The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described.
The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration.
All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains.
These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.
Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease characterized by reproductive failures in sows and respiratory syndromes in pigs of all ages . Since its first outbreak in the United States and Canada in 1987, the disease has been causing heavy economic losses to the pig industry worldwide ,. Although both inactivated and live-attenuated vaccines are available for PRRS control, these vaccines failed to provide sustainable protection against the disease, against heterogeneous viral strains in particular . Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped positive–sense RNA virus classified within the Arteriviridae family ,. In pigs, the virus targets the cells of monocyte/macrophage lineage ,, causing severe cell death, slow and weak antiviral responses, and/or persistent infections. In addition, PRRSV uses additional evasion strategies to escape the host innate and acquired immunity, including interference with antigen presentation, antibody-mediated infection enhancement, reduced cell surface expression of the viral proteins and shielding of the neutralizing epitopes. As a consequence, new PRRS vaccine development faces great challenges since they suffer from the immune evasion strategies of the virus and the highly antigenic heterogeneity of field viral strains .
PRRSV enters the target cells by receptor-mediated endocytosis . To date, at least three PRRSV receptors have been identified on porcine alveolar macrophages (PAMs), including heparan sulphate as the general attachment factor, sialoadhesin (Sn or CD169) for the viral binding and internalization, and CD163 for the viral genome release . Previous studies have shown that PRRSV infection of PAMs can be blocked partially by the Sn- or CD163-specific antibody or completely by a combination of two antibodies , and that adenoviral (Ad) vector-delivered soluble Sn and CD163 receptors have an additive effect against PRRSV infection . These data suggest that the two viral receptors are the useful targets for designing new strategies for PRRS control.
RNA interference (RNAi) is a post-transcriptional gene silencing mechanism conserved in eukaryotes ranging from worms to humans . Since its discovery in 1994 as an innate antiviral mechanism, RNAi has become a feasible strategy against a variety of viral infections . Two types of small RNAs, namely small interfering RNAs (siRNAs) and microRNAs (miRNAs), are the central players in RNAi process, both of which inhibit gene expression by binding to the target RNA molecules . A recent study has shown that viral vector-expressed artificial miRNAs (amiRNAs) are more effective than the conventional short hairpin RNA (shRNA) strategy ,. Among the viral vectors available, Ads have been used extensively as the gene transfer vectors for gene therapy and vaccine development with several advantages, including efficient gene delivery, transduction of both dividing and non-dividing cells, ease of propagation to high titers, and minimal risk of genomic insertional mutagenesis . In addition, Ad vectors have been used to deliver PRRSV-targeted shRNAs in vitro and in vivo . However, PRRSV targets the cells of monocyte/macrophage which are resistant to rAd transduction due to the lack of high affinity Ad receptor . More recently, it has been shown that the exosomes derived from human and mouse cells can serve as an efficient small RNA transfer vehicle . Therefore, we hypothesized that the co-delivery of Sn- and CD163-targeted amiRNAs by rAds and exosomes could become a novel strategy against PRRSV infection.
To test the above hypothesis, in this study we predicted the candidate miRNAs targeting Sn or CD163 receptor and validated them experimentally using a reporter assay. Two rAds expressing the effective amiRNAs were generated for further study. Cell transduction assays showed that the sequence-specific amiRNAs were expressed efficiently in and secreted from rAd-transduced pig cells via exosomes. In primary PAMs, the expression of two viral receptors was inhibited significantly by transduction with the amiRNA-expressing rAd and/or treatment with the amiRNA-containing exosomes. Furthermore, PRRSV infection of PAMs was inhibited significantly by transduction with the two amiRNA-expressing rAds and/or treatment with the two amiRNA-containing exosomes. These results supported the hypothesis that simultaneous knock-down of Sn and CD163 receptors may become a novel strategy against PRRSV infection. In addition, our findings suggest that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells. To our knowledge, this is the first study to explore an amiRNA strategy against PRRSV infection by targeting the two viral receptors.
Computational prediction represents an effective strategy for identification of the candidate siRNAs or miRNAs that can be validated experimentally. We analyzed the first 1751-nt sequence of porcine Sn mRNA and the 1511-nt sequence of porcine CD163 mRNA for candidate miRNAs. Ten top-scoring miRNA sequences for each target were reported and 18 of them (Table 1) were selected for amiRNA vector construction (Figure 1A). To facilitate the candidate miRNA validation, the first 1751-bp sequence of the Sn cDNA or the 1511-bp sequence of the CD163 cDNA was cloned in frame with the green fluorescent protein (GFP) coding sequence in pEGFP-N1 vector to produce the reporter vector pSn-GFP or pCD163-GFP (Figure 1B). NIH 3 T3 cells were co-transfected with each amiRNA expression vector and the reporter vector, and the cell culture was assayed for GFP+ cells by flow cytometry. By using pSn-GFP-transfected cells as the reference, transfection with each amiRSn expression vector plus the reporter vector resulted in GFP+ cell number reductions ranging from 39.6% to 96.3% (Figure 1C). Similarly, transfection with each amiRCD163 expression vector plus the reporter vector led to GFP+ cell number reductions ranging from 53.3% to 88.5% (Figure 1D). The most effective amiRSn-2 and amiRCD163-2, as well as an irrelevant amiRcon (Table 1), were selected for further study.
The candidate amiRNAs were predicted by following the instructions for the web-based Block-iTTM RNAi designer. The mature amiRNA and forward primer sequences for the detection of amiRNAs in rAd-transduced cells are indicated as italics.
Validation of Sn and CD163 receptor-targeted amiRNAs. (A) The schematic structure of amiRNA expression vector. PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; TK pA, TK gene poly(A) signal of human simplex herpes virus. (B) The schematic structure of the reporter vectors for amiRNA validation. PCMV, immediate early promoter of cytomegalovirus; Sn/CD163, porcine Sn or CD163 receptor cDNA; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (C or D) NIH 3 T3 cells were transfected with different amiRNA expression vector plus the reporter vector, and the GFP-positive cell numbers were measured by flow cytometry 24 h after transfection. The know-down efficiency of each amiRNA was expressed as the percent inhibition of total fluorescence in the cell culture co-transfected with the amiRNA expression vector and the reporter vector.
We subcloned amiRSn-2, amiR163-2 or amiRcon expression cassette into Ad vector pShuttle-IRES-hrGFP, and three rAds, namely rAd-amiRSn, rAd-amiR163 and rAd-amiRcon, were generated by transfecting AAV-293 cells with the rAd vectors (Figure 2A). Primary PAMs and PK-15 cells were transduced with different doses of rAds, and the cell cultures were analyzed for GFP+ cells since the GFP reporter gene was included in the rAd vectors. Compared to 100% transduction efficiency for PK-15 cells at MOI 100 (Figure 2B), only 62% transduction efficiency was achieved for PAMs at MOI 800 (Figure 2C), which was not increased further by using higher MOI. The different rAd transduction efficiencies for the two pig cell types were confirmed by fluorescent microscopy (Figure 2D). Therefore, the further experiments were carried out at MOI 800 for PAM transduction and MOI 100 for PK-15 cell transduction.
rAd transduction efficiencies for pig cells. (A) The schematic structure of Ad vector for amiRNA expression. ITR, inverted terminal repeat of adenovirus; PCMV, immediate early promoter of cytomegalovirus; miR, miR-flanking sequences of mouse BIC non-coding mRNA; Pre-amiRNA, double–stranded oligonucleotide for amiRNA; IRES, internal ribosome entry sequence; GFP, green fluorescent protein coding sequence; SV40 pA, poly(A) signal of SV40 virus. (B) PK-15 cells and (C) primary PAMs were transduced with different doses of rAd-amiRSn and the GFP-positive cell numbers were measured by flow cytometry 48 h after transduction. (D) Primary PAMs and PK-15 cells were mock-transduced or transduced with each rAd and analyzed by fluorescent microscopy (40×) 48 h after transduction.
To investigate whether the amiRNAs were expressed in rAd-transduced cells, primary PAMs or PK-15 cells were transduced with rAd-amiRSn, rAd-amiRCD163 or rAd-amiRcon, and the total RNA was extracted 48 h after transduction. At the same time, the exosomes were purified from cell culture medium and the total RNA was extracted for sequence-specific amiRNA detection. The expected 80-nt amiRNAs were detected in all of the three rAd-transduced cells (Figure 3A) and their exosomes (Figure 3B), but not in the mock-transduced cells and their exosomes.
Detection of amiRNA expression in the rAd-transduced cells and purified exosomes by RT-PCR. (A) Primary PAMs and PK-15 cells were mock-transduced or transduced with each rAd, and the total RNA was extracted for sequence-specific amiRNA detection 48 h after transduction. (B) The exosomes were purified from the cell culture medium of each rAd-transduced cells and the total RNA was extracted for sequence-specific amiRNA detection 48 h after transduction.
The sufficient knock-down of Sn and CD163 receptors is the precondition for the objective evaluation of anti-PRRSV effects of the receptor-targeted amiRNAs. We compared the knock-down efficiencies of the two viral receptors using three different strategies: rAd transduction, exosome treatment and rAd transduction plus exosome treatment. First, primary PAMs were transduced with rAd-amiRSn, rAd-amiR163 or rAd-amiRcon. After incubation for 48 h, the total RNA was extracted for mRNA detection by quantitative RT-PCR and the cell culture was analyzed for Sn+ or CD163+ cell number by flow cytometry. Compared to that in rAd-amiRcon-transduced cell cultures, Sn or CD163 receptor mRNA expression in the rAd-amiRSn- or rAd-amiRCD163-transduced cells was decreased by 0.51 or 0.54 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased from 48.7% to 22.7% and from 49.9% to 27.6%, respectively (Figure 4B). Next, primary PAMs were incubated with the exosomes (0.5 mg protein/ml) derived from rAd-amiRSn- or rAd-amiRCD163-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for quantitative RT-PCR and cell cultures were harvested for flow cytometry analysis as described. Compared to that in the two control groups, Sn or CD163 mRNA expression in the amiRNA-containing exosome-incubated cells was decreased by 0.41 or 0.46 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased to 33.4% and 36.5%, respectively (Figure 4B). Finally, primary PAMs were transduced first with each rAd, incubated for 48 h with each amiRNA-containing exosomes, the total RNA was extracted for quantitative RT-PCR and the cell cultures were harvested for flow cytometry analysis as described. Compared to that in the two control groups, Sn or CD163 mRNA expression was decreased by 0.73 or 0.73 fold (Figure 4A), while Sn+ and CD163+ cell numbers were decreased to 18.9% and 19.1%, respectively (Figure 4B).
Knock-down of Sn or CD163 receptor by the rAd- and/or exosome-delivered amiRNA. (A) Primary PAMs were transduced with each rAd and/or incubated with each amiRNA-containing exosomes derived from the rAd-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for Sn or CD163 mRNA detection by real-time quantitative RT-PCR. (B) The rAd-transduced and/or exosome-incubated cells were stained with Sn- or CD163-specific antibody and analyzed by flow cytometry.
We used two different quantitative assays to evaluate the additive anti-PRRSV effect between Sn and CD163 receptor-targeted amiRNAs: quantitative RT-PCR and viral titration. For the quantitative RT-PCR, primary PAMs were transduced with rAd-amiRcon, rAd-amiRSn and/or rAd-amiRCD163 as described, and infected with PRRSV strain VR2332 (MOI 0.2) 48 h after transduction. At 24 h post infection, the total RNA was extracted for quantitative RT-PCR using PRRSV ORF7-specific primers (Table 2). Compared to that (5.4 log10) in rAd-amiRcon-transduced cells, the ORF7 copy number in rAd-amiRSn and/or rAd-amiRCD163 transduced cells was decreased by 3.5, 2.3 or 2.6 log10 (Figure 5). Next, primary PAMs were incubated with the exosomes derived from rAd-amiRSn- and/or rAd-amiRCD163-transduced PK-15 cells, infected with PRRSV and the total RNA was extracted for quantitative RT-PCR as described. Compared to that (5.4 log10) in the control group, the ORF7 copy number in amiRSn- and/or amiRCD163-containing exosome-incubated cells was decreased by 3.3, 2.0 or 2.2 log10 (Figure 5). Finally, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes, infected with PRRSV and total RNA was extracted for RT-PCR as described. Compared to that (5.4 log10) in the control group, the ORF7 copy number in double rAd-transduced and double amiRNA-containing exosome-incubated cells was decreased by 4.2 log10, while the ORF7 copy number in single rAd-transduced and single amiRNA-containing exosome-incubated cells was decreased by 3.3 or 3.6 log10 (Figure 5).
Detection of PRRSV ORF7 copy numbers in rAd-transduced and/or exosome-incubated PAMs. Primary PAMs were transduced with single or double rAds and/or incubated for 48 h with single or double amiRNA-containing exosomes. The cells were infected with PRRSV strain VR-2332 and the total RNA was extracted 24 h post infection for ORF7 copy number detection by real time quantitative RT-PCR.
For the viral titration assay, primary PAMs were transduced with different rAds and infected with PRRSV strain VR2332 as described. At different time points post infection, the cells were harvested for PRRSV titration on MARC-145 cells. Compared to that (4.3 log10 TCID50) in the mock- or rAd-amiRcon-transduced cells, the PRRSV titer in rAd-amiRSn- and/or rAd-amiRCD163-transduced cells was decreased by 1.5, 0.9 or 1.0 log10 at 24 h post infection (Figure 6A). Next, primary PAMs were incubated with amiRSn- and/or amiRCD163-containing exosomes, infected with PRRSV and harvested for PRRSV titration as described. Compared to that (4.3 or 4.2 log10 TCID50) in the mock- or amiRcon-containing exosome-treated cells, PRRSV titer in amiRSn- and/or amiRCD163-containing exosome-incubated cells was decreased by 1.2, 0.6 or 1.0 log10 (Figure 6B). Then, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes, infected with PRRSV and harvested for PRRSV titration as described. Compared to that (4.3 or 4.4 log10 TCID50) in the two control groups, the PRRSV titer in double rAd-transduced and double amiRNA-containing exosome-incubated cells was decreased by 2.0 log10, while the viral titer in single rAd-transduced and single amiRNA-containing exosome-incubated cells was decreased by 1.1 log10 (Figure 6C). The similar additive anti-PRRSV effect between the two amiRNAs lasted for at least 96 h (Figure 6A,B or C). Finally, primary PAMs were transduced with different rAds, incubated with different amiRNA-containing exosomes and infected with PRRSV strain JX-A1, CH-1R or VR2332 as described. At 72 h post infection, the infected cells were harvested for PRRSV titration. Compared to that (4.9-6.2 log10 TCID50) in the two control groups, the PRRSV titers in three viral strain-infected cells were decreased by 2.7, 2.1 and 2.6 log10, respectively (Figure 6D).
Titration of PRRSV titers in rAd-transduced and/or exosome-incubated PAMs. (A) Primary PAMs were transduced with different rAds and infected with PRRSV strain VR2332 48 h after transduction. At different time points after infection, the cells were harvested for PRRSV titration on MARC-145 cells. (B) Primary PAMs were incubated for 48 h with different amiRNA-containing exosomes and then infected with PRRSV strain VR2332 before PRRSV infection and titration. (C) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes before PRRSV infection and titration. (D) Primary PAMs were transduced with different rAds and then incubated for 48 h with different amiRNA-containing exosomes. The cells were infected with three different PRRSV strains and harvested for PRRSV titration 72 h after infection.
As a natural antiviral mechanism, RNAi has become a feasible strategy against a variety of viral infections , but its in vivo use is limited by the low efficiency of small RNA delivery. Such drawbacks can be alleviated by using viral vectors for small RNA delivery. Among the viral vectors available, Ads have several advantages and thus have been used extensively as the gene transfer vectors for gene therapy and vaccine development . Furthermore, Ads have been shown to an efficient shRNA delivery vehicle for pig cells . Therefore, in this study we used rAd vectors as the amiRNA delivery vehicle. Cell transduction experiments showed that the human Ad5-based vectors could not only transduce pig cells (Figure 2), but also express the encoded amiRNAs efficiently (Figure 3). Unlike pig kidney PK-15 cells, however, primary PAMs were resistant to rAd transduction and only a maximal transduction efficiency of about 60% was achievable by using very high doses (MOI ≥ 800) of rAd vectors (Figure 2). This could be explained by the lack of high affinity Ad receptor on primary PAMs, which has been reported for the primary macrophages in other species .
As a natural mechanism in animal cells, the exosomes derived from human and mouse cells can serve as an efficient vehicle for small RNA transfer . In the light of resistance of PAMs to rAd transduction, this warranted us to explore the feasibility of pig cell-derived exosomes as a small RNA delivery vehicle, which has not been reported in livestock such as pigs. Our RT-PCR assay showed that the sequence-specific amiRNAs were easily detected in the exosomes purified from the cell culture medium of rAd-transduced pig cells (Figure 3B), indicating that the viral vector-encoded amiRNAs were secreted from the pig cells via exosomes. Furthermore, the sequence-specific amiRNAs were detected in the exosome-incubated PAMs (data not shown) and showed significant inhibitory effects against PRRSV infection (Figures 5 and 6). These results suggested that exosomes could also serve as an efficient small RNA transfer vehicle for pig cells.
PRRSV uses at least three receptors to enter PAMs, among which Sn and CD163 play essential but different roles . It has been shown that PRRSV infection of PAMs can be blocked partially by Sn- or CD163-specific antibody, or completely by a combination of two antibodies . Our previous study has also shown that the soluble Sn and CD163 receptors have an additive effect against PRRSV infection . These indicate that the simultaneous knock-down of two viral receptors is required to achieve a significant antiviral effect. In the light of resistance of PAMs to rAd transduction, we compared Sn and CD163 receptor knock-down efficiencies of three different strategies: rAd transduction, exosome treatment and rAd transduction plus exosome treatment. Both quantitative RT-PCR and flow cytometry analyses showed that, compared to the lower knocking-down efficiency of Sn or CD163 receptor by rAd transduction or exosome treatment, the rAd transduction plus exosome treatment resulted in significantly more knock-down of the two viral receptors at both mRNA and protein levels (Figure 4). These results suggested that exosomes could assist rAds to deliver small RNAs into pig cells, which was particularly important for the delivery of amiRNAs into PAMs that were resistant to rAd transduction.
Similarly, we used the three different strategies to investigate the additive anti-PRRSV effect between Sn and CD163 receptor-targeted amiRNAs. Quantitative RT-PCR showed that the simultaneous knock-down of two viral receptors resulted in more reductions in PRRSV ORF7 copy number than the single receptor knock-down (Figure 5). In addition, much more reduction in the ORF7 copy number was achieved by using rAd transduction plus exosome treatment (Figure 5). The similar results were obtained from viral titration assays (Figure 6,B and C). The additive anti-PRRSV effect between the two viral receptor-targeted amiRNAs was relatively long-lasting (96 h) and effective against all three different viral strains tested (Figure 6D). However, the complete inhibition was not achieved by using three different strategies and very high rAd doses (MOI ≥ 800) for cell transduction. This could be explained by the following reasons: first, Sn and/or CD163 receptor-targeted amiRNAs delivered by three different strategies were insufficient for the complete know-down of two viral receptors (Figure 4) due to the inherent incomplete knock-down of RNAi strategy , and/or the resistance of primary PAMs to rAd transduction . Second, the turnover of Sn and/or CD163 receptor was longer than that we tested (48 h) and thus the certain amounts of pre-expressed receptors were present on the rAd-transduced and/or exosome-incubated PAMs. Finally, there were alternative cellular factor (s) may be involved in PRRSV infection of primary PAMs. Among these, CD151 may be an important one since it plays an important role in PRRSV infection of MARC-145 cells and is expressed on primary PAMs ,. These warrant us to refine the amiRNA strategy by targeting more cellular factors and/or using more efficient amiRNA transfer vectors such as recombinant lentiviruses.
Both Sn and CD163 receptors are macrophage-restricted cell surface molecules that are conserved across mammals. Among these, Sn receptor is a member of the sialic acid-binding IgG-like lectin family of proteins which contributes to sialylated pathogen uptake, antigen presentation and lymphocyte proliferation , while CD163 receptor is a critical for the efficient extracellular hemoglobin clearance during hemolysis . Therefore, whether the knock-down of two receptors could influence the viability or compromise the cell functions of PAMs should be considered. In the light of the fact that expression of the two receptors are regulated by several factors such as glucocorticoids and IL-10, and that the expression levels vary significantly under different conditions ,, we speculated that the two genes were not vital for the cell viability. This speculation was supported by an additional fact that the viability of PAM cell lines is not compromised by the loss of Sn and/or CD163 expression . In any case, more studies are certainly needed to address the safety of our amiRNA strategy before its in vivo use.
In summary, in this study we generated two rAds expressing Sn or CD163 receptor-targeted amiRNA and investigated their anti-PRRSV effect using different strategies. The Ad vectors could not only transduce different pig cells, but also express the vector-encoded amiRNAs. The vector-expressed amiRNAs were secreted from rAd-transduced cells via exosomes not only, but taken up by other pig cells as well. A significant additive anti-PRRSV effect between the two amiRNAs was demonstrated by transduction of PAMs with the two rAds or by incubation with the two amiRNA-containing exosomes, which was enhanced further by co-transduction with the two rAds plus co-incubation with the two amiRNA-containing exosomes. According to our knowledge, this is the first study to explore an amiRNA strategy against PRRSV infection by targeting the two viral receptors. In addition, we provided new evidence that the exosomes could also serve as an efficient small RNA deliver vehicle for pig cells. Although the complete anti-PRRSV effect has not yet been demonstrated, this study may facilitate further works on development of more efficient strategies against PRRS.
This study demonstrated that Sn and CD163 receptor-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. In addition, we provided new evidence that the exosomes could serve as an efficient small RNA deliver vehicle for pig cells.
Mouse fibroblast cell line L-929 (ATCC CCL-1) was grown in Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F-12 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% non-essential amino acids. Pig kidney cell line PK-15 (ATCC CCL-33), mouse embryo fibroblast cell line NIH 3 T3 (ATCC CRL-1658), AAV-293 cells (Stratagene, USA) and African green monkey kidney cell line MARC-145 cells (ATCC CRL-12231) were grown in DMEM supplemented with 10% FBS and 1% non-essential amino acids. Primary PAMs were prepared from 6-week-old Lancrace pigs as described previously  and grown in DMEM/F-12 supplemented with 10% FBS, 1% non-essential amino acids and 20% L-929-conditioned medium. PRRSV strain VR-2332 (ATCC VR2332) is a prototype strain of North American genotype . PRRSV strain CH-1R is an attenuated vaccine strain derived from the traditional Chinese strain CH-1a . PRRSV strain JX-A1 is a highly pathogenic Chinese strain . The three PRRSV strains were propagated and titrated on MARC-145 cells.
The first 1751-nt sequence of porcine Sn mRNA (GenBank: AF505985) and the 1511-nt sequence of porcine CD163 mRNA (GenBank: NM_213976) were predicted using the web-based BLOCK-iT™ RNAi designer (Invitrogen, USA). Among 10 top-scoring sequences reported for each target, 18 of them were selected for miR RNAi design according to their higher knock-down probabilities (star rankings). The top- and bottom-strand oligonucleotides (Table 1) were synthesized, each pair of them (5 μl, 200 μM) was denatured at 94°C for 5 min and slowly annealed at room temperature to form double-strand oligonucleotide.
For construction of the amiRNA expression vectors, each double-strand oligonucleotide was cloned at the Bbs I site of amiRNA expression vector pcDNA-miR , in which the amiRNA expression cassette consists of the 5′- and 3′-miR flanking sequences of mouse BIC non-coding mRNA, followed by the poly(A) signal of human herpesvirus TK gene (Figure 1A). The recombinant vectors, namely pcDNA-miRSn (1–9), pcDNA-miRCD163 (1–9) or control pcDNA-miRcon, were used for amiRNA validation.
For construction of the reporter vectors, the cellular RNA was extracted from primary PAMs using RNAiso Plus (KaTaRa, Dalian, China) according to the manufacturer's instructions. The reverse transcription (20 μl) was performed using RevertAid™ Reverse Transcriptase (Fermentas, USA) by following the manufacturer's protocol. The PCR (50 μl) was performed using 5 μl of RT product and LA Taq DNA Polymerase (KaTaRa, Dalian, China) according to the manufacturer's manual. The first 1751-bp Sn cDNA segment was amplified using primers Sn For1 and Sn Rev1 (Table 2). The PCR was carried out at 94°C for 5 min; 30 cycles of 94°C for 45 sec, 60°C for 45 sec and 72°C for 2 min; and a final extension at 72°C for 10 min. The first 1511-bp CD163 cDNA segment was amplified using primers CD163 For1 and CD163 Rev1 (Table 2). The PCR was carried out at 94°C for 5 min; 30 cycles of 94°C for 45 sec, 50°C for 45 sec and 72°C for 1.5 min; and a final extension at 72°C for 10 min. Each PCR product was cloned into pMD18-T vector for sequencing and then at the Xho I/Bam HI site of pEGP-N1 vector (Invitrogen, USA) to produce reporter vector pSn-EGFP or pCD163-EGFP (Figure 1B). The expression of two GFP fusions was checked by transfection of NIH 3 T3 cells and fluorescent microscopy.
For construction of the rAd vectors, the amiRSn-2, amiRCD163-2 or amiRcon expression cassette was excised from the amiRNA expression vector by restriction digestion with Aat II and Pvu I, and cloned into pShuttle-IRES-hrGFP (Stratagene, USA) vector after linerization with the same enzymes. The pShuttle-IRES-hrGFP vector is rendered replication defective by deletion of the E1 and E3 genes , and by inclusion of humanized Renilla reniformis GFP (hrGFP) sequence after the internal ribosome entry site (IRES) for follow-up purpose. The resultant rAd vector was called pShuttle-amiRSn-IRES-hrGFP, pShuttle-amiRCD163-IRES-hrGFP or pShuttle- amiRcon-IRES-hrGFP (Figure 2A).
NIH 3 T3 cells were seeded in triplicates at 5 × 105/well on 24-well plates and grown to 70% confluent growth. The cells were transfected with pcDNA-amiRSn (1–9), pcDNA-amiRCD163 (1–9) or pcDNA-amiRcon (2 μg/well) plus pSn-EGFP or pCD163-EGFP (0.4 μg/well). The transfection was performed using Lipofectamine™ 2000 (Invitrogen, USA) according to the manufacturer's manual. At 24 h after transfection, the cells were trypsinized, washed three times with PBS, diluted to 106 cells/ml and analyzed (104 cells) for total fluorescence and cell numbers on BD FACSAia III (BD, USA). The effectiveness of each amiRNA was expressed as the percent inhibition of Sn-GFP or CD163-GFP fusion expression using pSn-GFP- or pCD163-GFP-transfected as the reference.
The three rAds, namely rAd-amiRSn, rAd-amiRCD163 and rAd-amiRcon, were generated by transfecting AAV-293 cells (Stratagene, USA) with the rAd vectors according to the instruction manual for AdEasy™ Adenoviral Vector System (Agilent Technologies, USA). The rAds were amplified on AAV-293 cells, purified using ViraBind™ Adenovirus Miniprep Kit (CELL BIOLABS, USA) by following the manufacturer's protocol, and titrated on AAV-293 cells as fluorescent formation units (FFU)/ml. For transduction of PK-15 cells, the cells were seeded in triplicates at 5 × 104 cells/well on 24-well plates, and grown to 80% confluent growth. After two time wash with PBS, the cells were transduced (37°C for 2 h) with different doses (MOI) of rAds. The transduction of primary PAMs was carried out as previously described . Briefly, the cells were cultured for 7 days in the growth medium conditioned with 20% of L-929 cell culture medium. The cells were trypsinized, diluted to 105cells/ml with the same medium, mixed with different doses of rAds and centrifuged at 2000 × g for 1 h at 37°C. After wash again with PBS, the cells were grown for additional 48 h in the fresh medium and assayed for GFP-positive cells on BD FACSAia III or by fluorescent microscopy.
Exosome purification was performed as previously described . Briefly, primary PAMs and PK-15 cells were seeded in T75 flasks and grown to 70% confluent growth in the medium supplemented with 10% exosome-depleted FBS. The cells were transduced with each rAd (MOI 800 for primary PAMs or 100 for PK-15 cells) and incubated for additional 48 h. The cell medium was collected and centrifuged at 4°C, 300 g for 10 min, 1000 g for 30 min, 10,000 g for 45 min to remove the cell debris. After filtration through a 0.22-μm filter membrane, the exosomes were precipitated by centrifugation at 120,000 g for 70 min and suspend in PBS for immediate use or stored at −80°C for later use.
The poly(A)-tailed RT-PCR for amiRNA detection was performed using One Step Prime Script miRNA cDNA Synthesis Kit (KaTaRa, Dalian China) by following the manufacturer's protocol. Briefly, the total RNA was extracted from rAd-transduced cells or purified exosomes for cDNA synthesis. Each amiRNA cDNA was amplified using the sequence-specific forward primer (Table 1) and the general reverse primer in the cDNA synthesis kit. The PCR was carried out at 95°C for 30 sec, 35 cycles of 95°C for 10 sec, 58°C for 30 sec, 72°C for 10 sec, and a final extension at 72°C for 5 min. The PCR products (8 μl) were analyzed by electrophoresis on a 3% agarose gel.
The real-time quantitative RT-PCR for Sn and CD163 receptor mRNA detection was performed as described previously . Briefly, primary PAMs were transduced in triplicates with each rAd and incubated in the medium with or without the exosomes (0.5 mg protein/ml) purified from the rAd-transduced PK-15 cells. At 48 h after incubation, the total RNA was extracted for reverse transcription using RevertAidTM Reverse Transcriptase (Fermentas, USA). The Sn and CD163 receptor mRNA expression was analyzed by SYBR green-based real-time quantitative PCR using Roche LightCycler Nano system. The mRNA copy number was normalized by comparing to house-keeping GAPDH copy number. The primer sequences are listed in Table 2.
The real-time quantitative RT-PCR for PRRSV ORF7 copy number detection was performed as previously described . Briefly, primary PAMs were transduced in triplicates with different rAds and/or incubated in the growth medium with or without the exosomes derived from rAd-transduced cells as described. At 48 h after incubation, the cells were infected with PRRSV (MOI 0.2), incubated for additional 24 h and total RNA was extracted for reverse transcription and real-time quantitative PCR as described. The standard curve was generated by comparing to PRRSV ORF7-containing plasmid . The primer sequences are listed in Table 2.
The flow cytometry for detection of Sn and CD163 receptor knock-down was performed as previously described . Briefly, primary PAMs were transduced in triplicates with different rAds and/or incubated for 48 h with or without the exosomes derived from rAd-transduced cells. The cells were stained with Sn- or CD163-specific antibody and analyzed by flow cytometry as described.
Primary PAMs were seeded at 2 × 105/ml on 24-well plates and grown overnight in the medium supplemented with 10% exosome-depleted FBS. The cells were mock-transduced in triplicates or transduced with different rAds and/or incubated for 48 h with or without the exosomes (0.5 mg protein/ml) purified from different rAd-transduced PK-15 cells. The cells were infected with PRRSV strain VR-2332, CH-1R or JX-A1 (MOI 0.2) and harvested at different time points for PRRSV titration on MARC-145 cells as previously described .
This work was financially supported by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the fund from the Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal infectious Diseases and Zoonoses.
LZ and HQS performed the experiments; XYZ and XLX provided important reagents; LZ wrote the manuscript; HCS provided overall supervision and financial support and edited the manuscript. All authors read and approved the final manuscript.


