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Retinal Cell Biology | May 2000
Differential Expression of Neuroendocrine-Specific Protein in Form-Deprived Chick Eyes
Shigeki Fujii; Michael F. T. Escaño; Kazuki Ishibashi; Motoko Fujii; Yoshibumi Sekiya; Misao Yamamoto; Kiyofumi Saijoh
Shigeki Fujii
From the Department of Ophthalmology, and the
Michael F. T. Escaño
Kazuki Ishibashi
Motoko Fujii
Laboratory of Microbiology, Kobe University School of Medicine; and the
Yoshibumi Sekiya
Misao Yamamoto
Kiyofumi Saijoh
Department of Hygiene, Kanazawa University School of Medicine, Japan.
Investigative Ophthalmology & Visual Science May 2000, Vol.41, 1533-1541. doi:
Shigeki Fujii, Michael F. T. Escaño, Kazuki Ishibashi, Motoko Fujii, Yoshibumi Sekiya, Misao Yamamoto, Kiyofumi Saijoh; Differential Expression of Neuroendocrine-Specific Protein in Form-Deprived Chick Eyes. Invest. Ophthalmol. Vis. Sci. 2000;41(6):1533-1541.
purpose. To identify genes that are highly expressed in form-deprived retina–retinal pigment epithelium–choroid tissues. Neuroendocrine-specific proteins were found to be highly expressed.
methods. mRNAs enriched in retina–retinal pigment epithelium–choroid tissues from 3-, 7-, and 14-day form-deprived chick eyes were isolated by differential display technique with cDNA library screening. Neuroendocrine-specific protein A and C were cloned in control and form-deprived eyes. mRNA and protein levels, with respective regional localizations, were examined by Northern blot, Western blot, and immunohistochemical analyses, respectively.
results. The isolated clone included an insert with a sequence homologous to both chick neuroendocrine-specific proteins A and C. The increases in mRNA and protein levels were confirmed by Northern and Western blot analyses, respectively. Immunohistochemical localization of neuroendocrine-specific proteins A and C was detected in the layer of photoreceptor inner segments, presumably in the cone cells. Northern blot analysis using negative lenses showed that levels of neuroendocrine-specific protein A and C mRNAs were not altered using negative lenses.
conclusions. The expression of both neuroendocrine-specific proteins A and C mRNAs in cone photoreceptor cells was upregulated within 14 days of form deprivation, but not in response to negative spectacle lenses. These data suggest that the increase in induction of neuroendocrine-specific proteins is not a secondary consequence of ocular elongation or myopic refraction. Induction of neuroendocrine-specific proteins in form-deprived eyes may be causally related to the development of myopia or may be an unrelated effect of form deprivation.
Form-deprivation myopia (FDM) in animal models has been used extensively in research on myopia. 1 2 3 4 In chicks, occlusion of the eye during the early postnatal ocular development period impairs form vision and leads to myopia. 5 6 7 8 9 Eye enlargement stems from vitreous chamber elongation, the molecular mechanism of which is not well understood. Recently, transforming growth factor (TGF)-β, 6 basic fibroblast growth factor, 10 11 and three nitric oxide synthase (NOS) isoforms, 5 inducible NOS, brain NOS, and endothelial NOS, were reported to be involved in the development of FDM.
Isolation and identification of genes expressed in form-deprived ocular tissues may aid in the molecular evaluation and understanding of possible mechanisms involved in FDM. Such an attempt also makes it possible to identify genes involved in postnatal ocular development.
To identify which mRNA transcripts are more abundant in retina–retinal pigment epithelium (RPE)–choroid tissues from form-deprived eyes, a differential display method 12 was used with cDNA library screening using the chick spinal cord. One clone, designated pM29, contained the cDNA insert with a sequence homologous to neuroendocrine-specific protein (NSP)-A and -C mRNAs. NSPs in human subjects were first cloned as genes with a neuroendocrine-specific expression pattern. 13 14 Although three isoforms were found in humans, only two isoforms, NSP-A and -C, were cloned in the embryonic chick brain tissues. 15 These isoforms were found to be associated with the endoplasmic reticulum, 14 16 17 although their physiological roles and distribution in chick ocular tissue has not yet been investigated.
We induced myopia using two well-known techniques, form deprivation by goggles and negative spectacle lenses, and examined the expression pattern of NSP and its possible relation to the development of myopia.
Induction of Myopia
Chicks (male white leghorns) were obtained from a local hatchery (Shiroyama Hatchery, Hyogo, Japan) and maintained at 30°C under a 12-hour alternating light:dark cycle (6 AM–6 PM light; 6 PM–6 AM dark). All the chicks were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Form Deprivation or Lens-Induced Myopia
Form deprivation was induced by fixing translucent plastic goggles over the right eyes of the chicks, as described elsewhere. 5 Briefly, hemispheric goggles cut from the bottoms of 15-ml plastic test tubes were attached to the right eyes with cyanoacrylate adhesive. The left eye served as the internal control.
Lens-induced myopia was accomplished according to a previously reported method. 18 Briefly, −15-D lenses made from poly(methyl methacrylate) (PMMA) material with a back optic radius of 7 mm and an optic zone diameter of 10 mm were used. Lenses glued between rigid plastic, and Velcro support rings were attached to the right eyes with cyanoacrylate adhesives. Lenses were cleaned every 3 hours during the 12-hour light cycle. The left eyes served as an internal control.
RNA Isolation
Ten chicks were killed between 10 AM and 1 PM. Eyes were enucleated and divided into anterior and posterior hemispheres. The vitreous and sclera were gently cleaned out. For differential display method, the retina–RPE–choroid tissues from both the control and 7-day form-deprived eye were used. For Northern and Western blot analyses, retinas including the RPE carefully separated from choroid were used. Total RNA was obtained by guanidine-isothiocyanate-cesium chloride precipitation, followed by phenol and chloroform-isoamyl alcohol purification and ethanol precipitation. 19 The final RNA preparations were dissolved in diethyl pyrocarbonate–treated water. The amount of the purified RNA was determined by spectrophotometry (optical density 260/280; ratio higher than 1.9).
Calibration of the Amount of Total RNA
A mixture of 0.1 μl total RNA (1 μg/μl) and 2.5 μl oligo(dT) (20 picomoles/μl) was incubated at 65°C for 10 minutes and then cooled on ice for 5 minutes. After 10 μl 5× reverse transcriptase buffer (Toyobo, Osaka, Japan), 5 μl 2.5 mM dNTP, 1 μl RNase inhibitor (Toyobo), and 1 μl 200 U/μl MMuLV (Toyobo) to a total volume of 50 μl were added, the mixture was incubated at 37°C for 1 hour and then at 95°C for 5 minutes to inactivate the reverse transcriptase. The amount of RNA was controlled by monitoring the intensity of ribosomal RNA bands on a 1% agarose-formaldehyde–ethidium bromide gel. Furthermore, variation in the amount of RNA was minimized by monitoring the expression ofβ -actin mRNA, a housekeeping gene. No alteration in β-actin mRNA in FDM was confirmed by reverse transcription–polymerase chain reaction (RT-PCR; data not shown) using a set of original primers designed from a published sequence (sense primer; TAAGGATCTGTATGCCAACACAGT and antisense primer; GACAATGGAGGGTCCGGATTCATC). 20 The primers were synthesized by the Biologica Co. (Nagoya, Japan) and purified by precipitation. Five-microliter aliquots of the RT mix were added to the PCR master mix consisting of 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 1 U rTaq polymerase (Takara, Shiga, Japan) and 0.2 μM each of β-actin primer. The PCR cycle (performed in a thermocycler; Takara) consisted of 1 minute at 94°C and 1.5 minutes at 72°C. The sequence of the amplified products was confirmed as corresponding to chick β-actin cDNA after force-cloning into pGEM-T (Promega, Madison, WI).
Differential Display
Differential mRNA display was performed using kits (RNAimage; GenHunter, Brookline, MA) according to the manufacturer's instructions. In both the reverse transcription and PCR steps all the reaction reagents were prepared as master mixes and then aliquoted equally into individual tubes to provide uniform reaction conditions and minimize intertube variation. Briefly, 0.2 μg total RNAs from retina–RPE–choroid tissues of control and 7-day form-deprived eyes were reverse-transcribed for 1 hour at 37°C and incubated for 10 minutes at room temperature in a final volume of 20 μl containing 25 mM Tris-HCl (pH 8.4), 2.5 mM MgCl2, 37.5 mM KCl, 5 mM dithiothreitol, 20 μM of each dNTP, and 100 U MMuLV reverse transcriptase with 2 μM of T12G, T12A, or T12C (GenHunter) as primers. Two-microliter aliquots of the RT mix were added to the PCR master mix consisting of 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 2 μM dNTP, 0.25 μl ofα[ 32P] dATP (2000 Ci/mmol; Amersham, Buckinghamshire, UK), 0.2 μM each of T12G, T12A, or T12C (GenHunter) as a 3′ primer, 0.2 μM each of the arbitrary 13mers (AP 49–56; GenHunter) as the 5′ primer, and 1 U rTaq polymerase (Takara) to a final volume of 20 μl. After an initial incubation of 7 minutes at 94°C, 40 cycles of PCR amplification were performed (94°C for 30 seconds, 40°C for 2 minutes, and 72°C for 1 minute, followed by a final primer extension step at 72°C for 7 minutes). Two microliters of formamide containing 0.01% bromphenol blue and xylene cyanol were added to 3.5 μl of each PCR mixture and incubated at 80°C for 2 minutes. The fragments were size-separated in a 6% denaturing polyacrylamide gel in TBE (Tris base, boric acid, Na2 EDTA 2H2O) buffer. 19 The gel was blotted on a piece of nitrocellulose hybridization filter (Schleicher & Schuell, Dassel, Germany) wrapped in a plastic sheet, vacuum dried, and exposed to x-ray film (Amersham) for 16 to 48 hours.
The fragments reproducibly showing higher expression in the samples from form-deprived eye than those from control were excised from the gel with a clean razor blade, soaked in 100 μl distilled H2O for 10 minutes, and then boiled for 15 minutes. The extracted PCR products were ethanol precipitated, and then dissolved in 10 μl distilled H2O. Four-microliter aliquots were subjected to reamplification using the same procedures mentioned earlier, except for the dNTP concentration of 20 μM. Aliquots (0.5 μl) of reamplified PCR products were separated on 1.5% ethidium bromide–stained agarose gel and were confirmed to consist of a single band. After unincorporated nucleotides were removed by a Sephacryl column, the remainder of the products were used as probes for cDNA library screening.
Library Screening
A commercially available primary chick spinal cord cDNA library (Stratagene, La Jolla, CA), specific for chick tissues, was used for screening of the posthatch chick eye. The library was diluted in a suspension medium buffer (according to the Stratagene protocol) and spread on a 150-mm plate with XL-1 Blue MRF′ cells (Stratagene) at a titer of approximately 500,000 plaque-forming units[pfu]/plate. The plaques were grown overnight at 37°C and were blotted on duplicate nitrocellulose hybridization filters (Schleicher & Schuell, Dassel, Germany). The filters lifted from each plate were denatured in 1.5 mM NaCl and 0.5 mM NaOH for 2 minutes, neutralized in 1.5 mM NaCl and 0.5 M Tris-HCl (pH 8.0) for 5 minutes, rinsed briefly in 0.2 M Tris-HCl (pH 7.5) in 2× SSC, 19 air dried, and ultraviolet cross-linked. The 32P-labeled PCR products were added to the filters in hybridization buffer nitrocellulose (NC). 19 Hybridization was allowed to proceed overnight at 42°C. The filters were washed twice with 2× SSC and 1% sodium dodecyl sulfate (SDS) at room temperature for 30 minutes and then twice with 0.1× SSC and 1% SDS at 65°C for 20 minutes. After drying, the filters were exposed to x-ray film (Amersham) for 16 to 48 hours. Positive clones were picked from the primary plates, transferred into suspension medium buffer with a drop of chloroform, vortexed, and subjected to secondary screening. Single plaques were selected and excised in vivo into phagemids (pBluescript; Stratagene) using SOLR cells and helper phages (ExAssist; Stratagene).
