Inflammatory bowel disease (IBD) is an intractable autoimmune disease, relatively common in cats, with chronic vomiting and diarrhea. Previous studies have reported that mesenchymal stem cells (MSCs) alleviate inflammation by modulating immune cells. However, there is a lack of research on cross-talk mechanism between feline adipose tissue-derived mesenchymal stem cells (fAT-MSCs) and immune cells in IBD model. Hence, this study aimed to evaluate the therapeutic effects of fAT-MSC on mice model of colitis and to clarify the therapeutic mechanism of fAT-MSCs.
Intraperitoneal infusion of fAT-MSC ameliorated the clinical and histopathologic severity of colitis, including body weight loss, diarrhea, and inflammation in the colon of Dextran sulfate sodium (DSS)-treated mice (C57BL/6). Since regulatory T cells (Tregs) are pivotal in modulating immune responses and maintaining tolerance in colitis, the relation of Tregs with fAT-MSC-secreted factor was investigated in vitro. PGE2 secreted from fAT-MSC was demonstrated to induce elevation of FOXP3 mRNA expression and adjust inflammatory cytokines in Con A-induced feline peripheral blood mononuclear cells (PBMCs). Furthermore, in vivo, FOXP3+ cells of the fAT-MSC group were significantly increased in the inflamed colon, relative to that in the PBS group.
Our results suggest that PGE2 secreted from fAT-MSC can reduce inflammation by increasing FOXP3+ Tregs in mice model of colitis. Consequently, these results propose the possibility of administration of fAT-MSC to cats with not only IBD but also other immune-mediated inflammatory diseases.
Inflammatory bowel disease (IBD) is the most common intestinal disorder in cats and has been shown to lead to vomiting, chronic diarrhea, and weight loss . Although the exact underlying mechanism remains unknown, possible contributory factors include genetic factors, infectious agents (including bacteria and parasites), allergies (dietary), and immune dysregulation [2, 3]. Treatment of IBD usually involves alteration of the diet and the use of medication, such as immunosuppressants and antibiotics . However, some feline patients do not respond to any of these treatments.
With recent advances in veterinary medicine, stem cell-based treatments have begun to be applied for the treatment of animal inflammatory and immune disorders. Accumulating evidence suggests that the therapeutic potential of mesenchymal stem cells (MSCs) may be attributed to their differentiation and integration into the injured site . Additionally, MSCs have the ability to secrete soluble factors, which functionally modulate the microenvironment of the host tissue to facilitate the endogenous process of immunomodulation [6–8]. Several soluble factors secreted by MSCs, including transforming growth factor-β (TGF-β), indoleamine-pyrrole 2,3-dioxygenase (IDO), nitric oxide (NO), and prostaglandin E2 (PGE2), have been proposed to mediate the immunosuppressive effect [9, 10]. Previous studies have demonstrated a prominent role of PGE2 in the immunomodulatory properties of MSCs [11, 12]; they have proven that PGE2 may induce anti-inflammatory activity of MSCs through modulation of regulatory T cells.
Although many studies in veterinary medicine have suggested that MSCs have immunomodulatory effects on activated immune cells [13–20], only a few studies in feline medicine have characterized the secretory factors from feline MSCs. Moreover, the crosstalk mechanisms between feline MSCs and immune cells have not been fully elucidated.
In this study, we examined whether feline adipose tissue-derived mesenchymal stem cells (fAT-MSCs) could alleviate inflammation in colitis in immunocompetent mice induced by dextran sulfate sodium (DSS). Additionally, we analyzed the immunomodulatory mechanisms of PGE2 secreted from fAT-MSCs. Our findings provide important insights into the immunomodulatory abilities of the soluble factors of fAT-MSCs.
The cultured cells isolated from feline AT had a fibroblast-like morphology. The immune-phenotypes of the cells included high expression of cluster of differentiation (CD) 9 and CD44 and low expression of CD34 and CD45 (Fig. 1a). The fAT-MSCs had multilineage plasticity, as demonstrated by their potential for adipogenic, osteogenic, and chondrogenic differentiation. Adipogenic differentiation was evaluated by Oil Red O staining following 3 weeks of adipogenic induction. Matrix mineralization was evaluated by Alizarin Red S staining of fAT-MSCs following 3 weeks of osteogenic induction. Proteoglycans in cells were revealed by Alcian Blue staining after 3 weeks of chondrogenic induction (Fig. 1b).
In this study, we first investigated whether fAT-MSCs exerted an anti-inflammatory effect on mice with DSS-induced colitis. On day 10, mice treated with DSS developed a severe acute illness, characterized by mild to moderate diarrhea, rectal bleeding, and depressed activity, accompanied by continuous weight loss (Fig. 2a, and b), and microscopic examination of the colon of the PBS group showed striking hyperemia, inflammation, necrosis, and shortening (Fig. 2c, and d) as well as histological changes with increased wall thickness, localized inflammatory cell infiltration, and epithelial ulceration (Fig. 2e, and f). However, the mice in the fAT-MSC-injected group showed amelioration of colitis compared with those in the PBS group (Fig. 2a, b, c, d, e, and f). After checking the degree of reduction in body weight for 10 days, there was no significant difference in weight loss between the PBS group and the fAT-MSC group from day 1 to day 9. However, on day 10, mice treated with fAT-MSCs showed a lower weight loss (P = 0.0322 by t -test comparison) than those treated with PBS (Fig. 2a). In addition, fAT-MSC-treated mice had lower clinical disease score (Fig. 2b). Moreover, on day 10, an autopsy was performed for histological evaluation of the colon. The results showed that fAT-MSC-treated mice had longer colon length (Fig. 2c, and d) than PBS-treated mice and showed significantly ameliorated colonic transmural inflammation, decreased wall thickness, reduced mucosal ulceration, and focal loss of crypts, all of which were associated with decreased disease scores and histological scores (Fig. 2e, and f).
Because pro-inflammatory cytokines play important roles in the development of DSS-induced colitis, a possible mechanism of fAT-MSC therapy is suppression of the production of these cytokines in the colon. Therefore, we next investigated the effects of fAT-MSCs on the mRNA expression of inflammatory cytokines that are mechanistically linked to colitis in the colon of the same mouse as in the above experiments. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, and IL-6 were markedly increased after DSS induction. However, the levels of these cytokines in colon tissues from DSS-induced mice that had been infused with fAT-MSCs were significantly lower than those in mice of the PBS group. The results indicated that the infusion of fAT-MSCs had inhibitory effects on the expression of TNF-α, IL-1β, IFN-γ, and IL-6, which are classically associated with DSS-induced colitis, in the colonic tissue. Conversely, the expression of the anti-inflammatory cytokines IL-4 and IL-10 increased in the fAT-MSC group relative to that in the PBS group (Fig. 3).
Previous studies have shown that PGE2 secreted from stem cells plays an important role in immune regulation [21, 22]. However, studies on PGE2 secreted by fAT-MSCs are insufficient. Therefore, to further assess the mechanisms underlying the fAT-MSC-dependent downregulation of pro-inflammatory cytokines and upregulation of anti-inflammatory cytokines in inflamed colon tissue, we established a fAT-MSC/feline peripheral blood mononuclear cell (fPBMC) co-culture protocol in vitro. Our findings confirmed that the concentration of PGE2 was increased in the supernatants of the fAT-MSC group cultured with concanavalin A (Con A)-stimulated feline PBMCs but was decreased in the group treated with NS-398, an inhibitor of the PGE2 synthesis-related enzyme, cyclooxygenase (COX)-2 (Fig. 4). To determine whether increased PGE2 was secreted from fAT-MSCs in co-cultured medium, the relative mRNA expression levels of COX-2 in fAT-MSCs and fPBMCs were confirmed by qRT-PCR and agarose gel electrophoresis. The results showed that COX-2 was highly expressed in fAT-MSCs co-cultured with Con A-induced fPBMCs (Additional file 1). This suggested that PGE2 was secreted from fAT-MSCs rather than fPBMCs.
We then evaluated the anti-inflammatory effects of fAT-MSCs; pro- and anti-inflammatory cytokines were measured at the mRNA level in Con A-stimulated feline PBMCs. The expression of the pro-inflammatory cytokines, TNF-α, IL-1β, IFN-γ, and IL-6, decreased when Con A-stimulated feline PBMCs were co-cultured with fAT-MSCs. In contrast, the expression of anti-inflammatory cytokines, i.e., IL-4 and IL-10, increased. Next, we examined how the decreased secretion of PGE2 affected the cytokine-modulating effect of fAT-MSCs. Notably, both the pro- and anti-inflammatory cytokine-modulating effects of fAT-MSC were reduced in the NS-398 treatment group (Fig. 5).
PGE2 is known to be related to changes in T-cell polarization, and regulatory T cells (Tregs) are known to play important roles in alleviation of colitis [23–25]. Therefore, we determined the changes in T-cell phenotypes in the presence of different PGE2 concentrations. Because FOXP3 is specifically expressed in naturally occurring Tregs, the extent of changes in FOXP3 mRNA expression was confirmed by measuring changes in PGE2 concentrations. The expression of FOXP3 mRNA increased with increasing PGE2 and decreased following treatment with NS-398 (Fig. 6).
Immunostaining was performed in the inflamed colon to examine whether the ratio of Tregs was also increased in vivo. CD3 and FOXP3 were stained separately but compared at the same sites in the same colon samples. Quantitative analysis of FOXP3+ and CD3+ cells, detected in colon tissue sections, showed that the extent of the increase was larger in FOXP3+ cells than in CD3+ cells in the fAT-MSC group compared with that in the PBS group (Fig. 7).
In this study, we aimed to determine whether administration of fAT-MSCs alleviated intestinal inflammation and whether regulation of inflammatory cytokines associated with colitis occurred through immune cell regulation via secretory factors from fAT-MSCs. Feline stem cells are immune privileged, partly due to the low expression of the major histocompatibility complex class II molecule [26–28]. Therefore, we performed in vivo experiments using immunocompetent mice to confirm that fAT-MSCs had anti-inflammatory effects through immune system control.
We showed that intraperitoneal administration of fAT-MSCs in mice with DSS-induced colitis alleviated the disease symptoms such as decreased activity, rectal bleeding, and stool consistency. In addition, the body weights of the mice, measured for 10 days, were not significantly different between the PBS and fAT-MSC groups from day 1 to day 9, but on day 10, the weight reduction in the fAT-MSC group was less than that in the PBS group. Although there was little difference in the weight between the PBS group and the MSC group, the clinical symptoms, colon length, and histological examination showed that the fAT-MSCs alleviated DSS-induced colitis.
Recent studies have shown that the expression of inflammatory mediators, such as cytokines, is an important factor in the progression of colitis . In this experiment, we demonstrated that injection of fAT-MSCs reduced the expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, IFN-γ, and IL-6. However, in the fAT-MSCs group, the levels of anti-inflammatory cytokines, such as IL-4 and IL-10, were upregulated in the injured colon. These results indicated a distinct correlation between the immunomodulatory potential of fAT-MSCs and their ability to ameliorate the inflammatory response in IBD.
In previous studies, MSCs were found to secrete certain cytokines, such as IDO, TGF-β, NO, and PGE2, among which, PGE2 has been shown to be pivotal for the anti-inflammatory effect of MSCs in several inflammatory disease models, including wound disease, brain disease, arthritis, lung injury, periodontitis, and colitis [30–36]. Such anti-inflammatory effects are mediated through immune regulation; in the intestinal tract, immunomodulation occurs mainly via T cells [37–40]. Therefore, in this study, we hypothesized that PGE2 secreted from fAT-MSCs plays an important role in immune regulation and that reduction of PGE2 secretion from stem cells decreases the immunoregulatory capacity of fAT-MSCs. For this experiment, NS-398 (a selective COX-2 inhibitor; which has not been used in fAT-MSCs but has been used in various cells) [41–43] was used as an inhibitor of PGE2 secretion by fAT-MSCs. To prove this, we directly monitored protein concentration of PGE2 secreted from fAT-MSCs in conditioned medium following co-culture of fPBMCs and fAT-MSCs. The result confirmed that the concentration of PGE2 was high in the medium of Con A stimulated-PBMCs co-cultured with MSCs, and conversely, in the group treated with NS-398, the PGE2 concentration was decreased. In addition, fAT-MSCs co-cultured with fPBMCs stimulated with Con A (a T-cell mitogen activator) showed reduced expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and IFN-γ. However, in the NS-398-treated group, the overall anti-inflammatory effects of fAT-MSCs were decreased, and especially, mRNA expression of IFN-γ was significantly increased in the inhibitor group than in the fAT-MSCs group. In the case of IL-10 and IL-4, known to be anti-inflammatory or immunosuppressive cytokines, Con A -induced fPBMCs showed an increasing tendency when cultured with fAT-MSC. However, the NS-398-treated group showed the opposite tendency. Taken together, fAT-MSCs have cytokine-modulating effects on immune cells, and PGE2 indirectly plays a major role in this regulatory effect.
Diverse regulatory mechanisms cooperate to maintain intestinal homeostasis [44, 45], and disruption of these pathways may lead to inappropriate immune responses to intestinal communities [46, 47], thereby contributing to pathogenesis. Several studies have shown that bowel homeostasis is closely related to Treg activation [48–50]. Moreover, colonic Tregs recognize and suppress immune responses against antigens, including commensal bacteria and food [51, 52]. In particular, FOXP3+ Tregs, most of which are CD4+ T cells, are potent mediators of dominant self-tolerance in the periphery and can suppress the activation, proliferation, and effector functions of a wide range of immune cells, including natural killer cells, B cells, antigen-presenting cells, and T cells . In addition, in in vivo and in vitro studies, IL-10 was significantly increased in the fAT-MSCs group when anti-inflammatory cytokines were measured, and many studies have reported that IL-10 is closely related to regulatory T cells [54, 55]. Therefore, understanding the mechanisms responsible for the development of FOXP3+ Tregs in the intestine of patients with IBD could provide new therapeutic options.
The mRNA expression levels of FOXP3, a Treg lineage-specification factor [56, 57], were further confirmed in vitro; FOXP3 expression increased in the fAT-MSC group but decreased in the COX-2 inhibitor group. In addition, in vivo, fAT-MSCs blocked the infiltration of CD3+ T cells and increased the FOXP3+ Treg population in the injured colons of DSS-treated mice. These results suggested that the increased number of colonic Tregs in the fAT-MSC-treated group was associated with PGE2 secreted from fAT-MSCs.
Although we could not rule out the possibility of the contribution of other factors secreted from fAT-MSCs to the FOXP3+ Treg proliferation in mice with colitis, our findings collectively suggested that fAT-MSCs inhibited inflammation by regulatory T cells via a paracrine mechanism and that PGE2 secreted by fAT-MSCs may play an important role in increasing Tregs in mice with DSS-induced colitis.
PGE2 released by fAT-MSCs alleviated DSS-induced colitis in mice by inducing an increase in the Treg population. Our data indicated that regulation of PGE2 production modulated Treg development and function, thereby suggesting attractive therapeutic strategies, such as targeting PGE2-activated Tregs in the treatment of IBD. Taken together, our findings suggested that fAT-MSCs may be potential candidates for cell-based clinical therapy in cats with IBD.
With the consent provided written of the owner, Adipose tissue was obtained from a healthy, adult, female, domestic short-haired cat (1-year-old, 5.5 kg) during ovariohysterectomy at Seoul National University Veterinary Medicine Teaching Hospital; MSCs were isolated as previously described . Briefly, the tissue sample was washed four times in Dulbecco's PBS (PAN-Biotech, Aidenbach, Germany) with 1% penicillin-streptomycin (PS; PAN-Biotech), cut into small pieces, and digested for 1 h at 37 °C with collagenase type 1A (1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA). The enzymatic activity was inhibited by Dulbecco's modified Eagle's medium (DMEM; PAN-Biotech) containing 20% fetal bovine serum (FBS; PAN-Biotech). Following centrifugation at 1200×g for 5 min, the pellet was filtered through a 70-μm Falcon cell strainer (Fisher Scientific, Pittsburgh, PA, USA) to remove debris; erythrocytes in the pellet were eliminated by adding 1 mL red blood cell (RBC) lysis buffer (Sigma-Aldrich), and the cell solution was incubated for 5 min at 25 °C. Pellets were resuspended in DMEM containing 20% FBS and 1% PS and transferred to 100-mm dishes at a density of 3000 cells/cm2. Transferred cells were incubated in DMEM containing 20% FBS at 37 °C in a humidified atmosphere of 5% CO2, and the medium was replaced every 2–3 days until the adhered cells showed a fibroblast-like morphology and reached 70–80% confluence. Thereafter, the cells were repeatedly subcultured under standard conditions. Cells were characterized by flow cytometry using antibodies against the following proteins: CD9, CD44 (GeneTex, CA, USA), CD34-phycoerythrin (PE), and CD45-fluorescein isothiocyanate (FITC; eBiosciences, San Diego, CA, USA). For CD9 and CD44, indirect immunofluorescence was performed using goat anti-mouse IgG-FITC and goat anti-rat IgG-PE (Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively [43, 59]. Characterization was conducted using FlowJo 7.6.5 software (TreeStar, Inc., Ashland, OR, USA). Cellular differentiation was evaluated using special kits (StemPro Adipogenesis Differentiation, StemPro Osteogenesis Differentiation, and StemPro Chondrogenesis Differentiation kits; Gibco/Life Technologies, Mulgrave, Australia) according to the manufacturer's instructions, followed by Oil Red O staining, Alizarin red staining, and Alcian blue staining.
Male C57BL/6 mice, aged 6–8 weeks, were purchased from Nara Biotech (Seoul, Korea). All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of SNU (protocol no. SNU-171121-5), and all protocols were in accordance with approved guidelines. Environmental conditions were maintained at a constant temperature of 25 °C and humidity of 50% with a 12-h light/dark cycle. For environmental enrichment, 3–4 mice were raised in polycarbonate cages (W324 × D221.5 × H130 mm) containing clean bedding (shavings; Nara Biotech), cardboard boxes, and tunnels. All the mice were supplied with sterilized maintenance mouse food and fresh water ad libitum. The studies were conducted using 22 animals, and mice were randomly divided into three groups, each containing 6–8 mice (n = 6 naïve, n = 8 PBS, n = 8 fAT-MSC). At the start of the experiments, the health status of the mice was evaluated by weight, vitality, and defecation, and the experiments were carried out with mice with no abnormal symptoms. Colitis was induced by administration of 3% DSS (36–50 kDa; MP Biomedical, Solon, OH, USA) in drinking water ad libitum from day 0 to day 7. On day 1, the following procedure was performed: fAT-MSCs (2 × 106 cells in 200 μL PBS) or an equivalent PBS volume was injected intraperitoneally into the mice. During housing, animals were monitored once daily for health status. The mice were sacrificed on day 10, and colon tissues were collected for subsequent processing. On day 10 of the study, all the mice were humanely euthanized with injection of xylazine and inhalation of CO2. A completed ARRIVE guidelines checklist is included in Checklist S1.
The severity of colitis was assessed by scoring the clinical disease activity, including body weight loss, stool consistency, rectal bleeding, and general activity (Table 1). The combined DAI ranged from 0 to 16.
Colon segments were fixed in 10% formaldehyde for 48 h, and paraffin-embedded sections were prepared for hematoxylin and eosin (H&E) staining. Histological sections of distal colon were scored blindly by an independent researcher. And Histological scores were assessed as means ± standard deviations of the different groups of colon segments. The severity of symptoms was calculated by scoring tissue inflammation and tissue damage grade (Table 2). The combined histological score for severity of colitis ranged from 0 to 6.
Blood samples were obtained from the jugular vein of two healthy adult cats with the consent provide written of the owners, and blood (5 mL each) was collected into sterile CPDA tubes. Feline blood was diluted with an equal volume of PBS and layered over Ficoll-Paque PLUS (GE Healthcare Life Sciences, Piscataway, NJ, USA) in a conical tube. After centrifugation at 780×g for 30 min, the buffy coat layer was carefully collected. The collected sample was resuspended with RBC lysis buffer and incubated at 25 °C for 5 min. After adding PBS, samples were centrifuged at 850×g for 10 min, washed, and centrifuged again; The fPBMCs were plated at a density of 1 × 106 cells/well in 6-well plates (SPL Life Science, Pocheon, Korea), resuspended in DMEM containing 20% FBS and 1% PS, and stimulated with 5 μg/mL Con A (Sigma-Aldrich) for 6 h before further experiments . Then, 2 × 105 fAT-MSCs were seeded onto 0.4-μm pore-sized Transwell inserts (SPL Life Science). Additionally, the PGE2 inhibitor NS-398 (5 μM; Enzo Life Science) was added to the medium in the inhibitor group. The appropriate dose of NS-398 in these experiments was determined based on a previous study on the effects of NS-398 in feline cells . After incubation for 48 h, total RNA and proteins were extracted from the PBMCs and fAT-MSCs following collection by scraping and 2 ml of the culture supernatant was collected for enzyme-linked immunosorbent assay (ELISA) for PGE2.
For in vivo experiments, six colon tissues were collected from each group, and for in vitro experiments, five replicates each were analyzed for fPBMCs and fAT-MSCs for each group. Total RNA was extracted from homogenized colon tissue, fPBMCs, or fAT-MSCs using an Easy-BLUE Total RNA Extraction kit (Intron Biotechnology, Seongnam, Korea) according to the manufacturer's instructions. Extracted RNA was converted into cDNA using LaboPass M-MuLV Reverse Transcriptase (Cosmo Genetech, Seoul, Korea) following the manufacturer's instructions. Samples were analyzed in duplicate using 10 μL AMPIGENE qRT-PCR Green Mix Hi-RO with SYBR Green dye (Enzo Life Science, Lausen, Switzerland), 7.4 μL PCR-grade dH2O, 0.8 μL forward and reverse primers (Bionics, Seoul, Korea; Table 3), and 1 μL template cDNA. Cytokine mRNA levels were quantified by comparison with that of glyceraldehyde 3-phosphate dehydrogenase.
Supernatants from fPBMCs and fAT-MSCs culture medium were obtained after 48 h of incubation and used for protein analysis. PGE2 secreted from fAT-MSCs in the conditioned medium was quantified using an ELISA kit (Enzo Life Science) according to the manufacturer's instructions.
Paraffin-embedded colon tissue sections were cut into 4-μm-thick sections. Sections were deparaffinized in xylene and rehydrated sequentially in 100, 95, and 80% ethanol solutions; antigen retrieval was carried out using 10 mM citrate buffer (Sigma-Aldrich). After washing, the sections were blocked with a blocking buffer containing 1% bovine serum albumin in PBST for 30 min. The sections were incubated overnight at 4 °C with antibodies against FOXP3 (1:50; Santa Cruz Biotechnology) or CD3 (1:50; Santa Cruz Biotechnology). After three washes, the slides were incubated with secondary antibody. The colon sections, stained with an antibody against either FOXP3 or CD3, were washed three times and incubated with fluorescein-conjugated secondary antibodies (1:200; Santa Cruz Biotechnology) or Texas red-conjugated secondary antibodies (1:200; Santa Cruz Biotechnology) for 1 h at room temperature in the dark. Colon sections, stained with antibodies against either FOXP3 or CD3, were washed three times and mounted in VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). The samples were observed using an EVOS FL microscope (Life Technologies, Darmstadt, Germany), and the immuno-reacted cells were counted in 20 random fields per group.
Data are shown as the mean ± standard deviation. Mean values among different groups were compared by one-way analysis of variance using the GraphPad Prism 6.01 (GraphPad, Inc., La Jolla, CA). A P-value < 0.05 was considered statistically significant.
This study was supported by the Research Institute for Veterinary Science, Seoul National University and Basic Science Research Program of the National Research Foundation of Korea. These funds contributed collect, analyze and interpret data for this study.
JHA conceived and designed the study, collected, analyzed, and interpreted data, and helped in writing the manuscript; WJS participated in the conception and design of the study, data collection, and manuscript writing; QL contributed to the conception and design of the study, and helped with data collection; SMK, JIY, MOR and ARN collected the data; DHB and YCJ provided administrative support and study material; HYY contributed to the conception and design of the study, data analysis and interpretation, and granted final approval of the manuscript. All authors have read and approved the final manuscript.
All Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of SNU (protocol no. SNU-171121-5), Republic of Korea, and all protocols were in accordance with approved guidelines.


