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The Mstncmpt-Dl1abc mutation impairs secretion of promyostatin
Viktor Vásárhelyi, Mária Trexler, László Patthy
doi: https://doi.org/10.1101/077412
Viktor Vásárhelyi
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, P.O. Box 286, H-1519, Hungary
Mária Trexler
László Patthy
Info/History
Hypermuscularity of Compact mouse is caused by a 12-bp deletion in the myostatin gene, but the molecular basis of decreased myostatin activity is unclear since the deletion does not affect the integrity of the growth factor domain. In the present work we show that the deletion causes misfolding and impaired secretion of myostatin precursor with concomitant decrease in myostatin activity. We suggest that some modifier genes that influence the expression of the Compact phenotype of myostatin mutant MstnCmpt-dl1Abc mice may exert their action through their role in the Unfolded Protein Response.
Myostatin, a member of the transforming growth factor-β (TGF-β) family of growth factors, is secreted as an inactive precursor, promyostatin [1]. Two molecules of promyostatin are covalently linked via a single disulfide bond present in the C-terminal growth factor domain; the active growth factor myostatin is liberated from promyostatin by proteolytic processing. Furin type proprotein convertases cleave both chains of the precursor at the boundary of the prodomain and the growth factor domain, but the two prodomains and the disulfide-bonded homodimeric growth factor remain associated, forming an inactive complex referred to as a latent complex [2]. Active myostatin is released from this latent complex through degradation of the prodomain by proteases of the bone morphogenetic protein −1 (BMP-1)/Tolloid family of metalloproteinases [3].
Myostatin is a negative regulator of skeletal muscle growth: McPherron et al. were the first to show that mice lacking myostatin are characterized by a dramatic increase in skeletal muscle mass [4]. Subsequent studies on several breeds of cattle selected for increased muscle mass have shown that hypermuscularity is caused by an 11-nucleotide deletion in the third exon of the myostatin gene which causes a frameshift that eliminates the myostatin growth factor domain [5, 6, 7]. In the case of a racing-dog breed it was shown that a 2-bp deletion in the third exon of the myostatin gene increases muscle mass and enhances racing performance since the reading frame shift and premature termination removes the major part of the growth factor domain [8]. In the case of a human child it was shown that muscle hypertrophy was caused by a homozygous mutation in the first intron of the myostatin gene that leads to mis-splicing, premature termination and complete loss of myostatin protein [9].
Studies on the hypermuscular Compact mouse, selected for high body weight and protein content, have shown that the Compact phenotype is caused by a 12-bp deletion in the myostatin gene [10]. This deletion, however, did not provide a simple explanation as to how it leads to loss of myostatin activity. Unlike the myostatin mutations mentioned above, this mutation (denoted MstnCmpt-dl1Abc) in the prodomain region of the myostatin precursor does not result in premature termination and does not affect the integrity of the growth factor domain [10]. In harmony with the notion that myostatin activity is not zero in homozygous Cmpt/Cmpt mutants, these animals have a less pronounced hypermuscular phenotype than homozygous mice in which the myostatin gene was disrupted by gene targeting [11]. Genetic analyses also revealed that full expression of the hypermuscular Compact phenotype requires the action of modifier loci in addition to MstnCmpt-dl1Abc [12, 13]. These studies also revealed that the modifier loci exerted their effects on muscularity only in the presence of MstnCmpt-dl1Abc [12].
In principle, the MstnCmpt-dl1Abc mutation present in the prodomain region might lead to decreased myostatin activity if it interferes with any of the steps leading to the production of mature myostatin from promyostatin: it might interfere with the folding of the precursor and/or the formation of the covalent homodimer, preventing its secretion or it might impair the liberation of mature myostatin from the precursor and/or the latent complex. In the present work we wished to examine these possibilities, therefore we produced and studied the molecular properties of promyostatins carrying the MstnCmpt-dl1Abc mutation.
Our studies on recombinant proteins expressed in bacterial expression systems have shown that the Mstncmpt-Dl1abc deletion causes misfolding of promyostatin. In mammalian expression systems misfolding of Mstncmpt-Dl1abc promyostatin impaired its secretion, suggesting that the hypermuscular phenotype of Compact mouse is due to a severe decrease in the level of extracellular myostatin precursor. Based on these findings we suggest that the modifier loci that affect the expressivity of the Compact phenotype [12,13] may encode constituents of the Unfolded Protein Response (UPR) as they may influence the fate of mutant promyostatin.
2.1. Expression of wild type and MstnCmpt-dl1Abc mutant murine promyostatin in Escherichia coli
The cDNA of murine promyostatin was amplifed with the 5′ GGACCGCGGTCAATGAGGGCAGTGAGAGAGAAG 3'sense and 5′ CGGGTCGACTTATCATGAGCACCCACAGCGG 3′ antisense primers from the pCMV6-Entry mouse myostatin plasmid (MR 227629 Origene,) and ligated into the SacII/SalI sites of pPR-IBA2 (IBA) expression vector, yielding the expression plasmid designated as pPR-IBA2_MU_promyostatin.
We used the QuikChange Site-Directed Mutagenesis method to introduce the Cmpt-dl1Ab mutation into the coding region of murine promyostatin. The mutagenesis reaction was performed with the 5′ CAAACAGCCTGAATCCAACTTCAAAGCTTTGGATGAGAATGGC 3′ sense and 5′ GCCATTCTCATCCAAAGCTTTGAAGTTGGATTCAGGCTGTTTG 3′ antisense primers and with the pPR-IBA2_MU_promyostatin expression plasmid as template. The mutagenesis deleted the AGGCATTGAAAT nucleotide segment (positions 672-684) of the murine promyostatin cDNA, deleting five amino acids (Leu-Gly-Ile-Glu-Ile, positions 224-228 of murine promyostatin) and inserting a single phenylalanine in the protein.
E. coli BL21(DE3) cells were transformed with the pPR-IBA2_MU_promyostatin or pPR-IBA2_MU_ promyostatin_Cmpt_dl1Ab vectors and expression of recombinant proteins was performed with the same method that was used for the production of the recombinant wild type human promyostatin [14].
2.2. Expression of MstnCmpt-dl1Abc mutant human promyostatin in Escherichia coli
In order to produce the MstnCmpt-dl1Abc mutant human promyostatin we used the QuikChange Site-Directed Mutagenesis method to introduce the Cmpt-dl1Ab mutation into the coding region of the expression plasmid pPR-IBA2_HU_promyostatin containing the cDNA of human promyostatin [14]. Mutagenesis was performed with the 5′ CAAACAACCTGAATCCAACTTCAAAGCTTTAGATGAGAATGGTC 3′ sense and 5′ GACCATTCTCATCTAAAGCTTTGAAGTTGGATTCAGGTTGTTTG 3′ antisense primers. As in the case of the MstnCmpt-dl1Abc mutant of murine promyostatin, the mutation replaced residues Leu-Gly-Ile-Glu-Ile (positions 223-227 of human promyostatin) by a phenylalanine residue.
Escherichia coli BL21(DE3) cells were transformed with the pPR-IBA2_HU_Promyostatin_Cmpt_dl1Ab plasmid and expression, purification and refolding of recombinant mutant promyostatin was performed with the protocol used for the production of the recombinant wild type human promyostatin [14].
2.3. Expression of wild type and MstnCmpt-dl1Abc mutant promyostatins in HEK 293 human embryonic kidney cells
The cDNAs of wild type mouse or human promyostatin were cloned into the pENTRY-IBA51 entry vector (IBA) and the resulting pENTRY-IBA_MU_Promyostatin and pENTRY-IBA_HU_Promyostatin plasmids were used as templates of the Quickchange mutagenesis reaction; mutagenesis was performed with the primers given in sections 2.2 and 2.3. After sequence verification the inserts encoding the wild type and mutant promyostatins were transferred to the pCSG-IBA142 mammalian expression vector.
The HEK 293 cells used to express promyostatins were cultured in DMEM medium containing 10% heat inactivated fetal bovine serum in the presence of 5% CO2 at 37°C. For transfection 3 ml aliquots containing 5 × 105 HEK 293 cells were seeded in each well of a 6-well plate and the cells were allowed to attach for 24 h. The cells were transfected with 3-3 µg of pCSG-IBA142-HU_Promyostatin, pCSG-IBA142-HU_Promyostatin_Cmpt-dl1Ab, pCSG-IBA142-MU_Promyostatin, pCSG-IBA142-MU_Promyostatin_Cmpt-dl1Ab or pCSG-IBA142-control plasmids with Fugene HD transfection reagent (Promega) according to the recommendations of the manufacturer. 24 h after transfection the culture media were changed to serum-free DMEM and transient protein expression was allowed for 48 h.
Culture fluids were concentrated to 200 µl, cells were lysed with 200 µl icecold RIPA buffer (50 mM TRIS, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS pH 8.0) containing proteinase inhibitors (2 μg/ml Aprotinin, 5μg/ml Leupeptin, 1 mM PMSF, 5 mM EDTA, 1 mM EGTA) and 15 µl aliquots were subjected to SDS-PAGE on 12 % gels. Western blot analysis of the gels was performed using antibody AF788, specific for the growth factor region of promyostatin (R&D Systems). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) used as loading control was visualized with Anti-GAPDH mouse mAb 6C5 (Calbiochem).
Secondary structure of wild type and mutant mouse and human promyostatin were predicted with Jpred3 (http://www.compbio.dundee.ac.uk/jpred) that provides a three-state (α-helix, β-strand and coil) prediction of secondary structure at an accuracy of 81.5% [15].
The homology models of murine and human promyostatin were generated with the automated protein structure homology-modelling server SWISS-MODEL Version 8.05 (http://swissmodel.expasy.org/ [16, 17]. The template was identified from the SWISS-MODEL template library (SMTL version 2014-07-09, PDB release 2014-07-03) with the automated template search of the pipeline. The models were based on the selected 3rjr.1.A file that contains the structure of porcine proTGFbeta1 [18]. The sequence identities of murine and human myostatin with porcine proTGFbeta1 were 31.53 % and 31.68 %, respectively over the aligned regions. The models generated by the pipeline were analyzed with Swiss-PdbViewer DeepView version 4.1 (http://spdbv.vital-it.ch/ [19]. For further details of homology modeling see the online Supplementary files 1 and 2 at http://journals.cambridge.org/grh.
The list of genes present in modifier loci that affect expression of the hypermuscular Compact phenotype of mice carrying MstnCmpt-dl1Abc [12,13] were identified using the Mouse Genome Informatics database (http://www.informatics.jax.org) and their possible effect on the Compact phenotype was evaluated based on the functional annotation of the proteins in the Swiss-Prot database (http://www.uniprot.org/uniprot/).
3.1. The Mstncmpt-Dl1abc mutation impairs the folding of promyostatin expressed in Escherichia coli
Wild type and Mstncmpt-Dl1abc mutant promyostatins were expressed in Escherichia coli BL21(DE3) according to the protocol described previously for wild type human promyostatin [14]. Although this protocol yielded biologically active, homodimeric proteins in the case of wild type [14] and K153R mutant human promyostatin [20], in the case of murine myostatin, neither the wild type nor the Mstncmpt-Dl1abc mutant protein were expressed. Since the cDNAs of murine and human promyostatin are only 91.8 % identical at the nucleotide sequence level, it seems possible that the codon usage of murine promyostatin cDNA is less optimal for expression in Escherichia coli, than that of the human protein.
To overcome this problem we have introduced the Mstncmpt-Dl1abc mutation into human promyostatin. Human promyostatin carrying the Mstncmpt-Dl1abc mutation was similar to the wild type protein in as much as it was also expressed in Escherichia coli BL21(DE3) and also accumulated in inclusion bodies. In the case of the mutant protein, however, the refolding protocol yielded covalent polymers (Fig. 1), indicating that the deletion has deleterious effects on the structure, folding and homodimerization of promyostatin.
Figure 1. The Mstncmpt-Dl1abc mutation impairs the folding of human promyostatin.
