In yeast, birds and mammals, the SAPK-interacting protein 1 (Sin1) gene product has been implicated as a component of the stress-activated protein kinase (SAPK) signal transduction pathway. Recently, Sin1 has also been shown to interact with the carboxyl terminal end of the cytoplasmic domain of the ovine type I interferon receptor subunit 2 (IFNAR2). However, the function of Sin1 remains unknown. Since SAPK pathways are ancient and the IFN system is confined to vertebrates, the organization of the Sin1 gene and the sequences of the Sin1 protein have been compared across a wide taxonomic range of species.Sin1 is represented, apparently as a single gene, in all metazoan species and fungi but is not detectable in protozoa, prokaryotes, or plants. Sin1 is highly conserved in vertebrates (79–99% identity at amino acid level), which possess an interferon system, suggesting that it has been subjected to powerful evolutionary constraint that has limited its diversification.C. elegans the gene consists of 10 exons and 9 introns organized distinctly from those of vertebrates. In yeast and insects, Sin1 is intronless.Sin1 possesses at least two unique sequences in its IFNAR2-interacting region that are not represented in insects and other invertebrates. Sequence alignment between vertebrates and insects revealed five Sin1 strongly conserved domains (SCDs I-V), but an analysis of any of these domains failed to identify known functional protein motifs. SCD III, which is approximately 129 amino acids in length, is particularly highly conserved and is present in all the species examined, suggesting a conserved function from fungi to mammals. The coding region of the vertebrate Sin1 gene encompasses 11 exon and 10 introns, while in The study reveals the phylogeny of a little studied gene which has recently been implicated in two important signal transduction pathways, one ancient (stress response), one relatively new . Saccharomyces cerevisiae [RAS2Val 19 mutation had elevated levels of cyclic AMP, impaired growth control and were acutely sensitive to heat shock. This phenotype was reversed when the yeast strain was transfected with a cDNA (clone JC310) that encoded a then unknown protein. Although the authors suggested that the inferred interaction between the JC310 product and RAS might be fortuitous, they favored the possibility that that the unknown protein either was a true inhibitor of RAS or that it was a RAS target protein, which when over-expressed, had a protective action. A S. cerevisiae protein encoded by the AVO1 gene showed distant similarity the human JC310 product [Sin1 was originally described as a human protein that modulated Ras function in  clone JC10 that eSchizosaccharomyces pombe cDNA library as a 665 amino acid protein that bound via polypeptide sequences in its C-terminal 244 amino acids to the Sty1/Spc1, stress activated MAP kinase (SAPK) [S. pombe to grow at 37°C, albeit very poorly. Moreover, fusion of the first 486 amino acids of yeast Sin1 (which does not restore growth) with the C-terminal 182 amino acids of the chicken Sin1 sequence protected against heat shock. Together, these data showed that Sin1 functions as a component of the stress-activated Sty1/Spc1 MAP kinase pathway in S. pombe and that a functional homolog of Sin1 exists in vertebrates.Approximately eight years after the identification of JC310 it was again identified, on this occasion in a yeast two-hybrid screen of a No further information concerning Sin1 appeared since the paper of Wilkinson et al.  until ouOvine Sin1, which is 88% identical in sequence to chicken Sin1, can be co-immunoprecipated with the IFN receptor subunit IFNAR2 and shows a similar subcellular distribution to the receptor protein when co-expressed in mammalian cells . AlthougAlthough the SAPK pathway is itself ancient and is found in all the species in which the Sin1 gene exists, the IFN system of receptors and ligands is restricted to vertebrates. We reasoned, therefore, that an analysis of Sin1 gene sequences might not only provide insight into the function of Sin1, but indicate how the protein evolved to interact with IFNAR2. The fact that the Sin1 gene appears to be expressed ubiquitously, that it is highly conserved across a wide range of taxa, and that it is a likely participant in several important signaling pathways, makes it an intriguing candidate for a functional/evolutionary analysis.Schizosaccharomyces pombe and Saccharomyces cerevisiae), the red bread mold (Neurospora crassa) and a number of other fungal species (not shown here), Caenorhabditis elegans, a mosquito species (Anopheles gambiae), fruit fly (D. melanogaster), frog (Xenopus laevis), two fish species (Fugu rubripes and Danio rerio), chicken , mouse (Mus musculus), rat (Rattus norvegicus), human (Homo sapiens), sheep (Ovis aries), cattle (Bos taurus), and pig (Sus scrofa)  are much longer than Sin1 from vertebrate and insect species, which are ~520 aa long. The regions of similarity among these three fungal proteins are confined entirely to the carboxyl termini of these molecules, although several gaps have to be introduced to align them. No similarities are detectable in the amino terminal extensions, which, in the case of S. cerevisiae, is 370 aa long. It is the carboxyl regions of the fungal proteins that can also be aligned with the Sin1 sequences from C. elegans, insects, and vertebrate species, including Ovis aries, the sheep  to 91.3% (sheep and rat)  Table . All thet) Table .There is considerable conservation of Sin1 from mammals to birds (~90%), amphibians ~85%), and fish (~80%) Table . The ins%, and ficonserved region in the middle.The region of the greatest identity between these divergent insect and vertebrate sequences is an acidic region placed in conserved SCD III Fig. , 4, 5. IC. elegans retains the highly conserved 36 amino acid SCD I and the 127 amino acid Domain III  immediately following the end of SCD V.When the ovine Sin1 sequence is scanned for functional motifs , the strThere are numerous motifs that are recognized as potential but weak sites for phosphorylation by either casein kinase II (CK2), protein kinase C, or protein kinase A (data not shown). None of the 17 CK2 sites, the 12 protein kinase C, or the 5 protein kinase A sites present in the ovine Sin1 primary sequence are conserved from mammals to fission yeast, although many are retained across all vertebrates. A weak site for myristylation  and exon 5 (SCD III). As noted above and in Figure S. pombe is comprised of only a single exon.The Sin1 gene from S. pombe gene product is longer than that of mammals, with an extension at its N-terminus, human Sin1 can rescue the stress sensitivity noted in the phenotype of a S. pombe strain that expressed a constitutively active form of RAS, indicating that function, as well as structure, has been conserved over hundreds of millions of years.Sin1 is a little studied gene product of unclear function found in species ranging from mammals to fungi. Although the Two facts should be considered when attempting to infer a role for Sin1 in vertebrates. The first, as discussed in the Background, is the known ability of type 1 IFN to activate MAPK/SAPK in mammalian cells. The second is the proven involvement of Sin1 in the yeast SAPK (Sty1/Spc1) pathway and its involvement in controlling transcription of stress-activated genes . The preS. pombe, S. cerevisiae, and N. crassa, emphasizing the evolutionary distance between these three species. The regions of similarity are confined to the ~600 amino acid C-terminal regions of the three sequences (data not shown), and it is this region that is also conserved in insects and vertebrates , which control cell growth in response to nutrients [S. pombe is involved in a stress response signaling pathway by interacting with Sty1 [S. cerevisiae, although certain landmark amino acids are retained (data not shown). Interestingly, Sin1 from insects and vertebrates, despite having only about 35% identity, are of similar length and possess the five regions of high identity. Conceivably, the SCD III domain is functionally essential in all the species, while SCDs I, II, IV, and V have evolved conserved function within the Metazoa. A not unreasonable assumption is that that Sin1 plays an evolutionarily conserved role in SAPK signaling across a broad range of taxa, including all metazoan and fungal species [The Sin1 gene is remarkably divergent in both length and sequence identity within the fungi ates see  &2. ThisAVO2, AVO and LST8utrients . Cells wAVO2, AVO and LST8AVO2, AVO and LST8In vertebrates Sin1 falls into a class of highly conserved gene products. Its conservation is lower than that of two structural proteins, histone H3 and β-actin, but is comparable to that of CDK1 , a little-studied but widely expressed gene product, is encoded by a single gene in fungi, nematodes, insects, and all vertebrates analyzed and shows modest conservation of amino acid sequence that is consistent with some degree of conserved function in stress-activated signal transduction pathways. Sin1 is highly conserved in vertebrates where it has been implicated in linking interferon responses to the SAPK pathway.C. elegans, S. pombe, and S. cerevisiae, were