You can hear one of the talks here, recorded in Montclair New Jersey and see the slides that were presented here.
In March 2016, over 50,000 refugees were trapped in Greece. Europe had shut its borders. They had been travelling for weeks looking for somewhere to rebuild their lives. Most were living in the open and had exhausted all their resources. The winter had been harsh. Almost half were children.
The state institutions were failing people who had lost almost everything so two businessmen, who had been volunteering to help migrants in Calais, went to Greece just to try and do something. And so Refugee Support Europe was born. Working with the Greek community, they established a 'free shop' on a derelict military site where 800 Syrian refugees were living in tents. Through the shop they organised the dignified distribution of essential food and hygiene products.
With the support of almost 600 volunteers from 40 nations and many thousands of individual donors, what started out as a single shop operation became something much bigger. At that first camp in Alexandreia they founded a clothes boutique, community kitchen, language school, playground and cafeteria.
Soon after starting, they began supporting two other camps with weekly fresh food parcels, then after 6 months they set up food and clothes shops at Filippiada camp for 400 Syrian and Afghan refugees. After a year they started to support two isolated camps at LM Village and Doliana and in December 2017 they were the first support organisation to help the 400 new arrivals at Katsikas camp. In addition to that, they also had a team based in Bangladesh for 4 months supporting a community of 6,300 Rohingya refugees with food and clothing.
April 3rd, 2018 marks two years of an incredible journey. John Sloan and Paul Hutchings founded and managed Refugee Support but all these achievements are because of the people from all over the world who have helped. It is a story of human solidarity with resilient and resourceful people.


