text:
The Biggest Show on Earth è un film muto del 1918 diretto da Jerome Storm e prodotto da Thomas H. Ince. La sceneggiatura di Julien Josephson si basa su un soggetto di Florence Vincent. Ambientato in parte nel mondo del circo, il film - di cui si conserva la pellicola negli archivi del Gosfilmofond - aveva come interpreti l'attrice australiana Enid Bennett, Bliss Chevalier, Ethel Lynne, Melbourne MacDowell, Jack Nelson, Earle Rodney e Carl Stockdale.

Trama
Roxie Kemp fa la domatrice di leoni in un circo gestito dal padre. Lui, per mantenere una promessa fatta alla moglie sul letto di morte, manda la ragazza al college. L'istituto, di alto livello, accoglie allieve dell'alta società: una di queste, Marjorie Trent, diventa amica di Roxie. Accolta in casa degli aristocratici Trent, Roxie si innamora, ricambiata, di Owen, il fratello di Marjorie. Ma la signora Trent, sospettosa, non vede di buon occhio quella relazione. Quando il circo arriva in città, durante uno spettacolo il domatore si rifiuta di continuare e, per impedire che il pubblico, infuriato, si sollevi, Roxie entra nella gabbia al posto suo. La signora Trent, che la riconosce, non vuole più avere a che fare con lei. Ma, avendo scoperto che suo marito è comproprietario del circo, finisce per accettare Roxie in famiglia.

Produzione
Il film fu prodotto dalla Thomas H. Ince Corporation.

Distribuzione
Il copyright del film, richiesto dalla Thomas H. Ince Corp., fu registrato il 17 aprile 1918 con il numero LP12324.
Distribuito dalla Famous Players-Lasky Corporation e Paramount Pictures, il film uscì nelle sale statunitensi il 28 aprile 1918, venendo recensito anche come The Greatest Show on Earth. In Francia, uscì il 26 dicembre 1918 con il titolo Gladys la dompteuse, in Danimarca con quello di Cirkus Mammut.  In Finlandia, fu distribuito il 21 novembre 1921.

Copia completa della pellicola si trova conservata negli archivi del Gosfilmofond di Mosca.

Note

Bibliografia
 The American Film Institute Catalog, Features Films 1911-1920, University of California Press, 1988 ISBN 0-520-06301-5

Voci correlate
Filmografia di Thomas H. Ince

Collegamenti esterni

Film drammatici


If you decide to glue the wallpaper, you may have heard that they can be painted. There are several options for applying paint on the wallpaper, which are determined by the choice of the paint itself. Of course, before starting to paint, you should find out which wallpaper you can apply paint on and which paint to choose for a particular type of wallpaper. For this reason, let's look at how to paint the wallpaper.
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It is also important how much time is needed to completely dry the paint, as well as its environmental friendliness.
The paint can be made on the basis of latex, alkyd based or white spirit. Separately, you need to say about alkyd and latex paints.
Latex Paints. Paints on this basis are very popular because they dry quickly enough, and most importantly, they are environmentally friendly. It does not emit any harmful odors when applied to wallpaper, it is quite stable and keeps well on the surface of the walls. The resulting film perfectly protects the surface of the walls from the appearance of harmful fungi and mold.
Alkyd paints. This paint is less popular. Unlike latex paint, it dries longer. However, such paints have their advantages. For example, when applying paint on the surface of the wallpaper does not leave traces of a brush or roller. Also, alkyd paint is much more expensive than latex. True, it has a pungent toxic smell, for this reason it is less popular.
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Tikkurila is one of the best, but at the same time expensive paints that can be used for wallpaper.
Dulux Trade Supermatt This type of paint has the perfect combination of price and quality. The end result is guaranteed to satisfy you.
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Beckers, unlike its counterparts, has high reliability and is quite durable.
So, we have looked at how you can paint the wallpaper. When choosing a paint, pay attention to its basic technical characteristics, because you can complete your repair on a positive note!


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If you have been thinking about performing electrical services in your home or commercial property, let the love of Valentine's Day guide you to the service that is best for your property needs.


The Oxford Brookes University GetPublished! Student Research Poster Conference is taking place on Friday 12 April 2019, 12:00-14:00 in the Forum, John Henry Brookes Building, Headington Campus.
Dr Shelly Coe, Senior Lecturer in Nutrition at Oxford Brookes University, has led a study into how a hot chocolate drink could be effective in tackling the fatigue associated with multiple sclerosis (MS).
Shelly Coe, Lecturer in Nutrition at Oxford Brookes, was interviewed on BBC Breakfast this morning (14 January 2019) to talk about the University's research with those taking part in the Indian Ocean Row 2018.
At the beginning of July, five members of the Department of Sport, Health Sciences and Social Work (Dr. John Jakeman, Dr. Roger Ramsbottom, Dr. Peter Wright, Dr. Alaaddine El-Chab and Dr. Greg Walsh) presented their research at the 22nd annual congress of the European Conference of Sport Science (ECSS) in Essen, Germany.
The latest edition of Research Forum, the magazine showcasing research excellence at Oxford Brookes University is now available.
Dr Astrid Schloerscheidt has been appointed as the new Pro Vice-Chancellor and Dean of the Faculty of Health and Life Sciences at Oxford Brookes University and takes on the role from 30 May 2017.
We're continuing our multi-million-pound programme of investment in the Sinclair building at the University's Headington Campus.
It's just 50 days until the Rio Olympic Games 2016 and as the Olympic Torch makes its way around Brazil - the last part of the journey before the Games begin in August - Brookes alumna Julia Hayes shares memories of being a London 2012 Olympic Torchbearer.
Oxford Brookes opened its doors on Wednesday evening (25 November) to 150 Year 12 students from across Oxfordshire who are taking part in Brookes Engage.
International students can now follow a life sciences foundation pathway if they do not yet meet the academic or English language requirements for a bachelor's degree.