Wild type and mutant promyostatins were expressed in Escherichia coli and refolded according to the protocol described earlier [14]. In the case of wild type promyostatin (lane 1) the majority of the recombinant proteins formed homodimeric promyostatin, but in the case of promyostatin carrying the Mstncmpt-Dl1abc mutation (lane 2) the majority of the proteins formed covalent polymers. Non-reduced (-DTT, panel A) and reduced samples (+DTT, panel B) were run on 12 % SDS-PAGE and were visualized by staining with Coomassie Brilliant Blue R-250. The numbers indicate the Mr values of proteins of the Low Molecular Weight Calibration Kit.
3.2. Effect of the Mstncmpt-Dl1abc mutation on the structure of promyostatin
Our observation that the Mstncmpt-Dl1abc mutation causes misfolding of promyostatin suggested that the mutation might affect a region that is critical for the structural integrity, folding and homodimerization of this protein. Although, the three-dimensional structure of promyostatin is unknown, its homology with pro-TGF-β1 suggested that pro-TGF-β1 and promyostatin have similar secondary structural elements in equivalent positions and that the spatial arrangement of these elements is also similar [18]. In harmony with this suggestion secondary structure prediction with Jpred 3 predicts similar secondary structural elements in equivalent positions with high confidence. Furthermore, homology modeling of human and murine promyostatin using the crystal structure of pro-TGF-β1 as template (Table 1.) confirmed the suggestion that the topology of promyostatin is similar to that of pro-TGF-β1 (see Supplementary file). Although the overall model quality (Global Model Quality Estimation score) of the homology models of human and murine promyostatin is limited by their moderate (<40%) sequence similarity with the template, the Local Quality Estimates of the conserved secondary structural elements are very high since their local sequence similarities are >80% (see Supplementary file).
Table 1: Summary of homology modeling of human and mouse promyostatin.
The homology models of murine and human promyostatin were generated with the automated protein structure homology-modelling server SWISS-MODEL Version 8.05.
In the crystal structure of homodimeric pro-TGF-β1 [18] (and the homology models of promyostatins), the N-terminal parts of the two prodomains, consisting of the α1 and α2 helices, provide the 'straitjacket' that encircles and shields each growth-factor monomer of the homodimer. The C-terminal parts of the two prodomains (that provide the 'arm domains' that connect the two prodomains in the homodimeric precursor) contain three distinct structural regions. A four-stranded β-sheet (consisting of β1, β3, β6 and β10 strands and buried by the α2, α3 and α4 helices) is in close proximity of the growth factor monomer of the same polypeptide chain, whereas a four-stranded β-sheet (consisting of β2, β4, β5 and β7 strands) and β-strands β8 and β9 extend to link the two arm domains of the homodimeric precursor in a 'bowtie' [18].
The Mstncmpt-Dl1abc mutation that deletes 12 nucleotides of the murine myostatin gene, results in the replacement of residues Leu-224, Gly-225, Ile-226, Glu-227 and Ile-228 by a phenylalanine residue [10]. Since residues 224-228 correspond to the five residues that form the β7 strand of the arm domain (Fig. 2) it is plausible to assume that deletion of this β7 strand may affect the formation of the four-stranded β-sheet that appears to be critical for the folding and homodimerization of promyostatin. It should be noted that secondary structure prediction with Jpred 3 predicts with high confidence the presence of the β7 strand in wild type murine and human promyostatin, but the β7 strand is missing from predictions for murine and human promyostatins carrying the Mstncmpt-Dl1abc mutation.
Figure 2. Position of the residues deleted by the Mstncmpt-Dl1abc mutation in the homology model of mouse promyostatin.
The figure shows the homology model of murine promyostatin highlighting the positions of the residues deleted in the Compact mutant (residues in red). Note that the Compact mutation deletes the β7 strand of the prodomain of promyostatin. The growth factor domain of promyostatin is shown in gold. The homology model was computed using the structure of pro-TGF-1 as a template.
Our observation that the Mstncmpt-Dl1abc deletion interferes with the folding and homodimerization of promyostatin is in harmony with the suggestion that the β7 strand is critical for the integrity of the four-stranded β-sheet formed by β2, β4, β5 and β7 of promyostatin. Nevertheless, the failure of Mstncmpt-Dl1abc mutant promyostatin to attain a native-like structure in vitro does not exclude the possibility that in mammalian cells Mstncmpt-Dl1abc mutant promyostatin may acquire native-like structure. To check this possibility we have studied the expression of Mstncmpt-Dl1abc mutant promyostatins in mammalian cells.
3.3. The Mstncmpt-Dl1abc mutation impairs secretion of promyostatin in HEK 293 cells
In our earlier work we have shown that human HEK 293 cells transfected with wild type or K153R mutant promyostatin secrete the precursor efficiently [20]. In harmony with these data, the culture fluids of HEK 293 cells transfected with wild type mouse or human promyostatin contained significant amounts of secreted promyostatin. However, the culture fluids of cells transfected with mouse and human promyostatin carrying the Mstncmpt-Dl1abc mutation did not contain detectable amounts of promyostatin (Fig. 3).
Figure 3. The Mstncmpt-Dl1abc mutation impairs secretion of promyostatin in HEK 293 cells.
HEK 293 human embryonic kidney cells were transfected with pCSG-IBA142 mammalian expression vector (control), or pCSG-IBA142 vectors harboring the cDNA of wild type human promyostatin (wt huPM), wild type mouse promyostatin (wt muPM), human promyostatin carrying the Mstncmpt-Dl1abc deletion (del huPM) or mouse promyostatin carrying the Mstncmpt-Dl1abc deletion (del muPM). Transient protein expression was allowed for 48 h in serum-free DMEM then reduced samples of the culture media (CM) and cell lysates (LYS) were subjected to SDS-PAGE on 12% gels. Promyostatin was visualized on Western blots using antibody AF788, specific for the growth factor region of promyostatin. The left hand panels show the results of the analysis of the culture media and the right hand panels show the results obtained by analysis of cell lysates. The arrows indicate the positions of ovalbumin (45 kDa) and phosphorylase b (97 kDa) of the Low Molecular Weight Calibration Kit. Note that wild type human and mouse promyostatin are secreted into the culture medium (wt huPM_CM and wt muPM_CM), but promyostatin could not be detected in culture fluids of control cells (control_CM) or cells transfected with promyostatins carrying the Mstncmpt-Dl1abc deletion (del huPM_CM and del muPM_CM). Also note that promyostatin is present in comparable amounts in the lysates of cells transfected with wild type or mutant promyostatin (cf. wt huPM_LYS vs. del huPM_LYS and wt muPM_LYS vs. del muPM_LYS). Top row: reduced samples; lower row: non-reduced samples. In the case of cell lysates GAPDH was used as a loading control (bottom panel).
Analyses of reduced samples of lysates of HEK 293 cells revealed that cells transfected with wild type and MstnCmpt-Dl1abc mutant promyostatins were similar in as much as they contained comparable amounts of intracellular promyostatin (Fig. 3). Analyses of non-reduced samples have shown that lysates of HEK 293 cells transfected with wild type promyostatins contained promyostatin dimers. Promyostatin dimers were, however, missing from lysates of cells expressing mutant promyostatins, indicating that the mutant proteins failed to form disulfide-bonded homodimers.
In the present work we have shown that the Mstncmpt-Dl1abc mutation that deletes 12 nucleotides of the myostatin gene causes misfolding and impaired secretion of myostatin precursor with concomitant decrease in myostatin activity.
Our observation that lack of secretion of MstnCmpt-Dl1abc mutant promyostatins did not lead to a significant increase in the amount of intracellular promyostatin (as compared with wild type promyostatin) was somewhat surprising since there is a general consensus that mutations that affect secretory signals usually lead to intracellular accumulation of the mutant proteins (e.g. [21, 22, 23]). The most plausible explanation for the lack of significant accumulation of mutant promyostatin is that terminally misfolded, improperly assembled MstnCmpt-Dl1abc mutant promyostatins are recognized by the quality-control system of the endoplasmic reticulum and are degraded by the proteasome [24].
In our earlier work we hypothesized that, although the Mstncmpt-Dl1abc mutation may seriously decrease myostatin activity, this activity is not zero in homozygous Cmpt/Cmpt mutants, therefore modifier genes may have a significant influence on the Compact phenotype [10]. The search for modifier genes affecting the expression of hypermuscularity in the Compact mouse was based on the assumption that the Compact line, in addition to achieving homozygosity for the MstnCmpt-dl1Abc mutation, also accumulated modifier alleles that increased the expression of hypermuscularity in the Compact mouse. These studies have revealed significant association with hypermuscularity for markers on chromosomes 1, 3, 5, 7, 11, 16, and X, the strongest association was found for markers on chromosomes 16 and X [12,13].
In principle, modifiers of the Compact phenotype may enhance hypermuscularity by increasing the penetrance of the MstnCmpt-dl1Abc mutation, through their influence on myostatin activity, their effect on downstream components of the myostatin signaling pathway or their effect on other genes regulating muscle development.
Earlier surveys of the genes present in the chromosomal regions showing strong association with hypermuscularity of Compact mice identified a number of candidate genes that might play some of these roles in the regulation of muscle mass [12,13] see Table 2. For example, it was pointed out that regions containing modifier loci on chromosome 7 contain gene MyoD1 of Myoblast determination protein 1, a key regulator of skeletal myogenesis, as well as gene Pcsk6 of proprotein convertase subtilisin/kexin type 6, an enzyme involved in the proteolytic processing of precursors of TGF-βfamily members. Similarly, it was noted that a region of chromosome 16 associated with hypermuscularity of Compact mouse contains gene Chrd of chordin, a protein known to bind members of the TGF-βfamily and sequester them in latent complexes. In the case of chromosome 1 a modifier of the expressivity of the Compact phenotype was mapped to the same region as Myog of myogenin, a downstream target of myostatin [13]. Along the same lines, it was emphasized that the chromosomal region corresponding to the strongest peak on chromosome X [12] contains gene Ar of androgen receptor and it was suggested that Ar might act as a modifier of the Compact phenotype through its influence on the expression of myostatin.
Table 2: Markers and selected genes near major modifier loci influencing the compact phenotype in the myostatin mutant (Mstncmpt-Dl1abc) compact mouse.
For each chromosome the markers closely linked to major modifier loci are in bold.
It must be emphasized, however, that the studies that identified regions on Chromosomes 1, 3, 5, 7, 11, 16 and X showing strong association with the hyper-muscularity of compact mice were of relatively low resolution, the association of modifier loci with selected markers showed rather broad (several cM wide) peaks (see [10,12,13]). The putative modifier genes mentioned above are usually separated by several cM from the markers showing strongest association with the modifier loci (Table 2). The problems caused by low resolution modifier-maps may be illustrated by gene Ar of androgen receptor that was suggested to act as a modifier of the Compact phenotype as it is found in the chromosomal region corresponding to the strongest peak on chromosome X [12]. More recent studies refuted this assumption: quantitative analyses of gene expression did not show differences between mRNA levels of androgen receptor in Compact and control mice, indicating that the androgen receptor gene is not the true X-linked modifier of the Compact phenotype [25,26]. Furthermore, finer mapping of the modifier loci on chromosome X has succeeded in narrowing down the modifiers to two regions that are distinct from the region containing the gene of androgen receptor [26].
