of the laser lowers when high doping is used. Small, the laser performance is improved. The 2.94 μm emission wavelength of Er: YAG with a doping focus of 50% lies at the water absorption optimal, which can be highly soaked up by water particles and also can also be absorbed by organic tissues.
Since the absorption coefficient of water at this wavelength is 100 times larger than that of 2 μm as well as 10,000 times larger than 1 μm, the 2.9 μm laser word has vital applications in the medical field. Er: YAG crystals have been used in medication, such as oral surgery, cosmetic surgery, as well as ophthalmology. And Er-YAG laser wavelengths can enhance various skin conditions, such as actinic photodamage, hyperpigmentation, terrible scarring, harsh skin structure, laxity, etc. Furthermore, with the advancement of laser innovation, Er: YAG pump lasers additionally have applications in the army area and melon optical interaction, such as lidar, telemetry, and active imaging.


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This year, we’re participating in the 100-Mile Thanksgiving.
Our reasons for moving to a farm are many, but the primary reason is that we could produce some of our own food. Those of you who know me know I have a thrifty streak, and obviously, if I can spend $.10 on a pound of homegrown leeks versus the $5 I pay for organic ones, I am liable to do it. Knowing how the food was produced and having a very short commute to the table also means this is the freshest, most nutritious, most personally gratifying produce possible. Our weekly boxes from the CSA when we lived in the city were great; this is even better.
But we are having 8 people and one small person to our table this Thanksgiving. So I have been hoarding, trying to keep all foods within a hundred miles from our table…and that means I am cooking (and freezing) that cauliflower soup now, baking that bread for stuffing now, while I can still get that fresh cauliflower and those freshly ground grains. The turkey has been ordered. I need to make butter from our raw milk cream. Oil and spices will somewhat come from far away. Other irreplacable things like celery in the stuffing will be substituted (lovage and lots more onions, I think). It is all a grand experiment. But it is also something I can do, and in so doing, hopefully I can teach others the importance of local food.


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A human rights lawyer in Harare has slammed what he called a deliberate 'crackdown' on the Zimbabwe Human Rights Association (ZimRights), which has been targeted by the state with arrests and intimidation.
The ZimRights offices in Harare and Bulawayo have both been raided by police in the past week, with one official from the capital being arrested last Wednesday. ZimRights programs manager Leo Chamahwinya has remained in detention at Harare remand prison since last week, after being charged with 'conspiracy to commit fraud'.
He was originally arrested on allegations that he was involved in 'illegal voter registration'. His lawyer, Trust Maanda, told SW Radio Africa on Thursday that Chamahwinya's case now also involves three other individuals that the state has dragged in to implicate him.
"We don't actually know who they are. They don't work for ZimRights. They were arrested two days before Leo and the state is alleging that they implicated Leo. But they don't even know him," Maanda said.
The other three individuals are Dorcas Shereni, Tanaka Chinaka and Farai Bhani who are all being accused of forgery, fraud and publishing 'false statements'. The state has alleged that the group forged voter registration certificates "to tarnish the name of the Registrar General."
"But this is clearly a way of targeting NGOs ahead of elections. There is a general crackdown on NGOs and this is a deliberate target on ZimRights," Maanda explained.
One of the three individuals arrested before Chamahwinya has told lawyers that he was beaten by 'violent youths' who ransacked his home early last week. Maanda said that these youths then dragged his client to the police, who then accused him of holding the forged documents.
All four of the accused deny the charges against them but still remain in detention. Maanda explained that a bail application was made this week, but the judge has reserved making a decision until Friday.