fever like no one else: "Such an intelligence," Laplace wrote, "would embrace in the same formula the movements of the great est bodies of the universe and those of the lightest atom; for it,... Chords for The Great Comet Music Video: No One Else. Play along with guitar, ukulele, or piano with interactive chords and diagrams. Includes transpose, capo hints, changing speed and much more. Play along with guitar, ukulele, or piano with interactive chords and diagrams.
See more What others are saying "Print and download No One Else sheet music from Natasha Pierre & the Great Comet of 1812. Sheet music arranged for Piano/Vocal/Chords, and …... The genre is soul + folk. Also quite a long melody, of 6:32, that reminds songs of prisoners who pass something to each other in the chain, melodiously humming a composition, to keep up the rhythm.
- No One Else (Natasha, Pierre & The Great Comet of 1812) ..D. Malloy 2. Anja Kelly - The Light In The Piazza (The Light In The Piazza)..A. Guettel 3. Julia Franson - In My Dreams (Anastasia) ..Flaherty/Ahrens 4. Jase Nelson - Empty Chairs at Empty Tables (Les Miserables) ..Boublil and Schoenberg 5. Madison Morin - My House (Matilda) ..Tim Minchin _____ Monday, April 16, 2018 …... the Earth, causing great Earthquakes. Luna – Our Moon, or is it really a former Comet? Many associated meteors were seen, and for the first time, ocean tides.
Music from the show. pierre; no one else (featuring Phillipa Soo) charming (featuring Amber Gray) sonya alone (featuring Brittain Ashford) letters *2017 Tony Award, Best Set Design of a Musical (Mimi Lien), Best Lighting Design of a Musical (Bradley King)*... Shop no one else smokie norful sheet music, music books, music scores & more at Sheet Music Plus, the World's Largest Selection of Sheet Music.
Natasha, Pierre & The Great Comet of 1812 by Dave Malloy Piano/Vocal Score NATASHA, PIERRE & THE GREAT COMET OF 1812 by Dave Malloy adapted from War & Peace by Leo Tolstoy PROLOGUE 1.
if a comet is passing very close to our planet, it will speed quickly across the sky and might emerge from the twilight into a posi- tion high in the dark sky within a matter of a day or thereabouts.