In view of our finding that the MstnCmpt-dl1Abc mutation leads to misfolding and impaired secretion of mutant promyostatin, we think that more attention should be paid to genes of proteins involved in the quality control system of the endoplasmic reticulum. We assume that some mutations affecting constituents of Unfolded Protein Response system may enhance the expressivity of the MstnCmpt-dl1Abc mutation if they increase the efficiency of the removal of MstnCmpt-dl1Abc mutant promyostatin. In other words, mutations are expected to increase hypermuscularity of Compact mouse if they decrease the activity (decrease expression and or decrease molecular activity) of proteins assisting protein folding (e.g. chaperones) of the MstnCmpt-dl1Abc mutant promyostatin or increase the activity (increase expression) of proteins assisting removal of the misfolded protein (e.g. constituents of the ubiquitin-dependent protein degradation system). Our survey of the modifier regions of chromosomes 1, 3, 5, 7, 11, 16, and X that show strong association with hypermuscularity of Compact mice [12, 13, 26] has indeed identified a number of genes that might affect the folding and rescue or degradation of MstnCmpt-dl1Abc mutant promyostatin (Table 2). It should be noted, however, that the extended chromosomal regions that are suspected to contain the modifier loci encode hundreds of genes, only a small fraction of which have a role in protein quality control.
Considering modifier loci on chromosomes 1, 3, 5, 7, 11, 16, and X individually, on chromosome 1 there is a clear maximum between the markers D1Mit309 and D1Mit33, close to D1Mit262 (Table 2). As pointed out earlier [13], this region contains the gene Myog for myogenin, however, in the vicinity of D1Mit262 (56.55 cM) and Myog (58.18 cM) we also find gene Ube2t (58.24 cM) of ubiquitin-conjugating enzyme E2 T (UniProt: Q9CQ37) that catalyzes covalent attachment of ubiquitin to other proteins, marking them for degradation.
On chromosome 3 a narrow region of strong effect was found on the segment defined by markers D3Mit206 and D3Mit224, close to D3Mit224 [12]. In the vicinity of D3Mit224 (20.54 cM) we find gene Hspa4l (at 19.51 cM) of heat shock protein 4 like (UniProt: P48722), a chaperone that aids protein folding.
On chromosome 5 a narrow region of strong effect was defined by markers D5Mit155, D5Mit239, D5Mit26 and D5Mit161, close to D5Mit26 [12]. In the vicinity of D5Mit26 (54.65 cM) we find gene Usp30 (55.96 cM) for ubiquitin specific peptidase 30 (UniProt: Q3UN04) and gene Ube3b (55.99 cM) for ubiquitin protein ligase E3B (UniProt: Q9ES34), two proteins involved in ubiquitin-dependent degradation of proteins. This region also contains gene Hspb8 (56.41 cM) for heat shock protein 8 (UniProt: Q9JK92), a protein with chaperone activity.
On chromosome 7 a modifier region was mapped close to D7Mit83 [12]. As noted previously [12], genes of Myod1 and Pcsk6 map to this region. However, we note that gene Ube3a (33.95 cM) of ubiquitin protein ligase E3A (UniProt: O08759) is even closer to D7Mit83 (32.84 cM) than Myod1 (30.03 cM) or Pcsk6 (35.36 cM). It should be pointed out that ubiquitin protein ligase E3A functions as a cellular quality control ubiquitin ligase by helping the degradation of misfolded proteins.
Broad regions of effect were found on chromosome 11 with two major peaks at D11Mit24 and D11Mit41 [12]. Close to D11Mit24 (31.95 cM) we find gene Hspa4 (31.91 cM) of heat shock 70 kDa protein 4 (UniProt: Q61316), a constituent of the Unfolded Protein Response that is involved in chaperone-mediated protein complex assembly. The same region also contains gene Canx (30.46 cM) of calnexin (UniProt: P35564), a protein that plays a major role in the quality control apparatus of the endoplasmic reticulum by the retention of incorrectly folded proteins, as well as Ube2b (31.82 cM) of ubiquitin-conjugating enzyme E2B (UniProt: P63147). A possible candidate gene in the peak region defined by D11Mit41 (54.34 cM) is gene Usp32 (at 51.34 cM) of ubiquitin specific peptidase 32 (Uniprot: F8VPZ3), an enzyme involved in ubiquitin-dependent protein degradation.
Chromosome 16 also has an extended modifier region with three peaks at D16Mit143, D16Mit136 and D16Mit93 [12]. In the vicinity of D16Mit143 (11.23 cM) we find gene Ube2l3 (10.63 cM) of ubiquitin-conjugating enzyme E2L3 (UniProt: P68037) that is involved in the selective degradation of short-lived and abnormal proteins, as well as Ufd1l (11.65 cM) of ubiquitin fusion degradation 1 like (UniProt: P70362), an essential component of the complex that exports misfolded proteins from the endoplasmic reticulum to the cytoplasm, where they are degraded by the proteasome. Note that these genes are closer to the D16Mit143 marker (11.23 cM) than Chordin (12.51 cM) that was suggested earlier as a potential modifier gene.
In the sharp peak defined by the D16Mit136 marker (27.82 cM) we find gene Fstl1 (26.48 cM) of follistatin-like 1 (Q62356) that, similarly to other follistatin-related proteins, is an antagonist of growth factors of the TGF-ΰfamily [27, 28]. Close to the peak at D16Mit93 (43.22 cM) we find gene Hspa13 (43.36 cM) of the chaperone, heat shock protein 70 family, member 13 (Q8BM72) and gene Usp25 (44.40 cM) of ubiquitin specific peptidase 25 (P57080), a constituent of the Ubiquitin Proteasome System.
Chromosome X showed a broad and exceptionally strong region of effect extending from DXMit126 to DXMit99, with a single peak at DXMit128 [12]. Although the gene of androgen receptor present in this region appeared to be an attractive candidate for the X-linked modifier of the Compact phenotype, subsequent studies refuted this assumption: 1. Neither the structure nor the level of expression of androgen receptor were different in Compact and control mice; 2. Finer mapping of the modifier loci on chromosome X excluded the androgen receptor gene as the major X-linked modifier of the Compact phenotype [25, 26]. These studies narrowed down the modifier regions to two sharp peaks and a third broader region between markers DXMit40 and DXMit116 [26]. The sharp peak showing strongest association with hypermuscularity is close to DXMit83 (27.99 cM), the second peak is bordered by DXMit130 (55.45 cM) and DXMit37 (59.10 cM) [26]. A survey of the genes located in the region of the peak at DXMit83 (27.99 cM) identified Usp26 (28.31 cM) of ubiquitin specific peptidase 26 (UniProt: Q99MX1), a component of the ubiquitin-dependent protein degradation system as a possible candidate that may modify the expressivity of the Compact phenotype. Our survey of the second major modifier region bordered by DXMit130 and DXMit37 failed to identify genes that might contribute to hypermuscularity of Compact mice, but in the region between DXMit40 and DXMit116, close to DXMit116 (49.58 cM) we find Ube2dnl1 (49.40 cM) and Ube2dnl2 (49.40 cM), ubiquitin-conjugating enzyme E2D N-terminal like 1 and 2 (UniProt: A2AFH1, and A2AFH2) that might function as constituents of the ubiquitin-dependent protein degradation system.
It should be pointed out that our suggestion that the phenotypic consequences of the misfolding of Mstncmpt-Dl1abc promyostatin are modified by constituents of the Unfolded Protein Response system is reminiscent of the conclusions drawn from studies on neurodegenerative diseases caused by misfolding of mutant proteins. In the last decade the search for modifier genes affecting susceptibility to neurodegenerative protein folding diseases has led to the conclusion that many of them encode proteins involved in protein folding and protein degradation [29, 30]. For example, the search for modifier genes of prion diseases led to the identification of Hectd2, an E3 ubiquitin ligase [31] and the chaperone Hspa13 [32] that is also a candidate modifier gene for the Mstncmpt-Dl1abc mutation (see above).
In the present work we have shown that the Mstncmpt-Dl1abc mutation impairs the folding and secretion of promyostatin. Nevertheless, the mutant precursor may give rise to some mature myostatin explaining why homozygous Cmpt/Cmpt mice have a less pronounced hypermuscular phenotype than homozygous mice in which the myostatin gene is disrupted [11]. This difference between Compact and myostatin-null mice may also be reflected in the spectrum of genetic loci that affect muscle mass: in the case of Compact mice components of the protein quality control system may increase the penetrance of the Mstncmpt-Dl1abc mutation (through efficient elimination of mutant promyostatin), but these proteins are not expected to have an influence on myostatin activity in myostatin null-mice. Our survey of the genes present in the chromosomal regions showing strong association with hypermuscularity of Compact mice has identified numerous members of the protein quality control system that might play a role in the elimination of Mstncmpt-Dl1abc mutant promyostatin.
In harmony with the difference between Compact mice and myostatin-null mice, the spectra of modifier loci identified in myostatin-null mice [33, 34] are markedly different from those defined using Compact mice [12, 13, 25, 26]: in the case of myostatin-null mice twelve Quantitative Trait Loci were identified on mouse chromosomes 1,3, 6, 7 that significantly interacted with the myostatin genotype [34], whereas in the case of compact mice hypermuscularity was associated with several unrelated markers, markers on chromosomes 16 and X showing the strongest association [12,13].
This work was supported by grant 108630 of the National Scientific Research Fund of Hungary (OTKA).
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More than 40% of adults experience tooth sensitivity, also referred to as dentin hypersensitivity. It occurs more frequently in women than in men. (1), (2), (3) Most people experience tooth sensitivity as a short, sharp pain originating from a tooth or teeth in their mouth.
Each tooth contains four parts. The enamel comprises the visible crown of the tooth and is the hardest layer of the tooth containing the highest level of calcium phosphate mineral). The dentin is the layer below the enamel in the crown of the tooth, and below the cementum in the root. It is softer than the enamel, but stronger than the cementum. The cementum is present on the root surface to help anchor the teeth into the jaw. The pulp is in the center of the tootth and contains soft connective tissue, blood vessels, and nerves).
Major factors that lead to tooth sensitivity are gum recession and loss of cementum, and loss of enamel. Gum recession (gum moving down from the gum line, exposing the root dentin surface) can be caused by loss of periodontal attachment as a result of periodontal disease or by brushing too hard causing gum abrasion. Loss of enamel, on the other hand, occurs when the toothbrush abrades or wears down the enamel surface and exposes the underlying dentin (the layer underneath the enamel). Enamel loss is more prominent when brushing occurs immediately before or soon after consumption of acidic foods (fruits, tomatoes) and drinks that cause tooth erosion.
What Happens in the Tooth to Cause Tooth Sensitivity?
When the enamel is worn down, or the gum line is recessed, exposing the dentin, tooth sensitivity can occur. The dentin contains tubules that run from the center of the tooth, i.e., the pulp, which contains the nerves of the tooth, to the exposed dentin surface. When the dentin is exposed, dentin fluid flows outward from the pulp. If this flow is perturbed by exposure to an external stimulus, such as heat, cold, change in pressure, sweet or sour foods and drinks, a signal id transmitted to the nerves which is perceived as pain.
Picture is of open dentin tubules.
If you think you are experiencing tooth sensitivity, it is recommended that you see your dentist who can correctly diagnose whether you are experiencing tooth sensitivity and provide a solution.
1 Irwin CR, McCusker P. Prevalence of dentine hypersensitivity in a general dental population. J Ir Dent Asso 1997;43:7-9.
2 Smith WA, Marchan S, Rafeek RN. The prevalence and severity of non-carious cervical lesions in a group of patients attending a university hospital in Trinidad. J Oral Rehabil 2008:35:128-34. 3 Addy M. Dentine hypersensitivity: New perspectives and an old problem. Int Dent J. 2002;52:375-376.