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text:
August 2015; 2 (4) ArticleOpen Access
Caspr2 autoantibodies target multiple epitopes
Abby L. Olsen, Yongjie Lai, Josep Dalmau, Steven S. Scherer, Eric Lancaster
First published July 2, 2015, DOI: https://doi.org/10.1212/NXI.0000000000000127
Abby L. Olsen
From the Department of Neurology (A.L.O., Y.L., J.D., S.S.S., E.L.), The University of Pennsylvania, Philadelphia; and ICREA-IDIBAPS (J.D.), Hospital Unit, University of Barcelona, Spain.
Yongjie Lai
Josep Dalmau
Steven S. Scherer
Eric Lancaster
Neurol Neuroimmunol Neuroinflamm Aug 2015, 2 (4) e127; DOI: 10.1212/NXI.0000000000000127
Make Comment
Info & Disclosures
Objective: To better understand the mechanisms of autoantibodies to the axonal protein contactin-associated protein-like 2 (Caspr2) by studying their target epitopes.
Methods: A plasmid for expressing Caspr2 was modified so that the various extracellular subdomains were deleted individually and in groups. Cultured cells were transfected to express these constructs and assayed by immunofluorescence staining with a commercial Caspr2 antibody and a panel of patient sera known to react with Caspr2. Western blotting was also performed. The role of glycosylation in immunogenicity was tested with tunicamycin and PNGase F treatment.
Results: Patient antibodies bound to the extracellular domain of Caspr2. Neither native protein structure nor glycosylation was required for immunoreactivity. Caspr2 constructs with single or multidomain deletions were expressed on the plasma membrane. All deletion constructs were recognized by patients' sera, although reactivity was significantly reduced with deletion of the discoidin-like subdomain and strongly reduced or abolished with larger deletions of multiple N-terminal subdomains. Caspr2 with all subdomains deleted except the discoidin-like domain was still recognized by the antibodies.
Conclusion: Caspr2 autoantibodies recognize multiple target epitopes in the extracellular domain of Caspr2, including one in the discoidin-like domain. Reactivity for some epitopes is not dependent on glycosylation or native protein structure.
Caspr2=
contactin-associated protein-like 2;
cDNA=
complementary DNA;
IRB=
institutional review board;
JPN=
juxtaparanodes;
nAChR=
nicotinic acetylcholine receptor;
NMDAR=
NMDA receptor;
VGKC=
voltage-gated potassium channel
Autoantibodies to the voltage-gated potassium channel (VGKC) complex are found in patients with acquired neuromyotonia (Isaac syndrome),1,2 limbic encephalitis, or Morvan syndrome.3 Although initial reports suggested that VGKC complex antibodies bind directly to Kv1.1/Kv1.2 potassium channel subunits,2,4 the antibodies primarily target the VGKC-associated proteins leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein-like 2 (Caspr2).5,6 Patients with Caspr2 antibodies may have encephalitis, acquired neuromyotonia, or both.7
Caspr2 is a transmembrane axonal protein of the Neurexin IV superfamily that organizes and concentrates VGKCs at the juxtaparanodes (JPN) of myelinated axons by a poorly understood mechanism.8 Caspr2 and its binding partners TAG-1 and Protein 4.1B are each necessary to concentrate Kv1.1/Kv1.2 potassium channels at the JPN,9,10 which is important for the proper electrical function of axons.11
Caspr2 has a large extracellular domain with 8 distinct subdomains and 12 potential N-linked glycosylation sites. It is not clear what the functions of these subdomains are or how the autoantibodies may affect the function of Caspr2 or its interactions with other JPN proteins. Autoantibodies to synaptic proteins, including the NMDA receptor (NMDAR) and the nicotinic acetylcholine receptor (nAChR), may target either a single dominant epitope or a major immunogenic region.12,–,14 For NMDAR and nAChR antibodies 3-dimensional structure is very important, but other autoantibodies, such as to glutamic acid decarboxylase 65 (GAD65), recognize linear epitopes under denatured conditions (e.g., on Western blot). We have therefore attempted to find a single dominant epitope for Caspr2 antibodies and to determine whether the autoantibodies require intact tertiary structure and/or protein glycosylation.
Standard protocol approvals, registrations, and patient consents.
Serum and CSF were collected with informed consent from patients with clinical features of Caspr2 autoimmunity (encephalitis and/or peripheral nerve hyperexcitability) under a tissue bank protocol approved by an institutional review board (IRB) at the University of Pennsylvania. A separate protocol approved by the University of Pennsylvania IRB was used to access the samples from the tissue bank for testing and to access the clinical information. Samples from patients found to contain Caspr2 antibodies using methods previously described7 were selected for further studies. The clinical characteristics of 8 of these patients have been published previously: 2 had encephalitis only, 1 had acquired peripheral nerve hyperexcitability only, and 5 had both encephalitis and peripheral nerve hyperexcitability.7 In addition, we studied samples from 2 additional patients who were subsequently identified: 1 with neuromyotonia only and 1 with both neuromyotonia and encephalitis. The number of patient samples included for the experiments was limited by the amount of sera available for testing. All experiments in this manuscript used a minimum of 4 patient samples to confirm results.
Creation of plasmids for expressing Caspr2 mutants.
Complementary DNA (cDNA) encoding full-length human Caspr2 in a Prk5 vector was obtained (gift from Dr. Elior Peles, Weizmann Institute of Science, Rehovot, Israel). This cDNA was used as a template for deleting individual subdomains of Caspr2 using the Quikchange XL Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA) with modifications described previously,15 following an approach used previously to study NMDAR antibodies.12 Primers for the various mutations were selected to include 27 bases before the cut site and 18 bases after the cut site on average for both the forward and reverse primers; these primers are listed in table e-1 at Neurology.org/nn. Clones were selected using standard microbiology techniques, and plasmid DNA was purified using QiaPrep spin miniprep and maxiprep kits (Qiagen, Venlo, the Netherlands). Clones were screened by sequencing across the area with the target excision to determine which contained the desired deletions; clones with successful deletions were verified to contain no unintended mutations by full-length sequencing. Plasmid DNA concentrations was determined on a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA).
Immunofluorescence staining.
HEK293 T cells were grown on poly-d-lysine–coated coverslips in MEM with 5% FBS and 1% P/S and were transiently transfected with full-length Caspr2 or Caspr2 mutants using JetPrime reagent (Polyplus Transfection, Illkirch, France), per the manufacturer's instructions. Cells were assayed 24–48 hours posttransfection. Cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% PBS-tween or 0.3% triton X-100 for 15 minutes, and blocked in 0.3 M glycine with 10% normal goat serum and 5% BSA for 1 hour at room temperature. Cells were singly or doubly stained with a commercial anti-Caspr2 primary antibody (ab33994, Abcam, Cambridge, United Kingdom) diluted at 1:200 and patient sera diluted 1:1,000 in blocking buffer overnight at 4°C. (ab33994 targets the intracellular domain of Caspr2 and would not be expected to compete with the human autoantibodies, which target the extracellular domain. The intracellular domain was also not modified in any of the deletion constructs.) Coverslips were washed with PBS and then secondary antibodies (Alexa Fluor 488–conjugated donkey anti-human IgG and TRITC-conjugated donkey anti-rabbit IgG at 1:400) were applied for 1 hour at room temperature. Slides were mounted in Fluoromount-G with DAPI (SouthernBiotech, Birmingham, AL) and visualized on a Leica DMRBE fluorescent microscope. Pictures were captured with a Leica DFC-350F digital camera and Openlab software (Improvision, Coventry, United Kingdom). Quantification of fluorescence was performed using NIH Image J and Adobe Photoshop software. Untransfected cells and cells in which only the secondary antibody was applied served as negative controls. Each experiment contained a minimum of 2 technical replicates per condition, and experiments were repeated a minimum of 3 times.
Quantification of immunofluorescence staining.
Quantification of fluorescence was performed using NIH Image J and Adobe Photoshop software. The intensity of staining of each single domain deletion construct by patients' sera was normalized by the amount of staining with the commercial antibody channel. A ratio was obtained for each construct.
Tunicamycin treatment.
HEK293 T cells were cultured and transfected as described above. At 4 hours posttransfection, growth media were replaced with media containing 0.2 μg/mL tunicamycin (Sigma-Aldrich, St. Louis, MO) or DMSO vehicle control. Cells were harvested 24–48 hours later and assayed as described above.
Western blotting.
HEK293 T cells were transfected as above and lysed on ice in RIPA buffer (50 mM Tris pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5% Na deoxycholate, 1% triton X-100, 2 mM EDTA) supplemented with complete protease inhibitor tablet (Roche, Basel, Switzerland). Lysates were centrifuged at 10,000 rpm for 10 minutes. Protein concentration in the supernatant was measured using Pierce BCA assay (Thermo Scientific) on a plate reader. The supernatant was diluted in sample buffer (Bio-Rad, Hercules, CA) and heated to 100°C for 5 minutes. Samples were run on a Bio-Rad electrophoresis system on 4%–20% gels, transferred to PDVF membrane, blocked in 5% milk for 1 hour, and incubated in a commercial anti-Caspr2 antibody (ab33994, Abcam) diluted 1:1,000 or patient sample overnight diluted 1:1,000 at 4°C. Secondary antibodies were alkaline phosphatase–conjugated donkey anti-human or anti-rabbit applied for 1 hour at room temperature at 1:10,000. Blots were developed using Tropix CDP-Star reagent (Applied Biosystems, Waltham, MA) and visualized on a Chemi-Doc imaging system (Bio-Rad).