text:
Create a free account to save articles for later, keep track of past articles you've read, and receive exclusive access to all RP resources.
Get Articles Delivered!
Articles, news,and reviews with a Biblical perspective to inform, equip, and encourage Christians delivered direct to your inbox!
Articles, news, and reviews with a Biblical perspective to inform, equip, and encourage Christians.
Save articles for later, keep track of past articles you've read, and receive exclusive access to all RP resources.
Check out our other sites!
RP Press
Our App!
By: Bassam Michael Madany
Wednesday October 17 – A living sacrifice
I appeal to you therefore, brothers, by the mercies of God, to present your bodies as a living sacrifice, holy and acceptable to God, which is your spiritual worship. – Romans 12:1
Scripture reading: Romans 12:1-2
Paul followed this pattern in his Letters to the Churches. After expounding the Gospel, he turned to its application in the life of the believers. While justification by faith in Jesus Christ is a once-for-all event, sanctification, that is the practice of the faith and the working out of God-centered living, is a life-long process. Paul followed the order of the Great Commission, as given by the risen Christ prior to His Ascension:
"Go therefore and make disciples of all nations, baptizing them in the name of the Father and of the Son and of the Holy Spirit, teaching them to observe all that I have commanded you. And behold, I am with you always, to the end of the age" (Matthew 28:19,20).
It wasn't only Paul that mentioned the importance of the Christian's walk during his earthly pilgrimage. The same emphasis exists in the New Testament Letters of Hebrews, James, I and II Peter, I, II, and III, John and Jude.
Followers of world religions have objections to Christian beliefs, but are impressed by the Christian life. Having grown up in the Middle East where Muslims who regarded Christians as unbelievers, still couldn't help but admire the life of their Christian neighbors. One of their sayings was, "A Christian doesn't lie." My father used to relay this anecdote that Muslims, who had plans to travel, used to "deposit" some of their money for safe-keeping at the home of missionaries whose conduct in life exemplified honesty and integrity!
Suggestions for prayer
Pray that the Lord will enable us to manifest the Christian Mind in all our dealings with others!
This daily devotional is available in a print edition you can buy at Nearer to God Devotional. Rev. Bassam Michael Madany has served as a missionary in Syria, and in 1958 he began a 36-year radio and literature ministry to the Arab world. After retirement Bassam & his wife Shirley began an Internet ministry, Middle East Resources, to provide a "Christian Response to the Global Challenge of Islam."
Tagged: Bassam Michael Madany, Nearer to God daily devotional
We Think You May Like
Soup and Buns
"Lord, how can I help?"
There's a difference between doing what is right because you have always been told to do so, and doing it from your heart. There's a difference betwee...
Movie Reviews, Sexuality, Watch for free
In His Image: Delighting in God's Plan for Gender and Sexuality
Reading films: are Christians as discerning as they used to be?
Documentary, Movie Reviews
Dragons or Dinosaurs? Creation or Evolution?
Documentary 84 minutes / 2010 Rating: 7 / 10 The Chinese lunar calendar cycle includes twelve animals, eleven of which are quite familiar to us: ...
Bench Press Theme by Evolve Digital & Third Floor Design Studio