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After serving in church ministry for 20 years, Stephen now writes, speaks and produces Christian films with his brothers, Alex and Shannon. Stephen produced the movies Overcomer, War Room, Courageous, Fireproof, Facing the Giants, and Flywheel and co-wrote the New York Times bestselling books The Love Dare, The Resolution for Men, and The Battle Plan for Prayer. He has been interviewed by "Fox & Friends," CNN, ABC World News Tonight, and serves on the boards of the Fatherhood CoMission and the Christian Film foundation. He and his wife, Jill, live in Albany, Ga. with their six children and are active members at Sherwood Church.
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Month: January 2018
Lindo Creek COI will begin to unravel tapestry of criminality from 2000-2008 -President Granger as Justice Trotman is sworn in
Georgetown, Guyana – (January 31, 2018) President David Granger, today, said that the establishment of the Commission of Inquiry (COI) into the Lindo Creek massacre, which claimed the lives of eight miners in 2008, will lead to the unravelling of the criminal network and exposure of operatives, who were responsible for the reign of terror, …
Guyana, Cuba to increase trade, cooperation -As President accredits new Ambassador
Georgetown, Guyana – (January 31, 2018) Guyana and Cuba are well-placed to further strengthen economic cooperation. The signing of an agreement for the export of Guyana's rice to Cuba and the signing of the Memorandum of Understanding (MoU) between the Georgetown Chamber of Commerce and the Chamber of Commerce of the Republic of Cuba has …
President welcomes UN's referral of Guyana-Venezuela border controversy to ICJ
Georgetown, Guyana – (January 30, 2018) The Government of the Cooperative Republic of Guyana has welcomed the decision of the Secretary General of the United Nations (UN), Mr. Antonio Gutteres to refer the controversy arising out of the claim by the Bolivarian Republic of Venezuela to Guyana's territory, to the International Court of Justice (ICJ). …
New land surveyors urged to guard against professional dishonesty
Georgetown, Guyana – (January 29, 2018) Noting Government's plans to accelerate physical infrastructural development, agricultural diversification, renewable energy projects, agro-industrial processing and affordable housing drives, Minister of State, Mr. Joseph Harmon, today, charged the eight graduates of the Sworn Land Surveyors Course offered by the Guyana Lands and Surveys Commission (GLSC), to uphold the tenets …
Transcript of the Address by His Excellency David Granger, President of the Cooperative Republic of Guyana, at the 69th Republic Anniversary of India, held at the Guyana Marriott Hotel January 26, 2017
President David Granger: Minister of State, Honourable Joseph Harmon, Former presidents, Mr. Donald Ramotar and Mr. Samuel Hinds, former Prime Minister, Mr. Hamilton Green, Ministers of the Government, Members of the National Assembly, His Excellency, Mr. Venkatachalam Mahalingam, Members of the Diplomatic Corps, Distinguished Invitees, Members of the Indian Community, Members of the Media, Ladies …
President remembers Forbes Burnham's commitment to working class, social cohesion -at Annual Fund-raising Dinner
Georgetown, Guyana – (January 28, 2018) President David Granger, last evening, hosted members of the Forbes Burnham Foundation at the Baridi Benab at State House, where they held their Annual Fund-Raising Dinner. This is the first in a series of activities that is being spearheaded by the Foundation to commemorate the 95th birth anniversary of …
President lauds service of MOTP staff members
Georgetown, Guyana – (January 27, 2018) President David Granger, yesterday, told members of staff at the Ministry of the Presidency that he trusts and likes them as a team of public servants as they have strived to live up to his expectations of integrity, impartiality, industry and intelligence. The Head of State was at the …
New Cheddi Jagan Int'l Airport must boast efficiency as entry point to a modern state -President Granger as he conducts site visit
Georgetown, Guyana – (January 27, 2018) President David Granger, this morning, charged the management and staff of the Cheddi Jagan International Airport (CJIA) to commit to ensuring that with a newly modernised airport comes efficiency, high standards and quality service, where an increasing number of investors, tourists and the travelling public can be in an …
President joins nonagenarian in birthday celebration
Georgetown, Guyana – (January 28, 2018) President David Granger, yesterday, journeyed to Belladrum, West Coast Berbice to join the relatives and friends of Mr. Sydney Grenville, one of Guyana's newest nonagenarians, in the celebration of this milestone. Mr. Grenville, a well-known resident of the village, marked this significant birth anniversary last Wednesday, January 24, 2018. …
"Once it has to do with the quality of life of our people, we will be there with you" – State Minister makes good on promise to Alexander Village residents
Georgetown, Guyana — (January 28, 2018) Minister of State, Mr. Joseph Harmon, yesterday afternoon, promised to work alongside neighbourhood constituencies in order to achieve an improved quality of life for all Guyanese. The Minister made this pledge while at a commissioning ceremony for three newly constructed bridges at Alexander Village in Georgetown. The construction of …