PNGase digestion.
Whole cell lysates from Caspr2-transfected or untransfected control HEK293 T cells were diluted in denaturing buffer and incubated at 100°C for 10 minutes, after which G7 reaction buffer, 10% NP40, and PNGase F (New England Biolabs, Ipswich, MA) were added per the manufacturer's instructions and incubated for 1 hour at 37°C. The samples were then prepared similarly to other lysates for Western blotting.
Caspr2 autoantibodies consistently recognize an extracellular epitope of Caspr2.
We studied a panel of 10 sera from patients with Caspr2 antibodies identified using methods previously described.7 In order to confirm that patients' Caspr2 antibodies consistently recognize a surface epitope of Caspr2, HEK293 T cells were transfected to express wild-type Caspr2 and immunostained live with patient sera (1:500). The cells were then fixed, permeabilized, and treated with a rabbit Caspr2 antibody targeting an intracellular epitope, followed by fluorescent anti-human and anti-rabbit secondary antibodies (4 representative cells are shown in figure 1). All 10 patients' sera recognized a surface epitope on transfected cells but not on untransfected cells.
Figure 1 Patients' sera recognize surface epitopes of Caspr2
HEK293 T cells were transiently transfected to express Caspr2, stained with a series of patient sera live, and then stained with a commercial antibody to Caspr2 after permeabilization and fixation. Nuclei of transfected and untransfected cells were stained with DAPI (blue). Surface epitopes of Caspr2 were recognized by all 10 patients' sera (4 representative samples [S1–S4] are shown). Scale bar = 10 μm.
Glycosylation and tertiary structure are not required for antibody recognition of Caspr2.
Caspr2 has 12 potential N-linked glycosylation sites in its extracellular domain.16 In order to determine whether glycosylation of these sites is required for recognition by Caspr2 autoantibodies, we transfected cultured cells to express Caspr2 while these cells were treated with tunicamycin (figure 2). (In these experiments, human antibodies were applied after fixation and permeabilization since Caspr2 was no longer membrane-expressed.) Tunicamycin treatment prevented cell surface expression of Caspr2 and resulted in a shift of the Caspr2 band on Western blot, but it did not prevent antibody recognition. Protein extract from cells transfected to express Caspr2 was also deglycosylated with PNGase F, resulting in a shift in the band on Western blot (figure 2B). This method of deglycosylation likewise did not prevent recognition of Caspr2 by the autoantibodies, even in the denatured state (i.e., on the Western blot). We further tested a panel of 6 sera for the ability to recognize Caspr2 under denaturing conditions (on Western blot), and all samples were able to do so (figure e-1). Caspr2 autoantibodies therefore recognize the protein under denaturing conditions and glycosylation is not required for antibody recognition.
Figure 2 Caspr2 recognition does not depend on glycosylation or tertiary structure
(A) Cells were transfected to express Caspr2 in the presence of tunicamycin or vehicle (DMSO). Tunicamycin treatment resulted in intracellular localization of Caspr2, but the transfected cells were still recognized by patients' sera and a commercial antibody to Caspr2 (ab33994). Scale bar = 10 μm. (B) Lysates from cells transfected with empty vector, cells transfected with Caspr2 treated with vehicle (DMSO), Caspr2 cells treated with tunicamycin (Tun), and Caspr2 lysates treated with PNGase F were probed on Western blot with a commercial Caspr2 antibody and patient sera.
Deletion of single Caspr2 subdomains does not entirely eliminate reactivity.
Caspr2 has a very large extracellular domain, comprising more than 1,200 amino acids. The extracellular domain has previously been subdivided into 8 regions based on homology to various protein domains. We have followed a previously proposed definition of these domains (http://www.uniprot.org/uniprot/Q9UHC6#section_seq). Starting from the N-terminus of the protein and moving to the transmembrane region, these domains are Discoidin (Disc), LamininG (Lam1), LamininG (Lam2), Egf (Egf1), FibrinogenC (FibC), LamininG (Lam3), Egf (Egf2), and LamininG (Lam4). There is a single transmembrane domain and a relatively small intracellular region with a protein 4.1-binding domain. We generated a series of plasmids for expressing Caspr2 with deletions of each of the 8 extracellular subdomains (figure 3A). We named these constructs with a delta symbol followed by the domain deleted. ΔDisc, for instance, has the Disc domain deleted.
Figure 3 Single-domain deletion constructs were recognized by patients' sera
(A) Single-domain deletion constructs were generated as shown. (B) Each of these constructs was recognized by patients' sera on Western blot probed with a commercial antibody to Caspr2 (Caspr2) and patients' sera (sera 1).
The single subdomain deletion constructs were all expressed when transfected into cultured HEK293 T cells. It is interesting that patients' sera reacted with each of these constructs on Western blot (figure 3B) and also by immunofluorescence (figure 4). All 10 tested patient sera were able to immunostain living cells expressing each of these single-domain deletion constructs, confirming that they were all expressed on the cell membrane (figure 4). This pattern of findings showed that no single extracellular subdomain was absolutely necessary for antibody recognition and that multiple epitopes must exist in the extracellular domain.
Figure 4 Some single-domain deletion constructs showed decreased labeling by patients' sera
(A) Cultured cells were transfected to express wild-type and single-domain deletion constructs. All constructs were expressed on the cell membrane and all were recognized by a panel of patients' sera. Scale bar = 10 μm. (B) The relative staining in the human and commercial channels was quantified for the various deletion constructs. Only ΔDisc differed significantly from full-length Caspr2 using this method.
The relative intensity of immunostaining of some constructs, particularly ΔDisc, was decreased by eye for all patients studied. This finding is quantified in figure 4B, where the relative intensity of staining with the human and commercial antibodies is compared for each of the single-domain deletion constructs. Using this method, only ΔDisc showed significant reduction in staining with the human antibodies compared to full-length Caspr2. These results suggest that the Disc domain may contain a target epitope.
The Disc domain is a common epitope for patients with Caspr2 antibodies.
In order to more definitively determine whether the Disc domain is sufficient for antibody recognition, a construct with only the Disc domain, the transmembrane domain, and the intracellular domain was generated (figure 5A). This construct was recognized by 9 of 10 serum samples (figure 5B). The Disc domain alone is therefore sufficient for recognition by the majority of tested patients. However, deletion of this single Disc domain (ΔDisc) did not prevent recognition by any of our samples, suggesting that another epitope (or potentially multiple other epitopes) outside the Disc domain exists. This experiment was repeated 4 times, and all experiments suggested decreased intensity of but not absence of immunostaining with deletion of the Disc domain compared to the wild type (figure 4B). Thus, there is a discrete epitope within the Disc domain that is sufficient but not necessary for reactivity.
Figure 5 The Disc domain contains a target epitope
(A) A construct (Δ182-Lam4) with all extracellular subdomains except the Disc domain was created. (B) This construct was recognized by 9 of 10 sera in transfected cells. Scale bar = 10 μm. (C) Western blot of lysates from cells transfected with an empty vector, wild-type Caspr2, or the Δ182-Lam4 construct were probed with human sera and then reprobed with a rabbit antibody to Caspr2. The Δ182-Lam4 protein is significantly smaller than wild-type Caspr2 but is still recognized by both human and rabbit antibodies.
Patients' sera also recognized the construct containing only the Disc domain (with all other extracellular subdomains deleted) on Western blot, suggesting that an epitope in the Disc domain is still recognized under denaturing conditions (figure 5C). Taken together with the ability of patients' sera to recognize ΔDisc on Western blot (figure 3B), it appears likely that there are multiple epitopes in the Disc domain and elsewhere that are not dependent on tertiary structure.
Other target epitopes are generally in the N-terminal half of Caspr2.
In order to determine which other subdomains (aside from Disc) might contain target epitopes, we constructed a new series of deletion constructs involving multiple domains of Caspr2 (figure 6A). In the case of constructs with a large deletion of the C-terminal domain of the protein, we could confirm that these constructs were expressed on the cell membrane by immunostaining live cells with patient sera (figure 6B). Although constructs with larger deletions of the N-terminal half of the protein were not recognized by most patients' sera (in live or fixed/permeabilized cells), these constructs also appeared to be expressed on the membrane (figure 6B). Testing with a panel of 7 sera showed that target epitopes for most patients (6 of 7) were confined entirely to the 4 subdomains on the N-terminal of the protein. Only 1 of 7 samples reacted (weakly) when these 4 subdomains were deleted (figure 6C). Taken together, these results suggest that most target epitopes are concentrated within the N-terminal half of the extracellular domain and that patients' sera only rarely target any epitope within the C-terminal half of the extracellular domain.