redpajama_set_name: RedPajamaCommonCrawl


Featuring a fleet of three catamarans more than 40 feet long, Santorini Sailing offers private and semi-private cruises to the Santorini Caldera. Limited to 10 passengers, the tours include gourmet lunch or dinner with linen table service, featuring local fare such as fresh seafood, prime steaks, pasta and premium wines. Featured on the Discovery Channel, the Travel Channel and the Food Network, the tour departs from Vlihada Marina, passing along the ancient city of Akrotirien route to the Red and White beaches. When the Venetian lighthouse comes into view, the five-hour cruise journeys into the caldera, where passengers visit private bays for a swim and snorkel. After a barbecue lunch in the bay, the cruise sets sail past the Aspronisi back to Vlihada Marina. Each catamaran offers numerous amenities, such as expansive deck space, private suites and a panoramic bridge deck.


"We heard the governor present a plan and a vision with many ideas that I believe Republicans can embrace. The idea of helping to fund and support workforce development and investment in our communities has a lot of support.
There is general agreement on his goals for education, health care, and infrastructure, but the manner in which he proposes to pay for them is certain to be the subject of further discussion.
I was pleased to see the Governor give recognition to Misericordia University's Women With Children program as a model to support and replicate across Pennsylvania.
I think we heard some very positive comments and some ideas we will certainly continue to look at. As the new chair of the Judiciary Committee, the idea of Justice Reinvestment, what we do to help rein in the costs of our corrections budget, those will be things I will continue to look at.
I think we set a positive tone on striking that hopeful balance between taxpayers and how we fund important programs.


This Asscher cut diamond appears to float inside a surrounding octagonal setting, held in place by signature sharp split claw prongs. The surrounding geometric bezel is covered on all sides by small white diamonds which contrast brilliantly against the gunmetal grey colour of the metal. The Frances ring makes a bold statement whilst remaining elegant and timeless.
Image shows the FRANCES ring made in Platinum with black plating holding a 0.92 carat F VVS2 asscher cut diamond with 0.63 carats of 1.3mm diamonds.
All of our designs can be modified to meet a range of different budgets and design preferences. The specific ring pictured is available for purchase for $11,500 AUD.


Hallstatt, a small village in the state of Upper Austria and part of the Salzkammergut region is often called the most photographed village in Europe and the world. Lake and mountain views charm tourists all around the year but there are also many other things to see and do. As a UNESCO World Heritage Site, Hallstatt knows how to treat travelers of all kind. You can book romantic winter holidays or photography trips to the roots of this beautiful European cultural village. In the morning, under 1000 citizens and just a few additional tourists make sure that you can truly enjoy the peaceful remote village.
When planning a stay here, you have to book accommodation early because there aren't many places to stay in the small village. If you are late and there is no accommodation left, consider staying in close-by resorts. You can see it completely in just one day or alternatively you can use several days to discover all the beautiful streets and attractions. The town itself doesn't have many attractions, but the architecture and locals make it worthy of visiting. The best time for trips is on early morning, because after 9am buses loaded with Chinese tourists (nothing against the Chinese) start to arrive and soon you have nowhere to step or sit down.
Hallstatt has gotten its place on UNESCO World Heritage list because of the important cultural scenery. The Alps, the lake and the tiny village make this place look like a scene from a fairytale. Officially it's in the list because of two reasons: first, its unique local traditions and secondly, due to the outstanding scenery. Austria has a total of nine UNESCO World Heritage Sites and this is one of the most popular ones.
When you travel there, the important thing is, of course, to walk around and see what all the hype is about. On this list of things to see and do I'll make you sure to enjoy the town completely and not to forget anything.
The word "Hallstatt" means "place of salt". Why? It's home to the world's first salt mine. Thanks to the salt industry, the town quickly progressed and became wealthy. Salt has always been an important commodity, more so in the pre-historic times as it was used to preserve meat.
In recent decades, though, Hallstatt's fame derives mostly from its natural beauty. Sandwiched between a lake and mountains (you could even say that it cuddles the lake), this perfect little town easily became a favorite tourist destination as well as a UNESCO World Heritage site I've talked about earlier.
The town may be compact –-you can walk from end to end in under half an hour – but there's much to see within the town and in the surrounding area. And that's what I'm going to talk about now.
The Hallstätter See, a spectacular mountain lake in Austria's Salzkammergut region, lies at the northern foot of the mighty Dachstein mountain range. Some 8.5 kilometers long and between one to two kilometers wide, the lake is up to 125 meters deep and is surrounded by steep wooded slopes that give it a fjord-like character. Of the lovely villages that dot its shoreline, the most popular are Obertraun, Steeg, and Hallstatt. The Hallstätter See is the most significant lake in the Alps in terms of history, and thanks to its many easily accessible beaches and stunning mountain scenery it has long been a draw for artists and outdoor enthusiasts, as well as those simply wanting some peace and quiet. Popular activities include fishing, diving, and boating on traditional flat-bottomed boats called Salzkammergut. The lake is also ringed by many excellent biking and hiking trails, some of which head up into the surrounding mountains. It's also home to a variety of diverse flora and fauna, including many rare species in the wetlands and moors in the south and north, including native orchids, waterfowl, and fish.
The little market village of Hallstatt, one of the most attractive places in the Salzkammergut, is idyllically positioned on the southwest shore of the Hallstätter See. Taking its name from the nearby salt mine, the village is home to a variety of attractions, including the 15th-century Roman Catholic Ascension of Our Lady Church with its Romanesque tower, three fine winged altars, Late Gothic frescos from around 1500, and the fascinating Bone House with its collection of old skulls stored here due to a lack of space in the church cemetery. Also of note is the 18th-century Evangelical Church of Hallstatt with its slender spire. One of the most famous views of the old village is from the popular Photo Point in the Römisches district, just a short walk away from the historic Market Square. Here, you'll find many other great reasons to stop, from its excellent terraced cafés and shops to the elegant Holy Trinity statue.
Really a number of excellent attractions in one, Hallstatt Salt World lies high above the town on the 1,030-meter-tall Salzberg (Salt Mountain). Accessible by cable car or a three-minute journey aboard the funicular railway, it's well worth of visiting for the wonderful views from historic Rudolf's Tower (Rudolfsturm), an old fortification built to defend the mines against invaders (now a restaurant and observation tower). Visitors should also check out the Skywalk, an incredible viewing platform extending over a sheer drop with Hallstatt 350 meters directly below. The star attraction, however, is the 7000 year-old salt mine itself. Highlights include the story of the preserved corpse found here in 1734 known as the Man in Salt, along with fascinating displays of ancient (and modern) mining methods, and a chance to visit the Subterranean Salt Lake. I would say kids of all ages, but after I saw it I have to say EVERYONE will want to try Mega Slide, Europe's longest wooden slide (also underground).
Hallstatt is also home to the World Heritage Museum (Welterbemuseum Hallstatt) with its displays dealing with the rich history of both the village and its surroundings over the past seven millennia, from the earliest days of its salt mines to its UNESCO World Heritage Site status. Other exhibits deal with later settlers such as the Celts, while the Studio of Epochs is a hands-on multi-media display of tools and artifacts from the village's history. The other museum over there, the Prehistoric Necropolis, is built over an ancient burial ground and provides an close-up look at ancient burial places dating back between 5000 BC and 1000 BC (more than 4,000 people were known to have been buried here) along with artifacts, including grave furnishings from the Iron Age.