Bharti Airtel has slashed the prices of its 4G Hotspot to Rs 999 from earlier Rs 1,950. Airtel's 4G Hotspot allows customers to simultaneously connect multiple devices - laptops, smartphones, tablets, smart devices (TV, speakers) - to high speed internet while on the move or inside their homes and offices. In addition, the device delivers a highly secure data experience over a fully protected network.
Airtel 4G Hotspot is available at all leading Airtel retail stores across the country. In addition, customers will soon be able to order the Airtel 4G Hotspot from Amazon India.


text:
"The guy from another league" by Kristina Gallo
"The guy from another league" by Kristina Gallo is a story of bad choices. Everyone makes mistakes. Even good, decent, and clever people.
Renata is unlucky in love. She is young, pretty, and intelligent, but the men she meets don't seem to treat her as she deserves. I think that because everyone expected her to meet her Mr Right really easily, she has made the wrong choice in love. Questions like "haven't you gotten married yet" maybe are asked more seldom than twenty years ago, but there are still those who think that it's appropriate to ask a young woman such things. These questions create pressure that makes girls think that they must find a husband as quickly as possible.
Renata's story reminded me of the relationship I had witnessed when I was a teenager. The married son of my aunt had a lover, an intelligent, gentle, and educated girl, just like Renata. I remember that even at that age I felt confused and couldn't understand why such a young woman agreed to such a relationship. The guy wasn't overly attractive or successful, and he was involved in some shadowy deals.
The story is raw and direct. It has a feeling of "a documentary" over it. I'd say that there are a few valuable lessons that might be learnt from "The guy from another league". Maybe some girl, who has gotten confused about her relationship with Mr Bad, would read it and see her situation from another perspective.
bookreview, contemporary
"A Week in Winter" by Maeve Binchy
"The Complexities of Love" by M.A. Quigley

redpajama_set_name: RedPajamaCommonCrawl


text:
from __future__ import absolute_import

import unittest

import bs4

import bokeh.embed as embed
from bokeh.resources import CDN, INLINE, Resources
from bokeh.plotting import figure
from bokeh.session import Session
from six import string_types