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Boutique Hotelier Ian Schrager's EDITION Brand to Open in Turkey
By Natalie White
Once an ancient city known as Halicarnassus of Caria, Bodrum was famous as the site of one of the Seven Wonders of the Ancient World.
Today, on the West side of the peninsula, in Yalıkavak Marina, The Bodrum EDITION — part of EDITION Hotels that is a partnership between boutique hotelier Ian Schrager and Marriott International — is tucked into a wind-sheltered cove, bringing with it a new wonder to the modern incarnation of the historic region.
Set within 22,000 sq metres, the Bodrum EDITION draws inspiration from local influences and native natural resources yet infuses each space with surprising, ultra-modern twists. The landscape, conjured as a Mediterranean oasis, reflects the peninsula's indigenous flora and fauna. Native olive trees that are so significant to the Turkish landscape as well as local stone have been used throughout the resort to create a sustainable and captivating scene, in keeping with its natural environment.
The jewel in The Bodrum EDITION's crown and its most significant characteristic is the magnificent white beach, spread out below the property and leading into the irresistible turquoise waters of the Aegean Sea.
As the horseshoe-shaped grounds slope downward towards the beach and jetty with its private cabanas, the pathways and leisure areas are interspersed by the hotel's 102 guest rooms, bungalows with private pools and sublime four bedroom private villa with pool.
Destined to be Bodrum's premium gathering place for sophisticated guests, the hotel's 100 metre long beach area will play host to one of the main social hubs of the resort, with its casual-cool beach bar and club welcoming resident guests to lounge in the private cabanas as well as yacht-transported visitors from the nearby Greek islands, just a 50-minute sail away.
The property also houses a unique, state-of-the art spa facility complete with Turkish Hammam, salt treatment room, sauna, steam room and plunge pool. 14 tranquil treatment rooms will offer world class treatments.
In the magical sanctuary of Bodrum, the jewel of the Turquoise Coast, The Bodrum EDITION is a secret village of paradise, a portal to a separate, more relaxed reality.
By understanding that a hotel is more than just a place to sleep—it's a hub, a meeting place, a centre culture, a site for relaxation, reflation, and hard-earned leisure time, and place for life and play—Schrager creates not just a hotel but a seamlessly integrated experience, a small city within a city. It's an international ethos with a local product: a more comfortable stay, and an elevated experience.
#TheTimeIsNow Breitling Unveils its Southeast Asia Squad
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Facebook Offered to Build Houses in Silicon Valley
San Francisco, USA (Gadgets Now) – Facebook Offered to Build Houses in Silicon Valley: The shortage of housing in California's Silicon Valley has gotten so severe that Facebook Inc on Friday proposed taking home building into its own hands for the first time with a plan to construct 1,500 units near its headquarters.
Facebook Offered to Build Houses in Silicon Valley.
The growth of Facebook, Alphabet Inc's Google and other tech companies has strained neighborhoods in the San Francisco Bay area that were not prepared for an influx of tens of thousands of workers during the past decade. Home prices and commute times have risen.
Tech companies have responded with measures such as internet-equipped buses for employees with long commutes. Facebook has offered at least $10,000 in incentives to workers who move closer to its offices.
Those steps, though, have not reduced complaints that tech companies are making communities unaffordable, and they have mostly failed to address the area's housing shortage.
"The problem with Silicon Valley is you don't have enough supply to keep up with the demand," said Sam Khater, deputy chief economist at real estate research firm CoreLogic.
With Facebook's construction plan, the company said it wanted to invest in Menlo Park, the city some 45 miles (72 km) south of San Francisco where it moved in 2011.
The company said it wants to build a "village" that will also have 1.75 million square feet of office space and 125,000 square feet of retail space.
"Part of our vision is to create a neighborhood center that provides long-needed community services," John Tenanes, Facebook's vice president for global facilities, said in a statement.
The 1,500 Facebook housing units would be open to anyone, not just employees, and 15 percent of them would be offered at below market rates, the company said.
Facebook said it expects the review process to take two years.
Alphabet has taken a smaller step, buying 300 modular apartment units for short-term employee housing, the Wall Street Journal reported last month.
Menlo Park Mayor Kirsten Keith said in an interview that there were concerns about whether the Facebook plan would increase traffic, a subject the city's planning department would study.
She said, though, that Facebook's plan fits with the city's own long-term plan for development, and that the city was excited about the additional housing.
Facebook's Tenanes said the density of the proposed development could also entice spending on transit projects.
"The region's failure to continue to invest in our transportation infrastructure alongside growth has led to congestion and delay," he said.
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Visual functioning and quality of life in the SubFoveal Radiotherapy Study (SFRADS): SFRADS report 2
M R Stevenson1,
P M Hart1,
U Chakravarthy1,
G MacKenzie2,
A C Bird3,
S L Owens3,
I H Chisholm4,
V Hall5,
R F Houston6,
D W McCulloch7,
N Plowman8
1Ophthalmology and Vision Science, Queen's University Belfast, UK
2Centre for statistics, Keele University, Keele, UK
3Institute of Ophthalmology, University College London, UK
4Eye Unit, Southampton University Hospitals Trust, UK
5Wessex Radiotherapy Centre, Southampton, UK
6Northern Ireland Radiotherapy Centre, Belfast, UK
7Department of Economics and Politics, University of Ulster, Jordanstown, UK
8Department of Radiation Oncology, St Bartholomews Hospital, London, UK
Correspondence to: Usha Chakravarthy Ophthalmology and Vision Science, Royal Hospitals, Belfast BT12 6BA, UK; u.Chakravarthyqub.ac.uk
Aims: To determine whether or not self reported visual functioning and quality of life in patients with choroidal neovascularisation caused by age related macular degeneration (AMD) is better in those treated with 12 Gy external beam radiotherapy in comparison with untreated subjects.
Methods: A multicentre single masked randomised controlled trial of 12 Gy of external beam radiation therapy (EBRT) delivered as 6×2 Gy fractions to the macula of an affected eye versus observation. Patients with AMD, aged 60 years or over, in three UK hospital units, who had subfoveal CNV and a visual acuity equal to or better than 6/60 (logMAR 1.0).
Methods: Data from 199 eligible participants who were randomly assigned to 12 Gy teletherapy or observation were available for analysis. Visual function assessment, ophthalmic examination, and fundus fluorescein angiography were undertaken at baseline and at 3, 6, 12, and 24 months after study entry. To assess patient centred outcomes, subjects were asked to complete the Daily Living Tasks Dependent on Vision (DLTV) and the SF-36 questionnaires at baseline, 6, 12, and 24 months after enrolment to the study. Cross sectional and longitudinal analyses were conducted using arm of study as grouping variable. Regression analysis was employed to adjust for the effect of baseline co-variates on outcome at 12 months and 24 months.
Results: Both control and treated subjects had significant losses in visual functioning as seen by a progressive decline in mean scores in the four dimensions of the DLTV. There were no statistically significant differences between treatment and control subjects in any of dimensions of the DLTV at 12 months or 24 months after study entry. Regression analysis confirmed that treatment status had no effect on the change in DLTV dimensional scores.
Conclusions: The small benefits noted in clinical measures of vision in treated eyes did not translate into better self reported visual functioning in patients who received treatment when compared with the control arm. These findings have implications for the design of future clinical trials and studies.
AMD, age related macular degeneration
CS, contrast sensitivity
DLTV, Daily Living Tasks Dependent on Vision
DVA, distance visual acuity
EBRT, external beam radiation therapy
NVA, near visual acuity
SFRADS, SubFoveal Radiotherapy Study
visual functioning
http://dx.doi.org/10.1136/bjo.2003.030445
A number of studies have suggested that the most commonly used conventional measure of visual function, distance acuity, does not adequately reflect visual functioning and ability to undertake vision dependent tasks.1,2 Historically, distance acuity has been measured on a high contrast letter chart containing letters of progressively smaller sizes, which subtend progressively smaller angles at the focal point on the retina, and which serves as a marker for the ability of the eye to resolve objects. It is easily and quickly determined, hence its popularity in the clinical setting. However, there are many other aspects of vision that are important such as contrast sensitivity, colour and binocular vision, reading ability and visual scanning, which are disregarded when high contrast distance acuity alone is measured. As they are time consuming, often requiring the use of complex equipment and an operator with special skills, these other aspects of vision are only likely to be measured in the research setting. Thus, there has been an increase in interest in the use of self reported visual functioning questionnaires, which are performance based and easily administered, in the hope that these will reflect more accurately functional vision in the individual.
A variety of questionnaires have been devised, validated, and used as outcome measures in the evaluation of cataract surgery,3–6 and a few studies have utilised visual functioning instruments to ascertain the impact of age related macular degeneration (AMD) on vision related quality of life.7,8 Currently there is much interest in the ability of a variety of therapies to limit or prevent visual decline in wet AMD. Thus far, all of the clinical trials that have tested the effect of interventions in exudative AMD have used "change in distance visual acuity" as the primary outcome variable.
Although treatment interventions in exudative AMD have had a limited degree of success in reducing the risk of moderate and severe vision loss, the value of the benefit remains questionable. Thus, for instance, a one line difference in mean acuity between treatment and control groups may not translate into improved visual functioning. The increasing tendency to use dichotomous end points such as the loss of 15 letters of vision presupposes that this difference is significant in terms of lost quality of life. However, this remains unproved.