The nucleotide sequence of the cDNA inserts of isolated clones was verified with the a dye terminator cycle sequencing core kit (ABI Prism; Perkin Elmer, Alameda, CA) with DNA polymerase (AmpliTaq FS; Perkin Elmer) on a DNA sequencing system (model 377; Applied Biosystems, Chiba, Japan). The sequences obtained were compared with the GenBank EMBL and DDBJ DNA database. The sequence analysis of clone pM29 was 100% identical with the hydrophobic domain of chick NSP-A and -C (Fig. 1) .
Northern Blot Analysis
For Northern blot analysis, 20 μg of each total RNA sample was size separated on a 1% agarose-formaldehyde gel, transferred onto a nitrocellulose filter by wicking, and fixed by ultraviolet cross-linking (Stratagene). The pM29 cDNA insert was excised by EcoRI (Toyobo) and EcoRV (Toyobo), which allowed the inserts to have no corresponding recognition sites in the vector sequences. It was then isolated by gel electrophoresis, eluted, and radiolabeled using a DNA labeling system (Megaprime; Amersham) withα[ 32P] dCTP (Amersham) before it was used as a probe.
Hybridization and stringency washes were performed as described except for the usage of the 32P-labeled insert of pM29 as a probe. After drying, the filters were mounted on an imaging plate (BAS-III; Fuji, Kanagawa, Japan), then exposed on an image analyzer machine (BAS-2000; Fuji). For quantity and quality control of the RNA samples, total RNA separated on an agarose-formaldehyde gel were visualized by ethidium-bromide staining and photographed under ultraviolet illumination (337 film; Polaroid, Cambridge, MA). To allow for the differences in the actual quantity of total RNA loaded on the gel, the filter was stripped for 10 minutes at 95°C using a solution containing 1 mM EDTA, 40 mM Na2HPO4 (pH 7.2), and 5% SDS. Hybridization was performed using the amplified product 32P-labeled chick β-actin cDNAs as probes. 20
To check whether increased NSP expression is involved in the development of myopia or may be a consequence of form deprivation, we looked at the time course of the NSP changes in both form-deprived and lens-induced myopic retinal tissues. For Northern blot analysis only retinal tissues were used to rule out the possibility that an increase in gene expression in one tissue may be masked by a decrease in another tissue.
Camera images from the Northern blot analysis data were captured by a black-and-white camera (ACMEL CRT, model M-085-D; Polaroid, Tokyo, Japan) and film (337; Polaroid). Images were saved and converted to Macintosh (Apple Computer, Cupertino, CA) tagged-image file format (TIFF) files (360 and 720 pixels with 8 bits of gray level). TIFF files of all the numerical densities (arbitrary units) were exported to the public domain NIH image 1.55 program for further analysis on a Macintosh (Apple) computer.
Western blot analysis was performed using monoclonal antibodies (MON-160, MON-161, and MON-162) against human NSP-A and rabbit antiserum POL-8 against human NSP-C (kindly provided by Wilm J. M. Van de Ven, University of Leuven, The Netherlands). Among the three monoclonal antibodies, MON-162 recognized 80% of the human amino acid residues 338 to 442 (GenBank accession number L10333), which is a highly conserved region in human and chick (Fig. 1) . MON-160 and -161 recognized 53% of the amino acid residues 174 to 337 (GenBank accession number L10333), which were also conserved among animal species with slightly lower homology. To check for the specificity of the anti-NSP-A antibodies, Western blot analysis was performed with MON-162 only and with a mixture of MON-160 and MON-161. These cross-reacted with an approximately 150-kDa chick retinal protein. No other bands were observed in the Western blot. POL-8 was raised against the first 20 unique N-terminal amino acids of human NSP-C, the amino acid sequence of which was not present in NSP-A and -B (Fig. 1) . This sequence (MQATADSTKMDCVWSNWKSQ), 14 however, shares 17 identical amino acids and 2 amino acids similar to the N-terminal amino acids of chick NSP-C (MQASADSTKMDCLWSNWKCQ). POL-8 was diluted 1:100 and preabsorbed overnight at 4°C with a 10−4-M concentration of the chick 20-amino acid peptide (Biologica). This complex was then used for incubation with the membranes followed by secondary antibody detection. POL-8 cross-reacted with a 23-kDa chick protein. The 23-kDa band was eliminated by preabsorption with the immunizing peptide synthesis. Therefore, both POL-8 and the monoclonal antibodies (MON-160, -161, and -162) seemed to be suitable for Western blot analysis and immunohistochemistry. Their cross-reactivities to chick NSPs were confirmed by Western blot analysis. That is, the retina–RPE tissues were sonicated in buffer containing 10 mM Tris-HCl (pH 7.4), 1% (wt/vol) NP-40, 150 mM NaCl, 1 mM EDTA, 20 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride at a concentration of 0.1 g wet wt/ml. Each preparation was boiled with Laemmli's solution containing 5% 2-mercaptoethanol and loaded into a 5% SDS-polyacrylamide gel containing 0.1% SDS. Separated proteins were transferred electrophoretically onto a polyvinylidine membrane (Millipore, Bedford, MA). The membranes were soaked for 30 minutes in phosphate-buffered saline (PBS) containing 5% (wt/vol) skim milk and 0.2% (vol/vol) Tween 20 and then washed with PBS. The membranes were then incubated in a 1:6 dilution of MON-160, -161, and -162 or 1:100 rabbit antiserum POL-8 17 for 24 hours at 4°C. This was followed by incubation for 2 hours at 4°C with the 1:1000 secondary anti-mouse IgG horseradish peroxidase (HRP)–linked antibodies (Amersham) for MON-160,-161, and -162 or 1:1000 anti-rabbit IgG HRP-linked antibodies (Amersham) for POL-8. For the detection of immobilized specific antigens conjugated to HRP-labeled antibodies, a Western blot detection system (ECL Plus; Amersham) was used. The filters were exposed to x-ray films (Hyperfilm MP; Amersham) for 16 to 48 hours. Images were digitized and processed as described earlier.
Immunohistochemical examination was performed as described elsewhere. 5 Briefly, enucleated eyes (control and form-deprived eyes) were fixed at room temperature for 24 hours in 4% paraformaldehyde containing 0.2% picric acid, washed with 30% sucrose in 0.1 M PBS, and frozen at −20°C. Five-micrometer-thick frozen sections were cut on a cryostat, mounted on poly-l-lysine–coated slides, and air dried. The sections were soaked in PBS containing 10% normal goat serum and 0.03% Triton X-100 for 20 minutes, then incubated in a 1:5 dilution of a mixture of MON-160, -161, and -162 antibodies or 1:100 rabbit antiserum POL-8 for 24 hours at 4°C. The sections were then washed with PBS.
For the immunoperoxidase procedure, sections incubated in MON or POL-8 antibodies were reincubated in 1:1000 HRP-conjugated goat anti-mouse immunoglobulins or HRP-conjugated horse goat anti-rabbit immunoglobulins (both from Vector, Burlingame, CA), respectively. After rinsing in PBS, sections were incubated for 10 minutes in 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in 0.05% TB (Tris-HCl buffer, pH 7.6).
For fluorescence immunostaining, the sections incubated in the MON antibody mixture were reincubated in fluorescein isothiocyanate (FITC)–labeled rabbit anti-mouse IgG antibody (Dako; Kyoto, Japan) for 2 hours at 4°C. Those incubated in 1:100 rabbit antiserum POL-8 were reincubated in 1:50 FITC-labeled swine anti-rabbit IgG (Dako) for 2 hours at 4°C. After washing in PBS, sections were mounted in 50% glycerol in PBS. Identification of cone photoreceptors was performed using FITC-labeled peanut agglutinin (PNA) lectin (Biomeda, Foster City, CA). 21 The sections were viewed and photographed under a microscope (Carl Zeiss, Thornwood, NY) equipped with epifluorescence and a standard automatic photographic attachment with film (ASA 400; Fuji). For the negative controls, procedures were the same as for the Western blot analysis.
The data obtained from the densitometric readings of the Northern and Western blot analyses were analyzed statistically with an unpaired Students' t-test. P < 0.05 was considered to be statistically significant.
Isolation of Differentially Expressed mRNAs Using Differential Display
When compared with the control group, 15 fragments reproducibly showed higher expression in retina–RPE–choroid tissues from 7-day form-deprived eyes. Secondary amplification by PCR was performed on these 15 fragments using the same set of primers. Ten of the 15 fragments were reamplified (Fig. 2) .
Library Screening and Sequence Analysis
The 10 reamplified cDNAs were mixed, purified, 32P-labeled, and used as probes to screen the chick spinal cord cDNA library. Secondary screening resulted in the appearance of 10 putative positive plaques/500,000 pfu. Although 32 clones were isolated, Northern blot analysis confirmed that only one clone, designated pM29, contained the mRNA inserts induced by form deprivation. Northern blot analysis using the inserts of the 31 other clones did not show significant differences in expression between retina–RPE–choroid tissues from control and form-deprived eyes.
pM29 had a cDNA insert size of 700 bp. Its sequence was 100% identical with the hydrophobic domain of both chick NSP-A and -C deposited in GenBank (EMBL and DDBJ DNA databases; Accession Nos. U17606 and U17605, respectively). Although three isoforms of NSP—namely, NSP-A, -B, and -C—were reported in humans, 14 the chick gene corresponding to human NSP-B has not been detected. Because multiple promoters rather than alternative splicing caused the diversity of NSPs, 22 these three isoforms shared 100% homology in the hydrophobic domain of the carboxyl-terminal and 3′ untranslated region (Fig. 1) . It is conceivable that the sequence of these regions in chick NSP-B, if any, was identical with those of NSP-A and -C.
The inserts of pM29 were radiolabeled and used as probes for Northern blot analysis to confirm which isoforms of NSP were highly expressed in retinal tissues from form-deprived eyes and to check for the existence of NSP-B in chick eyes. The inserts of pM29 were hybridized to mRNA species with sizes of 1.5 kb and 3.5 kb. Considering the differences in sizes of human mRNAs, they corresponded to chick NSP-C and NSP-A, respectively (Fig. 3A ). In the control retinal tissues NSP-C mRNA expression was higher than that of NSP-A mRNA (Fig. 3A) . In the 7-day retinal tissues from form-deprived eyes, expression of both NSP-A (Figs. 3B) and -C mRNA (Fig. 3A) species were significantly increased (Figs. 3A 3B) . The expression of β-actin mRNA did not appear to change with form deprivation (Fig. 3C) . Despite overexposure of the film, no apparent hybridization to a 2.3-kb mRNA species, which would be expected for the transcript of a chick NSP-B homologue, was observed (Fig. 3B) .
In the Northern blot analysis using retinal tissues from control, form-deprived, and lens-induced myopic eyes, the increase in retinal NSP-A mRNA expression was significantly higher at 3 days, followed by 7 days, and then 14 days of form deprivation (Fig. 3D) . The expression pattern of NSP-A mRNA in the retina was similar to that of NSP-C (Fig. 3E) . Although −15-D lenses induced increased ocular growth and myopia during 14 days of negative lens-induced myopic treatment, the expressions of NSP-A and -C mRNA in retinal tissues were not altered (Figs. 3D 3E) .