_embed_test_plot = None

def setUpModule():
    global _embed_test_plot
    _embed_test_plot = figure()
    _embed_test_plot.circle([1,2], [2,3])

class TestComponents(unittest.TestCase):

    def test_return_type(self):
        r = embed.components(_embed_test_plot)
        self.assertEqual(len(r), 2)

        script, divs = embed.components((_embed_test_plot, _embed_test_plot))
        self.assertTrue(isinstance(divs, tuple))

        script, divs = embed.components([_embed_test_plot, _embed_test_plot])
        self.assertTrue(isinstance(divs, tuple))

        script, divs = embed.components({"Plot 1": _embed_test_plot, "Plot 2": _embed_test_plot})
        self.assertTrue(isinstance(divs, dict) and all(isinstance(x, string_types) for x in divs.keys()))

    def test_result_attrs(self):
        script, div = embed.components(_embed_test_plot)
        html = bs4.BeautifulSoup(script)
        scripts = html.findAll(name='script')
        self.assertEqual(len(scripts), 1)
        self.assertTrue(scripts[0].attrs, {'type': 'text/javascript'})

    def test_div_attrs(self):
        script, div = embed.components(_embed_test_plot)
        html = bs4.BeautifulSoup(div)

        divs = html.findAll(name='div')
        self.assertEqual(len(divs), 1)

        div = divs[0]
        self.assertTrue(set(div.attrs), set(['class', 'id']))
        self.assertEqual(div.attrs['class'], ['plotdiv'])
        self.assertEqual(div.text, "")

class TestNotebookDiv(unittest.TestCase):

    def test_return_type(self):
        r = embed.notebook_div(_embed_test_plot)
        self.assertTrue(isinstance(r, str))

    def test_result_attrs(self):
        r = embed.notebook_div(_embed_test_plot)
        html = bs4.BeautifulSoup(r)
        scripts = html.findAll(name='script')
        self.assertEqual(len(scripts), 1)
        self.assertTrue(scripts[0].attrs, {'type': 'text/javascript'})

    def test_div_attrs(self):
        r = embed.notebook_div(_embed_test_plot)
        html = bs4.BeautifulSoup(r)
        divs = html.findAll(name='div')
        self.assertEqual(len(divs), 1)

        div = divs[0]
        self.assertTrue(set(div.attrs), set(['class', 'id']))
        self.assertEqual(div.attrs['class'], ['plotdiv'])
        self.assertEqual(div.text, "")

class TestFileHTML(unittest.TestCase):

    def test_return_type(self):

        class fake_template:
            def __init__(self, tester, user_template_variables=None):
                self.tester = tester
                self.template_variables = {
                    "title",
                    "plot_resources",
                    "plot_script",
                    "plot_div"
                }
                if user_template_variables is not None:
                    self.template_variables.update(user_template_variables)

            def render(self, template_variables):
                self.tester.assertTrue(
                    self.template_variables.issubset(
                        set(template_variables.keys())
                    )
                )
                return "template result"

        r = embed.file_html(_embed_test_plot, CDN, "title")
        self.assertTrue(isinstance(r, str))

        r = embed.file_html(_embed_test_plot, CDN, "title", fake_template(self))
        self.assertTrue(isinstance(r, str))

        r = embed.file_html(_embed_test_plot, CDN, "title",
                            fake_template(self, {"test_var"}),
                            {"test_var": "test"})
        self.assertTrue(isinstance(r, str))

class TestAutoloadStatic(unittest.TestCase):

    def test_invalid_resources(self):
        self.assertRaises(ValueError, embed.autoload_static, _embed_test_plot, INLINE, "some/path")

        dev_resources = (Resources("absolute-dev"), Resources("server-dev"),Resources("relative-dev"))
        for x in dev_resources:
            self.assertRaises(ValueError, embed.autoload_static, _embed_test_plot, x, "some/path")

    def test_return_type(self):
        r = embed.autoload_static(_embed_test_plot, CDN, "some/path")
        self.assertEqual(len(r), 2)

    def test_script_attrs(self):
        js, tag = embed.autoload_static(_embed_test_plot, CDN, "some/path")
        html = bs4.BeautifulSoup(tag)
        scripts = html.findAll(name='script')
        self.assertEqual(len(scripts), 1)
        attrs = scripts[0].attrs
        self.assertTrue(set(attrs), set(['src',
            'data-bokeh-modeltype',
            'data-bokeh-modelid',
            'async',
            'id',
            'data-bokeh-data']))
        self.assertEqual(attrs['async'], 'true')
        self.assertEqual(attrs['data-bokeh-data'], 'static')
        self.assertEqual(attrs['data-bokeh-modeltype'], 'Plot')
        self.assertEqual(attrs['data-bokeh-modelid'], str(_embed_test_plot._id))
        self.assertEqual(attrs['src'], 'some/path')

class TestAutoloadServer(unittest.TestCase):

    def setUp(self):
        self.sess = Session(load_from_config=False)
        self.sess.docid = 'docid10'
        self.sess.apikey = 'apikey123'
        self.sess.root_url = "http://foo"

    def test_return_type(self):
        r = embed.autoload_server(_embed_test_plot, self.sess)
        self.assertTrue(isinstance(r, str))

    def test_script_attrs(self):
        r = embed.autoload_server(_embed_test_plot, self.sess)
        html = bs4.BeautifulSoup(r)
        scripts = html.findAll(name='script')
        self.assertEqual(len(scripts), 1)
        attrs = scripts[0].attrs
        self.assertTrue(set(attrs), set([
            'src',
            'data-bokeh-docid',
            'data-bokeh-docapikey',
            'data-bokeh-modeltype',
            'data-bokeh-modelid',
            'data-bokeh-root-url',
            'async',
            'id',
            'data-bokeh-data',
            'data-bokeh-conn-string'
        ]))
        self.assertEqual(attrs['async'], 'true')
        self.assertEqual(attrs['data-bokeh-data'], 'server')
        self.assertEqual(attrs['data-bokeh-docapikey'], 'apikey123')
        self.assertEqual(attrs['data-bokeh-docid'], 'docid10')
        self.assertEqual(attrs['data-bokeh-modelid'], str(_embed_test_plot._id))
        self.assertEqual(attrs['data-bokeh-root-url'], "http://foo/")
        divid = attrs['id']
        self.assertEqual(attrs['src'], "%s/bokeh/autoload.js/%s" % ("http://foo", divid))

if __name__ == "__main__":
    unittest.main()

redpajama_set_name: RedPajamaGithub


Beautiful autumn swag with burlap banner displayed on an easel. Perfect for sympathy gift or for personal enjoyment. Swag is removeable for seasonal changes. This is a very unique gift sure to be treasured.


The American Society of Limnology and Oceanography invites the submission of abstracts for oral and poster presentations! In order to have your abstract considered for acceptance, you must submit before the abstract deadline of October 1, 2004.
The American Society of Limnology and Oceanography 2005 Aquatic Sciences Meeting is supported by The Nature Conservancy, the Geological Society of America, and the North American Benthological Society. Members of each of these societies and organizations are encouraged to submit abstracts or simply come to the meeting to discuss research with aquatic scientists with whom you may not normally interact. Each member of a supporting society will be able to register for the conference at the ASLO member rate.
New! Major update to this site completed 01/19/05. New information is available site-wide. Also, the PDF version of the printed program is available for download.
New! Browseable Session Schedules with abstracts are now available! Pick a session from a drop down list and see room numbers, presentation times, poster numbers aand full abstract citations..
New! Scientific Inquiry in the K-16 Classroom - a workshop for scientists and science educators trying to meet requirements by funding agencies to demonstrate the "broader impact" of their scientific research.
New! A Program Schedule with Author Index is now available for download in the files section.
New! Abstract submission is closed.
Special Note: Limnology and Oceanography, ASLO's premier aquatic journal, celebrates 50 years of publication in 2005!
Organizers of the ASLO 2005 Aquatic Sciences Meeting have designed this meeting to be interactive and to encourage discussion of new and cutting-edge research and applications in the aquatic environment. We invite you to participate through submissions to oral or poster sessions as well as to actively exchange ideas and information as a registered participant. We hope you will leave this meeting with a broader understanding of current research directions and concepts in aquatic sciences and make contact with new colleagues as you participate in this interactive process.
Following the trend at recent ASLO meetings, increasing emphasis is being placed on poster sessions with the overall goal of reducing the number of concurrent oral sessions and giving greater exposure to presenters at all sessions. We especially encourage the submission of poster presentations as a very effective means of facilitating discussion of research. Poster sessions will be scheduled at times when there are no conflicts from oral sessions or scheduled special workshops, field trips, or town meetings. The poster sessions include receptions to provide opportunities to make professional connections in a social setting.
The Sunday Opening session by ASLO President, Jon Cole, Institute of Ecosystem Studies, will be followed by a welcome mixer reception. Plenary lectures will be held Monday through Friday. Poster and oral sessions will be held throughout the meeting. Daily conference events will be held at the Salt Palace Convention Center. Please make plans to join us in Salt Lake City, Utah, USA, the site of the 2002 Winter Olympic Games!