retrieved from at NCBI Genome databases [Fugu rubripes) and zebrafish (Danio rerio) genome databases at the Ensembl site [Saccharomyces cerevisiae) ORF (open reading frame) database [Sin1 genomic sequences from human, mouse, rat, fruit fly, mosquito, atabases . Sin1 cDatabases . For fismbl site  were usedatabase  was usedPairwise global sequence alignment was performed by using either the BESTFIT or the GAP program from GCG . Multiple global sequence alignment was performed by using either the PILEUP program  and GeneDoc  or ClustSchizosaccharomyces pombe) and chicken : The two Sin1 sequences were published by Wilkinson et al. [Fission yeast : The BLASTp program was used to search the budding yeast ORF database for any protein sequence that had significant similarity to the fission yeast Sin1 protein. The obtained budding yeast Sin1 protein sequence had a GenBank link where its cDNA was available. The cDNA sequence was used to analyze its genomic structure at the NCBI yeast genome site.Budding yeast (Neurospora crassa): Sin1 protein was retrieved from the Neurospora crossa protein data base by searching (BLASTp) with the budding yeast Sin1 protein.Red bread mold (Caenorhabditis elegans): The Sin1 protein sequence was obtained from the C. elegans protein database by searching with ovine Sin1 protein. The cDNA sequence was then obtained from the GenBank link and used to determine the structure of the Sin1 gene.Worm (Drosophila melanogaster): The fruit fly Sin1 protein sequence was retrieved from the D. melanogaster protein database as above. The cDNA sequence was obtained from the GenBank link. Unexpectedly, querying the Drosophila genomic sequence with the C. elegans Sin1 sequence and vice-versa failed to yield a match in either case.Fly (Anopheles gambiae): The mosquito Sin1 protein sequence was retrieved from the Anopheles gambiae str. PEST protein database as above. The cDNA sequence was then obtained from the GenBank link.Mosquito (Fugu rubripes) and Zebrafish (Danio rerio): Both Fugu rubripes and Danio rerio genome databases, which are accessible at two websites, NCBI and ENSEMBL, were queried with Sin1 cDNA sequences from sheep, chicken, and frog. For both species, only the Ensembl site provided the complete genomic sequence. Although the Fugu rubripes genome sequence is incomplete, the exons of Sin1 cDNA can be retrieved and successfully assembled into the full length structure by alignment with other Sin1 cDNA and gene sequences. No GenBank entry was available for the Fugu rubripes Sin1 gene. When a similar method was used to retrieve the Zebrafish Sin1 cDNA sequence, the full length sequence could not be obtained because the region (~20 kb) covering one exon (exon 4) was incomplete. Therefore, the fish Sin1 protein sequence used here is from Fugu rubripes.Puffer fish (Xenopus lavis): The full-length cDNA sequence of Sin1 reported here was from African clawed frog, and was obtained by blasting the Xenopus EST database [Frog (database  with theMus musculus): The mouse Sin1 cDNA sequence was obtained by editing several ESTs, after performing a BLASTn search of the Mus Musculus EST database with the ovine Sin1 cDNA sequence. Searching the mouse genome database with the mouse Sin1 cDNA coding region then allowed the gene, down stream of its transcription start site to be located and its structure to be inferred.Mouse (Rattus norvegicus): The rat Sin1 cDNA sequence was retrieved from several overlapping ESTs, which were obtained by searching the Rattus norvegicus EST database with the ovine Sin1 cDNA sequence. The coding region of the rat Sin1 cDNA was then used to search the rat genome database at the NCBI website for the genomic structure of the gene.Rat (Bos taurus): The full length bovine Sin1 cDNA sequence was obtained from overlapping ESTs, which were obtained by searching the NCBI EST database with the ovine Sin1 cDNA sequence.Cattle (Sus scrofa): The swine Sin1 cDNA sequence was obtained as above by searching the NCBI EST database with the ovine Sin1 cDNA sequence.Pig (Homo sapiens): The sequence published by Colicelli et al. [Human : The sheep Sin1 cDNA sequence was cloned from a sheep endometrial cDNA library in a yeast two-hybrid screen [Sheep  is highlighted by a line above the sequence, and appears not so well conserved in the budding yeast as in other species. Abbreviations: S. pombe, Schizosaccharomyces pombe (fission yeast. GenBank accession No. AL136521). S. cerevisae, Saccharomyces cerevisae (budding yeast. GenBank accession No. NP_014563).Click here for fileAlignment of Sin1 proteins from the fission yeast and the red bread mold. The Bestfit program was used to align the two sequences. Black shading shows identical residues. Abbreviations: S. pombe, Schizosaccharomyces pombe (fission yeast. GenBank accession No. AL136521). N. crassa, Neurospora crassa (red bread mold. GenBank accession No. XP_322410).Click here for file


Scientists continue to argue the extent that human activities drive global warming, but few would argue that it exists. The International Panel on Climate Change predicts that greenhouse gases will increase global temperatures by 3.6 degrees F by 2100—a rise unprecedented over the past 10,000 years. What might the world look like as we approach that point? Wetlands will disappear. Floods, hurricanes, and droughts will become progressively more severe. Infectious diseases will increase in virulence and range. Montana's famed glaciers may all but disappear within 30 years. A quarter of species may vanish by 2050.While the effects of climate change on species' geographic ranges and population dynamics have been studied to some extent, scientists know little about how species respond to climate change at the genetic level. In this issue, Elizabeth Hadly and colleagues analyze three different dynamic processes—environmental change, population response, and gene diversity fluctuations—and present evidence that climate change influences variation in genetic diversity.Focusing on two mammal species—the Montane vole and the northern pocket gopher—Hadly et al. asked how the two species responded to historical climate-induced habitat alterations in northwestern Wyoming. They gathered fossils from Yellowstone National Park's Lamar Cave, which contains a treasure trove of carbon-dated deposits that mirror the community of mammals in the area today. Comparing genetic material extracted from fossil samples from different time points over the past 3,000 years to genetic data taken from contemporary animals, Hadly's team tracked genetic changes in populations of the two species and used this information  to estimate changes in effective population size over time.  The genetic and demographic data were then combined with environmental records to analyze the relationship between the factors.Studying these populations in space and time—an approach the authors call “phylochronology”—offers an opportunity to analyze the genetic diversity of a species against the backdrop of environmental fluctuation within an evolutionary time frame. It also suggests how microevolutionary forces—factors that affect genetic variation in populations over successive generations—shape genetic responses to climate change. Such evolutionary forces include mutation, genetic drift , and gene flow (changes in the gene frequency of a population caused by migration).The past 3,000 years includes two periods marked by dramatic climate change—the Medieval Warm Period and the Little Ice Age—that had different effects on local mammal populations depending on their habitat preferences. Habitat specialists, the vole and pocket gopher live in the wet mountain regions of western North America. Though both showed population increases during wetter climates and declines during warmer periods, Hadly et al. predicted the gene diversity fluctuations of the two species would differ based on their different ecological behaviors. And that's what they found: genetic response is tied to population size. Pocket gophers have low population densities, stick close to home, and are fiercely territorial, while voles live in high-density populations and range more widely. For the gophers, population declines resulted in reduced gene diversity; for the voles—which have a larger effective population size and greater dispersal between populations—population declines resulted in increased gene diversity. But what forces underlie these differences in genetic variation?A recent study suggests that migration (a primary agent of gene flow) is most common in and between low-density patches in vole populations, which implicates gene flow as the driver of gene diversity patterns. But the authors don't rule out selection as a possibility, and suggest how to go about resolving the question. Hadly et al. show that phylochronology opens a unique window onto the relatively recent evolutionary past and offers “the potential to separate cause from effect.” They also conclude that “differences in species demography can produce differential genetic response to climate change, even when ecological response is similar.” With a 3-degree temperature increase in just the past 50 years in the American West, conservation of biodiversity may well depend on such insights.