The expression of NSP-A and -C mRNAs was altered only with FDM treatment but not with lens-induced myopic treatment. The time course of the NSP protein expression was evaluated only in form-deprived myopic retinal tissues. Human anti-NSP-A antibodies (a mixture of MON-160, -161, and -162) cross-reacted with an approximately 150-kDa chick retinal tissue protein (Fig. 4A ). The size of this protein was almost equal to the sizes reported in humans, rats, and mice. Therefore, this seems to reflect chick NSP-A. POL-8 polyclonal antibody, an anti-human NSP-C, cross-reacted with a 23-kDa chick protein (Fig. 4B) , which is likely to be chick NSP-C. These bands were absent when the reaction was performed without the primary antibodies for NSP-A or by preabsorption with the immunizing peptide synthesis sequence for NSP-C. (Figs. 4A 4B) .
Form deprivation caused significant increases in the intensity of both NSP-A and -C immunoreactivity in retinal tissues during the 14 days of form deprivation (Figs. 4C and 4D) .
Immunohistochemistry of NSPs in the Chick Ocular Tissue
In the control eyes, immunoreactivity to anti-NSP-A and anti-NSP-C antibodies was detectable in the layer of photoreceptor inner segments (Figs. 5A 5B 5C 5D) . Because of pigmentation, it was difficult to determine whether RPE cells were also immunopositive. Immunoreactivity in the ganglion cell layer, basal layer of the RPE, and choroid was observed in the negative controls (no primary antibody) and therefore does not indicate specific NSP-A or -C immunoreactivity. FDM did not alter the localization of the NSP-A and -C in any of the retinal layers (data not shown).
Figure 6A shows the schematic representation of the four types of cone cells and a rod cell. PNA lectin-binding was observed in the cone cells (Fig. 6B) . The abundance and configuration of the PNA-positive cells suggest that cone and not rod photoreceptors in the photoreceptor inner segments were positive to the anti-NSP-A antibody (Fig. 6C) . Similarly, only the photoreceptor inner segment, presumably cone photoreceptor cells, was presumably positive to the anti-NSP-C antibody (Fig. 6D) .
Form deprivation in the chick eye induces axial elongation accompanied by growth and remodeling of the sclera. 5 6 7 8 9 10 11 Most researchers have focused on various factors that modulate myopic development, such as neurotransmitters. 2 3 4 Differential mRNA display has been developed to detect and characterize altered gene expression. 12 A combination of reverse transcription using anchored oligo-dT primer and PCR using arbitrary 5′ primers allows simultaneous identification of genes that are either upregulated or downregulated. Although differential display is an extremely specific and sensitive method, it could only amplify products with sizes of less than 300 bp. Therefore, cloning of products is not always sufficient to identify the amplified mRNA. In the present study, retina–RPE–choroid tissues from form-deprived eyes were compared with control eyes. Those showing higher expression in the form-deprived samples were used as probes for cDNA library screening.
Each probe was positive to the chick spinal cord cDNA library with a frequency of approximately 1/500,000 pfu, indicating that this differential display method could detect mRNA species amounting to approximately 0.001% of all mRNAs. Of 32 positive plaques, pM29 alone contained the cDNA insert whose sequence was 100% identical with the 3′ terminal and 3′ noncoding region of both chick NSP-A and -C. 15 Although the diversity of NSPs is based on multiple promoters rather than alternative splicing, 22 their C-terminals consist of identical hydrophobic domains. NSP-A contains unique N-terminal sequences of 404 amino acids, whereas NSP-C includes only 20 amino acids in its N-terminal. Thus, hybridization to the 1.5-kb and 3.5-kb mRNA species in Northern blot analysis corresponded to the NSP-C and NSP-A transcripts, respectively. The NSP-C transcript was more abundant than the NSP-A transcript in the control chick retinal tissue. It has been reported previously that expression of NSP-C mRNA is greater than that of NSP-A mRNA in several mammalian endocrine tissues and cell lines. 13 In human tissues the existence of NSP-B, which consisted of a unique 5′ terminal of approximately 500 bp and the same 3′-terminal half as NSP-A and -C, was found only in the neuroendocrine small-cell lung cancer cell line NCl-H82. 13 We did not detect any transcript corresponding in size to NSP-B in chick retinal tissues. This may be due to limitations in the sensitivity of our methodology. However, these findings demonstrate that the NSP-B promoter was silent, minimally activated, or otherwise absent, and that only NSP-A and -C were expressed in chick ocular tissues. Nonetheless, form deprivation caused a marked increase in both NSP-A and -C mRNAs. We determined whether such increases in mRNAs were responsible for the corresponding increase in NSP proteins.
Although the anti-NSP-A antibody was monoclonal and the anti-NSP-C polyclonal, both anti-human NSP-A and -C antibodies cross-reacted with their chick counterparts. The amino acid residues used to generate anti-NSP-A antibodies were completely conserved in chick, whereas those for anti-NSP-C antibodies were directed to a less well-conserved region accounting for the lower affinity of NSP-C compared with NSP-A in the Western blot analysis. Therefore, the apparent amounts of NSP-A and NSP-C were not comparable. However, both NSP-A and NSP-C proteins were apparently increased in FDM. These increments were associated with an increase in the mRNA levels mainly through upregulation of transcription.
We cloned the NSP-A and -C genes showing higher expression in the 7-day FDM samples by differential display method. Next, we evaluated the time course of NSP expression after initiation of form deprivation. The increase in expression of NSP-A and -C mRNAs was most apparent at 3 days after induction of form deprivation, followed by 7, then 14 days. Although form deprivation caused overexpression of NSP-A and -C through upregulation of transcription during 14 days form deprivation treatment, the increased NSP expression may be an effect of visual deprivation, completely unrelated to myopia. Therefore, we examined the effects of negative spectacle lenses, which also induce increased ocular growth and myopia. Negative lens-induced myopia did not alter the expression of either NSP-A or -C mRNA. This may suggest that increased expression of NSPs reflects morphologic changes in photoreceptors after periods of form deprivation, rather than enhancement of ocular elongation through intracellular signal transduction of the cone cells.
Our results indicate that the changes in NSP expression are specific for form deprivation rather than negative lens defocus and leaves open the question of whether NSP is part of the mechanism by which form deprivation causes excessive ocular elongation. The difference in NSP expression between form deprivation and negative lenses strengthens the possibility that the signaling pathways involved in enhanced ocular elongation brought about by wearing diffusers are different from those involved in the elongation brought about by lenses. The eye, particularly the amacrine cells, has been reported to have the ability to distinguish between form deprivation–induced and lens-induced blur in the development of myopia. 23
Because expression of NSPs in ocular tissues has not been evaluated in the past, its relationship to eye growth and development remains unknown. However, because NSPs have been reported to be upregulated in nerve cells in association with treatment with growth factors such asβ nerve growth factor, 24 the changes in NSP expression with form deprivation treatment may be an unrelated effect.
NSP-A and -C expression were reported to be restricted to neuroendocrine and neural tissues in human, 25 but the distribution of NSPs in human and chick ocular tissues has not yet been investigated. We investigated their distribution in the chick ocular tissues.
Both NSP-A and -C were found to be enriched in the granular endoplasmic reticulum of neuroendocrine and neural tissues. 14 16 17 These endoplasmic reticula lie between cytoplasmic channels of smooth reticula. The shape of the immunoreactive structures suggested that both NSP-A and NSP-C were presumably localized to the cones cells in the inner photoreceptor segment. 26
NSP-A was ubiquitously expressed in the mature rat brain, 15 whereas NSP-C expression was restricted to the cerebral and cerebellar cortex, excluding Purkinje cells. 27 Only NSP-C was found to be expressed in various peripheral nerves. 27 In contrast to such regional differential expression, the distribution of NSP-A and -C in ocular tissues is identical and may be restricted to cones, suggesting the existence of NSP genes having ocular tissue–specific regulation.
In conclusion, NSP-A and -C were constitutively expressed in chick ocular tissues, although NSP-B was conclusively absent. FDM caused upregulation in expression and translation of both NSP-A and -C in the photoreceptor inner segments, presumably to cone photoreceptor cells.
Further identification of genes that are highly expressed in both form-deprived and lens-induced myopic ocular tissues by differential display may provide new insights into the mechanisms regulating development of myopia.
Supported in part by Grants-in-Aid (C-07670398 and B2-07557110) from the Ministry of Education, Science, and Culture, Japan.
Submitted for publication February 24, 1999; revised September 8 and November 22, 1999; accepted December 1, 1999.
Commercial relationships policy: N.
Corresponding author: Shigeki Fujii, Department of Ophthalmology, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. sfujii@post.med.kobe-u.ac.jp
Schematic representation of the human and chick NSP transcripts and location of the pM29 inserts. (A) Corresponding sequences of human NSP-A, -B, and -C in the 3′ terminal of the coding region and 3′ untranslated region (open bar). Hatched areas indicate the 5′ terminals. NSP-C included a unique 5′ terminal (black area). The regions corresponding to the amino acids used to generate human anti-NSP-A antibodies, MON-160, -161, and -162, and anti-NSP-C antibody, POL-8, are underlined. (B) Whole open reading frames for chick NSP-A (3.5 kb) and chick NSP-C (1.5 kb) with location of pM29 cDNA insert.
Differentially expressed mRNAs (differential display) using total RNA from 7-day retina–RPE–choroid tissues of control and form-deprived eyes. Only 10 (numbers 1–10) of 15 fragments could be amplified by secondary PCR. Lanes A through G: differential display using primer sets shown below the lanes. The approximate band sizes (numbers 1–10) were 150 bp, 150 bp, 150 bp, 300 bp, 250 bp, 200 bp, 200 bp, 100 bp, 150 bp, and 400 bp, respectively. 1, control; 2, FDM.
Northern blot for the detection of NSP transcripts. (A) Hybridization to a 1.5-kb mRNA species corresponding to NSP-C and a 3.5-kb mRNA species corresponding to NSP-A. (B) Overexposure showing the increased expression of NSP-A retinal mRNA at 7 days from the initiation of form deprivation. (C) Expression ofβ -actin mRNA. (D) Time course of NSP-A mRNA expression in form-deprived or lens-induced myopic retinal tissues during 14 days of FDM treatment. NSP-A mRNA expression in the retinal tissue was highest at 3 days, followed by 7 days. Expression of NSP-A and -C retinal mRNAs was equal in the lens-induced myopic and control samples. (E) Expression of NSP-C mRNA in the retina was also upregulated by form deprivation in the same time course pattern as that of NSP-A mRNA. There was no difference in NSP-C mRNA expression in the lens-induced myopic samples. For (D) and (E), the white bar corresponds to control, hatched bar to the −15-D group, and the black bar to the FDM group. Values are means ± SD (arbitrary units, n = 10). *P < 0.05, significant difference in mRNA expression, **P < 0.05, versus 7 and 14 days.