text:
The Myths and Legends of Horse Racing
Did you know that the origin of horse racing goes way back? There is no concrete date that can be attributed to horse races, but it is believed that they originated during the Greek Olympic Games, around 700 to 40 B.C. The sport began as a series of bareback mounted races, and then spread to neighboring countries including North Africa and the Middle East. There is even a betting system for horse races. Read on to learn more about horse racing.
Initiation of horse racing
Horse racing is a global tradition that dates back to ancient times. Archeological records show that horse racing was practiced in ancient Greece, Rome, Babylon, Syria, and Egypt. Its use is also documented in mythology and legend. However, these records are not conclusive. Although horse racing has been a popular sport for centuries, some elements of it remain controversial today. Here are some common myths that are related to the sport.
Origin of the "Triple Crown"
The term "Triple Crown" first arose in the 1930s, when a journalist for Daily Racing Form wrote about horse racing. Hatton was often quoted as using the term "triple crown" to refer to the three most prestigious horse races of the American season, the Kentucky Derby, the Preakness Stakes, and the Belmont Stakes. This term grew in popularity as more owners prepared for these races, and in 1950, the Triple Crown was officially recognized in New York, with Sir Barton named as the first winner of the Triple Crown. The term quickly became a reality, and subsequent winners were honored at annual dinners.
Though the Triple Crown has been in existence since the late 1870s, the term was not widely used until the 1930s. It was first used to describe the Triple Crown, which is a race in which the winner receives a special trophy. The term was adopted to echo England's Triple Crown. The current Triple Crown drought surpasses the 25-year drought that lasted from 1948 to 1973, when the first Triple Crown winner was Seattle Slew.
Famous races that require horses to jump over fences
There are a number of famous horse races that require horses to jump over fence. The Cheltenham Gold Cup is a popular example. The Gold Cup has a prestigious prize purse and is considered the pinnacle of National Hunt racing. This race tests a horse's ability to run at a true gallop, jump over fences, and maintain stamina. It also includes a hill at the end of the race. It also requires jockey skill to ride a horse to the finish line.
Point-to-point races are popular among amateur race horses. They are usually run over two to four miles and can stretch up to 7.2 kilometers in new money. The races feature many different types of obstacles and at least eight of these obstacles. These races also help young horses develop their jumping skills and experience. Some of the most famous hurdlers of all time have come from the point-to-point division.
Betting system for horse races
If you have been betting on horses for many years, you may have heard about the best horse betting systems. But which ones really work? There are several reasons to backtest your horse race betting system. For one, you want to be sure that it produces the results you want, right? It's important to know the average price of winners and qualifiers in the race, as well as the strike rate. Moreover, you need to be patient, since it takes time to develop a winning system.
One of the most basic betting systems for horse races is to bet on the horses that are more likely to win. When you choose a horse that the racebook believes is less likely to win, you can still make money. The reason for this is that the odds of the horse are higher than the odds you're receiving from fixed odds sportsbooks. You can exploit this to your advantage and win big! So, how do you start a betting system for horse races?


Clickbank is one of the largest marketplaces for digital products. It acts like a bridge between digital creators or vendors and affiliate marketers which then promote the products to customers.
This means that people go there to sell, buy and promote pretty much any product as long as it is digital. Examples of products are usually e-books and software programs.
The first step to getting started with Clickbank is setting up an account. At this stage you need to know whether you want to promote other products or sell your own. If you want to buy products you don't need an account.
Setting up the account is fast and easy. Now, depending on the account type, you can start promoting or selling products.
Clickbank – How to buy?
The company has made buying surprisingly easy. The customer only has to select the product click pay and then select the payment method. The most common and simple is through Paypal. The process takes a couple of minutes at most and after purchase you get the product immediately.
Clickbank – How to sell?
After you set up your seller account, things are pretty easy when it comes to selling your products. Clickbank has given sellers all the tools needed to effectively sell their products. All they have to do is create their e-product and input it in the Clickbank selling process. That's pretty much it. Now Clickbank's own affiliate network will start finding the product and promote it. Depending on how good the product is, it will attract more promoters. Of course no one is stopping you from promoting it yourself if you want to, but the fact that there is already an established affiliate network at your disposal willing to promote it for you for a share simply mean you have enough time to start developing other products.
Clickbank offers a wide array of statistics to help you in your selling process. You can use it to optimize your product and ultimately increase your sales.
Clickbank – How to promote?
Promoting is very easy. Once you enter your account, all you have to do is browse the marketplace and find a product you like and then create the hop link. This link will be used by the affiliate marketer to promote the product. Once someone clicks it and then buys something, the promoter gets a percentage of the product price.
Getting started with Clickbank is very easy and is potentially the first step in building a successful affiliate marketing business.


text:
Q: Limit of $\frac{y^4\sin(x)}{x^2+y^4}$ when $(x,y) \to (0,0)$ I have to find the following limit when $x,y$ tend towards $0$. I think the limit doesn't exist (thanks to Wolfram Alpha), but all I can find no matter what path I use (I've tried $y=x², x=y^2, x=0, y=0$) is that the limit equals $0$ (because of the $sin(0)$). Any tips?
$$\frac{y^4\sin(x)}{x^2+y^4}$$

A: Using AM-GM then we get
$$\frac{x^2+y^4}{2}\geq |x|y^2$$ so $$\frac{y^4|x|}{x^2+y^4}\le \frac{y^2}{2}$$ and this tends to zero if $y$ tends to zero.
We also have used that $$|\sin(x)|\le 1$$

A: Let $|x|,|y| <1$,  $x,y$ real.
$0\le \left | \dfrac{y^4 \sin x }{x^2+y^4} \right | \le $
$\dfrac{y^4|x|}{x^4 + y^4} \le$
$\dfrac{(y^4+x^4)|x|}{x^4+ y^4}= |x| \le \sqrt{x^2+y^2}.$
Choose $\delta =\epsilon.$
Used : $|\sin x| \le |x|$

A: We have that
$$\frac{y^4\sin x}{x^2+y^4}=\frac{\sin x}{x}\frac{y^4x}{x^2+y^4}$$
then recall that $\frac{\sin x}{x}\to 1$.
To show that $\frac{y^4x}{x^2+y^4} \to 0$ we can use inequalities or as an alternative by $u=x$ and $v=y^2$
$$\frac{y^4x}{x^2+y^4}=\frac{uv^2}{u^2+v^2}=r\cos \theta\sin^2 \theta \to 0$$

redpajama_set_name: RedPajamaStackExchange


There is a connection between the broad decline of trust and the growing faith in internet messages that confirm our biases, and this has become a global trend.
The United States is no longer the only big kid on the geopolitical block, but has an opportunity to lock in its role as the world's pivotal power–still with a better hand to play than any of its rivals.
Both Beijing and Washington are contributing to the serious decline in U.S.-China relations under way today. Any true understanding of the relationship must accurately assess what is happening in both capitals.


For our clothing line, we source all our fabrics from a mill outside of Osaka, Japan. The mill has been weaving and making old school selvedge denim and linen for years. In the weaving process our mill uses almost no chemicals. Most of the processing is done through the use of soap and water allowing the natural material to go unaltered. This old school method increases our linen's overall quality and longevity. We chose to work with linen because it is one of the most sustainable fibers to produce and one of the hardest working fabrics. Linen is 100% biodegradable and hypoallergenic. It is also created from flax plants which require very little water or maintenance to grow.
The small amount of cotton in our linen blend allows the fabric to have a softer feel, more matte finish, and helps to mediate the wrinkles that are often associated with linen.
Hand or machine wash on gentle cycle with cold water. Tumble dry low or let air dry. We pre-wash all our clothing so it won't change size as you wash and wear them. You also do not need to wash your linen garments as much as your other cotton clothing. Linen contains a resin called Lignin that not only soothes the skin but is anti-microbial eliminating perspiration and odors. That means you can wear your garment longer without sweat stains or any odors from use.
Our fabric is made of 100% natural materials so it will become smoother and mold to your body overtime. Wear our linen rumbled for a natural look or smooth out with a medium hot iron for crisp clean lines. Be careful not to overheat your iron as the heat can break down the natural fibers causing them to become brittle.


text:
orderedset
==========

.. automodule:: ctmatching.orderedset
    :members:


Businesses are required to submit W2s to the SCDOR by January 31. Uploading W2s on MyDORWAY is the SCDOR's preferred method for W2 submission. MyDORWAY is free, secure, and available 24/7.
Choose your submission method: Select the method below that best suits your business, regardless of the number of employees you have. You can save your application after each W2 and return later. If you choose to upload a file, you can submit as many times as needed to achieve an error free submission.
Federal Identification (FEI) Number for the business.
The following information for each employee: Social Security Number (SSN), first name, middle initial, last name, suffix, location address (attention, suite, room number, etc.), delivery address, city, state abbreviation, zip code, zip code extension, employee's Income Tax reporting, employee's wages, tips, other compensation (dollars and cents), employee's state taxable wages (dollars and cents), and the employee's state Income Tax withheld (dollars and cents).
W2s are due to employees, the IRS/SAA, and the SCDOR annually by January 31.
If you submit W2s on MyDORWAY you do not need to file a WH1612 Transmittal Form by paper.
You can submit return information for multiple customers in a single file, which is beneficial for tax professionals and payroll companies who file on behalf of multiple clients. The service is also a great option for small business owners who prepare return(s) using tax preparation software.
W2s should be submitted for a shorter term if the business has opened or closed during the year.
If you change payroll providers during the year, only submit one filing at the end of the year. Incomplete filings will cause errors in the information provided for the business.