The development of antifilarial antibody responses is a characteristic feature of infection with filarial parasites. It should be possible to exploit this fact to develop tools to monitor the progress of the global program to eliminate lymphatic filariasis (LF); however, assays based on parasite extracts suffer from a number of limitations, including the paucity of parasite material, the difficulty of assay standardization and problems with assay specificity. In principle, assays based on recombinant filarial antigens should address these limitations and provide useful tools for diagnosis and surveillance of LF. The present multicenter study was designed to compare the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. Coded serum specimens were distributed to five participating laboratories where assays for each antigen were conducted in parallel. Assays based on Bm14, WbSXP, or BmR1 demonstrated good sensitivity (>90%) for field use and none of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections. Limitations of the assays are discussed. Well-designed field studies are now needed to assess sampling methodology and the application of antibody testing to the monitoring and surveillance of LF elimination programs. Wuchereria bancrofti, assessment of antigenemia offers the convenience of daytime testing and greater sensitivity than testing for microfilaremia; however, both microfilaremia and antigenemia develop from months to years after exposure, reducing their utility for detection of low levels of infection or recrudescence of transmission [The exponential growth of the lymphatic filariasis elimination program has highlighted the need for tools that can be used to monitor progress toward programmatic endpoints (e.g. when to stop mass treatment) as well as to conduct surveillance to detect any potential resumption of transmission. Measurement of microfilaremia, the recognized gold standard for demonstrating the impact of community-wide interventions, is not an optimal monitoring or surveillance tool because of the requirement for nocturnal blood collection in much of the world and because it is a relatively insensitive test for infection . For Wucsmission -6. Entomsmission . By provAntibody detection has served as the basis for diagnostic assays for filariasis for many decades. The best of these assays are sensitive for infection but are not specific, both because they cannot distinguish current infection from past infection or exposure to the parasite and because there is some degree of cross-reactivity with other helminth infections. On the positive side, prior studies have shown that virtually all residents of filariasis-endemic areas mount antifilarial antibody responses within the first few years of life. Thus, prevalence rates of antifilarial antibodies in children may be a useful index for assessing changes in transmission of the infection ,9.Antibody assays based on crude filarial extracts are limited by cross-reactions with other nematode antigens . RecombiBrugia malayi or Wuchereria bancrofti. Comparable results have been reported with assays for SXP [B. malayi cDNA library by antibody screening [B. malayi or B. timori microfilaremia were tested [Bm14 and WbSXP belong to a family of related genes that encode proteins that are strong immunogens in many parasitic nematode infections . SXP and for SXP ,16,17. Bcreening ,19. ELISe tested -22.The present study was designed to compare objectively the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. We report here the results of this multicenter evaluation.Serum specimens from patients of known infection status  was defined on the basis of agreement among at least 4 of 5 labs. If only 3 of 5 labs obtained the same result or if 3 did and one of the two remaining laboratories did not obtain an interpretable result, this was considered to represent a lack of consensus (recorded as 'NC' or no consensus in Tables W. bancrofti (91%) and Brugia (96%) infections, while the other two tests performed better with specimens from the homologous infections  in terms of its performance with W. bancrofti samples. Results were comparable for both the BmR1 dipstick and cassette (data not shown). Similarly, the WbSXP assay was positive for 30 of 33 (91%) serum specimens from W. bancrofti patients, but only 39% of Brugia cases.The Bm14 ELISA displayed comparable sensitivity for both Wuchereria and Brugia infections, and there was little reactivity with specimens from persons with O. volvulus, Loa loa or other helminths (e.g. Strongyloides). The Bm14 assay, and to a lesser extent, the WbSXP assay, appeared to function as a 'pan-filaria' assay, showing reactivity with the specimens from persons with W. bancrofti, B. malayi, L. loa and O. volvulus. None of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections  still remains. For mapping exposure ,25.B. malayi infection [Antibody assays almost certainly will find other uses in the context of lymphatic filariasis (LF) elimination programs. For example, antifilarial antibody tests may be sensitive markers of transmission intensity or provide evidence of ongoing exposure to filarial infection long before the development of antigenemia or microfilaremia. Primates develop antibody responses to Bm14 within 4–8 weeks following nfection . In a lonfection . Less isAs LF programs reach their planned end point (5 or more years of > 80% drug coverage in targeted populations), it will be necessary to determine whether or not transmission has been interrupted and whether mass drug administration can be stopped. Parasitologic testing, whether for microfilaremia or antigenemia, will require testing of thousands of persons to demonstrate that infection levels are below 0.1%, the level established by the Global Program as the end point for defining the elimination of LF. Since antibody responses develop in the absence of demonstrable infection, detecting incident antibody responses should provide a more sensitive measure of transmission than microfilaria or antigen detection. Children born following the cessation of transmission should be antibody-negative, while older children and adults may have evidence of residual antibody reactivity ,27. A teOperational use of antibody assays requires far more practical experience with the assays than we now have. Of greatest concern is the specificity of the tests employed. For example, ELISA tests often use a statistical approach to establish cutoff values for positive results. A test that is 99% specific will predictably have some false positive results if large numbers of samples are tested. Further work will be needed to establish rates of antibody positivity that exceed the number that are likely to occur by chance. In addition, it will be necessary to develop and validate algorithms for confirming the presence of infection or ongoing transmission in situations where low antibody prevalence rates are detected. Despite these caveats, we believe that antibody tests based on antigens like Bm14, BmR1, and Wb-SXP will prove to be useful tools that can be used to facilitate decision making by program managers in the context of filariasis elimination programs.GW, RN, and PK have relationships with companies interested in developing commercial applications of the Bm14, BmR1 and WbSXP assays, respectively.GW, RN, PK, and VBL were responsible for the initial development of the assays. All of the authors participated in the planning of this multicenter study. DG was responsible for coordinating specimen shipment and database management. Participating labs included PL and DG from CDC, RN from the Universiti Sains Malaysia, PK and VBL from Anna Center for Biotechnology, CS from NIH and GW from Washington University School of Medicine. PL and EO were responsible for coordinating the study. PL wrote the first draft of the manuscript, but all of the authors participated in the editing of subsequent versions.


Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine. Artificial prions - infectious, misfolded proteins - can be created by fusing the replication element of one prion to aggregation sequences from another Saccharomyces cerevisiae and Podospora anserina, prions are transmitted from cell to cell through mating and cell division, resulting in readily assayed phenotypes with a non-Mendelian pattern of inheritance , which “cures” cells of prions by inhibiting Hsp104p, a molecular chaperone needed for prion replication , also promoted the aggregation of other Q/N-rich proteins, emulating the effect of [+PIN]  and an aggregated prion state ([+PSI]) with diminished translation termination activity, which can be monitored by nonsense suppression of the mutant ade1–14 allele (–psi] strains form red colonies on yeast extract-peptone-dextrose (YEPD) medium and cannot grow in the absence of adenine, [+PSI] strains suppress the premature stop codon in ade1-14, and thus appear pink or white on YEPD medium and grow on adenine-free medium . The N or prion domain of Sup35p (residues 1-112) is required for [+PSI] formation but is dispensable for the translation termination activity of the carboxy-terminal C domain  supported a [+PSI]-like prion state termed [+NU] .We had earlier identified New1p as an uncharacterized protein with a Sup35p-like N-terminal domain; when fused to the M and C domains of Sup35p, the first 153 residues of New1p (New1ed [NU+] . Sup35p ed [NU+] . The pri] (sup35  and new11–153 into a [+NU] prion state (induction). Finally, we fused these constructs to the M and C domains of Sup35p (–M-C), introduced them in place of endogenous SUP35, and assessed whether these proteins could adopt stable prion states (maintenance).To understand the sequence requirements for the prion behavior of New1p, we constructed a series of truncated prion domains A and exa+NU]  aggregated and induced [+PSI] at levels comparable to the full prion domain, in agreement with +PSI], albeit with decreased efficiency. Although a construct lacking oligopeptide repeat 2 entirely (residues 1–49) did not induce [+PSI] de novo, this GFP fusion could nonetheless decorate preexisting Sup35p aggregates. Thus, while oligopeptide repeat 2 contributes to the aggregation of Sup35p, the primary determinants of prion induction reside in the amino-terminal Q/N-rich region and oligopeptide repeat 1.We found that the Q/N-rich tract and a small portion of the adjacent oligopeptide repeat are responsible for Sup35p aggregation and de novo [+PSI] aggregates. Constructs that did not form fluorescent foci could not retain [+PSI], suggesting that aggregation is a prerequisite for prion maintenance. However, aggregation is not sufficient for prion inheritance, as Sup35p constructs with deletions spanning oligopeptide repeats 3–5 could not support a prion state despite their ability to form aggregates and efficiently induce [+PSI]. Only the sixth (incomplete) oligopeptide repeat proved dispensable for [+PSI] maintenance, consistent with an earlier report  shows strong interference with [+PSI] in certain strain backgrounds through a poorly understood mechanism  to distinguish between these two functions. PNM2-1  yeast medium that selected for [+PSI] (SD-ade medium) but reverted to [–psi] at a high frequency in nonselective YEPD medium, resulting in sectored colonies  propagons per cell, the PNM2-1 strain had dramatically fewer: 41 of 50 cells had no [+PSI] propagons at all , and among the remaining nine [+PSI] cells, the median propagon number was six  in a PNM2-1 strain. Here, prion replication is inhibited by GuHCl treatment. As the cells divide, preexisting propagons are diluted but not destroyed. The number of propagons present in a colony arising from a single cell is then evaluated by removing the GuHCl prion replication block after a large number (10 or more) of cell divisions and counting the total number of , after transient overexpression of F-M-GFP. As with [+NU], [+F] induction did not require [+PIN] (data not shown). [+F] could be eliminated by GuHCl treatment  or aggregates as a result of the resident prion (pink/white). Consistent with the induction data, F-M-C and New11–153-M-C were not