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The National Safety Council is a 501 nonprofit, nongovernmental public service organization promoting health and safety in the United States of America. Headquartered in Itasca, Illinois, NSC is a member organization, founded in 1913 and granted a congressional charter in 1953. Members include more than 55,000 businesses, labor organizations, schools, public agencies, private groups and individuals. NSC is nonpolitical and does not contribute to or support any political party or candidate.The group focuses on areas where the greatest number of preventable injuries and deaths occur, including workplace safety, teen driving, cell phone use while driving and safety in homes and communities. Wikipedia.
The voluntary agreement with 20 car manufacturers means that the important safety technology will be available more quickly than if the government had gone through the lengthy process of issuing mandatory rules, said Mark Rosekind, head of the National Highway Traffic Safety Administration. However, some safety advocates have filed a petition asking the government to issue mandatory regulations. They say voluntary agreements aren't enforceable, and that since automatic braking is already available in some cars, issuing rules requiring the technology could be done faster than the six to eight years allowed under the agreement. Automatic braking systems use cameras, radar and other sensors to see objects that are in the way and slow or stop a vehicle if the driver doesn't react. It's the most important safety technology currently available that's not already required in cars. "A commitment of this magnitude is unprecedented, and it will bring more safety to more Americans sooner," Rosekind said. Deborah Hersman, president of the National Safety Council, said the agreement "has the potential to save more lives than almost anything else we can accomplish in the next six years." There are about 1.7 million rear-end crashes a year in the U.S., killing more than 200 people, injuring 400,000 others and costing about $47 billion. More than half of those crashes could be avoided or mitigated by automatic braking or systems that warn drivers of an impending collision, NHTSA has estimated. Of the 194 most popular vehicle models already on the market, 17 come with automatic braking as standard equipment. It is available as part of an options package in 71 other models. The reason automakers don't want to be required to put automatic braking into vehicles sooner than the six to eight years promised in the voluntary agreement is that they don't want to have to redesign vehicles and change production schedules sooner than planned, said safety advocate Joan Claybrook, a former NHTSA administrator. "This six- to nine-year lead time is all about the auto companies saving money," she said. The agreement requires that automatic braking be standard in most cars and light trucks with weighing up to 8,500 pounds no later than Sept. 1, 2022. The braking would have to be standard on nearly SUVs and pickup trucks with weighing between 8,501 and 10,000 pounds beginning no later than Sept. 1, 2025. NHTSA estimates that the agreement will make automatic braking standard on new cars three years faster than could be achieved through the formal regulatory process. During those three years, according to Insurance Institute for Highway Safety estimates, the technology will prevent 28,000 more crashes and 12,000 more injuries than without the agreement. However, the standards for how effective the brakes must be are set so low under the agreement that few if any lives will be saved, said Clarence Ditlow, executive director of the Center for Auto Safety. Explore further: Automakers commit to put automatic brakes in all cars
News Article | June 12, 2016
Vehicular accidents remain as one of the leading causes of accidental deaths in the United States, according to a new report by the National Safety Council (NSC). Many of these incidents can be blamed on drivers using their cellphone while behind the steering wheel. Cellphone-related vehicular accidents have prompted authorities to urge drivers not to use their mobile phones while driving but it appears that the threat holds true even for drivers using hands-free devices. Researchers of a new study found that talking on a hands-free phone while driving a car is just as hazardous as holding the device in the hands. For the study, published in the Transportation Research journal, study researcher Graham Hole, from the University of Sussex, and colleagues asked participants to perform video-based hazard-detection tasks. The researchers found that distracted participants, who listened to sentences and decided if these were true of false, were slower than undistracted participants when it comes to responding to hazards. Distracted participants also detected fewer hazards or were unable to see hazards despite being able to focus their eyes on them. The researchers likewise observed that the impairments were worse for those distracted by imagery-inducing statements suggesting that for those who drive while talking, the most dangerous kind of conversations are those that spark visual imagination. The findings suggest that phone conversation entails more use of the visual processing resources of the brain than earlier believed. "Telephone conversations may interfere with driving performance because the two tasks compete for similar processing resources, due to the imagery-evoking aspects of phone use," the researchers wrote in their study. The researchers said that anything that causes drivers to imagine something, which include passengers, may affect their driving performance. Nonetheless, passengers pose lesser risk compared with mobile phone conversations. "Chatty passengers tend to pose less of a risk than mobile phone conversations. They will usually moderate the conversation when road hazards arise," said Hole. "Someone on the other end of a phone is oblivious to the other demands on the driver and so keeps talking." The findings of the study support results of earlier studies on the dangers of using hands-free device in cars. A study conducted by AAA Foundation for Traffic Safety and the University of Utah found that in-dash systems up mental distraction which increases risk of accidents and that hands-free use of the iPhone's Siri causes high level of mental distraction among drivers. © 2016 Tech Times, All rights reserved. Do not reproduce without permission.
News Article | October 30, 2015
Traffic deaths in the United States rose by 14 percent in the first six months of 2015, according to the National Safety Council. If this trend continues, annual traffic deaths might exceed 40,000 for the first time since 2007. Over the next decade, experts anticipate that technology in the form of intelligent transportation systems (ITS) will increase traffic safety across the nation. ITS refers to advanced communications and information technology that connects vehicles to transportation agencies and systems. When combined with advanced sensors, driver assistance systems and smarter road design, ITS may be able to virtually eliminate traffic crashes. Currently, nearly every highway, toll, transit, airport and rail project in the United States incorporates some aspect of ITS. A good example is the variable messaging signs that now operate on a number of major highways, including the Garden State Parkway and New Jersey Turnpike. The Turnpike Authority installed 260 high-resolution, color LED signs, making New Jersey the first state to fully employ the high-tech signs for traffic management. Now, motorists know instantly from the sign’s color whether they are about to encounter congestion, receive general information or enter a construction zone. But coming soon – and likely by the end of the decade – is V2V (vehicle-to-vehicle) technology, which will allow transportation agencies to send data directly to your car or truck about accidents, approaching storms and alternate routes. V2V also will collect data about surrounding cars’ speeds and even traffic volumes to prevent crashes or redirect drivers to alternate routes. According to a recent America THINKS survey conducted by HNTB, an architecture, civil engineering consulting and construction management company headquartered in Kansas City, Mo., this is exactly what the public wants. Seventy-one percent of Americans believe emerging technologies should be used to improve safety and 53 percent want technologies to provide current information about traffic conditions. Many agencies already are preparing for the new technology. For example, the New Jersey Turnpike Authority recently buried a 48-strand fiber optic cable along a portion of the turnpike. In the future, when the Turnpike Authority needs that infrastructure to accommodate connected vehicles, it will be in place. ITS will keep drivers better informed than ever before. With human error a factor in nearly 90 percent of highway crashes, and 30 to 50 percent of peak-period delays caused by crashes, ITS has the potential to change highway travel, saving lives, reducing injuries and providing improved mobility for the traveling public.
News Article | October 27, 2015
School buses are the primary mode of transportation for many children during the school year. It’s a safer alternative than riding in a car, but it can still be dangerous. That’s why Amica Insurance is offering tips to help protect your child while riding the bus. According to the National Highway Traffic Safety Administration, nearly 500 students ages 5 to 18 die each year in passenger vehicles during school travel hours. Amica is sharing the following bus safety tips from the National Safety Council for parents to review with their children: About Amica Insurance Amica Mutual Insurance Co., the nation’s oldest mutual insurer of automobiles, was founded in 1907. The company, based in Lincoln, Rhode Island, is a national writer of auto, home, marine and umbrella insurance. Life coverage is available through Amica Life Insurance Company, a wholly owned subsidiary. Amica employs more than 3,400 people in 44 offices across the country. For more information, visit Amica.com. Media inquiries can be sent to MediaCenter(at)amica(dot)com.
News Article | August 30, 2016
One of the potential benefits of self-driving car technology, and certainly one of the benefits of mass transit options such as rail travel, is that auto collisions and fatalities can be reduced. How much they can be reduced is something of an open question, but considering how distracted, inebriated, and generally terrible many human drivers are, one would presume quite a lot. On that note, the all-time driving mileage record in the US for the first half of the year was recently broken — with Americans driving around 1.58 trillion miles between January and July 2016, the distance equivalent of 250 round trips to Pluto. This was accompanied by a 9% year-on-year increase in traffic fatalities (as compared to 2015). More specifically, roughly 19,000 people have been killed on US roadways since the year began (an 18% increase as compared to 2014) and around 2.2 million people were seriously injured in auto accidents during the first half of the year. This relates to around $205 billion in injury and death costs, according to the congressionally chartered nonprofit, the National Safety Council. (In 2014, the US fatality rate per 100 million vehicle miles was 1.1; in 2015 it was 1.2; and in 2015 it’s projected to be 1.3.) 2.2 million serious auto-accident-related injuries in just 6 months in a country the size of the US is pretty amazing when you think about it. That’s a substantial portion of the population that’s being affected (whether directly or indirectly). Would greater availability of self-driving taxis and high-speed (or low-speed) rail lead to a significant drop in these figures? Factoring in related savings, what’s the actual financial benefit of rail? Considering that the main driver of increased personal-vehicle travel in recent years has simply been low gas prices (along with a growing aversion to regional air travel), I would expect that, if self-driving taxi services were able to notably undercut the costs of travel by personal vehicle, that many people would simply switch over regardless of ingrained habits. Since the increasing rates of auto collisions per miles driven in the US seen during the last few years are likely correlated (this is pure speculation, I know) with an aging baby boomer population (which is often heavily medicated), trends could possibly be reversed simply by giving that population access to affordable self-driving taxi services. Notably, this is one of the main drivers behind the development of self-driving car technologies in Japan — the recognition that the aging population will be increasingly dangerous on the roads. The removal of much of the younger generations from the road, mostly through self-selection if attractive options other than personal vehicle ownership are available, would likely help in the reversal of this trend as well. The oldest and the youngest drivers out there are, after all, the most dangerous by a fair margin. (Amongst those that possess actual driver’s licenses anyways.) A couple of other background points to make: On the subject of why people are driving more … I’ll just direct your attention to this: a woman pushing her baby in a pram, while leaning out of a moving-car’s window. Also on that subject, I’ll relate a story: I recently saw a healthy woman in her 30s or so drive her BMW SUV a block-and-a-half back from the grocery store to her house, with only one bag of groceries. And, before you say it … I was in the park next to both places when she left and when she came back — the grocery store certainly wasn’t an intermediate stop on a longer trip; it was the whole trip. Obviously, this sort of trip, while ridiculous, isn’t the sort that leads to serious collisions or fatalities, but it does illustrate something of the current attitude towards the use of personal vehicles in the US. Take away from this what you will. … Drive an electric car? Complete one of our short surveys for our next electric car report. Keep up to date with all the hottest cleantech news by subscribing to our (free) cleantech newsletter, or keep an eye on sector-specific news by getting our (also free) solar energy newsletter, electric vehicle newsletter, or wind energy newsletter.