At WastePages, we strive to bring you the best service at the best price available. We have a wide selection of dumpster sizes in stock in Keo, AR, with flexible & timley pickup and delivery. Give us a call today for all your dumpster rental and roll off needs.
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The weather outlook may be with rain as well as snow and you also need to find the roof done within the next week. Thankfully you can find your materials quickly such as the shingles with the roofing along with a dumpster the next day.
Do-it-yourself or Home rehabilitation Projects: Spin Off dumpsters are a safe along with convenient technique to collect dust from any home remodeling or do-it-yourself venture. Collect raw wood or timber scraps, microsoft windows, drywall, flooring surfaces, old gates, fixtures, pipe joints, siding or another type of building or demolition dust. The items can be placed safely within a dumpster without having to stress about anyone as well as anything receiving injured through old rustic nails, sharpened metal edges or splintered timber.
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It is advisable to rent a dumpster along with wheels on so you can transfer it from your house to this barn to the garage area if needed.


text:
Things are calmer than they were in the book business, but change is a constant
May 25, 2016 by Mike Shatzkin 12 Comments
Among the shifts that have been taking place in publishing houses over the past decade is an increase in the head count dedicated to marketing and a decrease in head count dedicated to sales. This reflects the reduction in the number of bookstore accounts and the transfer of "discovery" from store shelves to digital search.
The reduction in bookstores and the concurrent and related reduction in print books sold in stores also affects how publishers view the economics of the sales departments and the entire support system for print distribution. The big houses still need sales forces and warehouses and sophisticated systems to track inventories and payments and returns but the "throughput" of print from their own publishing programs is declining. For many, that means that distribution clients are increasingly important. They provide the volume to support scaled operations without requiring the publisher to invest in publishing more titles. For at least four of the big five (HarperCollins being an apparent exception), distribution of other publishers' books, with or without providing the sales force effort, is a critical component of maintaining the volume that keeps unit costs in line.
But that adds risk. Distribution contracts vary in length, but they generally only extend two or three years out. With four major publishers plus Ingram, which has, effectively, five different full distribution options to offer, on the prowl for clients, there is a plethora of choices for any publisher seeking to shed their own fixed-cost distribution or to switch distributors. Indeed, the percentages being charged for distribution services have dropped drastically over the past two decades. The competitive environment is likely to perpetuate that trend.
While the big publishers doing distribution have (so far) tended to insist on fairly large clients, Ingram is using its multiple configurations to try to serve publishers of all sizes and entities that aren't primarily publishers at all. Today a publisher that is really a literary agency or, before long if not already, a bank, an advertising agency, or a not-for-profit with a mission, can put a book or a list of its own into the book publishing arena with sales and distribution capabilities competitive with the biggest and most experienced publishers. So a revolution that began with Amazon enabling indie authors, starting about ten years ago, to reach a big percentage of the total book market through Kindle and CreateSpace, is being dramatically extended. Going after real bookstore distribution definitely requires incremental investment and marketing savvy, even with the machinery in place to help.
But incremental investment and marketing savvy were always far easier to come by than the machinery has ever been for the small or occasional publisher.
While this levels the playing field in a major way, there are still distinct advantages to size and a B2B publishing brand. The diminishing bookstore shelf space has made the also-diminishing mass merchant (Walmart, Target) shelf space relatively more important. Between the chains — primarily Barnes & Noble and Books-a-Million — and independent stores, there are only about 1000 to 1200 points of purchase for books provided by bookstores. There were three to five times that many two decades ago. So the additional thousands of opportunities to put a book in front of the public through the mass merchants are critical, particularly to move bestseller quantities.
But relatively few titles can make the cut for those outlets and the pressure on them to perform quickly is immense. Returns are high. These slots are simply not available to publishers who aren't recognizable B2B brands with a solid reputation for backing their books effectively. These outlets represent the competitive advantage that remains for the Big Five publishers.
For the past few years, pretty much since the demise of Borders in 2011, the number of bookstores has been going up a bit each year. (It is not clear that the bookstore shelf space has been going up; indie stores seem to be smaller, on average, today than they were two decades ago, or at least there are fewer mammoth ones.) It could well be that, aside from Borders, the indie revival is also fueled by the reduction in shelf space for books at the mass merchants. If so, that is good for smaller publishers and it is good for backlist, both of which are seriously challenged getting in front of the public through mass merchants.
So, while it is definitely true that the dizzying pace of change we saw during the early years of ebooks has subsided, and it is true that the print format has not yielded much share, if any, to ebooks in the past couple of years, it is not time to celebrate a new stability. The marketplace itself is still changing; the online share when you combine print and digital is still growing and the ratio of shelf space available for backlist and slower-sellers is still declining. The smallest publishers are getting better and better market access and the biggest publishers are seeing escalating risk in how they place the books they publish and in the danger they'll face a sudden decrease in distribution volume that would turn their fixed costs into a burden.
This is a great time in the book business to be very big (among your peer group) or very small and focused. It is a challenging time to be anything else.
A very frequent point of contention when negotiating distribution arrangements is how Amazon will be handled and compensated. Amazon is almost always the single largest account and it is not uncommon for it to represent — on many books and even some publishers — 50 percent or more of the sales. Although sophistication definitely helps in dealing with Amazon, it is also true that Amazon provides incentives to give up the "other half" of the market and just work through them. Any sophisticated businessperson is likely to get more money out of Amazon working it themselves than any distributor can get for them, even before distribution fees. (IF, and this is a big if, you discount the marketing value of books throughout the supply chain which, counterintuitively but frequently, will raise the level of sales at Amazon from what they would have been without books broadly distributed.) In any case, being able to really add value to Amazon sales would be a Holy Grail. Right now, most of the time, distributing publishers really have to make the argument that you can't effectively split things and that they will add so much value in the rest of the world, and do the work around Amazon, that the overall relationship is worth the trade-off.
Filed Under: Atomization, eBooks, General Trade Publishing, Marketing, New Models, Scale, Self-Publishing, Supply-Chain, Unbundling Tagged With: Barnes & Noble, Books-a-Million, Borders, CreateSpace, HarperCollins, Ingram, Kindle


text:
— японская рок-группа из города Вакканай на Хоккайдо.

История

Galileo Galilei была образована в 2007 году. В 2008 году группа выиграла гран-при музыкального фестиваля
 и начала свою серьёзную музыкальную карьеру. 21 января 2009 года вышел первый альбом . В 2012 году Фумито Иваи и Кадзумаса Ногути покинули Galileo Galilei и в 2013 году сформировали группу FOLKS.

Участники
 Нынешние
  — вокал, гитара, автор песен.
  — бас-гитарист.
  — ударные, бэк-вокал. Младший брат вокалиста Юки Одзаки.
 Бывшие
  — гитара.
  — гитара, бэк-вокал.
  — синтезатор.

Дискография

Альбомы

Мини-альбом

Синглы

Примечания

Ссылки
  Galileo Galilei  профиль на SonyMusic
  Galileo Galilei  на сайте AniDB

Музыкальные коллективы, появившиеся в 2007 году
Рок-группы Японии
Инди-рок-группы
Инди-поп-группы

redpajama_set_name: RedPajamaWikipedia


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I want me some personalized m&ms. I don't know why but I do. Assorted colors, the regular array, not the pastels and not the stupid wedding bland colors. What to put on them, though? I think you can have 2 lines of up to 8 characters each.
How about "Kizz" or better, "Kizz!"
If I could get a variety I think I'd put the names of all three of my animals on them. That would be funny. No, actually it wouldn't, it would be boring and catapult me into crazy cat lady status.
If I could have a huge variety I'd just put inspirational words on them like: write, act, click, breathe, believe, just be, love, live, leap, twirl. That would be fun. I would really like to eat those because it would be like ingesting inspiration.
What would you put on your m&ms? You know, if they weren't so freaking expensive.
I would put Russell Crowe.
And I would chow down.
I love me some M&M's!
You beat me to it. I was going to say "Eat Me." What about emoticons? The old ascii character ones, not the new smilies.
one ofmy work girls had a baby and they gave away m&m's with the new baby name on it to guests at the hospital!