The Sub Foveal Radiotherapy Study (SFRADS) which examined the effect of 12 Gy of external beam in subfoveal choroidal neovascularisation of AMD included both standard tests of visual function (distance and near acuity, reading speed, and contrast sensitivity) that were described in the first SFRADS report9 and patient centred variables as outcome measures. There was a difference in favour of treatment in the primary outcome measure (distance visual acuity) at 24 months, which was marginally significant using longitudinal modelling techniques. Also, significant differences in the secondary outcomes measures of near acuity and contrast sensitivity were detected at 6 months and 24 months, respectively, which were in favour of treatment. The present analysis and report deals with outcomes in vision related and health related quality of life in treatment and control groups during the 24 month period after enrolment.
When the SFRAD trial was under development we considered the existing visual functioning questionnaires that had been designed for studies in ophthalmology. These questionnaires were mainly in the field of cataract surgery in which outcomes are typically perfect or near perfect restoration of vision. A significant proportion of tasks within existing instruments were devoted to extracting information on driving abilities and reading tasks. Because many of the subjects enrolled into studies of neovascular AMD have bilateral involvement, and driving and reading are adversely affected with even small degrees of vision loss in the better seeing eye, we noted a tendency to floor effects when using existing questionnaires. As it is over-optimistic to expect even small improvements following treatment in neovascular AMD and the best outcome envisaged was prevention of further vision loss in the treatment group, it was thought necessary to develop a new instrument that took into account the severe and central nature of the vision loss in neovascular AMD. This instrument which we termed the Daily Living Tasks Dependent on Vision (DLTV) was developed and validated concurrently with the study.
SFRADS inclusion and exclusion criteria, source of participants, intervention, randomisation, and masking have been described in detail.9 However, a synopsis of the trial design and methods is presented here. Patients were required to be 60 years of age or older and have evidence of subfoveal choroidal neovascularisation on a fundus fluorescein angiogram performed within 1 week of randomisation. Visual acuity at baseline was required to be equal to or better than logMAR 1.0 (6/60 or better) in the study eye. Patients with a presumptive diagnosis of subfoveal choroidal neovascularisation were screened in special study clinics in three UK hospitals in Belfast, London (Moorfields), and Southampton. Enrolment commenced in December 1995 and was completed in September 1998. In all, 203 patients were recruited into the study after giving informed consent and randomised to either the treatment (external beam radiotherapy) or control (observation only) groups. Four patients were subsequently removed from the analysis as they did not fit the entry criteria on grounds of age or visual acuity in the study eye. Patients were examined at baseline, 3, 6, 12, and 24 months after randomisation when standard efficacy and safety parameters were recorded. Visual functioning and quality of life data were collected at all time points except at the 3 month visit. The optometrists who undertook visual assessments and the interviewers who administered the questionnaires were unaware of the treatment status of the patients, although neither the treating physician nor the patient was masked.
Clinical measures of vision
The clinical measures of vision—distance and near acuity and contrast sensitivity—were tested by an optometrist trained to undertake the study protocol that was adapted from the Macular Photocoagulation Study manual of procedures.10 All measurements were performed on each eye of each patient. Before refraction distance visual acuity (DVA) in each eye was measured with patients wearing their usual glasses if any. The backlit Bailey-Lovie logMAR chart11 was used for all DVA measurements. Following standardised refraction, DVA was measured again and the line with the smallest letters in which at least three of the letters were correctly identified was entered as the line acuity for that eye. Near visual acuity (NVA) was tested with each eye with a + 4.00 addition on the Bailey-Lovie near reading chart at 25 cm and scored in logMAR notation. Contrast sensitivity (CS) was measured for each eye using the Pelli-Robson chart with the patient seated at the recommended distance of 1 metre.
Visual functioning and quality of life
An interviewer trained for the task in each centre administered the instruments. The visual functioning questionnaire used was the DLTV,12,13 and information on health related quality of life was obtained using the SF-36.14–16 We also developed an additional short instrument to assess self reported dependency and the use of social services. The results from this latter aspect of SFRADS are not included in this report.
A full description of the validation of the DLTV and the methods used to assign items to the dimensions is the subject matter of a separate publication.17 In brief, however, the DLTV was conceived as a 33 item questionnaire covering tasks relating to visual function (with and without the use of magnification aids) and general aspects of visual health. In the majority of instances, each item was scored on a 4 point ordered categorical scale where the minimum possible score was 1 (inability to do the task) and the maximum was 4 (no difficulty with the task). Some items were scored differently, and details of the items and scoring system used have been published previously.12,13 After the exclusion of items dealing with the use of magnification aids and those on general aspects of visual health, the DLTV contains 22 items that directly pertain to activities of daily living. Validation of the instrument in an independent large dataset of older adults with a clinical diagnosis of AMD in one or both eyes showed that these 22 items formed four dimensions of nine, eight, three, and two items, respectively (table 1). As there was redundancy in dimension 1, two items were omitted. The data from SFRADS for the present report were analysed using the validated domain structure,17 that is four dimensions containing seven, eight, three, and two items respectively (table 1). The scores from each of the items within a dimension were averaged and converted into a scale between 0 and 100 similar to those used in other studies. Where an individual indicated that a task was not applicable, this item was not scored and the percentage DLTV score was adjusted for the number of items answered.
Items comprising the original DLTV
The SF-36 was the generic instrument of choice which has been validated in older people and used along with visual functioning instruments in other studies. The SF-36 is generally analysed as eight multi-item dimensions of health.18
Questionnaire data were unavailable in a small proportion of clinic visits although visual outcome and angiographic data were acquired. This was usually in response to the participant's desire to minimise the length of time spent in the hospital clinics. The flow chart (fig 1) shows the route followed by patients during time on study, number of patients who completed each scheduled study visit and the percentage for whom questionnaire data were available.
Flow chart. (Figures in parentheses give the numbers of patients for whom self reported outcomes were collected at visit.)
Visual functioning indices have been shown to be most strongly driven by visual acuity in the better seeing eye.3,5–7,12,13 We examined the effect of treatment in the entire study population and then in a subgroup categorised by the status of the study eye relative to the fellow eye. Each study eye was allocated to the status of better eye or worse eye based on DVA. If DVA was identical in the two eyes, the eye with better near visual acuity was assigned the status of the better eye. If DVA and NVA were identical, CS was used to determine better eye status. Of the 199 study subjects, only one had identical DVA, NVA, and CS in both eyes and this patient was allocated to the group where the study eye was the better eye. We present summary statistics on the entire study population, and in the subgroup where study eye was the better eye at baseline.
We tested for differences in baseline characteristics between treatment and control groups using the independent samples t test and χ2 tests.
We then examined the changes in the summary scores for the four dimensions of the DLTV and the eight dimensions of the SF-36 at 12 months and 24 months using the independent samples t test with arm of study as grouping variable. Additionally, regression analysis was employed to test for confounding variables. These analyses were then repeated in the subgroup where the study eye was the better eye. As the data were subjected to multiple testing, only p values below 0.025 were considered as significant.
The DLTV has been previously validated for use in subjects with AMD in cross sectional studies. We exploited the longitudinal nature of the dataset to examine the relation between change in DLTV dimension scores and change in distance visual acuity. We included the status of the presenting eye (study eye) and arm of study as co-variates.
The SPSS version 10 was used for the analyses.
In total, there were 199 eligible patients of whom 99 were assigned to the treatment group and 100 to the control group. The mean (SD) age of treated patients was 75.3 (6.4) years and that of the control group was 75.2 (6.4).
Full details of the demography of the SFRAD study population have been reported previously.9 Sex and centre distribution, which are of relevance to the present report, are shown in table 2.
SFRADS participant details by sex and centre
We have already shown in the first SFRADS report that the treatment and control group were well matched with no significant differences in the clinical measures of vision in the study eye at baseline.9Table 3 shows the mean and range of distance and near acuity and contrast sensitivity in the study eye with the study population subdivided by whether the study eye was the better or the worse of the patients two eyes.
Clinical measures of vision at baseline in SFRADS participants when study eye is better eye or when study eye is worse eye by treatment status (figures in parentheses are standard deviations).
Patient centred outcomes
DLTV
Mean scores in dimension 1 were generally lowest compared with those of dimensions 2 and 3 in both treatment and control groups. There were no significant differences in mean scores in any of the dimensions of the DLTV between treatment and control groups (table 4). Reductions in mean scores in dimensions 1–3 of the DLTV were observed during time on study. These were more marked in the first 12 months when compared with the second 12 months of review. In dimension 4 that contained the two items on difficulty with light and dark adaptation, little or no change from baseline mean scores was seen at 12 months and 24 months in both the treatment and control group. Analysis of the change in scores in all dimensions showed that there were no significant differences between treatment and control groups at either 12 months or 24 months (table 4).
Mean DLTV dimension scores in treatment and control groups at baseline, and mean change in dimensional scores at 12 and 24 months for all study patients (figures in parentheses are standard errors)
Subgroup analysis
In the subgroup in which the study eye was the better eye at baseline (117), the change in mean DLTV dimension scores was not significantly different in treatment and control groups at 12 or 24 months (table 5). Similarly, also in this subgroup, changes in SF-36 dimension scores were not significantly different at 12 and 24 months in treatment and control groups (data not shown).