text:
{-# LANGUAGE DeriveDataTypeable #-}
module Data.Lambda.UnTyped where

import Data.Data (Data)
import Data.Typeable (Typeable)
import Test.QuickCheck
import Text.Read
import Control.Applicative

import Data.Name

-- $setup
-- >>> :set -XOverloadedStrings
-- >>> :set -XScopedTypeVariables

data Lambda
    = Variable Name
    | Lambda Name Lambda
    | Apply Lambda Lambda
    deriving (Eq, Ord, Data, Typeable)

-- | >>> (Variable "x" `Apply` Variable "y") `Apply` Variable "z"
-- x y z
-- >>> Lambda "x" (Lambda "y" (Variable "y"))
-- \ x y . y
-- >>> Lambda "x" (Variable "x" `Apply` (Variable "y" `Apply` Variable "x"))
-- \ x . x (y x)
-- >>> Apply (Lambda "x" (Variable "x")) (Variable "y")
-- (\ x . x) y
instance Show Lambda where
    showsPrec _ (Variable x) = shows x
    showsPrec d x@Lambda{} = showParen (app_prec < d) (showString "\\ " . f x) where
        f (Lambda y z) = shows y . showChar ' ' . f z
        f y = showString ". " . shows y
        app_prec = 10
    showsPrec d x@Apply{} = showParen (app_prec < d) (f x) where
        f (Apply y z) = f y . showChar ' ' . showsPrec (app_prec+1) z
        f y = showsPrec (app_prec+1) y
        app_prec = 10

-- | >>> read "x y z" :: Lambda
-- x y z
-- >>> read "\\ x y . y" :: Lambda
-- \ x y . y
-- >>> read "\\ x . \\ y . y" :: Lambda
-- \ x y . y
-- >>> read "\\ x . x y (y x)" :: Lambda
-- \ x . x y (y x)
-- >>> read "(\\ x . x) y" :: Lambda
-- (\ x . x) y
--
-- prop> \ (x :: Lambda) -> (read . show) x == x
instance Read Lambda where
    readPrec = parens (app <++ lam <++ var) where
        var = Variable <$> readPrec
        lam = prec app_prec $ lexP >>= isLambda >> f where
            f = Lambda <$> readPrec <*> ((lexP >>= isDot >> reset readPrec) <++ f)
        app = prec app_prec $ foldl1 Apply <$> some (step readPrec)
        isLambda (Punc "\\") = return ()
        isLambda _ = pfail
        isDot (Symbol ".") = return ()
        isDot _ = pfail
        app_prec = 10
    readsPrec = readPrec_to_S readPrec

instance Arbitrary Lambda where
    arbitrary = frequency
        [ (,) 3 $ Variable <$> arbitrary
        , (,) 2 $ Lambda <$> arbitrary <*> arbitrary
        , (,) 2 $ Apply <$> arbitrary <*> arbitrary
        ]

instance CoArbitrary Lambda where
    coarbitrary (Variable a) = coarbitrary a
    coarbitrary (Lambda a b) = coarbitrary a . coarbitrary b
    coarbitrary (Apply a b) = coarbitrary a . coarbitrary b

sCombinator :: Lambda
sCombinator = read "\\ f g x . f x (g x)"
kCombinator :: Lambda
kCombinator = read "\\ x y . x"
iCombinator :: Lambda
iCombinator = read "\\ x . x"
-- | y f -> f (y f)
yCombinator :: Lambda
yCombinator = read "\\ f . (\\ x . f (x x)) (\\ x . f (x x))"
-- | omega omega -> omega omega
omegaCombinator :: Lambda
omegaCombinator = read "(\\ x . x x) (\\ x . x x)"

redpajama_set_name: RedPajamaGithub


Exercise is an important part of a healthy lifestyle, however this needs to be backed up with healthy food choices and balanced nutrition. Our guide has been specifically designed to help you to understand the fundamentals of good nutrition and achieve healthier and quicker results.
Our Nutritional Fitness Guide is free to all Personal Training clients who sign up to training with 4 week block bookings.
If you have already downloaded our 14 Day Healthy Eating Plan then you'll receive a £2 discount off our regular priced guide of £10. The guide is in hard copy only and can be picked up at your Personal Training or Boot Camp session.
If you're not a training regular or live too far from Bristol, then no problem as your Nutritional Fitness Guide can be posted (P&P will be added). Simply email Jason your discount code from you health eating plan.
How your hormones can affect how much fat you store or burn.
How dieting alone can make you fatter.
Why exercise is so important and what type of exercise is best for you.
How you can eat more and still lose weight.
What G.I foods are and how they'll help you to lose weight.
Eat at the right times to increase energy levels.
Help to prevent cancer with cancer fighting foods.
Understanding your hormones to help perfect your training.
How carbohydrates can make you fat.
Written by Jason himself from his own personal fitness and nutritional experience and years of professional knowledge.


text:
Help us stop the asbestos trade
13 March, 2017IndustriALL Global Union is calling on affiliates to take action to help stop the deadly trade in asbestos. Please download the letter to send to your government.
Hundreds of thousands of workers die every year from diseases caused by exposure to asbestos. It is the world's worst industrial killer.
For years, many countries have been trying to list chrysotile asbestos as a toxic substance under the Rotterdam Convention, a United Nations treaty that requires dangerous substances on the list to be traded with prior consent and full knowledge of their hazards.
However, these efforts have been repeatedly blocked by a handful of nations that still benefit from the asbestos trade, notably Russia and India.
IndustriALL is asking trade unions to write to their governments as soon as possible to support a proposal to amend the Rotterdam Convention so that no single country has the power of veto to stop a dangerous substance being on the list.
The proposal, submitted by 12 countries in the African region, would mean that in the absence of a consensus, a substance could be listed as hazardous if a majority of 75 per cent of parties supported that action.
The proposal will be addressed at the upcoming Conference of the Parties to the Rotterdam Convention, taking place from 24 April – 5 May 2017 in Geneva, Switzerland.
Two million tonnes of asbestos is still being mined, sold and used across the world every year. And the deadly consequences will endure for decades to come. If countries have to give prior informed consent to import chrysotile asbestos, it is expected to reduce the trade in the fatal mineral.
Valter Sanches, IndustriALL's General Secretary, said:
Every five minutes someone dies from exposure to asbestos and millions more suffer disability and pain. So long as any one party can exercise a veto over good science and common sense, the Rotterdam Convention is discredited. We count on your support to correct this.
Download the letter to send to your government on this page and help us in the fight for a global ban on asbestos. Please don't forget to tell us when you have a sent a letter at [email protected]
Write to your government
Model letter to government - English
Model letter to government - French
Model letter to government - German
Model letter to government - Russian
Model letter to government - Spanish
The Rotterdam Convention