Figure 6 Multiple target epitopes exist in the N-terminal domains of Caspr2
(A) Multidomain deletion constructs of Caspr2 were generated to determine which domains contain target epitopes. (B) These constructs were transfected into cultured cells and immunostained with patient sera and a rabbit antibody to Caspr2 (ab33994). Scale bar = 10 μm. (C) The results for 7 patient sera are shown. See text for discussion.
These studies have shown that autoantibodies to Caspr2 target multiple epitopes on the extracellular domain of the protein. The large extracellular domain of Caspr2 may make it a particularly available target for autoimmunity compared with the other members of the VGKC complex. A major epitope is within the discoidin-like domain, and other epitopes appear to be concentrated within the N-terminal half of the protein. At least some of these epitopes are not dependent on tertiary structure (i.e., can be recognized on Western blot) and are not glycosylation-dependent. Since our single-domain deletions (including ΔDisc) were recognized on Western blot, it is likely that for most samples more than one epitope is not dependent on tertiary structure.
Other autoantibodies to neuronal surface epitopes target a range of ionotropic neurotransmitter receptors (NMDAR, AMPAR, glycine receptor, nAchR) or metabotropic neurotransmitter receptors (GABA-B-R, mGluR1, mGluR5). In the case of antibodies to the NMDAR and AMPAR, the antibodies cause cross-linking and internalization of the receptor and thus result in decreased receptor function.17,–,19 Since Caspr2 is a cell adhesion molecule and not a receptor, different autoimmune mechanisms must be considered, for example disruption of the interaction of Caspr2 with TAG-1, disruption of the ability of Caspr2 to cluster VGKCs, or internalization of Caspr2 (with or without associated VGKCs). Understanding the target epitope of the antibodies is a first step in examining these possibilities rigorously.
The extracellular domain of Caspr2 is known to interact with TAG-1 in cis (expressed on the axon) and in trans (expressed on myelinating cells apposed to the axon).20,21 It is known that the immunoglobulin-like domain of TAG-1 binds to the extracellular domain of Caspr2,22 but it is not known which subdomain(s) of Caspr2 are required for these interactions. Furthermore, it is not known whether the same area of Caspr2 is required for the cis and the trans interaction. One plausible explanation for the multiple epitopes identified is that some antibodies disrupt the interaction between Caspr2 and TAG-1 in cis, whereas others disrupt the interaction between Caspr2 in trans. Future studies will be needed to determine whether the antibodies affect the interaction between Caspr2 and TAG-1. Since the interaction between these 2 proteins is critical for the proper localization of potassium channels,8 this would be a plausible disease mechanism.
These autoantibodies represent valuable scientific tools for further understanding the basic biology of Caspr2. In addition, understanding the pathogenic mechanism of the antibodies opens up therapeutic possibilities. Autoantibodies target multiple epitopes on Caspr2, and it is possible that antibodies to certain epitopes may be more pathogenic than antibodies to other epitopes. For example, antibodies to the nAChR that target the major immunogenic region may be more pathogenic than antibodies to other parts of the receptor.23 An important avenue of future investigation is to determine which antibody-epitope interactions are most relevant for Caspr2 autoimmunity. If a particular epitope is the target of disease-causing antibodies, then specific immune therapies to direct the immune response away from this epitope may be possible. Luo and Lindstrom have recently used their knowledge of the target epitope of acetylcholine receptor autoantibodies to create this type of epitope-specific immunotherapy for experimental autoimmune myasthenia gravis.24
Abby L. Olsen: drafting/revising the manuscript, study concept or design, analysis or interpretation of data, acquisition of data. Yongjie Lai: analysis or interpretation of data, acquisition of data. Josep Dalmau: drafting/revising the manuscript, contribution of vital reagents/tools/patients. Steven S. Scherer: drafting/revising the manuscript, study concept or design, analysis or interpretation of data. Eric Lancaster: drafting/revising the manuscript, study concept or design, analysis or interpretation of data, acquisition of data, statistical analysis, study supervision, obtaining funding.
STUDY FUNDING
This work was supported by K08 NS075142 to E.L. E.L. was also supported by a grant from the Dana Foundation.
A.L. Olsen and Y. Lai report no disclosures. J. Dalmau is the editor of Neurology: Neuroimmunology & Neuroinflammation; is on the editorial board for Neurology UpToDate; holds patents for and receives royalties from autoantibody test, NMDA receptor autoantibody test; has patents pending for GABA(B) receptor autoantibody test, GABA(A) receptor autoantibody test, DPPX autoantibody test, and IgLON5 autoantibody test; has consulted for Advance Medical; and received research support from Euroimmun, NIH, NIMH, Instituto Carlos III, and AGUR. S.S. Scherer is on the scientific advisory board for the Charcot-Marie-Tooth Association; is on the editorial board for Cell and Tissue Research, Experimental Neurology, Glia, Journal of Neuroscience Research, and Journal of the Peripheral Nervous System; has consulted for Lundbeck, Scisive, Clarion Consulting, Pfizer, Guidepoint Global Advisors, Cydan, and Grifols; and received research support from NIH/National Institute of Neurological Disorders and Stroke. E. Lancaster received speaker honoraria from Grifols Inc; has consulted for Medimmune Inc; provided expert review for the Federal Vaccine Injury Compensation Program; and received research support from Talecris Inc, Lundbeck Inc, National Institute of Neurological Disorders and Stroke, and Dana Foundation. Go to Neurology.org/nn for full disclosure forms.
The authors thank Drs. Elior Peles (Weizmann Institute, Rehovot, Isreal), Domna Karagogeos (University of Crete Medical School, Heraklion, Crete, Greece), and Catherine Faivre-Sarrailh (Université de la Méditerranée Aix-Marseille II, Marseille, France) for sharing plasmid constructs related to Caspr2 used in some preliminary studies. The authors also thank the patients and their families for supporting this research by donating biological samples.
Funding information and disclosures are provided at the end of the article. Go to Neurology.org/nn for full disclosure forms. The Article Processing Charge was paid by the authors.
Supplemental data at Neurology.org/nn
Accepted in final form May 22, 2015.
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially.
Shillito P,
Molenaar PC,
Vincent A,
. Acquired neuromyotonia: evidence for autoantibodies directed against K+ channels of peripheral nerves. Ann Neurol 1995;38:714–722.
Hart IK,
Waters C,
. Autoantibodies detected to expressed K+ channels are implicated in neuromyotonia. Ann Neurol 1997;41:238–246.
Tan KM,
Lennon VA,
Klein CJ,
Boeve BF,
Pittock SJ
. Clinical spectrum of voltage-gated potassium channel autoimmunity. Neurology 2008;70:1883–1890.
Kleopa KA,
Elman LB,
Lang B,
Scherer SS
. Neuromyotonia and limbic encephalitis sera target mature Shaker-type K+ channels: subunit specificity correlates with clinical manifestations. Brain 2006;129:1570–1584.
Lai M,
Huijbers MG,
Lancaster E,
. Investigation of LGI1 as the antigen in limbic encephalitis previously attributed to potassium channels: a case series. Lancet Neurol 2010;9:776–785.
Irani SR,
Alexander S,
Waters P,
. Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan's syndrome and acquired neuromyotonia. Brain 2010;133:2734–2748.
Bar V,
. Investigations of caspr2, an autoantigen of encephalitis and neuromyotonia. Ann Neurol 2011;69:303–311.
Horresh I,
Poliak S,
Grant S,
Bredt D,
Rasband MN,
Peles E
. Multiple molecular interactions determine the clustering of Caspr2 and Kv1 channels in myelinated axons. J Neurosci 2008;28:14213–14222.
Traka M,
Goutebroze L,
Denisenko N,
. Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers. J Cell Biol 2003;162:1161–1172.
Gu C,
Gu Y
. Clustering and activity tuning of Kv1 channels in myelinated hippocampal axons. J Biol Chem 2011;286:25835–25847.
Zhou L,
Messing A,
Chiu SY
. Determinants of excitability at transition zones in Kv1.1-deficient myelinated nerves. J Neurosci 1999;19:5768–5781.
Gleichman AJ,
Spruce LA,
Dalmau J,
Seeholzer SH,
Lynch DR
. Anti-NMDA receptor encephalitis antibody binding is dependent on amino acid identity of a small region within the GluN1 amino terminal domain. J Neurosci 2012;32:11082–11094.
Raju R,
Foote J,
Banga JP,
. Analysis of GAD65 autoantibodies in Stiff-Person syndrome patients. J Immunol 2005;175:7755–7762.
Lindstrom J,
Luo J,
Kuryatov A
. Myasthenia gravis and the tops and bottoms of AChRs: antigenic structure of the MIR and specific immunosuppression of EAMG using AChR cytoplasmic domains. Ann N Y Acad Sci 2008;1132:29–41.
Makarova O,
Kamberov E,
Margolis B
. Generation of deletion and point mutations with one primer in a single cloning step. Biotechniques 2000;29:970–972.
Gollan L,
Martinez R,
. Caspr2, a new member of the neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K+ channels. Neuron 1999;24:1037–1047.
Hughes EG,
. Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 2008;7:1091–1098.
Peng X,
. Cellular and synaptic mechanisms of anti-NMDA receptor encephalitis. J Neurosci 2010;30:5866–5875.
. AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location. Ann Neurol 2009;65:424–434.
Chatzopoulou E,
Miguez A,
Savvaki M,
. Structural requirement of TAG-1 for retinal ganglion cell axons and myelin in the mouse optic nerve. J Neurosci 2008;28:7624–7636.
Theodorakis K,
Zoupi L,
. The expression of TAG-1 in glial cells is sufficient for the formation of the juxtaparanodal complex and the phenotypic rescue of tag-1 homozygous mutants in the CNS. J Neurosci 2010;30:13943–13954.
Tzimourakas A,
Giasemi S,
Mouratidou M,
Karagogeos D
. Structure-function analysis of protein complexes involved in the molecular architecture of juxtaparanodal regions of myelinated fibers. Biotechnol J 2007;2:577–583.
Luo J
. Myasthenogenicity of the main immunogenic region. Ann N Y Acad Sci 2012;1274:9–13.
Lindstrom J
. Antigen-specific immunotherapeutic vaccine for experimental autoimmune myasthenia gravis. J Immunol 2014;193:5044–5055.
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Bruce Arena's first national team camp since replacing Jurgen Klinsmann will present a huge opportunity for several of the players he has called in.
CARSON, Calif. — Wednesday morning marks the official beginning of Bruce Arena's second tenure as U.S. national team coach, as he kicks off the January camp some 18 years after the first time he convened players in his search for the squad to represent the country.
There is a different feeling this time around because of the circumstances that led to Arena's return. A pair of World Cup qualifying losses in November, followed by Jurgen Klinsmann's firing after more than five years as U.S. coach, has pressure hanging in the air. This January camp isn't casual, but rather the first step in what is essentially a rescue mission for Arena.
It also happens to be a chance to rescue a place on the national team for a collection of players who had either been ignored by Klinsmann or forgotten by him. As thorough as Klinsmann was in his own search for national team talent, he did develop a blind spot when it came to certain players, and Arena has essentially made a laundry list of just those types of players and brought them in for a chance to show what they can do.
In total, 31 players are in camp, which opens at StubHub Center. While there are some established regulars in the mix, as well as some older players still working to maintain their place under the new coach, the most compelling storylines will be the efforts of Klinsmann's forgotten players as they try to impress Arena.
The FC Dallas center back is coming off an MLS Defender of the Year campaign, and is widely regarded as one of the best — if not the best — central defenders in the league. Despite that, he wasn't in Klinsmann's plans even as he was helping lead FC Dallas to a U.S. Open Cup and Supporters' Shield last year.
Now, with Geoff Cameron's immediate future uncertain, and the competition at center back wide open, Hedges returns to the national team picture looking very much like a player who could make a push for a starting role with the full squad.
It was exactly one year ago when the Sporting Kansas City midfielder seemed to essentially give up on his national team future when he slammed Klinsmann for leaving him and others out of the picture. He had essentially accepted the fact Klinsmann would never give him another chance, and took the opportunity to rip into the German coach. Feilhaber said what many around American soccer were thinking when it came to what, at times, felt like an arbitrary list of players who couldn't get a look.
Arena wasted little time making it clear very early in his tenure he would give Feilhaber his chance to prove Klinsmann wrong, identifying the 2010 World Cup veteran as someone he would bring back into the mix. Now with a new January camp beginning, Feilhaber must play well, not only to justify the things he said a year ago but also to make a strong case for the role of playmaker, which appears to be wide open.
Garza's absence from the national team picture wasn't so much about Klinsmann not rating him as it was about injuries costing him his place just as he was beginning to look like a viable starting left back. In fact, it was Klinsmann who gave Garza a serious look shortly after the 2014 World Cup, at a time he was just breaking out at Club Tijuana.
After a frustrating two years of injuries and recoveries and time stuck on the bench with the Xolos, Garza enters 2017 poised for a rebound. He is joining MLS expansion side Atlanta United on loan, and begins this January camp poised to challenge for the same left back position he looked so good for back in 2014.
The defensive midfielder role is one that has needed new blood for some time, and even though Michael Bradley will likely slot into that role under Arena, there is still a need for depth at the position. Enter McCarty, who has been one of the best in MLS for several years, but had gone years since he's had a national team look.
It will be very interesting to see how he fares in a camp during which he'll have to compete with an imposing figure like Jermaine Jones, who doesn't seem ready to relinquish his national team place, even at age 35. Jones' freelancing ways haven't always made him a good candidate for the disciplined nature of the defensive midfield position, and if Arena decides to eventually move Jones along, then a strong January camp from the Red Bulls midfielder could see him benefit from a changing of the guard.
Quite a lot changed for the Portland Timbers midfielder in 2016. He began the year looking like someone who could push his way into a starting role under Klinsmann but quickly found himself stuck on the bench for much of the year. The two then clashed over Nagbe's reluctance to attend a camp in October, which led to him being left out of the November qualifiers. An exile from the national team seemed likely for Nagbe before Arena replaced Klinsmann.
Now, with a chance for a fresh start under a coach who knows all too well what Nagbe can bring to the table, the 26-year-old has an opportunity to really make a push for playing time, and potentially even a starting role with the full squad, if he can shine in this January camp.
A player who quietly revived his career in 2016, Agudelo earned a look from Klinsmann last fall after an impressive run with the New England Revolution, and more than seven years after his national team debut. Once considered one of the best young prospects in the American pipeline, Agudelo returns to the fold a more mature player and one who appears ready to make the most of his latest opportunity.
Agudelo returns to a competition at forward in which he brings different qualities than the other players in the pool. His technical ability on the ball, coupled with his ability to take on defenders, and combine size and strength, make him a handful to defend when he's on his game. Now he just has to put that all together in national team camp to earn a look with the full squad.
Another player who enjoyed a career revival, Chris Pontius was one of the pleasant surprises of 2016 with the way he played in his first season with the Philadelphia Union. The left winger flashed all of the dynamic attacking qualities he had shown at D.C. United before injuries took their toll. A healthy and motivated Pontius emerged as one of the best wingers in MLS and just the kind of player Arena needed to look at as he tries to revamp the U.S. team's wing options.
Pontius has an ability to generate his own scoring chances from the flank, working as much as a forward as a winger, and he could be an excellent option if Arena plans on using a 4-3-3. There aren't a ton of pure wing options in the national team pool and Pontius could climb the pecking order quickly with a strong camp.
As much as he would probably have gotten a look from Klinsmann, the LA Galaxy midfielder stands to benefit from the recent national team coaching change because Arena knows him well, and understands the different ways Lletget can help an attack.
Lletget began his time with the Galaxy working on the wings, but in 2016 he was finally able to play in his natural central midfield role — and he thrived. The 24-year-old provides a creative spark and confidence on the ball that could pose a real challenge to some of the older central midfield options in the player pool.
The best defensive rookie in MLS in 2016, and arguably one of the league's best right backs, Rosenberry is another player who probably would have been a part of this January camp even if Klinsmann were still the coach, but Arena won't be shy about giving him his opportunities.
The Philadelphia Union fullback will face some interesting competition in camp, from an older player in Brad Evans and a converted midfielder in Graham Zusi. Based on how well he played in 2016, Rosenberry could absolutely grab hold of a starting role in this camp, which would put him a step closer to a place with the full national team squad.
It may have looked like a typo at first when the Sporting Kansas City midfielder was listed as a defender on the initial January camp roster, but Arena made it clear soon after that he plans on trying to transform Zusi into a right back.
Arena has a track record of making just this sort of move, having converted Tony Sanneh from midfielder to right back ahead of a very successful 2002 World Cup, and more recently having converted Robbie Rogers from midfielder to fullback while coach of the Galaxy. Zusi has looked good at fullback in brief stints with Sporting KC, and if he takes to the right back position in this camp, he could quickly develop into a first-team option.