Mean DLTV dimension scores in treatment and control groups at baseline, and mean change in dimensional scores at 12 and 24 months for subgroup where study eye was the better eye at baseline (figures in parenthesis are standard errors)
Factors at baseline influencing DLTV outcomes at 12 months and 24 months
Treatment status did not influence outcome at either 12 months (table 6) or 24 months (table 7) in any dimension of the DLTV. Age and sex also did not influence outcome at 12 months or 24 months. Centre differences were detected solely in dimension 3 at 24 months (table 7). The visual status of the study eye (that is, when it was the better eye at baseline) was associated with a significantly larger change in dimensions 1, 2, and 3 at 12 months and dimension 3 at 24 months.
Regression model showing effect of baseline co-variates on change at 12 months in the three main dimensions of the DLTV
Regression model showing effect of baseline covariates on change in the three main dimensions of the DLTV at 24 months
Relation between DLTV and DVA
Regression analysis showed that only change in distance visual acuity in the better eye was highly significantly associated with change in the three main dimensions of the DLTV (table 8). The change in scores in dimension 4 was not statistically significantly associated with change in visual acuity.
Regression analysis showing relation between change in DLTV dimensions and change in visual acuity in the better eye
Analysis of the eight dimensions of the SF-36 showed that the treatment and control groups were well matched at baseline. The change in SF-36 dimension scores were not significantly different at 12 or 24 months between treatment and control groups (table 9).
Mean SF-36 dimension scores in treatment and control groups at baseline, and mean change in dimension scores at 12 months and 24 months for all study patients
Factors at baseline influencing SF-36 outcomes at 12 months and 24 months
As there are eight dimensions in the SF-36 leading to multiple testing, only differences which were significant at the 1% level or less are reported. Treatment status did not influence outcome at 12 months or 24 months. Geographical location impacted on outcomes in three of the dimensions of the SF-36. In these three dimensions (physical functioning, physical role, and energy and vitality) significant differences were seen between centres at both 12 months and 24 months (data not shown).
The value of generic quality of life measures as outcomes in clinical trials especially in the realms of cancer and cardiovascular disease are established.19–21 Disease or organ specific quality of life measures have also gained increasing acceptance. Both are now used extensively when considering the value of an intervention and for the purposes of resource allocation. To date, few published randomised controlled clinical trials in ophthalmology have used such self reported measures.22,23
To the best of our knowledge SFRADS is among the first major randomised controlled multicentre clinical trials in the field of AMD to evaluate visual functioning and quality of life as outcome measures.
Our analysis showed no significant differences between treatment and control groups in self reported visual functioning or wellbeing at the two main outcome points. In the initial report from the SFRADS study we described statistically significant differences in some measures of visual outcome (NVA and CS) in favour of radiotherapy treated eyes.9 However, these differences were small—namely, 0.6 lines of DVA, 1 line of NVA, and 0.3 log units of contrast that equates to two triplets in the Pelli-Robson chart.
An important and frequently asked question is whether or not such small differences in clinical measures of vision between two treatments when observed in trials translates into a meaningful difference in terms of visual functioning. Studies have shown that generic quality of life instruments are not significantly correlated with visual functioning in subjects with AMD.24 Even when improvements in function are dramatic such as those experienced following cataract surgery, generic instruments demonstrate poor relations with visual functioning.25,26 This is especially relevant to an elderly population where substantial co-morbidity has the potential to either swamp or drive the responses to an instrument. However, it was unknown whether self reported visual functioning or clinical measures would be more sensitive to change in the visual status. The protocols used to measure clinical measures of vision ensure reproducibility and hence small differences may be reliably detected. However, visual functioning instruments are inherently more prone to greater variation and minor differences may not be detected.
Our experience with SFRADS suggests that there are several factors that have to be taken into account when patient centred outcomes are used to assess the value of an intervention. SFRADS was powered to detect change in the primary outcome variable—namely, DVA. Examination of our data suggested that in order to detect a difference in summary scores of any one of the DLTV dimensions as statistically significant with 90% power, the study would have had to be increased in size to 600 subjects with 300 in each arm. As vision is mediated by a paired organ, it should be recognised that visual functioning is primarily driven by the better eye. This must be taken into account while designing trials with such an outcome measure. In this context, one third of the SFRAD study sample had good visual function in the non-study fellow eye. Therefore, change in the visual status of the study eye in this part of the sample would not have impacted on visual functioning provided that the fellow eye remained the better seeing eye during time on study. Scrutiny of our data showed that change in study eye status relative to the fellow eye occurred in 44 cases (22%) of the sample during the course of the study but the distribution by treatment allocation was not significantly different between groups. To overcome such considerations enrolment criteria would have had to be modified to allow recruitment of subjects whose study eye was the better eye at baseline and remained so for the rest of the study which is clearly impractical. Therefore, we accounted for any potential imbalance in the characteristics of the cohort by adjusting all analyses by the baseline covariates of sex, centre, and study eye status relative to the fellow eye. Finally, it is also possible that the difference between treatment and control groups that was detected in the measures of vision was so small as to be insignificant in patient centred terms.
Although we have used the DLTV in subjects with AMD and shown it to be sensitive to differences in clinical measures of vision,12,13 these validation studies were undertaken on cross sectional data. We therefore examined the relation between the DLTV and change in visual status in the SFRADS population and extended the scope of the present study to establish concurrent criterion validity in the context of a structured longitudinal study. In the three main dimensions of the DLTV the sensitivity to change in visual status was clearly evident as shown by the regression analysis presented in table 6. Even in dimension 4, where the mean change was small over time, a similar trend was seen. We are therefore confident that any meaningful differences in visual functioning as a result of treatment would have been captured by the DLTV.
The present study also examined changes in quality of life with the SF-36. Although treatment status and sex did not influence SF-36 scores in any of the dimensions, geographical location did have an effect. While the difference by geographical location was unexpected, our studies were generally in accord with previous reports where the SF-36 has been used in older adults.14–16
The impact of treatment on visual functioning and quality of life were considered to be important secondary outcome parameters in SFRADS. We were unsure how differences in traditional measures used to describe vision would affect patient centred outcomes especially in a longitudinal analysis. Our results suggest that the small benefit noted in these measures when treating subfoveal choroidal neovascularisation by low dose external beam radiotherapy is too small to impact materially on visual functioning and health related quality of life.
This study was supported by a strategic project grant from the Medical Research Council of the United Kingdom, International Standardised Random Control Trial Number (ISRCTN 84737434).
The authors thank the following: Miss M McClure, principal optometrist, Royal Hospitals Belfast, Ms K Grigg, senior optometrist, Moorfields Eye Hospital, London, Mr B Ashleigh, senior optometrist, Southampton University Hospitals Trust, Southampton, Ms P McEvoy, interviewer, Health and Health Care, Queen's University Belfast, Ms G McGoldrick, study coordinator, Queen's University Belfast, Mr A Brannon, study coordinator, Moorfields Eye Hospital, London, Ms K Parrish, study coordinator, Ms S Davis and Ms S Bryant, Eye Unit, Southampton University Hospitals Trust, Southampton, Belfast, Ms K Andrews, data processing supervisor, Epidemiology and Public Health, Queen's University Belfast.
Elliott DB, Hurst MA, Weatherill J. Comparing clinical tests of visual function in cataract with the patient's perceived visual disability. Eye 1990;4:712–17.
Cincotti A. Evaluation of indications of cataract surgery. Ophthalmic Surg 1979;10:25.
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Mangione CM, Phillips RS, Seddon JM, et al. Development of the activities of daily vision scale. A measure of visual function status. Med Care 1992;30:1111–25.
Steinberg EP, Tielsch JM, Schein OD, et al. The VF14, an index of functional impairment in patients with cataract. Arch Ophthalmol 1994;112:630–8.
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Mangione CM, Gutierrez PR, Lowe G, et al. Influence of age-related maculopathy on visual functioning and health related quality of life. Am J Ophthalmol 1999;128:45–53.
Scott IU, Smiddy WE, Schiffman J, et al. Quality of life of low vision patients and the impact of low vision services. Am J Ophthalmol 1999;128:54–62.
Hart PM, Chakravarthy U, Mackenzie G, et al. Visual outcomes in the subfoveal radiotherapy study: a randomized controlled trial of teletherapy for age-related macular degeneration. Arch Ophthalmol 2002;120:1029–38.
Macular Photocoagulation Study Group. MPS manual of procedures. Springfield, VA: National Technical Information Service, January, 1991.
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Hart PM, Chakravarthy U, Stevenson MR, et al. A vision-specific functional index for use in patients with age-related macular degeneration. Brit J Ophthalmol 1999;83:1115–20.
McClure M, Hart PM, Stevenson MR, et al. Do conventional measures of vision equate to performance of daily living tasks dependent on vision. Br J Ophthalmol 2000;84:244–50.
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Hart PM, Stevenson M, Montgomery A, et al. Further validation of the DLTV—identification of domains. Br J Ophthalmol. (in press).
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Copyright 2005 British Journal of Ophthalmology
BJO at a glance
Creig Hoyt
British Journal of Ophthalmology 2005; 89 931-931 Published Online First: 15 Jul 2005. doi: 10.1136/bjo.2005.bjaug05at%20a%20glance