text:
Kat Dennings Wiki – Movies and TV Shows, Net Worth, Age, Height, Thor Love and Thunder
Kat Dennings is a Phenomenal actress who started her career at an age of 16 in the TV series "Sex and the City" for her role as Jenny Brier. Kat Dennings is Best Known for her the Films like "The 40-year Old Virgin", and Thor, Thor: The Dark World. To get more Insights and Shocking Secrets about Kat Dennings do read the Blog.
Kat Dennings had an interest in Acting since she was a child and started her career by featuring in Commercials and Making Cameos on Small and Big Screens. Today she is famous as Best Sarcastic Max Black on CBS's 2 Broke Girls. Kat Dennings was Home Schooled by her parents, they didn't let her go to School and thus she completed her high school education at an age of 14 years. While she was Growing up studying in a small room with her parents she was not able to bear the situation, thus she took charge of her Studies. She took control of her studies and got graduated earlier than his.
Dollface — "Homecoming Queen" – Episode 208 — The girls face family dynamics. Jules helps her parents pack up her childhood home. Madison sees a new side of Ruby when she meets her music-industry-mogul father. Stella joins Liv for her son's family day at camp. Izzy takes a trip. Jules (Kat Dennings), shown. (Photo by: Jessica Brooks/Hulu)
After her graduation Kat decided to pursue her Career in Acting, her parents didn't give her approval at the beginning but decided to permit him to her Career in Acting. She started her career by doing a Debut on the Show "Sex and the City". Thus being a teenage star she had made her place in everyone's hearts.
In one of the Interviews, she also told that she remains away from all kinds of addictive substances and doesn't need any Foreign Stimulating substance to act in a certain way. Thus along with being an actress, she is a Talented Scriptwriter she has written a Script along with her brother Goeffrey Litwack on the 2008 Black List of "The Hottest Unproduced Screenplays of 2008.
Kat Dennings was born in Bryn Mawr, Pennsylvania on June 13th in the year 1986. Thus she was Home-Schooled by her parents and grew up in the United States.
kat dennings family
Her Mother's name is Ellen Judith Litwack who is a Poet and a Speech Therapist while her Father Gerald J.Litwack was a Molecular Pharmacologist, a College Professor, and a Chairman. Kat also has one brother named
Thor: The Dark World.
Nick and Norah's.
The 40-year-Old Virgin.
The House Bunny.
Big Momma's House.
Thor: Love and Thunder.
Suburban Gothic.
Charlie Bartlett.
Daydream Nation.
Raise Your Voice and more…
Kat Dennings is having the Networth of about $25 Million according to the year 2022 and thus she charges about a few million for her Role in any Movie while earning a massive amount of money through Brand Endorsements and Social Media Platforms. But as the Networth keeps on Changing based on one's Work.
Kat Dennings has about 4.9M followers on Instagram and thus the Instagram Followers keep on Changing, these numbers of followers are based on August 2022.
A post shared by Kat Dennings (@katdenningsss)
How old was Kat when she became famous? Find out the age at which Kat started her career and what is her age currently. Thus she was just 16 years of age when she got her first Debut role and according to the year 2022, her age is 36 years. Kat Dennings was born in the year 1986, thus her age according to the year 2022 is 36 years.
See Also – Who is Gama Pehlwan – Life Story, Kushti Video, Family Tree and Gama Pehlwan vs Dara Singh
Kat Dennings has dated several people some of them are:
1. Ira David Wood IV
From 2005-2008 Entertainer Ira David Wood IV is known to be the main serious sweetheart of the entertainer. The two were together for something like three years. The entertainer should be visible in the series One Tree Hill.
2. Matthew Gray Gubler
In 2008 After isolating from Ira David Wood IV, Dennings bthe egan a sentiment with the entertainer, movie producer, and model Matthew Gray Gubler. The man is generally known for featuring in the TV series Criminal Minds. The couple dated from January to August of that year.
3. Ryan Gosling
In 2009 The next year, the entertainer was supposed to have a throw with entertainer Ryan Gosling. The pair had been spotted out on the town at Disneyland. Probably, it was their first and the last date as no serious affirmation was given by both of them after their excursion.
4. Tom Hiddleston
Then, Dennings was again reputed to have hook-ups with another entertainer, Tom Hiddleston. It was the point at which the two of them joined the cast of the film Thor. Hiddleston featured as Loki there. Once more, not even one of them remarked on their conceivable issue.
5. Scratch Zano
Dennings and Nick Zano were sincerely connected to one another for a considerable length of time and a half. Zano is famous for his jobs in motion pictures, for example, Minority Report and One Big Happy. The two met on the arrangement of 2 Broke Girls and before long began dating. In any case, their affection wasn't bound to be everlasting and the entertainers separated, in actuality, similarly as their particular characters concluded a heartfelt connection was not so much for them.
6. Drake 2013
At the point when she was still involved with Zano, Kat was spotted going out on a sushi date with rapper Drake. In any case, was it a date? Nobody knows.
7. Josh Groban
When she split from Nick Zano, the entertainer began her sentiment with vocalist and musician Josh Groban. The two were acquainted with one another by her 2 Broke Girls co-star Beth Behrs. Dennings and Groban uncovered their relationship in October 2014, when they went to the Carousel of Hope Ball in Beverly Hills together. The caring birds were a particularly gorgeous couple, yet they threw in the towel eighteen months after the fact. It is known that Kat and Josh stayed in friendly conditions from that point forward
See Also – Stacey Park Milbern Wiki – Age, Height, Husband, Cause of Death and much more
2 Broke Girls.
Dollface.
Wanda Vision.
Big Mouth.
Raising Dad.
Dallas and Robo.
Thus this was all about Kat Dennings and her life, hope you enjoyed the Blog.
Tags: kat dennings 2 broke girls, kat dennings age, kat dennings boyfriend list, kat dennings instagram, kat dennings movies and tv shows, kat dennings net worth, kat dennings shows, kat dennings thor, kat dennings thor love and thunder
Who is Gama Pehlwan – Life Story, Kushti Video, Family Tree and Gama Pehlwan vs Dara Singh
Stacey Park Milbern Wiki – Age, Height, Husband, Cause of Death and much more
Famous Birthdays: Famous People, Celebrity, Sports Birthdays
The Daily Quirk
Your Daily Dose of Knowledge
Copyright 2018 - 23


text:
Q: using .find to find the space in a line Python im currently using this code
position= line.find(" ")

to help me find a position for example in the case of a name: Coner John Smith
how do i single out the smith, currently my line of code wont help because it positions itself on the first space it finds, how do rewrite the code to find the second space in a line?

A: Use find() twice:
firstspace = line.find(" ")
if firstspace != -1:
    secondspace = line.find(" ", firstspace+1)

The second argument to str.find() is the position to start searching from.

A: If you want the last space, use rfind or if you really want the last word, .rsplit() for it
>>> "Coner John Smith".rfind(" ")
10
>>> "Coner John Smith".rsplit(" ", 1)[-1]
'Smith'

If you want the Nth space, you can repeatedly locate with .find(), which can accept a start arg for where to begin finding!
source_text = "Coner John Smith"
N = 2

index = -1  # search from index 0
for _ in range(N):
    index = source_text.find(" ", index + 1)
    # opportunity to discover if missing or `break` on last, etc.

A: The answer to your literal question is:
position= line.find(" ", line.find(" ") + 1)

What this does is find the first space in line, and then find the first space in line, starting the search on the position of the first space, plus one. Giving you the position of the second space.
But as @barmar points out, this is probably an example of an XY problem
Do you want the second space? Or the last space (e.g. 'Mary Jo Suzy Smith')?
Do you want the position of the space? Or are you really just after the last word in the string? Do you care about any interpunction following the last word in the string (e.g. 'John Smith!')
Every case has a slightly different better answer.
To get the last word, you could:
last_word = line.split(' ')[-1]

To find the last space, if you need it for something else:
last_space = line.rfind(' ')

Etc.

redpajama_set_name: RedPajamaStackExchange


This year's workshop in Kenya is customised for modern businesses looking to stay at the top of their game.
Our session focuses on enabling your business's digital journey across multiple industries with a focus on financial services, insurance, telecommunications industries, and more. We also give our attendees the opportunity to share the innovations they are eager to see in the future.
We have invited Diamond Trust Bank to discuss their digital journey case study with attendees, and have also included our partners AIS, Impressions, and Wi-Group to share their exceptional offerings in the realm of business digitisation.
We are excited to join our Kenyan Team at this unique digital journey workshop and looking forward to sharing some insights from the event with you soon.


A quality card of a Hare with a pale blue background taken from Julie Weir's original painting titled 'Holding back the 'ears' . The card comes blank inside for your own message and has a white envelope. The size is 7" x 5".


These are all images I have created with Adobe programs. They are combinations of images I have made from my camera, the internet, and illustrations. These images were made in and out of my class assignments.


text:
Jimmy Fallon and Andy Samberg Kick off Facekickers, Brand New Game on 'Tonight Show'
Published September 11, 2015 • Updated on September 11, 2015 at 6:59 am
In honor of the NFL opener on Thursday, "Tonight Show" host Jimmy Fallon by playing a brand new game with guest Andy Samberg.
Facekickers involved a mock football field, six footballs and six tiles hanging over the goal post - three with Samberg's face, and the other three featuring Fallon's likeness.
The object of the game was to be the first one to smash all his opponent's faces by field kicking the footballs.
The "Tonight Show" host gave Samberg the first kick.
It wasn't until his third and last kick that Samberg managed to hit a tile. But it was his own. Fallon made the next kick - only to hit his own tile, too.
Fallon then invited Carrie Underwood to take a shot. She missed her first three kicks, and even missed when she tried to throw the football at a close distance.
Fallon declared Samberg the very first Facekickers winner.


Located in The Arcade, Barnsley oozing traditional charm, Aroma Café Bar invites you into its modern, chic surroundings, enticing you with a freshly cooked, mouth-watering menu of delicious food and wide selection of refreshments!
Having built an enviable reputation for its relaxing atmosphere and excellent service, Aroma Café Bar is everyone's favourite choice whatever the occasion! Business meeting, catch up with friends or a celebration? Make it memorable, relax and enjoy!
P.S. Check out our tapas menu served every Friday and Saturday and watch out for our live music nights, they promise to be a fabulous treat!
Summer is fast approaching and the Aroma Café Bar are getting into the Mediterranean mood with our all new Tapas menu.


Greenwich University is duly recognized by the Higher Education Commission (HEC), Pakistan, as an autonomous degree-awarding university and by the Tertiary Education Commission (TEC), Mauritius, as an accredited University.
Singles. The album's lead single and the title track "Love After War", was released on October 11, 2011. It officially impacted U.S. Urban radio on October 18, 2011.
C-COR Broadband is the key supplier of products and services to all major Australian Telcos and MSOs as well as majority of electrical wholesalers.
Names. Abu Bakr al-Baghdadi is the nom de guerre of an individual who has had various names and epithets attributed to him, including Abu Du'a (أبو دعاء ʾabū duʿāʾ), Al-Shabah (the phantom or ghost), and Abu Bakr al-Baghdadi al-Husseini al-Hashimi al …Abdullah Al-MattrodTraduire cette pagehttps://www.mp3quran.net/eng/mtrod_english.htmlAudio Quran library :A 22 language site contains a large variety of reciters, with direct high quality links to download any surah.In addition to radio broadcast, torrent service, direct file play, Mecca live channel and Surah Al-Khaf.
Previewing the launch of NASA's "InSight" mission to Mars on Sky News "Sunrise", 5 May 2018 I was interviewed on BBC 3 Counties Radio on the evening of InSight's landing on Mars on November 26.
Audio Quran library :A 22 language site contains a large variety of reciters, with direct high quality links to download any surah.In addition to radio broadcast, torrent service, direct file play, Mecca live channel and Surah Al-Khaf.
Pakistan-based militant group Jaish-e-Mohammad claimed the Pulwama attack which killed 46 soldiers.
Deciphering Daesh-Origins,Impact and Future Daesh's Organisational Structure. Mosul's takeover by Daesh, also known as the Islamic State or IS, prompted a flurry of questions on reasons for the group's overwhelming military triumphs against official state armies as well as armed militias.
"Trombone Shorty" illustration by Bryan Collier. Black males continue to be underrepresented, misrepresented, or invisible in all forms of children's media, including children's books.
Article by Wael Wael Abdelgawad is an Egyptian-American living in Fresno, California. He is the founder of several Islamic websites, including Zawaj.com and IslamicAnswers.com, and also of various technology and travel websites.


A bill (SB-114) to remove all state tests but those required by the federal government was passed by the Senate today and will now go to Gov. Perdue's desk for her signature.
The measure, spearheaded by Senator Austin Allran (R-Catawba) recently passed the House. The bill would eliminate four end-of-course tests for high school students – U.S. History, Civics and Economics, Algebra II and Physical Science.
The issue has created some interesting coalitions. Republicans who have been traditionally supportive of strong testing and accountability measures are leading the charge to eliminate the testing requirements. Teaching to the test is the most often heard criticism of the current system. Representatives from the North Carolina Association of Educators have joined Republicans in supporting the measure.
Proponents of the bill say they agree some testing is necessary to gauge academic progress. However, they aren't sure the current ABC testing system is the right one. Another common complaint against many North Carolina tests is that they are not nationally-normed.
Meanwhile, many Democrats are lining up against the measure, saying testing is necessary to identify which schools are failing so resources can be distributed accordingly.
Interestingly, Wake County Superior Court Judge Howard Manning has said that dumping the state tests would violate a student's rights under the state Constitution. Manning authored the famous Leandro v. State decision that all children have a constitutional right to a "sound, basic education. Manning has presided over a serious of cases that have sought to add clarity to the decision.
Complicating the current debate is North Carolina's Race to the Top grant. Many believe, as currently written, the grant requires the state to continue using the current testing system. Under terms of the grants, North Carolina will receive approximately $350 million over four years.
It is not known whether Gov. Perdue will sign the bill.