Giving a child a tea set is a great way to encourage pretend play. Whether you choose plastic, porcelain, or a specialty set, this toy is sure to become a beloved item in any child's playroom.
Tea sets are good selling toys, and for good reason. They offer plenty of design options and encourage creative play. Look around at a number of shops to find the perfect set.
Some will also come with a basket or other carrying case, linens, spoons, dessert plates, tea tray, and other items.
Ritter's Teacup Gallery offers traditional floral designed, Victorian and English looking tea sets for children. All are FDA approved for use. Additionally, this company offers the option of mailing a check for purchase to the company, instead of ordering online. Find their address on the Company information page.
Blue Moon Tea has several tea sets available, from traditional ones in baskets to an art deco design set. You can also order plenty of delicious teas from this company. Gift wrapping is available, perfect for sending a gift over the miles. Sign up for their newsletter to stay on top of their current promotions.
The Teapot Shoppe has a wide variety of tea sets in a large price range. Choose from the traditional porcelain sets to unique ones featuring classic characters like teddy bears, Alice in Wonderland, fairies, or even Hummel figurines. Luxury sets, like the Lenox Butterfly Meadow Teen Tea Set, to affordable options, like the Lauren's Tin Tulip Tea Set, make this a great place to find whatever kind of set you desire. There's even a set you can personalize for the special child in your life.
There are plenty of tea sets to choose amongst when it comes to picking one out for your little one. Before you decide on styles and patterns, consider which type of tea set will suit the needs of both you and your child.
Plastic tea sets are best for pretend play, as some plastics may not be FDA approved for use as a food container. They are much cheaper than porcelain, china, or specialty sets, and wear well for their relative cost. You can often find them with popular trademarked cartoon characters from your child's favorite television programs and movies, like the Disney Princess Cinderella version at Amazon. These tea sets are also best for younger children who may be hard on their toys.
Porcelain tea sets are a good option for older children who want a more authentic look to their tea set. They come in a variety of finishes, with beautiful designs that range from flowers to favorite fairy tale characters. Plain designs work well for either gender, like the Small World Toys It's a Party Tea Set available at Target.
However, these sets can be more expensive than plastic play tea sets, and can also break or shatter. Give this type of set to a mature child who is sure to cherish it.
Don't limit yourself to traditional tea sets. You can find them made of out tin, wood, or even fabric, like the tea set and case from Lillian Vernon. A specialty set is a good choice for a child who collects tea sets, or for a child with unique tastes. Specialty sets may be harder to come by than traditional plastic or porcelain versions, so check local children's boutiques and stores for these sets.
Make tea time with your child even more special by baking small cookies and making sandwiches together. Add some water, lemonade, or flavored tea to the teapot and you are set for a true tea party! Your child won't forget sharing a cup of tea with their parents, siblings, and friends.