Identification of molecular forms of immunoreactive NSP-A and -C in the Western blot analysis of chick retinal tissue proteins. (A) Cross-reaction of human anti-NSP-A antibody, a mixture of MON-160, -161, and -162 with an approximately 150-kDa chick protein in the control (lane 1) and FDM groups (lane 2) during 7 days of form deprivation. Lane 3: negative antibody control. (B) Cross-reaction of anti-NSP-C antibody POL-8 with an approximately 23-kDa chick protein in the 7-day control (lane 1) and FDM groups (lane 2). Lane 3: negative control. (C) Time course of NSP-A protein expression in retinal tissues from form-deprived eyes during 14 days of form deprivation. (D) Time course of NSP-C protein expression in retinal tissues from form-deprived eyes during 14 days of form deprivation. Values are means ± SD (arbitrary units, n = 10), *P < 0.05, significant difference in protein expression, **P < 0.05, vs. 7 and 14 days.
Immunohistochemical localizations of NSP-A and -C in the chick retina. (A) Immunoreactivity of NSP-A in the control eye detected mainly in the inner segments of the photoreceptor layer. (B) Immunoreactivity of NSP-C in the inner segments of the photoreceptor layer. (C, D) Negative antibody controls for NSP-A and NSP-C, respectively. Scale bar, 50 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer; RPE, retinal pigment epithelium; CHO, choroid.
Localization of NSP-A and -C immunoreactivity in the inner segments of the photoreceptor layer. (A) Types of visual receptor cells in the chick retina. (B) PNA lectin showing binding to cone photoreceptor cells. (C) Immunoreactivity to anti-NSP-A localized in the inner segments of the photoreceptor cell layer, presumably to cone cells. (D) Anti-NSP-C showing similar binding sites. Scale bar, 100 μm. R, rods; PC, principal cones; AC, accessory cones; SCI, single cones type I; SCII, single cones type II; P, paraboloid; OLM, outer limiting membrane.
Crewther DP, Crewther SG. Pharmacological modification of eye growth in normally reared and visually deprived chicks. Curr Eye Res. 1990;9:733–740. [CrossRef] [PubMed]
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Wildsoet CF, Pettigrew JD. Kainic acid-induced eye enlargement in chickens: differential effects on anterior and posterior segments. Invest Ophthalmol Vis Sci. 1988;29:311–319. [PubMed]
Fujii S, Honda S, Sekiya Y, Yamasaki M, Yamamoto M, Saijoh K. Differential expression of nitric oxide synthase isoforms in form-deprived chick eyes. Curr Eye Res. 1998;17:586–593. [CrossRef] [PubMed]
Honda S, Fujii S, Sekiya Y, Yamamoto M. Retinal control on the axial length mediated by transforming growth factor-β in chick eye. Invest Ophthalmol Vis Sci. 1997;37:2519–2526.
Rada JA, Thoft RA, Hassell JR. Increased aggrecan (cartilage proteoglycan) production in the sclera of myopic chicks. Dev Biol. 1991;147:303–312. [CrossRef] [PubMed]
Rada JA, Matthews AL. Visual deprivation upregulates extracellular matrix synthesis by chick scleral chondrocytes. Invest Ophthalmol Vis Sci. 1994;35:2436–2447. [PubMed]
Rada JA, Brenza HL. Increased latent gelatinase activity in the sclera of visually deprived chicks. Invest Ophthalmol Vis Sci. 1995;36:1555–1565. [PubMed]
Rohrer B, Stell WK. Basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-β) act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res. 1994;58:553–562. [CrossRef] [PubMed]
Rohrer B, Tao J, Stell WK. Basic fibroblast growth factor, its high-and low-affinity receptors, and their relationship to form-deprivation myopia in the chick. Neuroscience. 1997;79:775–787. [CrossRef] [PubMed]
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Roebroek AJM, van de Velde HJK, Van Bokhoven A, Broers JLV, Ramaekers FCS, Van de Ven WJM. Cloning and expression of alternative transcripts of a novel neuroendocrine-specific gene and identification of its 135-kDa translational product. J Biol Chem. 1993;268:13439–13447. [PubMed]
van de Velde HJK, Roebroek AJM, Senden NHM, Ramaekers FCS, Van de Ven WJM. NSP-encoded reticulons, neuroendocrine proteins of a novel gene family associated with membranes of the endoplasmic reticulum. J Cell Sci. 1994;107:2403–2416. [PubMed]
Ninkina NN, Baka ID, Buchman VL. Rat and chicken s-rex/NSP mRNA: nucleotide sequence of main transcripts and expression of splice variants in rat tissues. Gene. 1997;184:205–210. [CrossRef] [PubMed]
Senden NHM, van de Velde HJK, Broers JLV, et al. Cluster-10 lung-cancer antibodies recognize NSPs, novel neuro-endocrine proteins associated with membranes of the endoplasmic reticulum. Int J Cancer Suppl. 1994;8:84–88. [PubMed]
van de Velde HJK, Roebroek AJM, van Leeuwen F, Van de Ven WJM. Molecular analysis of expression in rat brain of NSP-A, a novel neuroendocrine-specific protein of the endoplasmic reticulum. Mol Brain Res. 1994;23:81–92. [CrossRef] [PubMed]
Wildsoet C, Wallman J. Choroidal and scleral mechanisms of compensation for spectacle lenses in chicks. Vision Res. 1995;35:1175–1194. [CrossRef] [PubMed]
Davis L, Kuehl M, Battery J. Preparation of stock solutions. Davis L Kuehl M Battery J eds. Basic Methods in Molecular Biology. 1994; 2nd ed. 745–751. Appleton and Lange East Norwalk, CT.
Kost TA, Theodorakis N, Hughes SH. The nucleotide sequence of the chick cytoplasmic β-actin gene. Nucleic Acids Res. 1993;11:8287–8301.
Blanks JC, Johnson LJ. Specific binding of peanut lectin to a class of retinal photoreceptor cells. Invest Ophthalmol Vis Sci. 1984;25:546–557. [PubMed]
Roebroek AJM, Ayoubi TAY, van de Velde HJK, Schoenmakers EFPM, Pauli IGL, Van de Ven WJM. Genomic organization of the human NSP gene, prototype of a novel gene family encoding reticulons. Genomics. 1996;32:191–199. [CrossRef] [PubMed]
Fischer AJ, McGuire JJ, Schaeffel F, Stell WK. Light- and focus-dependent expression of the transcription factor ZENK in the chick retina. Nature Neurosci. 1999;2:706–712. [CrossRef] [PubMed]
Hens J, Nuydens R, Geerts H, et al. Neuronal differentiation is accompanied by NSP-C expression. Cell Tissue Res. 1998;292:229–237. [CrossRef] [PubMed]
van de Velde HJK, Senden NHM, Roskams TAD, et al. NAP-encoded reticulons are neuroendocrine markers of a novel category in human lung cancer diagnosis. Cancer Res. 1994;54:4769–4776. [PubMed]
Morris VB, Shorey CD. An electron microscope study of types of receptor in the chick retina. J Comp Neurol. 1967;129:313–340. [CrossRef] [PubMed]
Senden NHM, Timmer EDJ, Boers JE, et al. Neuroendocrine-specific protein C (NSP-C): subcellular localization and differential expression in relation to NSP-A. Eur J Cell Biol. 1996;69:197–213. [PubMed]
Copyright 2000 The Association for Research in Vision and Ophthalmology, Inc.
Bone Morphogenetic Protein-7 Is an Antagonist of Transforming Growth Factor-β2 in Human Trabecular Meshwork Cells
Hsp25 and -90 Immunoreactivity in the Normal Rat Eye
Changes in Retinal and Choroidal Gene Expression during Development of Refractive Errors in Chicks
Role of Transforming Growth Factor-β in Transdifferentiation and Fibrosis of Lens Epithelial Cells
Differential Gene Expression in Pseudoexfoliation Syndrome
Validity, Usefulness and Cost of RETeval System for Diabetic Retinopathy Screening
Development of Visual Field Screening Procedures: A Case Study of the Octopus Perimeter
A double dissociation of the acuity and crowding limits to letter identification, and the promise of improved visual screening
Nonantibestrophin Anti-RPE Antibodies in Paraneoplastic Exudative Polymorphous Vitelliform Maculopathy
Oculometric assessment of dynamic visual processing
Retinal Cell Biology
Visual Psychophysics and Physiological Optics
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home > space > saturn
Here are the latest posts about Saturn on Science, Space & Robots:
NASA Video Shows Cassini Spacecraft's Saturn Dive
NASA releases video showing Cassini spacecraft's dive through the gap betweeen Saturn and its rings. (May 4, 2017)
NASA: Ingredients For Life Found at Saturn's Moon Enceladus
NASA has found the ingredients for life at Saturn's moon Enceladus. NASA's Cassini spacecraft discovered hydrogen in the particles spraying from the moon. (April 13, 2017)
NASA Reveals New Images of Saturn's Moon Pan
NASA has shared new images of Saturn's moon Pan. The shape has been compared to a fried egg, ravioli and empanada by space writers. (March 11, 2017)
NASA's Cassini Spacecraft Samples 36 Grains of Interstellar Dust
NASA's Cassini spacecraft has sampled interstellar dust. The dust grains were sampled at Saturn and have interstellar origins. (April 16, 2016)
NASA's Cassini Researchers Identify Titan's Tallest Peaks
Researchers with NASA's Cassini mission have identified the highest point on Saturn's moon Titan. It is located in a trio of mountainous ridges called the Mithrim Montes. (March 26, 2016)
Cassini Returns Views of the North Pole of Enceladus
NASA's Cassini spacecraft has returned views from its flyby of the north pole of Saturn's moon Enceldaus. The images include one dubbed the Saturnian Snowman. (October 15, 2015)
Cassini Images Saturn's Moons Dione and Hyperion
Cassini took images of Saturn's moon Dione and Hyerion over the past few weeks. (June 21, 2015)
Toxic Cloud Hovers Over Titan's South Pole
Saturn's largest moon Titan has a giant toxic cloud hovering over it. It contains frozen particles of hydrogen cyanide. (October 6, 2014)
Saturn's Auroras Caused By Its Collapsing Magnetotail
Saturn's auroras are caused by its collapsing magnetic tail say University of Leicester researchers. (May 19, 2014)
NASA's Cassini Spacecraft Captures an Image of Uranus
NASA's Cassini spacecraft has captured an image of Uranus. The planet appears as a blue dot in the distance. (May 4, 2014)
Possible Moon Named Peggy Forming in One of Saturn's Rings
A possible moon, named Peggy, appears to be forming in one of Saturn's rings. (April 15, 2014)
NASA: Saturn's Moon Enceladus Harbors Underground Ocean of Liquid Water
NASA announced today that Saturn's moon Enceladus harbors an underground ocean of liquid water. (April 3, 2014)
NASA's Cassini Spacecraft Finds Plastic Ingredient Propylene on Titan
NASA's Cassini spacecraft has found propylene, an ingredient of household plastic, on Saturn's moon Titan. Cassini's Composite Infrared Spectrometer detected the propylene. (September 30, 2013)
Image of Saturn Moons Mimas and Pandora Taken by NASA's Cassini Spacecraft
Here is an amazing image of Saturn Moons Mimas and Pandora taken by NASA's Cassini Spacecraft (July 31, 2013)
NASA's Cassini Spacecraft Takes Image of Earth From Saturn Orbit
NASA's Cassini spacecraft took an image of Earth from Saturn orbit on July 19th. (July 23, 2013)
Saturn's Moon Dione May Have Once Had Subsurface Ocean
Saturn's moon Dione may have had and may still have a subsurface ocean. The ocean could be under a mountain named Janiculum Dorsa (June 2, 2013)
NASA's Cassini Spacecraft Captures Close-Up View of Saturn's Massive Hurricane
NASA's Cassini spacecraft has captured close-up views on the massive hurricane around Saturn's north pole. (April 29, 2013)
Saturn Ring Rain is Greater Than Previously Thought
A new study has found that there are more charged water particles falling into the atmosphere of Saturn from its rings than previously thought. (April 10, 2013)
Cassini Returns Images of Saturn's Moon Rhea
NASA's Cassini spacecraft captured new images of Saturn's moon Rhea during its close flyby of Rhea on March 9, 2013. (March 12, 2013)
NASA's Cassini Spacecraft Captures Backlit View of Saturn
NASA's Cassini spacecraft shared this stunning new image of Saturn. (December 22, 2012)
Cassini Spots Giant Storm on Saturn
NASA's Cassini spacecraft has spotted a giant storm swirling on Saturn's north pole. (November 30, 2012)
Hazy, Swirling Vortex Spotted Over Saturn's Moon Titan
NASA's Cassini spacecraft has captured images of a high-altitude haze and a vortex materializing at the south pole of Saturn's moon Titan. (July 12, 2012)
Half-Mile Wide Snowballs Seen Punching Through Parts of Saturn's F Ring
Images from NASA's Cassini spacecraft have revealed half-mile-sized objects punching through parts of Saturn's outermost ring (F ring). (April 29, 2012)
Next Saturn Stories