incorporated into [+PSI] aggregates; likewise, Sup35p did not interact with [+F] or [+NU] aggregates. However, [+F] prions recruited New11–153-M-C and, to a lesser extent, [+NU] recruited F-M-C. Thus, F and New1p prion domains can cross-interact during de novo induction and at normal levels of expression, indicating that the NYN repeat is sufficient to specify homotypic interaction between two otherwise distinct prion domains.We next explored the specificity of [e [PSI+] E. Howeveon assay  ; conversely, Sup35p did not enter [+Q] aggregates.Can a simple aggregation-prone sequence such as polyglu-tamine  be turne+] cells B. Transi+] cells C. Severareatment D, and direatment E. We fur] strain F. The Q6A number of epigenetic traits in fungi result from the stable inheritance of self-propagating, infectious protein aggregrates (prions) . Prion iAggregation underlies the growth phase of the prion replication cycle A and occ+NU] but requires an adjacent oligopeptide repeat to form a minimal heritable New1p prion, [+NU]mini. Similarly, in Sup35p, the Q/N-rich amino terminal region mediates aggregation whereas most of the oligopeptide repeats are needed for the inheritance of [+PSI] propagons. Oligopeptide repeats 1 and 2 appear to contribute to both growth and inheritance, consistent with earlier reports that expansion and deletion within this region modulate in vitro polymerization of Sup35p and the appearance of [+PSI] in vivo  within its prion domain has an amino acid content reminiscent of the oligopeptide repeat sequence   . Only twesidues) , but it esidues) , it is u+F] prion cross-interacted with New1p but not Sup35p. Like [+NU], [+F] could be induced in the absence of a prion-promoting (PIN) factor. Finally, [+F] could itself act as a PIN factor, as does [+NU] (data not shown). Notably, the NYN repeat of New1p functions as an aggregation module apparently without regard to its position within a protein; this sequence induced prions when overexpressed by itself or with oligopeptide repeats at its N-terminal (in New11–153 and New150–100) or C-terminal regions (in the F chimera). These results suggest that aggregation sequences are portable and functionally separable from the oligopeptide repeat, perhaps constituting a structurally discrete amyloid core. Indeed, a peptide derived from the amino-terminal region of Sup35p forms a self-seeding amyloid in vitro , followed by loss of the maintainer spontaneously or through 5-FOA counterselection. The PNM2-1 strain in +PSI]. HIS3-marked oligopeptide repeat truncations and PNM2-1 maintainers were from –f] strain was generated by “gamma” chromosomal integration of pRS306 F-M-C into the SUP35 locus of YJW 584; excision of the wild-type gene was confirmed by PCR of Ade- colonies arising from subsequent growth on 5-FOA. The [–q] strain was made by “omega” chromosomal gene replacement (SUP35 with a C.glabrata HIS3-marked –M-C variant (with or without oligopeptide repeats) into the SUP35 locus of a diploid [+PSI] [+PIN] strain. After sporulation, gene replacement was confirmed by PCR and by loss of [+PSI] in half of the haploid progeny. Yeast culture methods were according to standard procedures . Unless otherwise noted, cultures displaying 10% or more cells with foci were scored as positive. For induction, dilutions of the above cultures were plated onto SD-ade and YEPD media to determine percentage of Ade+. In qualitative assessments, strains were scored as positive if 5% or more of plated cells grew on SD-ade medium after 5 d. In [+NU] maintenance experiments, strains with indicated maintainers were tested for the ability to support an Ade+ state following New11–153-GFP overexpression. In [+PSI] maintenance experiments, strains that began as [+PSI] were tested for Ade+ after plasmid shuffle gene replacement with the indicated maintainer. For decoration, a [+PSI] [+PIN] strain was transformed with the indicated inducers, grown in selective medium with 50 μM CuSO4, and examined by fluorescence microscopy during midlogarithmic phase. Propagon counts were performed as described in For aggregation, inducers were overexpressed by growth of cells in selective medium with 50 μM CuSOCentrifugation was performed as described in Data S1(30 KB ZIP).Click here for additional data file.The GenBank accession numbers for the proteins discussed in this paper are Hsp104p (NP_013074), New1p (NP_015098), Rnq1p (NP_09902), Sup35p (NP_010457), Ure2p (NC_014170), YDR210W (NP_010496), and YBR016W (NP_010319).


Health policy and systems research (HPSR) is an international public good with potential to orient investments and performance at national level. Identifying research trends and priorities at international level is therefore important. This paper offers a conceptual framework and defines the HPSR portfolio as a set of research projects under implementation. The research portfolio is influenced by factors external to the research system as well as internal to it. These last include the capacity of research institutions, the momentum of research programs, funding opportunities and the influence of stakeholder priorities and public opinion. These dimensions can vary in their degree of coordination, leading to a complementary or a fragmented research portfolio.The main objective is to identify the themes currently being pursued in the research portfolio and agendas within developing countries and to quantify their frequency in an effort to identify current research topics and their underlying influences.HPSR topics being pursued by developing country producer institutions and their perceived priorities were identified through a survey between 2000 and 2002. The response to a call for letters of intent issued by the Alliance in 2000 for a broad range of topics was also analyzed. The institutions that were the universe of this study consisted of the 176 institutional partners of the Alliance for Health Policy and Systems Research producing research in low and middle income countries outside Europe. HPSR topics as well as the beneficiaries or issues and the health problems addressed were content analyzed. Topics were classified into 19 categories and their frequency analyzed across groups of countries with similar per capita income. Agendas were identified by analyzing the source of funding and of project initiation for projects under implementation.The highest ranking topic at the aggregate level is "Sector analysis", followed by "Disease burden" and "Management and organization". Categories at the bottom of this ranking are "Equity", "Policy process", "Economic policy and health" and "Information systems". "Disease burden" is more often funded than other topics for which there is more demand or perceived priority. Analysis suggests few although important differences across priorities, demand for funding and actual project funding. The donors' agenda coincides most with the ranking of research topics overall.Ranking across country income groups shows important differences. Topics that gain prominence in low income countries are "Disease burden" and "Accessibility". In lower middle income countries "Insurance" gains prominence. In upper middle income countries "Decentralization/local health systems", "Equity" and "Policy process" are more prominent. "Program evaluation" is the most consistently ranked topic across income regions, showing a neutral influence by donors, governments or researchers.The framework proposed offers a basis to identify and contrast research needs, projects and products at the international level and to identify the actor agendas and their influence. Research gaps are suggested when comparing topic ranking against the challenges to health system strengthening and scaling up of disease control programs. Differences across per capita income groups suggests the need for differentiated priority setting mechanisms guiding international support. Data suggests that stakeholders have different agendas, and that donors predominate in determining the research portfolio. High-level consensus building at the national and international levels is necessary to ensure that the diverse agendas play a complementary role in support of health system objectives.The Ministerial Summit for Health Research to be held in Mexico in November 2004 should be an opportunity to analyze further data and to commit funding for priorities identified through sharing and discussion of agendas. Countries and international agencies have made a qualitative leap in the funding of the global disease challenges. The Global Fund for AIDS, TB and Malaria has received pledges totalling over US$ 2 billion. Bilateral donors are also making important funding contributions. In this context, strengthening of health systems has become a critical issue. Research can play a major role to identify the best policies to channel massive efforts, to ensure that vertical approaches do not fragment fragile health systems and to monitor and evaluate progress. How relevant is the research effort being undertaken in developing countries, and how is the agenda being driven?WHO is organizing the Ministerial Summit on Health Research, to be held in Mexico City, 23 to 26 November, 2004. The main theme will be the role of health research in meeting the Millennium Development Goals. Health policy and systems research (HPSR) will have a prominent role in the context of the scaling up of efforts against major diseases and child and maternal mortality. Looking towards the Summit, WHO established a task force to identify HPSR priorities as an effort to advocate for major funding in this area. The Alliance for Health Policy and Systems Research, an initiative of the Global Forum for Health Research in collaboration with WHO, has been promoting since its inception in 2000 the identification of research priorities among producer institutions in developing countries.This paper proposes a conceptual framework and a methodology to think about the HPSR research portfolio, the agendas that influence it and the priority setting process. The paper makes the case for the formulation of HPSR priorities at national, international and global levels. A previous article  looked aPriority setting efforts are often bogged down because of inadequate methods of categorization of the research that is undertaken and the influences shaping it. These influences, in turn, are often not adequately understood, including the nature and role of priorities. The conceptual framework proposed here strives to offer some simple definitions of the research portfolio and its various influences, as well as indicators to measure and relate these concepts.The nature of overall health research has been defined in terms of basic, applied and strategic research. These categories are useful to guide investment decisions which might maximize benefit ,3. HowevTo identify the object of research the concept of the research portfolio can be useful. The HPSR portfolio can be defined as the current set of research projects on health system structures, functions, processes and results at national, international and global levels. Projects, as distinct from plans or priorities, would include the commitment of resources towards a specific, time-bound aim and a set of objectives.It has been proposed that the research portfolio should be analyzed along three dimensions of strategic importance: where to make investments, that is, the identification of the object of research or thematic areas in which investments are made; the type of investment research and development (R & D) instrument used and the resources spent through each area and type of instrument . While oThe Ad Hoc Committee on Health Research proposed three types of R & D instruments: discovery oriented research to develop new health products and interventions; innovation research to adapt efficacious but unaffordable interventions to make them cost-effective, and implementation R & D to achieve greater efficiency in the use of existing interventions . HarrisoWith regard to funding, there is a need not only to consider investments, but also funding sources and mechanisms. Four broad types of sources