On Wednesday 30 July Folksy Theatre will bring their open-air production of Shakespeare's The Taming Of The Shrew to Marwood Hill Gardens. The performance forms part of their national tour.
Set in 1950's Britain, the famous comedy is filled with doo wop, combed back hair and fedoras; it's The Shrew as you've never seen it before!
Using minimal set, Folksy remain true to the roots of open-air theatre, relying on the natural beauty of Marwood Hill Gardens.
Bring along a picnic and your family and friends to enjoy the wonderful production.
The show begins at 6.30pm.
Tickets cost from £8 if pre-booked and from £10 on the door.


After the turkey and stuffing are put away on Thanksgiving night, bring your out-of-town guests to Historic Downtown Clarksville for family entertainment sure to get everyone in the holiday spirit.
Altra Federal Credit Union proudly presents Meredith Willson's MIRACLE ON 34TH STREET at the Roxy Regional Theatre, opening Thursday, November 22, at 7:00pm. In keeping with the theatre's traditional pay-what-you-can opening night, all tickets not pre-sold at the regular ticket price will go on sale at 6:30pm that evening for a $5 minimum donation.
Adapted into a musical by the creator of THE MUSIC MAN, Meredith Willson's MIRACLE ON 34TH STREET follows Kris Kringle as he takes on the cynics in this beloved holiday classic. Single mother Doris Walker doesn't want her six-year-old Susan's head filled with romantic notions. When their neighbor, Fred Gaily, tries to woo Doris by charming Susan and taking her to see Santa Claus at Macy's, where Doris works, Doris is not impressed. But when it turns out that Macy's Santa may, in fact, be the real Kris Kringle, a wave of love spreads across New York City that melts even the most cynical hearts.
Starring Mia Bergstrom as Doris Walker, Stephen Shore as Fred Gaily, Brian Best as Kris Kringle and Ellie Thomas as Susan Walker, MIRACLE ON 34TH STREET also features Demi Ahlert, Ian Alexander Erbe, Sebastian Fenton, Emily Goad, David Graham, Rhyen Kretz, Robert Murray, Rachel Musson, Rachel Sherman, Mikquala Skelton and Kyle Smith, with piano accompaniment and music direction by Matthew McNeill.
On Friday, December 7, patrons are invited to ring in the season at the eighth annual "Christmas Over The Cumberland" at F&M Bank's Franklin Room, featuring a holiday dinner, festivities and meet-and-greet with the cast. The evening's menu includes all the holiday favorites: turkey and dressing, ham, sweet potato casserole, green beans, rolls, salad and assorted holiday desserts. Doors open at 6:00pm, with dinner served at 6:30pm, followed by the 8:00pm performance of MIRACLE ON 34TH STREET at the Roxy Regional Theatre. Tickets are $40, including the show. Reservation deadline is November 30.
Directed by Ryan Bowie, MIRACLE ON 34TH STREET is based on the 20th Century Fox picture, with story by Valentine Davies and screenplay by George Seaton, and is presented by special arrangement with Music Theatre International (MTI).
In addition to Altra Federal Credit Union, funding support for MIRACLE ON 34TH STREET has been provided by Kay & Randall Haase, Randy & Su Whetsell, John & Cathy Stanton, Barbara & Larry Goolsby, James G. Holleman, Dr. Ben & Erin Webber and Elite Primary Care.
Performances run November 22 through December 22 on Thursdays at 7:00pm, Fridays at 8:00pm and Saturdays at 2:00pm and 8:00pm. There is no 8:00pm performance on Saturday, December 22.
Tickets are $25 (adults) and $15 (ages 13 and under) and may be reserved online via the below button, by phone at (931) 645-7699, or at the theatre during regular box office hours (9:00am to 2:00pm, Monday through Friday, and one hour prior to curtain). APSU students, military and CitySaver coupon holders can receive two tickets for the price of one to Thursday performances during the run.


text:
Q: Хедер в одну линию В хедере должна быть картинка, текст и меню с ссылками, меню и текст встали в одну линию, а вот картинка находится выше их, хотя они все находятся в одном блоке div и должно работать display:inline-block;
Что я делаю не так?

.slider {
  background-image: url('images/slider-bg.jpg');
  width: 1920px;
  height: 1024px;
}
.header {
  overflow: hidden;
  width: 100%;
  height: auto;
  margin-left: 150px;
}
.header div {
  display: inline-block;
}
#logo h1 {
  color: black;
}
#navigation {
  margin-left: 50px;
}
#navigation ul {
  list-style-type: none;
  margin: 0;
  padding: 0;
  overflow: hidden;
}
#navigation li {
  display: inline;
  color: white;
  text-align: center;
  text-decoration: none;
  padding: 10px;
}
#navigation a:link {
  color: black;
  text-decoration: none;
}

Waxom

        Home

        About Us

        Portfolio

        Features

        Blog

        Pricing

        Shortcodes

        Contact

A: Свойство display не наследуется, поэтому для дочерних элементов его надо задавать.
В вашем случае его на задать для #logo h1

.header {
  overflow: hidden;
  width: 100%;
  height: auto;
  margin-left: 150px;
}
.header div {
  display: inline-block;
}
#logo h1 {
  color: black;
  display: inline-block;
}
#navigation {
  margin-left: 50px;
}
#navigation ul {
  list-style-type: none;
  margin: 0;
  padding: 0;
  overflow: hidden;
}
#navigation li {
  display: inline;
  color: white;
  text-align: center;
  text-decoration: none;
  padding: 10px;
}
#navigation a:link {
  color: blue;
  text-decoration: none;
}

Waxom

        Home

        About Us

        Portfolio

        Features

        Blog

        Pricing

        Shortcodes

        Contact


text:
Q: Precise anchoring of shadows on entities I would have a question to ask, hoping for your advice. In my project I'm working on the different entities that will be present, one of them is the "TreeEntity". It is visibly composed of four parts. "Leaves, Shadow, Stump, Trunk". All four parts have only one id and are randomly chosen when a new "TreeEntity" is called. In addition, leaves and shadow each have four different states, based on the age of the tree. The shadow will have to rotate around the trunk based on the time. Now, since the engine of my project would like to create it as universal as possible, so that it can also be used in other projects or other people, I am facing a dead end.
I have this result at the moment (premise, all the images used are for testing purposes only):

As can be seen, the shadow is very detached from the trunk. This, theoretically, would be possible to fix it, modifying the image used:

Or write a huge dictionary with all the precise anchors written for each image that make up leaves, shadow, stump and trunk.
But doing so would be detrimental to my goal. So, the advice I would like to ask you, is whether in pyglet or otherwise, regardless of the shadow used (which in this case is random) is it possible to choose an anchor point that places it in the right place? Or do you have to modify the image or create a dictionary?
My question arises from the fact that I have seen several times images for example of shadows or something else that is like the one I inserted here, but in that case the shadows were in their correct place. The same game from here I took the pictures to test and I'm inspired by one or two mechanics has the shadows placed in the right place. So I don't know which way to go sincerely.
At the moment, all four parts have a universal anchor dictated by a small dictionary:
__statdict = {
        "leaves": {
            "cell": (3, 1),
            "anchor_x": "center",
            "anchor_y": "bottom"
        },
        "shadow": {
            "cell": (4, 1),
            "anchor_x": "left",
            "anchor_y": "bottom"
        },
        "stump": {
            "cell": (1, 1),
            "anchor_x": "center",
            "anchor_y": "bottom"
        },
        "trunk": {
            "cell": (1, 1),
            "anchor_x": "center",
            "anchor_y": "bottom"
        }
    }

And when loading resources, anchor is assigned:
if self.__statdict[name]["anchor_x"] == "center":
    ancx = texture.width // 2
elif self.__statdict[name]["anchor_x"] == "right":
    ancx = texture.width
else:
    ancx = 0
if self.__statdict[name]["anchor_y"] == "center":
    ancy = texture.height // 2
elif self.__statdict[name]["anchor_y"] == "top":
    ancy = texture.height
else:
    ancy = 0
texture.anchor_x = ancx
texture.anchor_y = ancy

I have tried to be as complete as possible. Thanks for your help. Maybe maybe I'm having problems for nothing and the solution is simpler than I thought, so I apologize if this were the case.
Update (1):
Is it possible, via pyglet or a module to accompany it, given the direction of a sprite (in this case on the right), to know what is the position of the first pixel that is not an alpha? Something like this: "The direction of the SpriteShadow is to the right. The first pixel that has an rbg value with alpha equal to 255 is ay = 20 (relative to the width of the sprite). Then I move the SpriteShadow to the left with respect to the general anchor of 20 pixels "