redpajama_set_name: RedPajamaCommonCrawl


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Q: Adding a Submit button to dynamically uploaded PDF document PDF documents are uploaded by the dealers to the site. There is no standard whatsoever to these documents. We want to present editable PDF forms to the users which later can be saved in the system after user filled up/edited data in these sheets. I am able to read the pdf, populate it whatever fields can be pre-populated, and also render the editable PDF to the browser. Problem is pdf does not have any Submit button and as much as I know, this can be added only by opening the pdf in acrobat.
 I tried

*

*rendering the pdf in iframe and a submit button in different frame (as much as foolish it was)

*Converting the read pdf as Text and presenting all the form fields as HTML fields to user but got stuck on anything other than input type text (no way to identify if it is checkbox, radio etc)

A: You have to create the PDF templates with the Submit button included. You'll need to create the PDF using a full copy of Adobe Acrobat, not the reader. You can't edit with the reader. You'll also have to learn how to write JavaScript inside of Acrobat in order to do validation and small interactions in the PDF if needed.
You're not going to save a copy of the populated PDF, you're going to save the data that was submitted via the PDF form. You can then populate the file for printing, if that's what they want, but the submit button will be part of the PDF that's printed.
When you need to use a PDF form, just link to it in the browser. When you submit the PDF form, the processing CFM will redirect to the next page in the workflow. We include a cross-site request forgery token as part of the PDF form data for security. If we don't get the token as part of the FORM scoped data, then we don't process it.
We have a mix of HTML and PDF forms. Generally, our clients hand us existing documents that they want converted for the web. In some cases, we create HTML forms, in others, they prefer we keep the formatting of the document so we use PDFs. You can't dynamically convert the PDFs to HTML forms, you'll have to have someone do that manually to ensure the correct form controls are used (and populated) throughout the form.

redpajama_set_name: RedPajamaStackExchange


I've recently took out a stud wall and in it was s double plug socket, which I connected up using a junction box to remove the switch (2 lots of wires).
My question is; can I spur off that to create a new double socket and does it have to be 2 wires or one? Or could I crest 2 new double sockets?
The idea of two cables is to make a ring, one in and one out if you like.
Then you run a new cable from one of the new sockets, to the other new socket.
So that each socket has two cables.
This way the new sockets are not spurs, but part of the ring.
Do they need to be extended by wago's or can I use connector blocks (chick blocks)?
You can use choc blocks if they are in a suitable enclosure and accessible.
Now what if the circuit is a radial or that bedroom socket is a spur from a ring feeding a spur!
But you know as well as I do that, chances are its a ring, and short of the OP tracing / metering the cables (Like that is going to happen) we will never know.
I would expect anyone doing basic stuff such as adding new sockets to do a continuity check on the ring. If they cannot do that then they should not be doing the work. And I do not mean go out and buy an expensive tester, this simple test could be done with a cheap multimeter.
So a simple LL NN EE ohms reading to show that it is a ring and look out for open circuits or high resistance values.
I agree with your sentiment, but its not going to happen. Look how many times I have asked / said (not to you) have you a multi meter? and they don't even know what one is, let alone have one.
Well you can always read em's bs.
What are the basics tests I need to carry out and look out for?
I do have an electrician that's going to do any final checks!
Should be minimal resistance if it's a ring, infinite resistance if it's a radial or spur.
The test isn't a big job to do, and has saved me cocking up in the past. I knew there was a ring, but the particular socket I opened was actually a spur, with another spur (naughtily) taken off it - making it look like the ring. Testing found that, but otherwise I could have added yet another spur, then totalling 3x double sockets hung off one 2.5mm spur..


Congratulations – you've put down the deposit on a new office lease or you've signed the paperwork for your new home. Moving into a new place is exciting. But the actual move can be the opposite, with the moving experience likely to be stressful and hard work. But if you take a closer look at why the move is stressful, and how you can combat the stress, you will have a much more enjoyable moving day as well as a less stressful period of time leading up to the move. If you are planning a move in Gloucester and the surrounding area, here's what you need to do to lower your stress levels and move more happily.
You can help yourself a lot by recognising the simple fact that a Gloucester move is going to be stressful, whether you are moving out of the county or just around the corner. By mentally visualising the potential stresses and preparing your mind to react to them you can make it easier to cope.
By far, the greatest thing you can do to cut stress on moving day is to hire a removals company. Trying to do it alone is a recipe for stress and sore backs. When it comes to removals Gloucester offers a good amount of choice. Make sure you select a company that is reputable and professional so that you can relax knowing your possessions are in good hands.
By planning ahead of the moving day you reduce a lot of the stress associated with it. You feel more in control when you monitor the tasks you need to do in the run-up to the move, and you can feel more secure that you haven't forgotten anything important, according to www.andrews-removals.com.
Moving day can be the ideal excuse to declutter and get rid of things you no longer want or need. Don't just blindly move everything – as you pack, throw things out, donate them, or give them away. The less you have to move, the less stressful the process.
On moving day, dress in your most comfortable (old) clothes and wear closed toe shoes. Put a change of clothes in a bag so you can change into something fresh when you finally get everything out of the lorry and into your new home.
Have a compact toolbox close at hand when you unpack so that you can quickly put together essentials like the dining room table or beds. You don't want to be rummaging through boxes at midnight trying to find a screwdriver so that you can assemble your bed.
Or, if you can't afford it, get a relative or friend on board to take the kids to the park or otherwise occupy them while you get everything in the van. It is considerably more stressful to move when you also have to watch what the youngest ones are doing in the next room.