can be recognized: bilateral and multilateral donors, government commissioning, private commissioning, and funding through resources available to research institutions as part of their budgets. Each will have different implications for the kind of knowledge produced and for its possible influence on the health system . This suPrevious analyses have revealed a complex heterogeneity along which researchers classify HPSR in developing countries, which is not surprising in an interdisciplinary field . However• concepts reflecting the health system, such as policy and financial structures, regulatory functions, processes such as technology evaluation and quality monitoring, and results such as satisfaction and health gain• the levels of the health system, such as the households and the community, first level facilities and hospitals• the issues or problems pertaining to the health system such as priorities, equity and the public private mix• the populations addressed by the system, such as children, mothers and the elderly, or rural and urban populations• the health needs addressed, whether in terms of risks or disease.While these dimensions can be useful to characterize the research portfolio, it is clear that there will be overlaps; for example, equity is both an issue and an attribute of the health system, particularly if it has been integrated in monitoring and regulation. In order to make use of these dimensions it is proposed to consider as the project topic the first dimension of concepts pertaining to the health system structures, functions, processes and results. The topic could then be classified following normative or theoretical frameworks or by using the categories researchers apply in their own research. The other four dimensions can be used to qualify the research topic as to provide a more detailed description. These four dimensions could be selectively used or aggregated to facilitate description according to the needs at hand.Each of the five project dimensions can be analyzed in terms of the range of items considered. The ranking and emphasis of each item can also be revealed by analyzing its frequency. A research portfolio at any level can be very focused and comprise a narrow set of topics. Or it can be wide-ranging across many health system functions, structures, processes and results. It has been argued that research portfolios should be more focused on improving immediate health problems through operations research in low income countries, where funding and human resources are very limited . HoweverThe level at which the portfolio is analyzed is important, as it will have different characteristics and uses. At national level, the HPSR portfolio would be the set of projects addressing health and health system problems within the confines of national borders and governmental authority as well as sector-wide and inter-sectoral issues. Examples would be the impact on equity of decentralization policies, or the roles played by conflicting policy actors in scaling up of services. The international research portfolio would be topic areas which are common across a number of countries or regions. Identification of the international portfolio could serve, among other purposes, to fund research at regional or international levels, to strengthen the critical mass of research available to inform country policy making and lesson learning across countries, and to extend the range of methodological approaches through comparative research . The HPSThe HPSR portfolio at any level is influenced by factors within and outside the research system . Within It is clear that if actor agendas have few areas in common there will be no consensus and therefore no overall priorities. Significant overlap of interest, on the other hand, can lead to the identification and formulation of shared research priorities. Research priorities are therefore defined as the explicit areas of agreement on, and ranking of, the object of research across diverse actor agendas. Priorities can then become policy instruments to coordinate diverse agendas towards a common end without forcing a single research agenda.The characteristics of agendas and of priorities can be identified through the same kinds of dimensions and indicators as the research portfolio. That is, preferences can be classified in terms of topics, issues or beneficiaries, and health problems. The characteristics of the HPSR agendas and priorities can also be studied in terms of the range of topics, issues, levels, populations and health problems. They can also be ranked and their emphasis revealed by frequency analysis. In this way, the agendas can be compared across themselves, priorities can be identified as common topics and issues and with similar ranking and emphasis, and the influence of agendas and priorities on the actual research portfolio can be assessed.It has been argued that co-ordination of the various influences shaping the HPSR portfolio can increase the impact of research on equity and can contribute to its strategic role for development . Co-ordiHPSR portfolio change through co-ordination would involve a gradual process of adjustment to new priorities, project completions, maturation of research capacity and funding opportunities. Coordination requires interfaces and mechanisms such as Research Forums and Essential National Health Research Mechanisms  to develIn sum, the HPSR portfolio can encompass a differing range of topics with diverse rankings and emphases; can be more or less coherent with respect to researcher, funding and policy maker agendas; and can be more or less co-ordinated along a set of shared priorities. The interplay of these dimensions can give rise to a number of scenarios, of which three are here illustrated.a) Co-ordination could lead to focusing the HPSR portfolio on a few, highly cost-effective topics in a situation of few resources and well identified, high priority needs. There would be, eventually, a high degree of coherence across the portfolio and agendas held by various actors. The risks here would be lack of diversity to foster innovation and healthy criticism.b) In a situation of plentiful resources the research portfolio and its driving agendas could be wide-ranging and have little overlap. Specific portfolio segments would correspond with particular agendas, thus satisfying multiple interests. However, co-ordination through overarching mechanisms could ensure integration of knowledge around high level priorities. In this manner a unified, although highly diverse, HPSR field of enquiry would be obtained.c) In a situation of lack of coordinating mechanisms, with low resources, the portfolio could focus on a reduced set of topics, each satisfying a particular agenda and thus fragmenting resources and hindering support to health system development. If resources are more plentiful, lack of coordination could lead to a rich but highly dispersed and inefficient research portfolio with little impact on development.Irrespective of the availability of resources, co-ordinating mechanisms are likely to be important to ensure an efficient use of research resources.Two sources of information were used to illustrate the conceptual framework proposed. The first was a survey of Alliance-HPSR partner institutions in developing countries detailing research priorities and project information. Researchers reported here on the priorities they had received from policy makers in the course of diverse consultations in the past year. The second source was a database of letters of intent (LOI) submitted to the Alliance for funding, where projects were justified on the basis of priorities negotiated by researchers and policy makers or service managers.This proceeded in several steps. A preliminary list with 24 research topics was identified through an inductive analysis using the research statements expressed in the LOI, which were the most detailed. This list was then used to classify research topics in the projects and priorities expressed in the Alliance partners' profiles. Beneficiaries/issues and diseases/health problems addressed by the LOI, projects and priorities were also categorized and classified at this stage. While projects may have contained more than one topic or beneficiary/issue, the most prominent one was selected. In a few cases where several topics were considered this was indicative of a sector-wide analysis and classified accordingly.The beneficiaries/issues of the research were classified to include any of four alternative dimensions: a) the demographic group: elderly or children/adolescents, b) level of care: community, primary or hospital, c) the geographical focus: urban or rural, and d): gender, equity/poverty, indigenous populations, and public-private mix. Whenever more than one dimension or aspect was applicable , a decision was made to include the most prominent.The concept "equity" was classified both as a topic and as an issue. It was assigned as a topic whenever equity was the main objective of the research and it was addressed through a number of health system attributes such as financing, access, and service delivery. Equity/poverty was considered as an issue when the poor were identified as the main subjects of research or when the equity implications of research directed mainly to another topic were highlighted as a major concern.The distribution of statements was analyzed according to country income group: low income, lower middle income and upper middle income. These income groups correlate highly with geographical regions, with LI being mostly in Africa and Asia, LMI mostly in Asia and with a particular weight by China, and UMI in Latin America and the Caribbean table .Two researchers classified all statements independently and disagreements were discussed and resolved. The 24 topic categories were reduced to 19 to avoid groups with less than 2% of the total number of statements while maintaining topic coherence. Table The frequency of responses by country for all types of statements is generally proportional to country population, with China, India, Brazil and Bangladesh at the top of the frequency. However, countries with a strong health systems research presence are over-represented, such as Colombia, Argentina, Philippines, Thailand, South Africa, Uganda, Ghana, Cuba, Costa Rica, Benin, Jamaica and Tanzania.The range and emphases of the HPSR portfolio and agendas were mapped through topic content and frequency analysis Figure . ProjectTwo methods were used to assess the coherence between the HPSR portfolio and the agendas held by researchers, policy makers and international donors and partners. The first method used the survey data to infer the agendas by observing the topic frequency of: projects proposed and funded by researchers without external assistance; projects initiated and funded exclusively by government, and projects initiated and funded exclusively by international stakeholders or research partners.The second method compared the research portfolio against the agenda expressed by policy makers. The policy maker agenda was observed through the survey as reported by researchers and through the LOI as negotiated with researchers. A negligible influence by the donor, in this case the Alliance-HPSR, would be expected in the LOI given that the call requested priorities within the generic definition of HPSR presented above.Each of the three modes of identification of agendas could have method-specific biases. In the first method, preferences are derived from the portfolio itself, that is, from research projects in implementation. Furthermore, the method isolates the preferences expressed by each actor. As such, this method could be deemed to reveal most objectively preferences behind each actor funding or initiating a project. However, projects under implementation may hide topic preferences that are not translated into projects or topics that were generated and funded through joint actor participation.The observation of negotiated priorities expressed in LOI captures the mix or balance of researcher-side influences and policy maker needs. It will therefore