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Q: Getting the address of an inline defined friend function Consider the following code:
#include <iostream>

struct foo {
    friend void bar(foo) {}

    void foobar() {
        std::cout << &bar << '\n'; // error
    }
};

int main() {
    bar(foo{}); // ok, visible through ADL
    foo{}.foobar();
}

gcc gives me this error:

main.cpp: In member function 'void foo::foobar()':
main.cpp:7:23: error: 'bar' was not declared in this scope
         std::cout << &bar << '\n'; // error
                       ^~~

That's because bar is a friend function defined in the class itself, making it invisible in the global namespace. The only way to access it is through ADL, but I have not found a way to use ADL to get the address of bar.
So my question is, how can I take the address of bar? Is there any other way than to define bar outside of foo?

A: You could declare the friend function in the enclosing namespace scope in a different translation unit:
In foo.hpp:
#include <iostream>
struct foo;
void (*get_bar_address())(foo);
struct foo {
  friend void bar(foo) {}

  void foobar() {
      std::cout << get_bar_address() << '\n'; // no error
  }
};

In foo.cpp
#include "foo.hpp"
void bar(foo);
void (*get_bar_address())(foo){
   return bar;
}

A: It seems to be impossible to get exactly what you want. The problem is that an inline friend declared function will only be found by argument-dependant lookup, as defined in the C++ standard 11 and 14 in

7.3.1.2
3 Every name first declared in a namespace is a member of that namespace. If a friend declaration in a non-local class first declares a class or function the friend class or function is a member of the innermost enclosing namespace. The name of the friend is not found by unqualified lookup (3.4.1) or by qualified lookup (3.4.3) until a matching declaration is provided in that namespace scope (either before or after the class definition granting friendship). If a friend function is called, its name may be found by the name lookup that considers functions from namespaces and classes associated with the types of the function arguments (3.4.2). If the name in a friend declaration is neither qualified nor a template-id and the declaration is a function or an elaborated-type-specifier, the lookup to determine whether the entity has been previously declared shall not consider any scopes outside the innermost enclosing namespace.

That means it is only ever be found by argument dependent lookup. Revising the rules, argument dependent lookup is only applied within a function call expression. Hence you will not be able to retrieve the address of the function as any such an expression will evaluate to the result of the call.

In case you only need a pointer referring to a function with the same functionality as bar, you could however utilize lambdas to get a pointer to such a function nevertheless.
void foo::foobar() {
    using fntype = void(*)(foo);
    std::cout << (fntype)[](foo f){ bar(f); } << '\n'; // error
}

The downside is that any other lambda will likely result in an intirely different address. You could however offer the address in a different (static) member function if the uniqueness is of importance.

A: Did you try something like this? I find it interesting that you would declare a friend function from inside the scope of the struct/class but hey.
#include <iostream>

// forward declare foo so you can forward declare bar :)
struct foo;

// forward declare bar so an address is available on the line that references it.
void bar(foo);

struct foo
{
    friend void bar(foo)
    {
       std::cout << "bar called" << std::endl;
    }

    void foobar()
    {
        std::cout << "address of bar " << &bar << '\n'; // error
    }
};

int main()
{
    bar(foo{}); // ok, visible through ADL
    foo{}.foobar();
}

Tried it and it works LOL not sure why:
$ ./test.exe
bar called
address of bar 1

though address of 1 is suspicious. :) but hey it compiled and did something. maybe you can take it further or someone can explain why this compiled/worked in the first place.

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