text:
Events of 2019 in Colombia.

Incumbents
 President: Iván Duque Márquez
 Vice President: Marta Lucia Ramirez

Events

January
Blacks and Whites' Carnival
2019 Bogotá car bombing: A vehicle-bound suicide bomb attack in Bogotá, Colombia, kills 22 people and injures 68 others, making it the deadliest attack on the Colombian capital since 2003.

February
2019 Tour Colombia
2019 shipping of humanitarian aid to Venezuela

March
Barranquilla's Carnival
Cartagena Film Festival
 March 30: A 14-year old "hit man" is arrested in Medellin after killing victims #11 and #12 in cold blood. The victims were businessman Darío Alexis, 42, and a messenger, Mateo C, 20.

July

 28 July – 22-year-old cyclist Egan Bernal won the 2019 Tour de France.

August

September

October

November

December

Births

Deaths

August
 August 13 – Cecilia Caballero Blanco, socialite and First Lady of Colombia (b. 1913)

September
 September 3 – José de Jesús Pimiento Rodríguez, Roman Catholic cardinal (b. 1919)

See also

 2019 Pan American Games

References

Colombia
2010s in Colombia
Years of the 21st century in Colombia
Colombia

redpajama_set_name: RedPajamaWikipedia


text:
Q: Connecting with remote SQL Server DB Internet browsing has suggested me following connection string to access remote DB, attached to remote SQL server.
"Data Source=190.190.200.100,1433;Network Library=DBMSSOCN;Initial Catalog=myDataBase;User ID=myUsername;Password=myPassword;"

I want to access the DB over internet, not over LAN.
My question is about the IP address and port.
1)What IP address I should use instead of 190.190.200.100?  I mean how I can know what IP address my server has.
2) How to check the what port my server is using?  Is it always 1433?
Thanks

redpajama_set_name: RedPajamaStackExchange


Each day of practice, the Kansas City Chiefs publish an official injury report leading to the next match. Chiefs travel to face the Oakland Raiders this Sunday.
After missing more than 100 days of training and games, safety Eric Berry was back in training for the Chiefs on Wednesday. The Mitch Morse Center, which had not trained since a concussion in Week 6 against the New England Patriots, also returned to training. Reid on the walrus: "Austin (Reiter) did a hell of a job. One thing we have discovered is that we have full confidence in Austin. Again, let's just see how Mitch (Morse) is doing here. It would be another in this same mix. "
Outback Sammy Watkins, who Reid said had a "soft" foot on Monday, did not leave. Watkins played five times against the Rams before leaving the match.
On the Raiders side, WR Martavis Bryant (left PCL), CB Leon Hall (back) and DT Maurice Hurst (ankle) all missed training. Bryan is expected to miss a long time due to his injury, while Hall missed the last game due to a back injury.
Want news of the NFL and interesting links in your inbox every morning on weekdays? Rise 'n grind with us.


Ruskin recognised the site, and noted that its 'rocks are lovely with lichen, the banks with flowers; the stream-eddies are foaming and deep. But Turner has attempted none of these minor beauties, and has put into this single scene the spirit of Scotland.'5 In a lecture, he regarded it as '... an extreme wonder how Turner could have made so little of so beautiful a spot', but declared that the 'essential character of Scotland is that of a wild and thinly inhabited rocky country, not sublimely mountainous, but beautiful in low rock and light streamlet everywhere; with sweet copsewood and rudely-growing trees.' Referring also to the Liber composition Dumblain Abbey, Scotland (see Tate D08157; Turner Bequest CXVIII C), he asserted: 'The chief element in both is the sadness and depth of their effect of subdued though clear light in sky and stream.'6 As has been pointed out, in the engraving the kilt which Turner often used to emphasise the Scottishness of Scottish views replaced the angler's breeches of the drawing,7 and his nondescript headgear became a tam-o'-shanter bonnet.
Davis 2002, pp.10–11 and fig.2 (photograph of the site).
Liber Veritatis; or a Collection of Prints after the Original Designs of Claude Le Lorrain ..., London 1777, vol.I, pl.84; from 1644 original drawing by Claude Lorrain (British Museum, London, 1957–12–14–90: Michael Kitson, Claude Lorrain: Liber Veritatis, London 1978, pp.105–6, reproduced pl.84).
See for example Andrew Wilton, J.M.W. Turner: His Life and Work, Fribourg 1979, p.361 nos.539 and 540, reproduced.
Francesca Irwin, Andrew Wilton, Gerald Finley and others, Turner in Scotland, exhibition catalogue, Aberdeen Art Gallery and Museum 1982, p.38; but see Michael D.C. Rudd, 'A Lonely Dell Rightfully Restored to Wharfedale', Turner Society News, no.79, September 1998, pp.7–9, relating D03431 to A Lonely Dell, Wharfedale (Leeds City Art Gallery: Wilton 1979, p.362 no.542, reproduced, as 'A Rocky Pool, with Heron and Kingfisher'), as suggested in Finberg 1909, I, p.158, and discussed in Martin Butlin, Andrew Wilton and John Gage, Turner 1775–1851, exhibition catalogue, Royal Academy, London 1974, p.83.
'Notes on Educational Series' in Catalogue of Examples ..., in Cook and Wedderburn XXI 1906, p.135.
'Lectures on Landscape' in ibid., XXII 1906, pp.35, 38, 39.
Anne Lyles, Turner and Natural History: The Farnley Project, exhibition catalogue, Tate Gallery, London 1989, p.64.
Rawlinson 1878, pp.59–68; 1906, pp.69–79; Finberg 1924, pp.105–24.
Townsend 1996, I, p.379; Townsend 2001, p.285.
Townsend, circa 1995, Tate conservation files.