On my old Toshiba Satellite A100 Antergos crashes with the subject message above after downloading all the packages for a kde install… help!
But I want it I want it I want it!
Just something I've discovered during install from Australia at least. The repos aren't sorted by speed of download before CNCHI starts downloading packages. Our internet speeds down here aren't fantastic so every little bit of tweaking counts.
Anyone else found this to be an issue?
Then it installs much faster as the closest sources are being tried first.
So perhaps after the country location is chosen reflector could run on these parameters before the install begins?
Other then that it's an awesome way to get the sleekest linux distro without the pain of wiki wasting a day away.
Just a little something I found out when trying to install Antergos on my old Toshiba A100 using a Geforce Go 7300 IGA , it trys to use the most recent nvidia driver which of course doesn't work. Solution is to uninstall the current nvidia driver + mesa and a few of it's dependencies after installing Antergos and going to the terminal (ctrl + alt + f2). Or chrooting into it with the disc.
I'm sorry I don't have exact details as I did it a few months ago.
Perhaps this could be adjusted in the install script for Antergos to deal with older hardware?
I think I was using a copy of Antergos I'd burnt several months ago.


Green Party Animal Welfare spokesperson Keith Taylor MEP has welcomed his colleagues "overwhelming support" on a vote to improve the welfare standards of chickens farmed for meat across the EU.
The vote comes just days after a new report from AgriBusiness Consulting found that intensive broiler farming contributes to the current increase in antimicrobial resistance and causes environmental degradation, while also being intrinsically linked to poor animal welfare.
The report added evidence to claims from animal rights advocates that EU legislation on broiler welfare fails to guarantee for the welfare of broiler chickens.
In response to the report, Keith Taylor MEP pushed for a resolution to address the issues identified. The resolution, supported by 409 MEPs and rejected by just 19, tackles the practice of breeding chickens to grow at unnaturally fast rates, concerns about 'stocking density', and the need to end the overmedication of chickens with medicines vital for human health.


Generous, virtuous, fair, and brave, Yet he fills a drunkard's grave.
A tireless student, pensive, pore O'er hist'ry's page, or some noble theme, That poets have sung in classic lore.
I saw an old man, whose locks were grey, Silver'd by care and the length of years; Unmoved by these signs of speedy decay, And by his children's frequent tears.
The young, the old, and the brave are there, The proud and the humble together sleep ; The father, caught by intemperance' snare; And his son, who once could o'er him weep. The rich�the poor�the free�the slave, Go alike to the drunkard's grave.
T HEY say the goblet's crown'd with flowers, And round its brim do brightly shine, Like gems, remember'd joys and hours, The treasures of immortal wine.


France's parliament has pledged to crack down on a national epidemic of food waste by passing a law banning supermarkets from destroying unsold food. They will instead be required to give all unsold food to charities or put it to other uses such as animal feed.
The national assembly voted unanimously on Thursday evening in favor of the measure, proposed by the Socialist deputy Guillaume Garot, a former food minister. "It's scandalous to see bleach being poured into supermarket dustbins along with edible foods," he said.
The law explicitly bans the practice of supermarkets deliberately spoiling unsold food so it cannot be eaten. Bigger supermarkets will be obliged to sign formal contracts with charities by July next year, or face penalties including fines of up to €75,000 (£53,000) or two years in jail.
The measures are part of wider official efforts to halve the amount of food waste in the country by 2025. According to official estimates, the average French person throws out 20-30kg of food a year, at a combined national cost of up to €20bn.
The law will also introduce an education program about food waste in schools and businesses. It follows a measure in February to remove the best-before date on fresh foods.


text:
Q: How are these "supplementary" or "satellite" Helmholtz coils used? Three mutually orthogonal pairs of Helmholtz coils can be used to produce a fairly uniform magnetic field of arbitrary direction within a certain volume, and/or to null/cancel an external field (e.g. Earth's) to make a nearly field-free region.
In this answer to my question Why is there a large wooden ball on Mariner 3's magnetometer? I link to the NASA video MAVEN Magnetometer which includes some clips of the MAVEN spacecraft's three-axis fluxgate magnetometer being tested in a large workspace surrounded by the standard three pairs of Helmholtz coils.
However, when I look closely I can see another six coils, smaller in diameter and at larger distances. I've annotated a screen shot below to make this clearer.
Is there a name for this 12-coil configuration? Do the extra coils improve the uniformity within the center, or extend the useful size of the uniform area, or both, or neither?
If possible a link to the math is appreciated, but mostly I'd like to know if this is an established technique and what it's called.
I've done my best to indicate the primary three pairs and at least some of the secondary coils separately. I think one or two have been removed for access - so things can be wheeled in and out of the test volume.

A: This arrangement of four coils per axis is called a Braunbek coil, a modified and improved Helmholtz coil developed in 1934 by Werner Braunbek.
Each of the four coils has the same number of windings and the coils are connected in series, the same current flowing through all four coils.

The Braunbek configuration chosen for the coil system affords a large
volume of  homogeneous field relative to its physical dimensions,
provides easy access to  the work space without the need for moving
and repositioning any coil loops,  and assures the simplest circuit
for maintaining a precise relationship among loop currents since all
loops on any given axis are connected in series.
Each of the  three orthogonal coil sets has two large inner coil loops
and two smaller outer  coil loops.  The orthogonal coil loops differ
slightly in diameter to avoid physical  interference. Each coil set
has seven separate windings differing in function,  number of turns,
and wire size.

Picture and quotes from this NASA paper about the

Goddard Space Flight Center Spacecraft Magnetic Test Facility (SMTF)
was constructed in the 1960's for the purpose of simulating
geomagnetic  and interplanetary magnetic field environments.
The facility includes a three axis Braunbek coil system consisting of
12 loops, 4 loops on each of the three orthogonal axes; a remote Earth
field sensing magnetometer and servo controller; and a remote power
control and instrumentation building. The inner coils of the Braunbek
system are 42-foot in diameter with a 10-foot by 10-foot opening
through the outer coils to accommodate spacecraft access into the test
volume. The physical size and precision of the facility are matched by
only two other such facilities in the world.
The facility was used
extensively from the late 1960's until the early 1990's when the
requirement for spacecraft level testing diminished. New NASA missions
planned under the Living with a Star, Solar Terrestrial Probes,
Explorer, and New Millennium Programs include precision,
high-resolution magnetometers to obtain magnetic field data that is
critical to fulfilling their scientific mission.
It is highly likely
that future Lunar and Martian exploration missions will also use
precision magnetometers to conduct geophysical magnetic surveys. To
ensure the success of these missions, ground testing using a magnetic
test facility such as the GSFC SMTF will be required.


The NFL has warned all 32 teams stating its displeasure with the release of contract terms before free agency begins Tuesday.
"The memo makes clear that we will look into any potential violations of the rules," NFL spokesman Greg Aiello said Monday.
The NFL sent its memo after news leaked, beginning Sunday, of several agreements between high-profile free agents and teams, including Ndamukong Suh with the Dolphins, Byron Maxwell and Frank Gore with the Eagles and Jeremy Maclin with the Chiefs.
NFL free agents are permitted to officially sign with other teams Tuesday at 4 p.m. ET, but a three-day negotiating period began Saturday during which teams could start exchanging written summaries of their pitches to agents for players on other teams. Teams can negotiate based on an agent's response but are not permitted to describe their pitches as offers.
Teams also are not permitted to agree to contracts with players on other teams during the negotiating period.


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It's no secret that I'm an avid mobile shooter on a near-daily basis. While I continue to reach for my Nikon D3X and a sling bag of lenses for my documentary assignments, I use my iPhone camera to document and share my own life. I might capture spontaneous expressions or curious moments inspired by my children, create abstractions that move me, reveal my feelings through found objects or simply pause experiences I want to remember. Because my iPhone is always with me, unobtrusive to my subjects and easy to use, I reach for it instinctively when I feel the desire to quickly preserve a moment, no matter where I am.
Several years ago, before the first photography app was available in the App store, I made a commitment to myself to complete Project 365, a Flickr-inspired photography project that required me to shoot and select one image to represent each day consecutively for 365 days. At the time, I was seeking an area of focus as a professional photographer and felt that the freedom of subject matter selection coupled with the discipline of shooting on a daily basis would guide me down the path.
Determined—and inspired—I hauled my first DSLR, a Nikon D80, and a lens or two in a backpack throughout each day. While carrying the camera and shooting daily wasn't always convenient, it prompted me to master that camera and its settings in a variety of environments. It also uncovered my love of documentary as an area of focus. And perhaps, most importantly, it taught me to move through each day with my eyes open and ready to receive images. On day 366, I remember thinking, "I can't stop."
Using my iPhone and a collection of photography apps, I now shoot, process, publish, share and exchange feedback on my personal photographs, the documentary of my own life, with a community of viewers around the world. It fulfills me in a way that shooting daily with my DSLR didn't because of the powerful community aspect of mobile photography.
With my iPhone and the Instagram app, I can experience life through the eyes of mobile photographers I follow all over the world. As I stand in line at the grocery story, I can visit New York City through the eyes of Sion Fullana (@sionfullana) and Anton Kawasaki (@anton_in_nyc). Leaning on the kitchen counter as I wait for the water to boil, I might travel to Singapore with AikBeng Chia (@aikbengchia) or follow David Alan Harvey (@davidalanharvey) on the road in Rio. While I'm curled up on the sofa, I can check in on my Shutter Sisters, including Tracey Clark (@traceyclark), Jen Lemen (@jenlemen), Meredith Winn (@camershymomma) and Kate Inglis (@sweetsalty). Interestingly, as a follower of avid mobile photographers, I'm finding that I feel less attached to a single photograph and more attached to the photographers who choose to express themselves through visual documentary of their own lives.
The mobile experience of processing my personal photographs in real time, titling them in intriguing ways and sharing them with my viewers in Instagram and Twitter on my iPhone, has become almost as appealing as the act of shooting. Sharing my personal documentary regularly online has been a way for me to gradually open myself up and experiment with photography as a form of expression—to learn what it feels like to expose who I am, what I see and how I feel. It has helped me gain perspective on what it feels like to be the subject of a documentary study, a practice of letting my guard down and getting comfortable with a certain amount of exposure and vulnerability. Consequently, it has helped me gain a new level of appreciation and sensitivity for my subjects, and keeps me focused in the present moment.


text:
Булзештій-де-Жос () — село у повіті Хунедоара в Румунії. Входить до складу комуни Булзештій-де-Сус.