The Federal Democratic Republic of The Federation of Kendor is a colossal, safe nation, ruled by Martha Leyner with a fair hand, and notable for its multi-spousal wedding ceremonies, ubiquitous missile silos, and absence of drug laws. The compassionate, democratic, cheerful, devout population of 7.332 billion Kendorians are free to do what they want with their own bodies, and vote for whomever they like in elections; if they go into business, however, they are regulated to within an inch of their lives.
The medium-sized government juggles the competing demands of Education, Industry, and Healthcare. It meets to discuss matters of state in the capital city of Baxter. The average income tax rate is 99.3%.
The frighteningly efficient Kendorian economy, worth a remarkable 2,236 trillion Kendorian Dollars a year, is broadly diversified and led by the Information Technology industry, with major contributions from Tourism, Book Publishing, and Beef-Based Agriculture. State-owned companies are common. Average income is an amazing 305,097 Kendorian Dollars, and distributed extremely evenly, with little difference between the richest and poorest citizens.
Unemployed taxi drivers ironically use self-driving cabs to reach employment offices, the government has instituted 'traveller reservations' across the country, Kendorian husbands are dead good at fathering kids, and artists attempt to convince the Hardship Fund that their unmade bed is a masterpiece. Crime is totally unknown. The Federation of Kendor's national animal is the Eagle, which is also the nation's favorite main course, and its national religion is Catholicism.
The Federation of Kendor is ranked 290th in the world and 6th in the Pacific for Most Inclusive, scoring 496.71 on the Mandela-Wollstonecraft Non-Discrimination Index.
12 days ago : The Federation of Kendor was ranked in the Top 1% of the world for Lowest Crime Rates.
14 days ago : The Federation of Kendor was ranked in the Top 1% of the world for Lowest Crime Rates.
28 days ago : The Federation of Kendor endorsed The Emirate of Schwartze.
29 days ago : The Federation of Kendor was endorsed by The Emirate of Schwartze.
35 days ago : The Federation of Kendor's influence in The Pacific fell from "Squire" to "Page".
37 days ago : The Federation of Kendor's influence in The Pacific rose from "Page" to "Squire".
40 days ago : The Federation of Kendor was ranked in the Top 1% of the world for Lowest Crime Rates.
41 days ago : The Federation of Kendor's influence in The Pacific fell from "Squire" to "Page".
58 days ago : The Federation of Kendor was reclassified from "Civil Rights Lovefest" to "Left-wing Utopia".
61 days ago : The Federation of Kendor was ranked in the Top 1% of the world for Lowest Crime Rates.
Endorsements Received: 5 » Aaronian, 14Greycliffe, East Durthang, Pierconium, and Schwartze.


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Invitation Email Requirement Could Cause Inbox to Be a Bust
Earlier this week, Google launched its new smartphone alternative to emailing: Inbox. Currently, the app is available by invitation only and its goal is pretty straightforward. Google wanted users to be able to organize, sync, group and bring together all the important content that the user requires in any given day. And such an idea is uniquely appealing to many people, especially since the email is considered to be the old horse that just keeps on galloping. Anyone from small business owners to self-employed, multi-taskers or just casual email users can benefit from Google's apparent spectacular new app.
There is a twist, however, to this seemingly perfect Google story. Many of those quite enthusiastic to try out Google inbox have already begun to question the legitimacy of the tech giant's initiative because of the mandatory invitation. Inbox requires an invitation to join, regardless of the fact that it is already available in App Store and in the Play Store.
As such, some wonder whether Google has lost touch with what is important to its users. And while they may not know precisely what you particularly want, they have a general concept about what the average email user desires. Google believes that the goal is organizing rather than prioritizing (despite the fact that marketing of the app has been unclear in that regard, letting many believe that Google and the Inbox app will eventually believe to make choices for the user).
But the fact is that all Google attempted to do was to employ its Google Now technologies into a user-friendly email application that would modernize the email experience and synergize various types of features.
The goal of the app wasn't to replace Gmail, but to rather put a modern spin on the modern man's necessity, the email, and transform it into an app that could also meet his changing needs.
Jack Gold, an independent analyst, studied Google's app and believes that this may actually be a slippery slope for the tech giant, if Inbox fails to keep up with its end of the bargain and efficiently organize.
"The issue is, how does an overseer sort through all the communications and accurately predict what's important to the user? It's not an easy task."
Gold said on Thursday.
There have been some to argue that Inbox will learn the behavior of the user, but that speaks to what Google Now is already about.
"It will learn which contacts are important to you and which topics you tend to actually read and respond to rather than delete."
Rob Enderle, an analyst for the Enderle Group said.
Regardless, only time will tell if users will appreciate the process of requesting to be brought into the app via App Store or Play Store and then having to request to join via email.
Filed Under: News Tagged With: app, app store, feature, google app, google application, google inbox, Google Play, inbox, inbox app, play store


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City Funds Approved
We hope to soon have a diverse collection of articles, videos, audio files, and other resources available online. Until then, here is a sampling of local history education goodness.
The Bentonville School History 1841 - 1921
Elsie Mistie Sterling
From Overland Stage to Postal Truck: A History of the Mail Service in Rogers, Arkansas
Ladies of the Loom
Living History Videos
Rebels, Federals and Bushwhackers
Remembering Rogers
Research Library
The research library in the Key Wing, consists of magazines and newspaper articles, books, research manuscripts, maps and photocopies of historical documents and local directories, as well as Museum-generated research reports and oral histories. Objects in this collection are frequently used for public and staff research. Also includes modern prints, slides, and negatives that can be, with permission, used for programs, publicity, research and exhibits. Hours are Monday - Saturday 10 a.m. to 4 p.m.
Research Library Topic List
Explore historical topics of interest in the Museum's Research Library.
Hailey Building
Monday - Saturday,
10 a.m. to 5 p.m.
Key Wing
Hailey Building Exhibit Galleries
313 South Second Street, Rogers, AR 72756
Historic Hawkins House, Education & Research Library at the Key Wing
Eversole Collections Building
300 W. Poplar, Rogers, AR 72756
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In Burnaby North-Seymour, a giant on the hillside
Posted in Climate, Election - Canada 2019, No Tankers
Written by Laura Benson
Trans Mountain looms large over this election. Where do our local candidates stand?
"I'm really into politics," my eight year old has declared.
I guess it's not surprising, given that his mom works for Dogwood. But Leo's newfound interest and his older brother Henry's growing interest in joining other youth fighting for climate action is putting the stakes of this year's federal election in stark relief.
We live on Burnaby Mountain, unceded territory of the Tsleil-Waututh, Squamish, Musqueum, Qayqayt, Stó:lō, Stz'uminus and Kwikwetlem peoples, within two kilometres of the Trans Mountain oil tank farm. For Leo's entire life, the TMX expansion proposal has been a threat to our family's health and safety, to our region's air, land and water and to the future of our warming planet.
A personal betrayal
After spending so much time and effort along with my Indigenous and non-Indigenous neighbours using every tool available to us in our society to fight TMX, it felt like a personal betrayal when Prime Minister Justin Trudeau approved the project in 2016 saying "If I thought that this project was unsafe for the B.C. coast, I would reject it."
But in a federal election, voters don't get to choose a prime minister. We vote for our local MP. So, Leo, Henry, Jeremy and I will be scrutinizing our candidates to decide who might best represent us and try to keep our neighbours safe in Burnaby-North Seymour.
Here's a look at the candidates' records over Leo's lifetime. (For more on these candidates or the folks running in your riding visit our Vote Local website).
The ones opposed
Amita Kuttner is running for the Green Party. Climate action is one of their top priorities. Tragically, Amita lost their mother in a landslide on the North Shore in 2005, so they want to do whatever they can to avoid the catastrophic impacts of climate change on real people. They recently returned to the community after many years away earning a Ph.D. in Astrophysics, so Amita hasn't really been involved in the TMX until now.
Svend Robinson is making a comeback run for the NDP. During my eleven years in Burnaby, Robinson has been known largely as a legend — the first openly gay MP in Canada, who was an outspoken advocate for environmental causes like stopping clearcut logging, as well as international human rights.
On the campaign trail, Robinson has taken a strong stand against the Liberal government's buyout of Kinder Morgan and pushed even his own party to do more to fight climate change and uphold Indigenous rights. During much of the TMX fight, though, Robinson was out of the country working for Public Services International on issues of climate change, pensions and trade and with the Global Fund to Fight AIDS.
The ones in favour
Conservative Heather Leung first made a name for herself in our community in 2011 as part of a group called Parents Voice which spoke out against policies to make schools safer and more inclusive of LGBTQ+ students. Since then Leung has run and lost campaigns for Burnaby school board (2014) and city council (2018) on her social conservative agenda. Her website suggests she supports the Trans Mountain expansion project, but she has been unreachable and largely invisible.
There is a candidate for the People's Party of Canada. But I'm not going to legitimize a party that uses hate speech in its platform and harmful images of the Canadian government's internment of Japanese Canadians in its campaign propaganda.
The one we're still not sure
That leaves our current MP Terry Beech, running for re-election as a Liberal. I've watched Beech closely and met with him a few times in person since 2015. During the 2015 campaign, the Liberals promised to redo the review of the the pipeline expansion proposal, and overhaul the National Energy Board.
After they won government the Liberals allowed the NEB to proceed as planned, while creating a toothless public panel that took testimony from thousands of trusting people. Beech attended the panel events and presented a detailed report of his constituents' concerns, but never took a clear stance personally on whether he supported or opposed the expansion proposal.
When Henry and I met with him in June 2016, Beech seemed genuinely blindsided by the Liberal government's approval of the proposal. But aside from a single symbolic vote on the matter in Parliament, he did not take a stand to oppose his government's position. In 2018 when the Liberals doubled down and bought the project with taxpayer money, Beech's constituents called on him to take a stand against the $4.4 billion bailout. Again, he chose not to take a personal stance and stood behind his party.
A cynical distraction
There's a lot more in Beech's record, but I'll highlight one thing that really sticks out for me. Remember that my family and our neighbours live with the fear that an accident, mistake or natural disaster at the Trans Mountain tank farm could put our lives in danger from fires, toxic fumes or oil spills — and that the expansion project would greatly compound those risks. Beech has heard these concerns ad nauseam from his constituents.
His response was to propose that the tank farm be moved to some mythical, empty "industrial area" to make way for affordable housing on the current tank site. This proposal was immediately criticized as essentially impossible by people who actually know something about land use policy, zoning, contaminated sites and housing development. But that hasn't stopped Beech from repeating the idea and raising false hopes among my neighbours.
Webinar: Adam Olsen on the passage of B.C.'s UNDRIP law
12.18.19 | Climate, Democracy, In Depth, Media, Mining, Podcast | Kai Nagata
How might the Declaration Act affect forestry, mining, Site C or other projects on the B.C. landscape? After Bill 41 became law, we had a chance to sit down for an hour with Adam Olsen, Tsartlip Nation member and MLA for Saanich North and the Islands. Topics included: How Indigenous languages offer a different perspective […]
Jonathan Wilkinson: the Minister of Greenwash
12.11.19 | Campaigns, Climate, Election - Canada 2019, No Tankers, Op-Ed | Cheryl Cameron
After working for ExxonMobil and Shell, it looks like his job now is to grow Canada's oil sands After the 2019 federal election, Alberta premier Jason Kenney laid out his demands for Ottawa. He wanted Environment Minister Catherine McKenna, whom he called "anti-pipeline," replaced with someone the oil companies could trust. Prime Minister Trudeau tapped […]
Op-Ed: Declaration Act a long overdue reset
11.26.19 | Climate, General, News, Op-Ed | Guest Writer
Originally published in the Times Colonist on November 22, 2019 Re: "Indigenous Peoples bill has good goals but overpromises," editorial, Nov. 3. I confess I was gobsmacked — and not in a good way — to read the unsigned editorial in the Times Colonist claiming that "the unifying authority of government has been fragmented by […]