Thanks for your interest in getting help with your Google My Business marketing.
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George from my office will be giving you a call to set up a time for us to talk.
Before we meet, I'll take a look at your Google My Business Listing.
During our meeting, I'll ask you questions about your marketing and the results you are looking for to see if this is right plan for your objectives.


text:
What can we do to salvage Pakistan's Covid-19 response?
Zulfiqar A BhuttaPublished November 23, 2020
It is likely that the dreaded second surge of Covid-19 is upon us and if the evidence from elsewhere and the great flu pandemic from 1918 is anything to go by, this will be both severe and sustained. — AFP/File
THERE is something unreal about public discourse and response in Pakistan these days, akin to watching a crash landing in slow motion. One watched in abject horror the blunt refusal by mainstream politicians to reconsider their plans for mass rallies and protests. This was topped by scenes from a mass gathering on Friday for a person who from available descriptions, almost certainly, succumbed to an acute illness eerily similar to Covid-19. Of the several hundred thousand people who assembled at the gathering from all over the country, available images show that fewer than five per cent wore masks and physical distancing was non-existent.
It is likely that the dreaded second surge of Covid-19 is upon us and if the evidence from elsewhere and the great flu pandemic from 1918 is anything to go by, this will be both severe and sustained. Fortunately, while many of the earlier doomsday models of Covid-19 burden and deaths in Pakistan failed to materialise, a major factor was the excellent work and oversight by agencies and the National Command Operations Centre. This may now all be undone. Premature celebrations and accolades have helped feed a false sense of security that somehow Pakistanis are immune and hence resistant to the effects of a virus that has to date infected over 58 million people and killed close to 1.4m globally.
Premature joy over lower epidemic burden fed a false sense of security that somehow Pakistanis are immune
Despite occasional exhortation, there has been a visible decline in the use of face masks, physical distancing and precautions culminating with the politicians repeatedly encouraging and leading mass rallies and meetings in all major cities. While the prime minister warned people of the need for care, he himself led mass meetings in Islamabad and more recently in Gilgit-Baltistan, thus making it difficult to selectively ban the opposition's meetings and political assemblies.
We now face a situation where once again the current limited small-scale "lockdown" measures could well escalate to larger scale closures. A decision on closing schools for an extended winter break might be taken on November 23. If implemented, this will be an abject tragedy. Children have just returned to schools after a prolonged closure and this will now be tantamount to the potential loss of a full academic year for many and an irremediable learning gap for young children at a very sensitive part of their development. There could also be an increase in school dropout rates for adolescent girls, some of whom may end up in early marriages. This will be a national tragedy with major long-term consequences. As a nation if we have to choose between keeping our schools open or politicians in the streets, most of us would make a clear choice to do the former.
What can be done immediately to address the potential crisis that confronts us and salvage Pakistan's gains to date in responding to Covid-19?
First and foremost, we must debunk the widespread misperception that Covid-19 is a relatively harmless disease and will somehow spare Pakistan. It is a potentially lethal virus and is now also known to be associated with significant long-term consequences even among those infected with negligible or mild symptoms. It would be wonderful if the electronic media could replace the interminable political squabbles in their prime-time programmes with real information and discussion on the virus and the risks it poses. Our media currently gets a D minus for its inability to lead with examples of appropriate mask use and public education. Meaningful discussions of issues at hand such as equitable deployment of limited supplies of appropriate Covid-19 vaccines when they do become available, or myths and misperceptions would have helped reduce some of the widespread disinformation about the disease.
The potential second wave of the virus should be treated as a national emergency. There is still time to stem its exponential increase, but this will mean putting politics aside and broker an urgent moratorium on public congregations, and complete ban on large political rallies. Strict imposition of section 144 in all cities should be a starting point and will likely need stronger non-partisan measures for implementation.
Protective measures such as the use of masks, hand hygiene and physical distancing must be universal with no exceptions. Even if a vaccine were to somehow arrive and become available, the virus will be among us for much of 2021. With rich nations of the world racing to procure and deploy the limited supplies of effective vaccines as they come on line, the likely scenario for Pakistan is that between 20-40 million doses of the vaccine might be made available by mid-2021 with support from the global vaccine alliance and open market purchasing. That is barely sufficient for less than a fifth of our population and likely to go to first line health workers and those who have power and access to claim first right. I predict that most politicians will be first in queue touting their indispensability to the nation. Notwithstanding the vaccine, protective measures will be needed for another 1-2 years at least and this should be the key message to all. Measures such as the use of face masks and shields in markets, assemblies and communal prayers should be mandatory.
Rebuilding the economy requires getting our institutions and businesses working safely. So far there is little evidence that there is systematic implementation and monitoring of key responses and most measures seem sporadic and reactive. In addition to well-designed regular sero-surveillance studies, few of which have been made public, a regular situational assessment of schools, day-care facilities, businesses and factories would help ascertain what is needed to reduce the risk of infections.
Finally, we need a national discourse on how best to prepare ourselves for future emergencies of this nature. Investments in our scientific research institutions related to basic immunological research and vaccine development a few decades ago could have placed us in a much better position than we find ourselves today. At the very least the current crisis should help us think through on what structural investments and measures are needed to prevent and address the next pandemic.
The writer is founding director of Institute for Global Health & Development, Aga Khan University
Published in Dawn, November 23rd, 2020
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Kashif Sangrasi
Precise and to the point, description of state of affairs resulting from mass political activities, both from traditionally-liberal and conservative parties..
Syama
Need smart lock down.
Soofia
So true. We as a nation have to rise from our happy sleep and have to realize situation.
@Syama, smart but micro/mini lockdown.
The best bet to effectively fight the rapidly expanding, ever increasing, fleetingly growind and almost unstoppable coronavirus is to follow in letter and spirit the "Three Ws" model (Wear Mask, Wash Hands and Watch Distance) presented by world-famous public health experts, doctors, immunologists, medical scientists, bio-Engineers, physicians, pharmacists, researchers and practitioners.
Where was this fraud media when Imran Khan attacked Parliament & PTV. You & PTI are no one to lecture anyone when you & PTI supported rioting and vandalism of public property.
Ibrahim S
Wait, I thought our smart lock down set by beloved PM IK was an exemplary in the world. I guess it depends what's the meaning of SOP in Pakistan and who should observe smart lock down and social distancing by their own SOP. The reality on the ground is very clear.