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Total Running Time: 34:18
File Size: 405MB
Sal Piper 6' 173 29yo
Jon Laracco 5'8" 175 27yo
Sal (grey shorts, no tattoos) is a strong, aggressive bodybuilder with attitude enough for three wrestlers. Jon (grey shorts and 2 tattoos) is just a nice guy. Jon is not a trained wrestler, just an athlete who has done the usual amount of fooling around with friends in High School and College. What Jon doesn't realize is that he is a very tough guy, strong and aggressive. This match was wrestled in our "CHICAGO REC ROOM".
Sal is in great shape, you can tell by looking at him, especially his legs. He works out at least 5 times a week and is in excellent cardio-vascular shape. He too is not trained in wrestling, but has always been an athlete. He was totally confident and cocky that he could handle Jon.
He got a big surprise, and wasn't happy about it. Jon was all over him. This is the closest to an Squash Job that you will find. The body builder takes a beating. Jon has Sal in a body scissors every time there is an opening, and Sal just can't get out of it. When Sal tries a leg lock submission, Jon turns it around and clams the same on Sal. Just when it seems that Sal is going to get Jon on his back, Jon powers over and Sal is down again. Not only is Sal frustrated that there is nothing he can do to get control, he's pissed too.
At one of the breaks Sal tells us "I should be killing this guy". It's just the other way around. But it's really awesome to watch this tough body builder take such a beating. Jon has his way and, although he doesn't flaunt it, he says at the end "bring on the next sucker".