reflect a consensus position across each actor. However, it will exclude the influence of funding opportunities, will not reveal actor-specific preferences and will be limited by the constraints placed on the LOI (see below).Priorities based on consultations between researchers and policy makers and expressed by researchers through a survey will reveal the understanding and conceptual framework of researchers and may underplay policy-maker needs. Furthermore, these priorities will be influenced by the research projects under execution and reported in the same instrument.The analysis of relationships or influences across the portfolio and each of the actor agendas, as well as of similarities or differences between agendas, was undertaken by correlating topic frequencies across lists and by undertaking a qualitative analysis of changes in rank order and emphasis.Analysis of the range and rank of topics across groups of countries by income was undertaken by aggregating project and priority topic data into a reference list representing the combined set of influences on the agenda-setting process, including the portfolio itself. The aggregation of data into a reference list was mainly a strategy to increase the observations and make the analysis more reliable, although it may have validity if it describes the overview of the agenda-setting factors at play. That is, the actual portfolio can be conceived as a force shaping the agendas, together with other factors.The survey of HPSR producer institutions in developing countries was described in detail elsewhere  and inclBiases in the partner database could have occurred as a result of preferences by certain type of institutions in joining the Alliance HPSR and in answering the questionnaire required from partners. Over-representation at both levels could have occurred of more competitive and productive institutions with larger project portfolios and funding, and more interest in international funding. On the other hand, larger institutions may have been discouraged from responding given the larger number of projects to be reported, although they would also have more capacity to respond. Furthermore, the response rate could have been lower among institutions where producing HPSR is not a main function.The LOI database has 403 submissions for research funding in response to a call by the Alliance-HPSR in 2000. Applicants requested funding for one year projects in high priority areas identified jointly by them and national policy makers and stakeholders. A limitation of this database is the exclusion of funding requests for projects over one year as well as topics that would be formulated solely by researchers. Analysis of the partners' database indicates that 24% of projects are of longer duration and that up to 34% of projects undertaken are initiated by the research institution without stakeholder collaboration.The frequencies of statements for each income region were expanded proportional to population to make comparisons across regions possible. The frequency of statements across the three types of statements  was standardized to give each equal weight when aggregating them to analyze the combined representation of the research portfolio and the agenda-setting process as a whole.Responses show a distribution across income regions proportional to population in some cases and with significant biases in others table . The lowThe frequency of statements on priorities is as would be expected for the population in each region. However, project statements and demand for funding are biased in favour of UMI, with 25% and 27% of the statements, respectively, against 12% of population share.This was done for each topic or category within the topic by rounding percentage differences to integers and grouping in the same rank all categories falling within the same percentage.This article does not attempt to provide an exhaustive analysis given the fact that data is limited to Alliance partner producer institutions in the South. The purpose here is to illustrate the potential of the proposed methodology and to present the most robust findings. HPSR topics are first presented and analyzed aggregating in a single list the topics in the research portfolio as well as in the policy maker and researcher agendas. This aggregate representation is then analyzed by groups of countries according to their per capita income. The influence exerted on the HPSR portfolio by various actors is then analyzed.HPSR producer institutions are generally small with an average of 3 projects, 8 researchers and a project portfolio worth $155,226 [A total of 19 research topics were identified when aggregating portfolio (project) and priority (voiced preferences) data into the reference list. Topics ranged in frequency from 2% to 11% and were ranked in 8 classes Table . The higThe fact that "Equity" appears so low in the aggregated ranking could be partly attributable to the fact that this topic was defined to include only projects and priorities having equity as the central topic and measuring it through multi-dimensional approaches such as health conditions, access to services and financing. A subsidiary analysis was thus undertaken to include under "Equity" those projects or priorities addressing equity or poverty as a secondary, qualifying, role of research on other topics. This broadened topic "Equity" climbs to fourth rank, at the same level as "Accessibility", "Program evaluation" and "Research to policy".Public and private institutions show no significant changes in topic ranking (corr = 0.70). "Community participation" and "Accessibility" are the only topics with major differences, ranking higher among private institutions.Out of the total topics classified in the reference list, only 38% 404) were sufficiently focused or detailed to be able to attribute a beneficiary or specific issue (Table 04 were sThe topics with least identification of beneficiary or issue were "Costing and cost effectiveness", "Policy process" and "Research to Evidence", with 79% to 96% in this situation. By contrast, "Community participation" and "Management & Organization" were the topics least frequently unidentified with beneficiary/issue, at between 30% and 41%.The most frequent beneficiary/issues were Community, Equity/poverty Hospital, Gender Primary care and Rural areas. The three beneficiary/issues least identified were Urban areas, elderly and Indigenous peoples/traditional medicine.The analysis of the correlation between beneficiary/issue and topic is tentative at this stage given the low frequency in many of the cells of the 19 by 11 matrix. Two beneficiaries/issues account for a large part of the focus of research topics: Community and Hospital as a focus on levels of care. At the community level the topics of community participation, financing, health insurance, decentralization, policy process, information systems and human resources are all prominent. At the hospital level the topics of costing and cost effectiveness, pharmaceutical policy, quality of care and management and organization are most prominent. By contrast, the topic of program evaluation is fairly widely spread across several issues or beneficiaries.The following topics show also a fairly discreet relationship to beneficiaries/issues: Research on accessibility is mostly focused on rural areas. Research on disease burden is prominent among the elderly and children. Economic policy and health focuses on children. Gender is also an important component of these three topics. Equity is focused on indigenous populations. The topic of information, education and communication is prominent among children. Sector analysis focuses mainly on the public private mix.The differences in ranking of the topics in the reference list across income regions are shown in the first three columns of Table The overall ranking of topics in the reference list was compared against the ranking of topics in projects initiated by each actor. The relationship between actor preferences and the reference list was assessed through an analysis of their rank congruence.As described in a previous paper in more detail , the resThe agendas across actors differ substantially, and none can be said to be close to the other. As a result, the reference list shows marked difference with respect to each actor's agenda. Government initiation shows preference for financing and cost-effectiveness as compared to the overall ranking. Government initiation tends to give lower regard to disease burden studies and for research on accessibility. International donor initiation matches best the reference list but gives somewhat less preference to sector-wide analyses. Research institution initiation is more marked for equity and community participation and less so for management and organization, accessibility and program evaluation. International donor initiation preferences are associated to the top ranking topics in the overall listing, suggesting a predominance of their agenda on the reference list.The analysis of the research portfolio and priorities at the international level shows a widely diversified set of topics, ranging from sector wide issues to more focused program evaluation. The emphasis on sector wide issues reflects the challenges to health systems today and suggests that countries consider as important the macro-level analysis as the micro. Micro approaches with a focused attention to beneficiaries or specific issues are well identified, particularly under the topic of program evaluation.However, the evidence also suggests a gap between the research that is actually being undertaken and the challenges for strengthening and scaling up of disease control programs. Such a gap is evident in the low emphasis given to research on human resources, policy process, equity, economic policy and health and information systems.By contrast, the analysis suggests a high degree of attention at the community level, although much attention is also given at the hospital level. Primary care thus seems to be under-emphasized. Considering the disease focus, whenever this was made evident, the data do not suggest a bias towards problems that would not be evidently important at country level.The fact that the public or private character of research institutions is insignificant for the agenda suggests the capacity of diverse institutions to work within a common agenda.There are significant differences in the research portfolio across groups of countries based on per capita income, suggesting the need for priority setting mechanisms at both national and international levels that reflect such diversity. The greater congruence between donor preferences and the international research agenda highlight the importance of consensus building between national and international actors. While it is appropriate for governments and international donors to fund different aspects of the research portfolio, this requires high-level priority setting and consensus mechanisms to ensure they complement each other rather than lead to fragmentation.More research is required to establish the relationships between actors' agendas and the research portfolio at the international level. There is also a need to discuss the most desirable balance of influences and to increase the voice of developing country actors. Evidence-based HPSR priorities emerging through such a process would then be able to support scaling up of research efforts on a par with scaling up of health system strengthening and disease control. Regional and global meetings, such as the Ministerial Summit for Health Research to be held in Mexico in November 2004, are good opportunities to present and discuss the evidence and to commit funding accordingly. Attention must be given to encouraging consensus building on research priorities within regions comprising countries with similar needs. The interests of donors, governments, health workers, the community and researchers must all be taken into consideration so that research funding leads not only to fund relevant research but to build the necessary interfaces for utilization.None declared.