Some good news to report regarding consumer protection. And news that it is illegal to imply that other therapeutic goods, including vaccines, are harmful.
Unfortunately, it has become all too common commonplace that SCAMs (Supplementary, Complementary and Alternative Medicine) make unsubstantiated claims about their products and services.
It has also become commonplace that they get away with making such claims, seemingly without consequence.
"Health practitioners not to make claims to cure certain serious illnesses. (1) A health practitioner must not hold him or herself out as qualified, able or willing to cure cancer and other serious illnesses".
So it was a surprise to see this happen today.
This is the work of a sceptical colleague, Michael, who issued a lengthy complaint to the Complaints Resolution Panel of the Therapeutic Goods Administration some time ago. Recently he received a 9 page facsimile advising that his complaints about Arnica Montana, a purveyor of homeopathy, regarding breaches of section 4(1)(b), 4(2)(a), 4(2)(b), 4(2)(c), 4(2)(d), 4(2)h, 4(2)(i), 4(7) and 5(2) of the advertising code were deemed valid.
Arnica Montana Enterprises Pty Ltd were instructed to comply with the following sanctions: Withdrawal of Advertisement, Withdrawal of Representation and Publication of Retraction. This involved publishing a retraction on the website which appears below.
The advertiser was given an opportunity to respond to the criticisms, and rejected the allegation that there was "anything deceptive or misleading" in the advertisement/website. The advertiser claimed that the website was "intended to be an information website to educate the public about the use of homeopathic remedies" but not to the exclusion of any other system of medicine.
But The Panel found that the advertisement breached sections of the code which prohibit advertisements for products that "abuse the trust or exploit the knowledge of consumers" and was "likely to arouse unwarranted and unrealistic expectations of product effectiveness".
This was based on the finding that "the advertisement included claims of benefits in relation to burns, scalds, pain, traumatic shock, severe burns….no persuasive evidence was provided that the advertised products could have benefits in relation to the wide range of conditions referred to".
Evidence? Why do you need evidence? The advertiser stated "controlled trials cannot be used for homeopathy but [that] there is a mass of unpublished evidence" and stated that they "would be grateful to know which section of the Code requires published scientific evidence as a precondition to advertising". Besides they have testimonials all over their website…but wait, they were called into question too.
The Panel stated that the advertiser did not provide evidence that any of the testimonials in the advertisement were genuine.
"the calendula cream I make is specific to cancer skin keratoses and I have successfully treated many of these and established melanonas by using the sage cream at night and the calendula during the day".
Interesting that it unlawful to publish testimonials that are not genuine or "atypical". This could apply to many a woo website.
If you search the website for vaccination, you will get hits for the Australian Vaccination Network (AVN), Meryl Dorey and other misinformation that we have come to expect from the anti-vaxers. (Who would have thought, a homeopath condemning vaccination). I wonder where does the AVN website sit with respect to this? Would their website be deemed an advertisement? After all, they do sell literature and DVDS, but I suspect these products do not come under the auspices of "therapeutic goods".
An advertisement is defined by The Code "…to include any statement, pictorial representation or design, however made, that is intended, whether directly or indirectly, to promote the use or supply of the goods".
This is why the entire arnica montana website was deemed an advertisement; the Panel noted that it was clear that the website offered a range of products for sale, and was satisfied that the website as a whole constituted an advertisement for therapeutic goods.
Interestingly, the complaint summary cites the subject matter of the complaint as "website advertisement" and the sanctions as "withdrawal of advertisement", thus implying the entire website is to be withdrawn. If you go to the website, you will see the published retraction, but the remainder of the website still functions as normal.
And if you think there's no harm in homeopathy, then see this story.
« A register for quacks and bad science journalism.


text:
08448 733 525 info@businessgrowthclub.co.uk
Be a Tipper
Small Business Development and Strategy Expert
Want to be a visitor?
info@businessgrowthclub.co.uk
Small Business Development and Strategy Expert starts at 7.00, 11 October, 2019
Mark Kiteley - Business Development & Strategy - 7am until 9:30am October 11, 2019 Abbey Hill Golf Centre
'10 Minute Tip' by Mark Kiteley - Business Development & Strategy - 7am until 9:30am
Mark Kiteley, started his first company in electronics in 1978 in the hamlet of Hulcote and gave it the very original name of Hulcote Electronics. He sold it in 1992, stayed on as a divisional director and saw the business sold again in 1999.
Mark then worked for a number of other companies building them up from very little to quite a lot, until he was appointed a director of Wallgate Services which in April 2008 was an AIM listed company with a value of £38M. By November it was bust!
It was at that point that Mark realised he had been running companies for over 30 years and had no idea what he was doing. So Mark went back to school and now holds the Diploma in Company Direction from the Institute of Directors.
Mark is and experienced Executive Director with expertise in Service Management, Reverse Logistics, Quality Management Systems, Internal Audit processes, and Business Development. Mark particularly enjoys lateral development of a core business to build strength and sustainability in the rapidly changing business environment.
Mark is also an experienced Non Executive Director in a number of businesses, including the public sector. He has over 10 years experience in Audit, Corporate Governance and Strategic Planning.
Mark currently runs his own company, Garmar Services and divides his time between "making stock disappear", and business consulting on Strategy, Risk, Audit and Business Development.
Mark has helped SME's from, one person bands up to £50,000,000 organisations with 200 or so employees. He's worked with owner managers, board directors and those with responsibility for Strategy. He's also consulted with non-Exec's needing independent insight into their businesses.
on your attendance costs »
About the conversation:
Imagine you're sitting on a 3 legged stool. The seat of that stool is your business and you're on top of it.
You're contemplating the success of your business and how it supports you. You're in perfect harmony with your work life balance. Leading and managing your business is effortless.
The three different legs of your stool each have name. They're called Strategy, Risk and Audit. If you take any one of those legs away, you'll become unbalanced. Let's face it, you probably started out unbalanced which is why you went into business in the first place.
Now you'll have to work really hard and use lots of energy just to avoid falling over. You'll be flailing your arms around like mad trying to stay balanced. Eventually, you'll have to put your legs down to restore your balance. At that point you are no longer on top of your business, it's on top of you. Your business won't be supporting you. You will be supporting your business.
In his 10 minute tip Business Development and Strategy Expert, Mark Kiteley will demonstrate the close relationship of these three legs of your business, Strategy, Risk and Audit. He will explain how executing these three key activities will enable you to stay on top of your business and remain calm and in harmony with your world.
Business Networking Meeting
Past events »
Be a '10 Minute Tipper »
Difference in business networking » About business networking »
Our highlighted member:
Stellar Building Consultancy Ltd
: Is it worth writing a business or self help Book? 7 months ago
Mark Orr: Remember, with Business Networking in Milton Keynes you're not alone! 7 months ago
Mark Orr: Don't Sh** on Your Own Doorstep 7 months ago
Copyright 2015 - web: Addmonte Ltd.


text:
Next_TV_Black_Vertical Copy Created with Sketch.
Broadcasting + Cable
Multichannel News Multichannel News
Search Multichannel News
MCN Wonder Women
MLB Clubs 'Crying Poor?'
Peacock Plucks Free Tier
Margaret Brennan's Influence
MCN Wonder Women of NY
Hispanic Television Summit: Salinas: An Impassioned Advocate
By MCN Staff
New York -- At the 11th annual Hispanic Television Summit here on Oct. 2, Multichannel News and Broadcasting & Cable honored longtime Univision journalist and co-anchor Maria Elena Salinas with the Outstanding Achievement Award in Hispanic Television.
The following is an edited version of her speech during the awards luncheon at the New York Marriott Marquis, which followed a video tribute to the veteran newscaster..
I wasn't expecting to see my daughters up there; that's a tough one for me. My oldest daughter, you can tell, is in her dorm and she probably just woke up (laughter). It's probably two o'clock in the afternoon. This is the first time we separate; she's at college. It's very special. Those of you have teen-aged kids know how difficult it is for them to say. 'Mom, I'm proud of you.'
I'm really, really honored to be here, and getting this recognition for doing something I really, really love and have been doing for a long time. Thirty years. You can tell, my hairdos, different styles (laughter). It's actually 32 years, but you know how people take off their age. Well, I'm trying to do that, to get you to think that I started when I was eight-year-olds (laughter). When I read the article that Laura Martinez so kindly wrote -- it was very, very nice -- one of the things that jumped out at me was the notion of advocacy journalism.
It's something I've had to defend so many times and I don't understand why I need to defend it, as if it were bad or immoral, as if it were unethical journalism. I have never seen it that way. As journalists, of course, our job is to seek the truth, to question those in power, to inform in an objective manner. Or should I say to be truly fair and balanced in our reporting. And I think that's what we've done with Spanish-language communications and at Univision, I know we do that.
But we go beyond that, because our audience has additional needs. Yes, they care about what's going on in their city, their state, in their country of origin if they're immigrants, or their newly adopted country, which is here. So I learned very early on in my career that we had a very special audience with very special needs. And I knew my job was going to be different than those who worked in mainstream media.
Like many journalists, I learned by being out in the streets, covering stories and taking advantage of life lessons in the field. It was many years ago in the early '80s, covering a local election that the Latino community would have the first opportunity in decades to elect someone who would represent them, as a Latino. At that time, Latinos were 25% of the population in Los Angeles. When I went out to speak to the people on the street, out of the 16 people I interviewed, 15 of them weren't voting. They weren't voting because they were not citizens, or weren't registered or because they didn't know there was an election that day. I remember going back to my office and talking to my news director, Pete Moraga -- rest in peace, he taught me so many things -- and I said, 'I can't do this story, nobody's voting.' 'Well, he said, 'there's your story, it's right in front of you. Nobody's voting because they're so disenfranchised from the mainstream. They feel so disconnected that they're not participating in the political process.'
That was one of those 'ah, ha' moments, where I realized that it was a wake-up call. That part of my job at KMEX at that time, and for those working in Spanish-language media, was to give our audience the information. So they could feel they are part of the society, so they would know their rights, but also their civic responsibilities.
In other words, the political empowerment of the Latino community became my mission. That's why it's so important to report on those things. It's not that my colleagues and I are taking a position on immigration reform or advocating for open border policy. It's that we're giving a voice to those who otherwise would not have a voice. You would not hear their voice in mainstream media. I'd like to say we contribute up to a certain extent to the democracy in this country, because we're contributing to creating a healthy dialogue for immigrants in this country. Otherwise what you would hear would be a monologue blaming immigrants for all the ills of this country.
Is my mission accomplished? I'm not going to say my mission is accomplished. I want to continue to work (laughter). But what I would say is that we have come a very long way. It's interesting because when I started there were 14 million Latinos in this country, now there are over 52 million Latinos. And I think the Spanish community, the Spanish-language media and my own career have grown together, hand in hand. We've grown hand in hand and I have this recognition to show for it.
Then, if I've been accused of doing advocacy journalism, I don't mind. Thank you (applause).
Multichannel Newsletter
The smarter way to stay on top of the multichannel video marketplace. Sign up below.
MCN Staff
Thank you for signing up to Multichannel News. You will receive a verification email shortly.
Season 10 of 'The Blacklist' Will Be Final One
Six Nations Rugby on Peacock and CNBC Starting February 4-5
GOP Senators Seek Info From DirecTV on Newsmax Drop
Bad Timing: YouTube TV Blackout of MLB Network Compounds Baseball's TV Distribution Problems Entering Spring Training
T-Mobile Adds 524K FWA Customers in Q4, But Growth Suddenly Slows
Multichannel News is part of Future plc, an international media group and leading digital publisher. Visit our corporate site (opens in new tab).


Cambridge Assessment International Education(CAIE) is the world's largest provider of internationally recognized academic qualifications for 14 to 19 years old, which incudles IGCSE, GCE O-Level and GCE A-Level. St. John's International School is registered CAIE school and examination centre, which offer the CAIE Examination, examination infosheet, registration materials and important examination notices.
Starting 2019, SJIS has open Malay Foreign Language Examination with Oral to all private candidates.
All original copy of Statements of Entry will be available for collection for May–June 2019 session starting 25th February 2019.
Official Certificate & Statement of Results for CAIE May-June 2018 session is available for collection.
The Exam Venue for Candidates sitting for AS Paper 35 Practical paper are finalised. Please contact Exam department for details.