domain: sports


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Antimicrobial activities of Aerva javanica and Paeonia emodi plants
Type of Spiritual Experience
A description of the experience
Pak J Pharm Sci. 2012 Jul;25(3):565-9.
Antimicrobial activities of Aerva javanica and Paeonia emodi plants.
Mufti FU1, Ullah H, Bangash A, Khan N, Hussain S, Ullah F, Jamil M, Jabeen M.
- 1Department of Biotechnology & Genetic Engineering, Kohat University of Science & Technology, Kohat, Pakistan. email@example.com
Aerva javanica and Paeonia emodi plants extracts were studied for antibacterial activity against Escherichia coli (NCTC 10418), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Staphylococcus epidermidis (NCTC 11047) and Methicillin Resistant Staphylococcus Aureus (MRSA) (NCTC 13143) and antifungal activity against Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Fusarium solani.
Extracts were obtained by using methanol, n-hexane, chloroform, ethyl acetate and aqueous fraction.
The extracts of Paeonia emodi and Aerva javanica showed significant antibacterial activity but only Salmonella typhi was resistant to Aerva javanica. Moreover, the antifungal activity of Aerva javanica was very poor but the fractions of Paeonia emodi showed sufficient inhibition against fungal strains.
The source of the experiencePubMed
Concepts, symbols and science items
Activities and commonsteps
Fungal infection treatments
Pseudomonas aeruginosa infection

domain: chemistry


Colorado Gov. Jared Polis heard about economic development plans and aspirations in Fort Morgan during a visit with city officials on Friday, April 26.
Polis was in Fort Morgan conducting a regional tour focused on agriculture but carved out a brief visit to Fort Morgan City Hall to hear of some of the exciting projects in the preliminary stages in the city.
A press release from the governor’s office said Fort Morgan’s economic development efforts can be a model for effective and responsible rural growth.
After a discussion with city officials including Mayor Ron Shaver, City Manager Jeff Wells, Chief of Public Safety Paul Schultz, Economic Development Specialist Sarah Crosthwaite and Community Development Director Ty Hamer, Polis and several of his staff went to the CentrePointe Plaza development site, east of the Fort Morgan Police station.
The city says this is “where infrastructure is being installed and streets built to accommodate what city officials hope will be a large mixed used development.”
One potential element of that development is a multi-family housing project for which the Morgan County Economic Development Corporation recently began a feasibility study using a grant from the state Office of Economic Development and International Trade.
MCEDC Executive Director Greg Thomason was also on hand for Polis’ visit.
Fort Morgan City Manager Jeff Wells said, “the ongoing growth and development in the City of Fort Morgan is the result of cooperation and coordination among city, county and state organizations.”