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Foreign Relations of the United States, 1969–1976, Volume III, Foreign Economic Policy; International Monetary Policy, 1969–1972
Document 153
153. Paper Prepared in the Department of the Treasury1
Washington, May 9, 1971.
U.S. NEGOTIATING POSITION ON GOLD
The official dollar gold price would not change
The United States would make clear at the outset of negotiations that it does not regard any change in the official price of gold as part of the negotiations. This posture will be necessary because the French, with varying degrees of support from countries such as Belgium, Switzerland and certain elements in the United Kingdom, may seek a devaluation of the United States dollar in terms of gold. French private citizens hold large amounts of gold on which they would like to make a profit, and Swiss bankers have substantial holdings for their clients and have been managing South African gold sales and on occasion, trying to stimulate gold speculation by their actions and speeches. Both French and British financial interests hold large blocks of South African gold mining stocks. A rise in the official gold price would help the private holders by raising the private market price and enlarging the scope of that market, in the judgment of the French and Swiss.
Fortunately the Germans and Italians do not favor an increase in the gold price, so that the French are unlikely to achieve a unified position in the European Community. The Germans and Italians hold very large reserves in dollars, on which they would gain no profit if the official gold price rose. Their officials might even incur criticism because other countries had profited by holding a larger share of their reserves in gold. The Japanese and even the Canadians could also be embarrassed.
The French argument
The main argument that the French may make is that the United States as the deficit country should devalue, because we have sinned and should expiate. Other countries (France) have devalued and the United States should behave like other countries and do so. Surplus countries should not have to revalue because the United States does not wish to change the price of gold, particularly since these surplus countries have followed virtuous anti-inflation policies and kept their balances of payments in order.
The French suggest a moderate increase in the official dollar price, while other countries maintain their existing gold prices or raise them to a lesser degree than the United States thus effecting a change in IMF parities (which are stated in gold) that would depreciate the dollar exchange rate in terms of their currencies.
In effect, the French attitude boils down to the suggestion that if the United States does penance and helps the private gold holders to make a bigger capital gain, this would ease the discomfort of appreciating their exchange rate and becoming less competitive with the U.S. in the U.S. market and world markets.
U.S. counter arguments
Against this point of view, the United States would make the following counter arguments:
The deficit is only partly due to U.S. economic policies. While the United States at some periods has augmented its balance of payments deficits by inflationary policies, our record of price stability was much better than all major countries except Belgium and Germany in 1950-65, and slightly better than these two. U.S. deficits have also been forcefully affected by (a) our heavy external costs for military expenditures to protect Europe and Japan, (b) by trade practices abroad that have cut back the growth trend in our agricultural and other exports, and (c) especially by the undervalued exchange rates which were fixed in the forties and fifties, at a time when foreign productive capacity was abnormally low relative to foreign demand, and which have been jealously preserved for trading advantage. Moreover, the recent large short-term flows have been due to divergent monetary policies in the U.S. and Germany—both needed for their domestic situations.
Other countries decided to peg to the dollar long ago. Other countries decided long ago to use the dollar as a reserve currency and to peg their currencies to the dollar. By doing so, they took to themselves the responsibility for fixing exchange rates, leaving the U.S. in a passive position, and have prevented their currencies from rising in response to natural market pressures that would have caused these currencies to [Page 429]appreciate. Some have devalued whenever their competitive position was weakened, and this fact raises questions as to whether they could be counted on to maintain their initial appreciation vis-à-vis the dollar under the French scheme.
A moderate gold price increase would be unstable. A moderate increase in the official gold price would be analogous to the "Munich settlement"; it could quite likely last only a short time—perhaps less than a year. It would establish a strong presumption that the prescription would be repeated if speculative pressures again became strong, and dollar reserves built up abroad again. Because central banks holding gold would have gained a gold profit, while holders of dollars would not, many central bankers would face public criticism for holding dollars. This would be likely to induce substantial requests for conversion of dollars into gold, especially by smaller central banks. U.S. gold reserves could shrink fairly quickly, offsetting or more than offsetting the devaluation gold profit (which itself would be about matched by a write-up in our gold guaranteed liabilities to the IMF and other international agencies). This factor of additional drain on U.S. reserves for conversion arises from the re-emphasis on gold inherent in the change in the U.S. official gold price. It would not be present if exchange rates are adjusted without a change in the dollar price of gold.
SDRs are better reserves for the future than gold. It is highly desirable to continue the trend toward greater reliance on SDRs than gold for international reserves. The fact that there has been a private commodity market for gold, in competition with monetary use of gold, subjected the monetary system before the two-tier system to speculative drains of gold to private hands, which weakened all currencies. The SDR and the two-tier gold system recognized that gold was no longer satisfactory as a source of reserve growth, and, if so, there is no logic to increasing the official gold price. It would be turning back the clock.
A gold price increase would be hard on those who have held dollars. An increase in the gold price is discriminatory and inequitable. It enlarges the reserves of those countries that hold gold, while leaving unchanged reserves held in the form of dollars. This is unfair to some of our closest cooperators, and benefits a few large gold holders who do not need reserve writeups.
It could weaken the two-tier gold system and benefit gold producers and speculators. The psychological reaction of raising the commodity price of gold would benefit Russia, South Africa and other producers as well as private hoarders and speculators. The spread between the official and private price might well widen, and this could make it harder to maintain the two-tier system. The system was regarded as favoring [Page 430]the United States rather than the French gold philosophy, and a gold price rise would sow new doubts.
Differential rather than a uniform exchange rate adjustment is needed. The Japanese adjustment, for example, might need to be steeper than the European, and all the Europeans might not be the same. To cover this spread the U.S. change in gold price could be undesirably large.
The U.S. would convert dollars into SDRs instead of gold. With major modifications in exchange rates and other improvements that should make the future drain on U.S. reserves manageable, the U.S. is prepared to resume responsibility for conversion of dollars into international reserve assets, which it alone has carried since 1945. But it would convert official dollars into SDRs or other claims on the Fund, rather than gold. We would use gold as needed to acquire these assets from the IMF. This procedure would continue the gradual evolution of the monetary system toward SDRs as the basic reserve asset, with gold gradually declining as a portion of world reserves. This process has already gone a long way, since gold reserves represented only 40 percent of global reserves at the end of 1970, as compared with 70 percent in 1948. Turning back the clock would be a very serious decision, and would be resisted in many quarters of the academic, banking and Congressional world.
Congressional opposition could be strong. A number of influential members of Congress (Patman, Widnall, Senator Long, Reuss, Senator Bennett) seem likely to be doubtful or critical. Some don't like to pay out gold to foreigners. Some want to de-emphasize gold. Many are likely to feel that the Executive should have specific Congressional authority, and a Congressional debate could lead to very serious market uncertainty and speculation. The attached correspondence with Congressman Reuss has been accepted by Congressman Reuss as indicating that action would not be taken without Congressional approval. If this were not the Treasury view, he had indicated that he would submit legislation making this quite specific. (See attached correspondence.)2
A massive gold price increase would be generally rejected in a worldwide inflationary period. A doubling of the price of gold at one swoop, as recommended by Rueff of France for many years, is probably not likely to be put forward now. We understand Rueff himself no longer puts it forward, as it is too unrealistic. It would be resisted very widely as it [Page 431]would enlarge world reserves by about $40 billion all at once. The 1970 addition to world reserves was $14 billion. Such a large increment to world reserves, even though initially immobilized in central bank reserves, would have a marked inflationary potential. It would probably be resisted by most members of the Fund, and by U.S. public opinion. One danger of a moderate change in the gold price is that by a series of crises, the same result might be approached.
Source: Washington National Records Center, Department of the Treasury, Deputy to the Assistant Secretary for International Affairs: FRC 56 83 26, Contingency Planning 1971. Confidential. A typed note at the top of the page reads: "2nd Draft 5:00 p.m."↩
Not printed. Attached were a February 6, 1969, letter from Secretary Kennedy to Congressman Reuss reaffirming the position taken in a January 23, 1968, letter from Under Secretary Barr to Reuss, and a copy of Barr's letter. It was Barr's view that the $35 per ounce price of gold notified to the IMF could only be changed by Congressional action.↩
Foreign Economic Policy; International Monetary Policy, 1969–1972
Office of the Historian Press Release
Foreign Economic Policy 1969-1972; International Monetary Policy, 1969-1972 (Documents 1-244)
International Monetary Policy, 1969-1972 (Documents 109-244)
Bennett, Jack F.Kennedy, David M.Reuss, Henry S.
FRCIMFSDR


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text:
Suraj Thapa | Biography, Girlfriend, Wiki, Age, Height, family, Affairs
Biofamous — October 15, 2017 in Singer 11 Likes 570 Views
One of the prominent contestants of Nepal Idol, Suraj Thapa is most popularly known as the matured singer of Nepal Idol. Being top five of the show he was able to prove himself as the top singer of Nepal. Many people state that he must have been the winner of the show as he had a very good voice and a very good personality. He although didn't win the title but was able to win the heart of viewers and of all the Nepalese people.
Suraj Thapa
Family Name Thapa
Profession Singer
Birth Place Nepalgunj, Nepal
Birth Sign Leo
He was the hope of audience and all the people all over Nepal. He was the only person who has done his higher studies in music and one of the most dedicated people of the reality show.
Born in a beautiful city of Nepal, Nepalgunj he spent his childhood in his hometown and later moved to the capital city for further progress of his singing career. Joining singing school along with his higher studies, he builds the craze for singing. Nepal idol opened the gate for his career and thus he is in this place.
The reality show became the path opener and best platform for many of the people. We have seen many people around us who have been working years and years in music industry and it has always been difficult for them to achieve fame and success. But for him within weeks he is a well known public profile and audience have high expectation from him.
Affairs And Relationships
The popular actor has not revealed any information regarding his personal affairs and relationships. He might be single as he is busy in his career and has so many things to do in his life. He is currently busy with his songs and has got many things to do in his life and thus he is single as of Today. He even mentioned in the Nepal Idol's stage he does not have any girlfriend as of that day.
He belongs to a middle family background and even these things affected him in the Voting system of the show. Many people raised the question that the show like "Nepal Idol" is not for poor people and always rich people win the show. But the winner of the show Buddha Lama turned all them down. He also belonged to a family of weak background and he was able to win the title.
Singer Suraj-Thapa