S100B belongs to a family of calcium-binding proteins implicated in intracellular and extracellular regulatory activities. This study of serum S100B in primary progressive multiple sclerosis (PPMS) is based on data obtained from a randomized, controlled trial of Interferon β-1a in subjects with PPMS. The key questions were whether S100B levels were associated with either disability or MRI findings in primary progressive MS and whether Interferon β-1a has an effect on their S100B levels. Serial serum S100B levels were measured using an ELISA method. The results demonstrated that serum S100B is not related to either disease progression or MRI findings in subjects with primary progressive MS given Interferon β-1a. Furthermore there is no correlation between S100B levels and the primary and secondary outcome measures. S100B belongs to a family of calcium-binding proteins implicated in intracellular and extracellular regulatory activities . IntraceSeveral studies suggest that S100B has a role in the pathogenesis of multiple sclerosis (MS). Phenotypically and functionally similar T cells specific against S100B can be detected in the peripheral blood of MS patients making S100B a putative candidate auto-antigen in MS . FurtherInterferon-β (IFN-β) is effective in reducing relapse rate in relapsing-remitting ,14,17 anIn this clinically negative phase II study , we asse®, Biogen) and were assessed three monthly over a study period of 2 years. Fifteen of these patients were treated with IFNβ-1a 30μg intramuscularly (im) weekly (IFN30), 15 received IFNβ-1a 60μg im weekly (IFN60) and 20 with placebo. IFNβ-1a was reduced to half dose in 5 subjects receiving 60μg im weekly, and in 2 subjects receiving IFNβ-1a 30μg im weekly. Seven subjects withdrew from treatment [Fifty patients with PPMS were recruited in a phase II trial of IFNβ-1a . Progression was defined as a sustained (3 months apart) increase of at least 1.0 on the EDSS scale between 0 to 5 and 0.5 for subjects with EDSS score of 5.5 and above.Fourteen healthy subjects served as controls.All subjects provided informed consent prior to their inclusion in the study. This study was approved by the ethics committee and has therefore been performed with the ethical standards laid down in the 1964 Declaration of Helsinki.MRI was performed at baseline and 6 monthly for 2 years. Only baseline and year 2 data were included in this study. Brain and spinal cord atrophy, ventricular volume, T1 and T2 lesion load were measured as described elsewhere .Serum samples were centrifuged and stored at -20°C. Serum S100B levels were quantified using a modified ELISA method as previously described by Green et al. . Ninety-Median, interquartile range and significance of group differences (Mann-Whitney U tests) were evaluated. Changes of serum level over time were examined using variance components regression models of serum response variable on time as predictor, with random subject-specific intercepts and fixed common slopes. Curvature was assessed using a quadratic term in time; modification of curve over time by treatment was assessed using additional terms for treatment and treatment by time interaction in the model. Two sets of treatment terms were used: i) indicators of assigned weekly dose ii) average weekly dose over follow-up (including changes to dose regime) as continuous variable. Modification of the curve over time by MRI variable values were similarly examined using terms for MRI variable and MRI variable by time interaction.Direct associations between serum level and MRI/clinical variables were examined by regression models of 24 month serum on 24 month MRI variable, adjusting for baseline serum and MRI values (this type of model takes into account change from baseline), with additional terms for treatment and treatment by MRI variable interaction, the latter to assess possible modifications of the relationship by treatment.Software used were the SPSS software package (version 11.0 for Windows) and Stata 7.0 .The median and interquartile ranges for all subjects are described in Table There was no change over time in the serum S100B levels. The shape of the serum trajectory did not vary between the treatment regimes, i.e. placebo vs. IFN30 vs. IFN60.There was no evidence that the 24-month serum S100B values were associated with either changes in the T1 or T2 loads, or ventricular or cord volumes at 24 months, after adjusting for the baseline values of each subject. There was no correlation with disease progression on the EDSS. There was also no evidence that these relationships were modified by treatment assignment  (Table These results suggest that serum S100B levels in patients with PPMS were not affected by intramuscular IFNβ-1a and that there was no observable change in S100B over time. Furthermore, we did not observe any correlation between S100B levels and clinical disability or between S100B and quantitative MRI measures.This study therefore suggests Although there is evidence that S100B elevation in MS is related to inflammatory activity ,13, thisIt would be valuable to identify surrogate markers of clinical progression in PPMS to aid the development of effective therapeutic intervention, since clinical trials with a disability endpoint are very large and resource consuming. It is possible that such markers would need to be less related to acute inflammation and more dependant on other neuropathology such as axonal loss and regeneneration.


The colour of animals' skin, fur, feathers or cuticula has been estimated in a large number of studies. The methods used to do so are diverse, with some being costly and not available to all researchers. In a study to measure plumage colour in a bird species, a new method of creating a colour chart was developed. While colour-charts have their own limitations, these can be minimised when they have the following properties: 1) being readily available to the majority of biologists, 2) containing a large array of colours to allow accurate recording and differentiation of subtle colour differences, 3) low cost, 4) adhering to a world-wide standard, and 5) being available in both hard-copy and digital formats to allow for various analytical methods. The method described below satisfies all of these requirements.Colour charts estimated to fit the range of the species' plumage colours were created on the computer screen using web software that allowed for HTML-coding (in this case Dreamweaver™). The charts were adjusted using feathers from dead specimens until a satisfying range of darker and lighter colours were found. The resulting chart was printed out and was successfully used in the field to determine the plumage colour of hand-held birds.Access to a computer and printer, and the software to enable the creation of a chart, is within the reach of the vast majority of biologists. The numbers of colours that can be generated should suit most studies, with the advantage of the method being that the chart can be individually tailored to the species under study. HTML colour coding is a worldwide standard, thus the colours used in studies can be described in the methods section of journal articles using the six-digit alphanumeric code. We believe this method is very useful as a low-tech method for future estimation of individual colour. Beagle expedition . Us. Us19]. The method of colour-chart creation utilising HTML code satisfies the criteria listed above. Access to a computer and printer, and the software to enable the creation of a chart, is within the reach of the vast majority of biologists. The numbers of colours (and patterns) that can be generated should suit most studies, with the advantage of the method being that the chart can be individually tailored to the species under study. HTML colour coding is a worldwide standard, thus the colours used in studies can be described in the methods section of journal articles using the six-digit hexadecimal code. Comparisons are not limited to a printout of a colour chart, digital images can also be compared and their colours scored using this method. Drawbacks may include identifying the right colours for the chart to accurately match the animals as encountered in the field, a problem equivalent to other printed colour charts. It is also possible that when printing, the printed colours differ from the colour range as displayed on the computer screen. With some people still using older computers, which are not able to display colours coded in newer machines, there is a risk that there is a discrepancy between computers in the colour presented on the screen. This may be a problem when the aim is to compare specific colours between different studies, but not an issue within studies. Fortunately, this problem will decrease with more computer system being able to display the full range of 16.7 million colours. When using the colour-charts in the field, the usual care of indexing individuals under the same lighting conditions should be taken . As the JM came up with the initial idea of using web designer tools for creating colour charts. ÅB developed the colour charts using HTML coding and used them in the field as a research tool. ÅB and JM wrote the manuscript, with ÅB doing the major part. All authors read and approved the manuscript.