Село розташоване на відстані 331 км на північний захід від Бухареста, 45 км на північ від Деви, 86 км на південний захід від Клуж-Напоки, 129 км на північний схід від Тімішоари.

Населення
За даними перепису населення 2002 року у селі проживали  осіб, усі — румуни. Усі жителі села рідною мовою назвали румунську.

Примітки

Села повіту Хунедоара


Engage Victoria is a digital way for the public to participate and to have a say in the development of government policies and programs.
The Family Violence Reform Implementation Monitor holds government and its agencies to account for implementing the family violence reform, following the Royal Commission into Family Violence.
Details of the Victorian Government's implementation plan in response to the official Hazelwood Mine Inquiry 2014-16.
Victoria's China Strategy set four ambitious 10-year targets from 2016 to 2026, across trade, investment, visitors and students.
The Victorian Government's strategy for developing our relationship with India over years 2018 to 2028.
By working together, we can create a safe and resilient Victoria to resist, respond and recover from acts of terrorism.
Read about what's being done to make Melbourne safer, including bollards and other protections, a public address system and upgraded CCTV.


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BECOME A MEMBER Choose your Membership level - Click Here
Home / The Legend / The Man
Robert Redd is rumored to have gone in search of adventure. Through the recently uncovered memoirs it is revealed that Redd may, in fact, have encountered many a fine lady in China, India, Egypt, England, France and across the roaring high seas in South America- to name a few locations. Without divulging too many details (for the study of these materials has only just begun, and admittedly the dates seem a bit…shifty), it might also be said that Redd found inspiration with the most notorious French Madams of Europe. There are other perspectives to be considered as well; from Lady Redd his immortal beloved to Wasser his trusty Valet. They are all represented in these manuscripts and therein lies the challenge to determine the true Legend of Robert Redd™.
Of the man and the Legend this is what is known… on a hazy summer afternoon when a striking ray of sun was falling just so across a rather dashing self-portrait of Robert Redd, it was noticed that the century-old painting that hung in the library of Robert Redd's home had lost its luster to dust and smudging. The once cool hazel-blue of his eyes now peered out through a droll gray, and the white shirt collar around his neck was yellowed.
"Robert Redd could use a little restoration," it was declared. In the hopes of recalling the buoyant sparkle to Robert Redd's façade, the large painting was taken down with great care. To the surprise of many, a small door was present in the wall behind the painting, and tucked away inside were a variety of accoutrements, comprised of various trinkets, documents, valuables, maps and Redd's insightful journals, spanning over a century of experiences, adventures and evolution. Of the leather-bound notebooks filled with the above-par penmanship of Robert Redd, he seems to have written his memoirs in no particular order, occasionally scribbling provocative words and phrases in the margins, and carefully drawing out the events of other days in meandering, lavishly detailed accounts. The incomparable happenings of Redd's life are only now being properly strung together.
Click the right arrow below to learn more about the Sticky Situations Redd found himself in.
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text:
'Cruz' Missile Absolutely Obliterates Hosts Of 'The View' Over Hypocrisy Of Abrams, Clinton Claiming Illegitimate Elections
By michael October 24, 2022
If there's one thing we've come to know and love about Texas Republican Sen. Ted Cruz, it's that he's not only a walking Constitution (he's memorized most, if not all, of the document), he's the kind of guy who will ask the hard-hitting questions and call the radical left out on their hypocrisy. We are seeing more and more folks in this country taking this kind of approach as they are sick and tired of watching their nation being ripped apart by radical leftists.
Cruz recently made an appearance on the super left-wing program, "The View," where he called out the co-hosts of the show for their hypocrisy in being completely okay with both Hillary Clinton and Stacy Abrams claiming their elections were illegitimate, but turning around and accusing former President Donald Trump of being an insurrectionist and plotting a coup for daring to say the same thing.
"[Hillary Clinton and Stacey Abrams] sat here and said [their elections] were illegitimate and you guys were fine with it," the Texas senator said.
Whoopi Goldberg then responded by saying, "And it was!"
Cruz fired back, "So it's illegitimate when Republicans win, but not Democrats?"
Cruz: "[Hillary Clinton and Stacey Abrams] sat here and said [their elections] were illegitimate and you guys were fine with it."
Whoopi: "And it was!"
Cruz: "So it's illegitimate when Republicans win but not Democrats?" pic.twitter.com/KgdBKitEv6
— Greg Price (@greg_price11) October 24, 2022
For those of you who might not remember, back in 2016, during the presidential election showdown between then-candidate Donald Trump and former secretary of state Hillary Clinton, the latter lost and then claimed that the reason she was defeated was because there was collusion between Trump's campaign and Russia.
As we all know by now, thanks to years of investigations, there's no evidence whatsoever to suggest this was indeed the case. In fact, it's quite the opposite. We have tons of information that points to the Steele Dossier, the document Hillary used to bring about some sort of "evidence" for her claims of collusion, being completely fabricated.
And then, of course, there's Abrams, who refused to concede her loss to Brian Kemp several years back. She has claimed on numerous occasions that she didn't lose fair and square, but that suppressive election tactics were employed by her opponent to prevent her from cinching a victory.
Apparently, it's perfectly acceptable for these two to make such claims and even file lawsuits based on them, but for Trump to take any sort of action like that is blasted as an attempt to overthrow the current process. It's funny how there's one standard of conduct that the Democratic Party has for itself and another they use for Republicans.
Apparently, according to The New York Post, this wasn't the only part of the program that featured fireworks.
During the episode on Monday, a bunch of climate protesters hijacked the live taping of the show, shouting over the interview with Cruz, chanting, "Cover climate now!"
Cruz tried to just talk over the chants until one woman screamed, "F*** you Ted Cruz!"
This last line was all Whoopi could take.
"Ladies, ladies! Excuse us! Let us do our jobs," Goldberg fired back at the protesters. "We hear what you have to say, but you gotta go! You gotta go, you gotta let us do our job."
a climate protest?? just interrupted the view's interview with Ted Cruz 🥴 pic.twitter.com/aa6n9bEPwF
— Déjà The View (@dejatheviewpod) October 24, 2022
Co-host Ana Navarro then took an opportunity to issue an apology to the Texas senator, saying: "I've been very vocal and critical of you, but I'm sorry that this has happened in our house."
Protesters in the audience of The View reportedly shout "F*ck you" at Ted Cruz in the middle of the show.
pic.twitter.com/XtvClZGW18
— Benny Johnson (@bennyjohnson) October 24, 2022


text:
Q: SaaS Multitenant Architecture i just arrived on this architecture, am doing a lot of research and i understood how it work in general but it's all theorical.
I decided to separate each step for the development of this architecture to start implementing so i can understand better these steps.
The first that i wanted to learn was the tenant provisioning, i wanted to apply it on AWS to mirror a production software example.
So, starting on that the common AWS service that i see most people using is AWS Cognito, but it's not clear in my mind the steps of the implementation, like how should i get the tenant data to onboard him in my app? Assuming it's tier based.
Should i have one database to store all tenants data separate from the application database?
I want to use microservices on this one because i think is better to onboard the tenant with different tiers and much more benefits.
Which AWS services should i use to make this process work? I'm not really asking about the implementation itself but a path to understand which services to use and how it connects with each other.
I hope i was clear about my doubts, english is not my mother tongue, sorry about that!

A: You are thinking in the right direction. However, there are decisions you need to make before diving into any saas service stack. I would start with

*

*Planning my infrastructure - how many tenants/group.

*the kind of tenant onboarding system you want

*How will tenants onboard their users and manage authorization/authentication

*Multitenant architecture, which needs to account for several things at the least like - DB model, shared vs isolated, data privacy, design keeping in mind industry data security standards

*what will be your tenant deployment model. Remember one of the disadvantages of multitenancy is also slow time to market.

*Your API stack needs to account for which apis needs to be multitenant and which are generic product offerings.

*operational tool to monitor app health, client analytics.

*how will you meter and bill the client and other non-functional decisions.

AWS offers good documentation to get started here : https://aws.amazon.com/blogs/apn/building-a-multi-tenant-saas-solution-using-aws-serverless-services/