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you are here: science media centre > roundups for journalists > expert reaction to modelling study looking at SARS-CoV-2 transmission during COVID-19 vaccination campaigns and likelihood of emergence of vaccine-resistant variants
expert reaction to modelling study looking at SARS-CoV-2 transmission during COVID-19 vaccination campaigns and likelihood of emergence of vaccine-resistant variants
A modelling study published in Scientific Reports looks at how rates of SARS‑CoV‑2 transmission and vaccination might impact the fate of vaccine‑resistant strains.
Prof John Edmunds, Professor in the Centre for the Mathematical Modelling of Infectious Diseases, London School of Hygiene & Tropical Medicine, said:
"This modelling study looks at the conditions which increase the risk of the emergence and establishment of a vaccine resistant variant. One of the key results is that the risk is highest when a large fraction of the population has been vaccinated, but transmission is also high. This is the situation we find ourselves in at the moment. It has to be remembered that this is a modelling study. The real world is always much more complex than can be represented in a model and chance will always play a key role. Nevertheless, studies like this can highlight threats and help us work through potential risk-mitigation strategies."
Dr Edward Hill, Research Fellow in Mathematical Epidemiology, University of Warwick, said:
"In this peer-reviewed modelling study, the authors use a model with SARS-CoV-2 like parameters to assess the potential emergence of vaccine-resistant variants, and the role of the impact of the rate of vaccination and non-pharmaceutical interventions to reduce the probability of the establishment of vaccine-resistant variants in the population.
"As stressed by the authors, the mechanism to adjust the transmission rate reflects the strengthening or relaxation of a package of non-pharmaceutical interventions. The model in the study does not attribute the effect of individual intervention measures on virus transmission rates.
"One of the authors' conclusions is that a global vaccination effort is necessary to reduce the chances of a global spread of a resistant strain, which corroborates other modelling work presented in a preprint1 (not yet peer-reviewed) suggesting vaccine sharing between nations could be a powerful tool to decrease the potential for antigenic evolution. Therefore, there could be additional benefits to pursuing an equitable vaccine distribution worldwide beyond the protection provided by the vaccines to those receiving it.
"Models of infectious disease transmission are one tool, as part of a collective response, that can assess the impact of options seeking to control a disease outbreak. To achieve robust public health outcomes requires an interdisciplinary approach, with multiple sectors communicating and working together.
"Whilst the modelling can provide general principles (under stated model and parameter assumptions) for a vaccine-resistant variant having an increased chance of emerging and becoming established, the model can not precisely tell us when such an event may happen or where it may happen.
"Therefore, it is crucial we have situational awareness of how the virus is mutating and identifying variants that are of concern; whether that concern be due to the variant being more transmissible, causing a rise in severity, immunity derived from vaccines or infection by another variant offering less protection against the new variant, or a combination of these. The World Health Organization advocates strengthening surveillance and sequencing capacity, and a systematic approach to provide a representative indication of the extent of transmission of SARS-CoV-2 variants. Another potential benefit of having fewer infections is a higher proportion of overall infections can then be sequenced, enhancing the chance of earlier identification of emergent variants that are of concern.
"Vaccination remains a crucial tool in protecting us from COVID-19 illness and severe outcomes. As the vaccine rollout advances, it is important to continually monitor in the population (through observational studies using surveillance data) the impact of vaccination on epidemiological outcomes such as preventing infection, symptomatic disease, hospitalisation, death and onward transmission. Also, obtaining high vaccine uptake in all regions and eligible demographic groups is key to lessen the chances of there being parts of the population that have less immunity, reducing the potential for outbreaks to spread and lowering the chance of the emergence and establishment of a vaccine-resistant variant.
"With SARS-CoV-2 being a relatively novel virus, there is still uncertainty in its epidemiological, virological and immunological characteristics. As we accumulate new information over time our ideas and understanding will develop."
1 Wagner et al. (2021) Vaccine nationalism and the dynamics and control of SARS-CoV-2. medRxiv 2021.06.02.21258229. https://doi.org/10.1101/2021.06.02.21258229
Dr Nick Davies, Assistant Professor of Mathematical Modelling, London School of Hygiene and Tropical Medicine, said:
"Modelling studies of pathogen evolution, such as this one from Rella and colleagues, can help us to tease out how certain aspects of policy decisions might impact, in relative terms, upon the risk of a new SARS-CoV-2 variant emerging. Rella and colleagues find here that, when vaccine coverage is high, the risk of a vaccine strain emerging increases when the prevalence of infection is also high. This makes sense, since the probability of a vaccine-resistant strain emerging depends upon two factors: one, the strength of natural selection for a vaccine-resistant strain, which is stronger when the frequency of vaccinated individuals is higher in a given population; and two, the rate at which potential vaccine-resistant mutations may emerge, which of course depends upon how many active infections there are in the first place, as each additional infection represents an additional opportunity for random mutation to produce a vaccine-resistant strain. Additionally, a vaccine-resistant strain is less likely to randomly die out during a period in which transmission is higher.
"The qualitative result—basically, that the risk of a vaccine-escape mutant emerging will be lower if we keep cases down as much as possible—certainly makes sense. However, I don't think we really know enough about the fundamental processes of SARS-CoV-2 mutation and selection to make solid quantitative predictions—i.e., about how much any specific policy increases the risk of a vaccine mutant emerging and spreading, in absolute terms. With regard to UK policy, one could make the argument that by releasing restrictions now as opposed to in a few months' time, the probability of a vaccine escape mutant emerging in the UK might actually be lower, because seasonal factors and school closures will help to dampen transmission and lessen the probability of an escape mutant spreading widely.
"Every country should do their part to avoid creating fertile grounds for viral evolution where possible, which, thankfully, is a goal largely in alignment with public health and economic goals, as they all involve controlling transmission. But ultimately, dealing with the emergence of a vaccine escape strain is really a global issue, not a national one; as soon as a vaccine escape mutant emerges somewhere, it becomes everybody's problem. So I would think that the most important decisions for policy makers in the UK to minimise the risk of a vaccine escape strain involve: strengthening global genomic surveillance; having an effective plan for border surveillance and controls when needed; continuing to contribute to the scientific work of monitoring the properties of new variants, regardless of where they emerge, to look for early evidence of immune escape; and helping to build the global capacity for manufacturing and deploying both existing and updated vaccines quickly and equitably."
Dr Peter English, Retired Consultant in Communicable Disease Control, Former Editor of Vaccines in Practice, Immediate past Chair of the BMA Public Health Medicine Committee, said:
"There has been a lot of discussion about what the consequences of lifting "non-pharmaceutical interventions" – restrictions on mixing and measures such as mask-wearing and improved ventilation – intended to reduce the transmission of Covid-19.
"This paper uses mathematical modelling to predict the consequences; in particular, the likelihood that a vaccine resistant strain of the virus will emerge.
"There is a saying that "All models are wrong, but some are useful". Modelling an issue like this is highly complex, and often a small change to the assumptions used can make a dramatic difference to the outcome of a model.
"Nevertheless, the assumptions made in this model appear robust.
"Furthermore, there is intuitive validity to its conclusions: "…we found that a fast rate of vaccination decreases the probability of emergence of a resistant strain. Counterintuitively, when a relaxation of non-pharmaceutical interventions happened at a time when most individuals of the population have already been vaccinated the probability of emergence of a resistant strain was greatly increased. Consequently, we show that a period of transmission reduction close to the end of the vaccination campaign can substantially reduce the probability of resistant strain establishment. Our results suggest that policymakers and individuals should consider maintaining non-pharmaceutical interventions and transmission reducing behaviours throughout the entire vaccination period."
"You would expect that as more people are vaccinated, and if the vaccines are effective at preventing infection and transmission (which they are1), there will be less transmission of the virus.
"The more transmission there is, the more likely it is that significant "variants of concern", including vaccine escape variants, will arise.
"Yet it is also the case that in a relatively unvaccinated population, vaccine escape variants will have little competitive advantage, and are unlikely to become prevalent. In moderately- or highly-vaccinated populations, however, variants that can still be transmitted to and by fully vaccinated people will have a considerable competitive advantage.
"So there is intuitive validity in the idea that using all means to minimise transmission, when a high proportion of the population is vaccinated, is important. Allowing high rates of transmission at this time means that fully vaccinated people are more likely to be infected; and this means that vaccine escape variants are more likely to arise and spread, undermining the value of the vaccination programme (at least until a "tweaked" vaccine that is effective against the variant can be rolled out).
"Modelling studies such as this should always be viewed with caution; but the findings from this one are highly consistent with other ways of looking at the same question, and thus provide useful support for them."
Public Health England. COVID-19 vaccine surveillance report: 22 July 2021 (week 29). COVID-19 vaccine surveillance reports 2021; Updated 22 Jul 2021; Accessed: 2021 (29 Jul): (https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/1002580/Vaccine_surveillance_report_-_week_28.pdf or via https://www.gov.uk/government/publications/covid-19-vaccine-surveillance-report).
'Rates of SARS‑CoV‑2 transmission and vaccination impact the fate of vaccine‑resistant strains' by Simon A. Rella et al. was published in Scientific Reports at 10.00am UK time on Friday 30 July 2021.
All our previous output on this subject can be seen at this weblink:
www.sciencemediacentre.org/tag/covid-19
Dr Peter English: "Dr English is on the editorial board of Vaccines Today: an unpaid, voluntary, position. While he is also a member of the BMA's Public Health Medicine Committee, this comment is made in a personal capacity."
None others received.


The company, in addition to this plan, hopes to verify sources of water by that time.
SpaceX announced its long-term goals of landing at least two cargo missions to Mars in the next five years. Elon Musk, the company's chief announced its plans at the International Astronautical Congress (IAC) in Australia.
Musk further spoke about SpaceX's intentions to use the BFR, the massive rocket it is developing to transport Martian settlers to their new abode. BFR could also be used to fly people anywhere on the Earth within an hour's time.
The company, in addition to this plan, hopes to verify sources of water as well as the potential hazards at the same time.
The plans to begin a mining, power and life support system by 2022 are also on the cards for SpaceX so that the first batch of settlers can start living by 2024.
According to Elon Musk, SpaceX intends to take aboard the first batch of settlers to Mars by 2024 in a two crewed flight. The required supplies will be ferried in two separate cargo flights, that same year.
The first set of settlers are bestowed with a responsibility to set up a base for the future batch of settlers. The aim is to work on further expansion and terraforming their new home.
Now, these may sound like some really ambitious goals, even unrealistic maybe but when it's Elon Musk, we can sure place our bets on him!