Obesity is the most common health problem in developed countries. Recently, several physicians' organizations have issued recommendations for treating obesity to family physicians, including instructions in nutrition, physical activity and medications. The aim of this study was to examine if effective weight-reducing treatment can be given by a family physician. It compares regular treatment with intensive treatment that include close follow-up and orlistat treatment.The study was conducted in three primary care clinics. 225 patients were divided into three groups according to their choice. Group A received a personal diet with fortnightly meetings with the family physician and dietitian and orlistat treatment. Group B received a general diet, monthly meetings with the family physician only and orlistat treatment. Group C received a personal diet, monthly meetings with the dietitian only and no drug treatment. The primary endpoint was reduction of at least 5% of the initial weight during the study period.A greater percentage of patients in group A achieved their weight reduction goals than in other groups . There was a significant reduction in triglycerides in all groups, a significant reduction of low density lipids (LDL) in groups A and B and no significant difference in high density lipids (HDL) in any group.Significant weight reduction was obtained in a family physician setting. Further research is needed to evaluate if, by providing the family physician with the proper tools, similar success can be achieved in more clinics. It is aabove 25 . The ratabove 25  and has above 25 . In Israabove 25 .Obesity is associated with increased prevalence of many serious chronic diseases such as diabetes mellitus, hypertension, dyslipidemia, and coronary heart disease ,9. It maDiet and exercise have limited effectiveness on long-term maintenance of weight loss . Within The intervention of primary physicians during a ten minute physician/patient encounter and telephone consultation with a community dietitian resulted in a significant decrease in the weight of patients . RecentlThe purpose of this study was to examine if more efficient and effective weight-reducing treatment can be given in the family doctor setting. The study compare a non-pharmacological intervention with drug intervention (orlistat) and compare regular management with more intensive family physician based management..The study was conducted in three primary care clinics in an urban area in central Israel. The family physicians who took part in this study participated in 80 hours CME course dealing with obesity treatment in Israel.®) is a lipase inhibitor for obesity management that acts by inhibiting the absorption of dietary fats.The patients were divided into three groups according to their choice. Patients in groups A and B were treated with orlistat at 120 mg TID. Orlistat  patients of either sex or patients with a BMI above 27 plus two or more cardiovascular risk factors, aged 20–75 years, were eligible for the study. Patients were excluded if they were pregnant or lactating or if they had any contraindication against using orlistat .Before the intervention, patients underwent an initial screening that included recording of a complete medical history, measurement of vital signs, body weight and height, and calculation of the BMI. Laboratory analysis included a lipid profile. The readiness of the patient to receive treatment was assessed. At the beginning of the intervention, all participants were in the third stage of readiness ("the preparation stage") according to the Transtheoretical Model of Behavior Change.An adverse event of any dose of orlistat was considered serious if it resulted in death, was life-threatening, required hospitalization, or resulted in significant disability.The main measure was weight loss. Each patient was weighed during each meeting. The primary endpoint was reduction of at least 5% of the initial weight during the study period (six months). Achievement of this goal was defined as successful treatment. Another measure was improvement in the lipid profile.The second lipid profile was done to half of the patients, only those who had dyslipidemia in the first profile had been offered a second profile.Data on patients' background and weight-reduction results between groups were compared using the Chi square test. Continuous data of the groups, i.e., measurements at the beginning of the study and continuous background data, were compared using one-way tests (ANOVA). When significant statistical differences were found among the three groups, the Tukey Post Hoc test was used to examine the statistical difference between each group of two. ANCOVA was also used and, if there was a difference amongst the groups, the significance (for the measured parameter) was checked using the Bonferonni technique.Two hundred and twenty-five patients participated in the study. Their demographic characteristics, history of cardiovascular disease, and initial lipid profile are shown in Table p = 0.03).Patient-reported adverse events in the orlistat-treated groups were all related to the gastrointestinal tract. The most commonly reported events were flatulence with discharge (9.2%), fatty or oily stool with increased defecation (9.2%), feeling of fullness in the stomach (4.6%), and constipation (1.7%). There were no statistically significant differences in side effects between groups A and B. In group A, 11 patients (17.7%) stopped the treatment for the following reasons: cost of the medication (47%), lack of time (33%), or dissatisfaction (20%). In comparison, ten patients (8.9%) in group B stopped the treatment, mainly because of the cost of the treatment (65%) or low motivation (23.5%). The reasons for stopping treatment significantly differed between groups A and B  of their initial body weight, patients in Group B reduced 7.8 kg (range 10–12 Kg.) from their initial body weight and patients in Group C reduced 3.12 kg (range 5–6 Kg.) of their initial body weight.Obesity is a worldwide problem. Treatment of a patient for obesity involves two processes: evaluation of the severity of the obesity and general health condition of the patient, and management which includes guidance in how to gradually reduce weight and maintain the new weight together with imparting healthy lifestyle habits and keeping track of improvement.In this study, we examined weight-reduction techniques that can be carried out in the setting of a community family practice. Intense treatment, combining frequent counseling by the family physician and a dietitian with medications (group A), resulted in the best weight reduction and lipid profile improvement in the short period of this study, as was reported earlier in special weight reduction clinics -27The effectiveness of interventions in primary care setting are controversial -30. BeerHauptman et al  in a stuThere are many advantages to a program involving cooperation between the family physician and the dietitian. The family physician is acquainted with the patient for a longer time than a dietitian. The physician is familiar with the patient's health condition, medications taken by the patient, the patient's environment and lifestyle and can recommend a treatment suitable to the patient's personality and lifestyle. The family physician is knowledgeable about weight-reducing drugs and possible side effects. The patient trusts and has confidence in the family doctor. Together with the dietitian, a personal diet appropriate to the drug treatment can be created.Obesity is a chronic disease. The physician and the patient must recognize that obesity treatment is a prolonged process that extends a lifetime. Since family physicians are usually familiar with their patients as well as with the patient's family and environment for many years, family physicians know what changes the patient can achieve. They can recruit family members to support the necessary lifestyle changes. Also, because of their training, family physicians are the most suitable professional to holistically treat obesity and its complications.There were several limitations in our study. One limitation was the non-random division of patients into groups A, B and C. It is possible that the patients who chose drug treatment differed from those who chose a diet only. Perhaps they were more ready for the weight-reduction process and to expend money for the drug . The rate of women amongst the patients seeking treatment was higher than the rate of men, as in other reports ,27. HencThe study periods for groups A and B (which received orlistat) were shorter than for group C, partly because there is significant co-payment for orlistat and partly because the targeted weight had been achieved earlier. The drug was well tolerated with minimal gastrointestinal side effects.The patients were treated by family physicians that were orientated toward and trained in weight reduction. Results might have been less successful with other physicians. Hence, family physicians should be trained in the subject by increasing both their knowledge and their treatment skills. Healthcare funds must recognize the importance of weight reduction so that they will allocate the necessary additional time and resources of their physicians, clinics and multi-field staff.This study evaluated only the weight reduction period. Long-term results and whether or not the patient maintained the lifestyle change for a long period were not examined. Further studies should examine the best program for maintaining the new weight.In conclusion, this study showed that within the setting of the family practice it is possible to carry out an effective program of weight reduction and achieve significant weight loss. The addition of orlistat can further improve results. We believe that by providing family physicians with the proper tools, similar success can be achieved in many more clinics.The author(s) declare that they have no competing interests.AF and SP conceived and designed the study, participated in the collection, analysis and interpretation of data and drafted the manuscript. SV Participated in the statistical analysis, interpretation of data and draft of the manuscript. MS and EF participated in the design of the study, data collection and interpretetion. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: