S. cerevisiae the β-1,4-linked N-acetylglucosamine polymer, chitin, is synthesized by a family of 3 specialized but interacting chitin synthases encoded by CHS1, CHS2 and CHS3. Chs2p makes chitin in the primary septum, while Chs3p makes chitin in the lateral cell wall and in the bud neck, and can partially compensate for the lack of Chs2p. Chs3p requires a pathway of Bni4p, Chs4p, Chs5p, Chs6p and Chs7p for its localization and activity. Chs1p is thought to have a septum repair function after cell separation. To further explore interactions in the chitin synthase family and to find processes buffering chitin synthesis, we compiled a genetic interaction network of genes showing synthetic interactions with CHS1, CHS3 and genes involved in Chs3p localization and function and made a phenotypic analysis of their mutants.In CHS1, CHS3, CHS4, CHS5, CHS6, CHS7 and BNI4 in a synthetic genetic array analysis we assembled a network of 316 interactions among 163 genes. The interaction network with CHS3, CHS4, CHS5, CHS6, CHS7 or BNI4 forms a dense neighborhood, with many genes functioning in cell wall assembly or polarized secretion. Chitin levels were altered in 54 of the mutants in individually deleted genes, indicating a functional relationship between them and chitin synthesis. 32 of these mutants triggered the chitin stress response, with elevated chitin levels and a dependence on CHS3. A large fraction of the CHS1-interaction set was distinct from that of the CHS3 network, indicating broad roles for Chs1p in buffering both Chs2p function and more global cell wall robustness.Using deletion mutants in CHS3 are involved in the amelioration of cell wall defects and in septum or bud neck chitin synthesis, and we newly assign a number of genes to these functions. Our genetic analysis of genes not interacting with CHS3 indicate expanded roles for Chs4p, Chs5p and Chs6p in secretory protein trafficking and of Bni4p in bud neck organization.Based on their interaction patterns and chitin levels we group interacting mutants into functional categories. Genes interacting with  Saccharomyces cerevisiae, chitin, a linear polymer of β-1,4-linked N-acetylglucosamine (GlcNAc) residues, is selectively concentrated at the bud neck and is also found as a minor component of the mature lateral cell wall. Chitin is also the main constituent of the primary septum, a structure that separates mother and daughter cells (for reviews, see [In vegetatively growing cells of ews, see ).CHS3, is responsible for synthesis of the chitin ring at the bud neck and for chitin in the lateral wall. CSII synthesizes the chitin of the primary septum, and is encoded by CHS2, a gene that is essential in many strain backgrounds [CHS1, is localized to the plasma membrane and to chitosome vesicles [CTS1 required for cell separation suppresses the chs1 lysis phenotype, leading to the suggestion that Chs1p is involved in chitin repair at cytokinesis [Polymerization of UDP-GlcNAc to chitin is catalyzed by a family of three membrane-associated chitin synthases (CS) with specialized activities. CSIII, encoded by kgrounds . CSI, envesicles  and mutavesicles . Disruptokinesis .BNI4, CHS4/SKT5, CHS5, CHS6 and CHS7. Chs7p is required for exit of Chs3p from the endoplasmic reticulum [The precise deposition of chitin is achieved through spatial and temporal controls on each chitin synthase which determine their localization and activity. CSII is expressed in a cell cycle-dependent manner, and is transported to the bud neck through the secretory pathway, and subsequently degraded in the vacuole ,9. CSI aeticulum , while Ceticulum ,13. Chs3eticulum .CHS3 is essential for maintaining the cell integrity of several cell wall mutants, such as fks1 or gas1 [CHS2 and CHS3 [Although accounting for only 1–2% of the wild type cell wall under vegetative growth, chitin can contribute up to 20% of the cell wall under the conditions of cell wall stress found in cell wall mutants or on drug exposure . Indeed, or gas1 -17. Simiand CHS3 .To further explore the relationship between chitin synthesis and other pathways, we assemble a network of 316 synthetic interactions of 163 genes with genes involved in the regulation of chitin synthesis. The relationship of these genes with chitin synthesis was analyzed by measuring the chitin content of the 156 viable deletion mutants and by testing for Calcofluor white sensitivity phenotypes of the 116 deletion mutants in non-essential genes of the CSIII network.BNI4, CHS1, CHS3, CHS4, CHS5, CHS6 or CHS7 using the SGA methodology [BNI4, CHS1, CHS3, CHS4, CHS5, CHS6 and CHS7 are connected to 22, 57, 63, 47, 71, 25 and 31 genes, respectively . Thus, this set of 44 genes defines a core group of multiply interacting genes. In addition, the "core" genes account for 57 of 74 synthetic lethal interactions of the CSIII network [The 123 genes engaged in the 259 interactions of the CSIII network were grouped by function Figure  interact network , highligCHS1 . Thus, the deletion mutants of group 1 genes are highly sensitive to CSIII pathway perturbation.Group 1 has 33 mutants with an altered chitin level and a requirement for Chs3p function for optimal growth Figure . All butCHS3 .Group 2 is composed of 19 and 2 mutants with an increased and a decreased chitin level, respectively, but whose optimal growth does not require 3 Figure . A largeCHS3  interact uniquely with SHC1. The pho85 null mutant was not further analyzed due to its very poor growth. The amount of chitin produced in the 4 remaining mutants was measured and found to be similar to the wild type  show a delay in exit from mitosis and mutants in ASC1, IES6, LGE1, RPL20B, RPS8A or VAM7 exhibit altered cell size, a phenotype frequently reflecting defects in cell-cycle checkpoints [CHS3 likely result from a failure to fully synthesize both the primary septum (as a consequence of a defect in cell-cycle progression) and a remedial septum  to the bud neck are essential for growth of mutants with low CSIII activity. Thus, we identify these genes as candidates for involvement in Chs2p localization and in bud neck integrity , suggesting multiple additional roles for Chs5p and Chs6p in protein targeting. Interestingly, a number of these interacting mutants have elevated chitin levels and fall into group 2. For example, the mutants ira2 and pde2 are synthetic with CHS5 and CHS6 and CHS5, respectively and make 76% and 35% more chitin than the wild type, respectively  some of which show elevated chitin levels. These mitochondrial proteins may play indirect roles in late-Golgi trafficking; for example, the secretory apparatus and mitochondria exchange lipids [CHS5, mitochondria may be poorly transferred to daughter cell with their efficient functioning being essential for optimal growth.e lipids , and a dCHS5 and CHS1 , and in protein recycling through the endocytic pathway (VPS5, VPS17, VPS29 and VPS35), all could be required for Chs2p function . CHS1 also shows synthetic interactions with YEL033W and YNL179C, that overlap with and are alleles of YEL034W/HYP2 and YNL180C/RHO5, genes that play a role in balancing cell integrity [YOR322C which has a role in signaling through the cell integrity pathway [BCK1, BEM4, CNB1, ECM21, FPS1, GRS1, HBT1, HIT1, NUC1, PDA1, PFK2, SPF1, TYR1, YGL081C, YGL110C and YPL261C) that show synthetic interactions with the FKS1, GAS1 or SMI1 genes involved in β-1, 3-glucan synthesis [In our data we also find interactions with mutants in a number of genes that are singly prone to lysis or show phenotypes consistent with osmotic imbalance  show many CHS3-independent interactions and these greatly expand the range of trafficking functions for these genes, especially for the heavily interacting CHS5. In contrast to its currently assigned minor auxiliary role, CHS1 shows an extensive web of genetic interactions, most of which are distinct from the CSIII network and which do not trigger the chitin stress response. One set of these identifies components of endocytosis, budding and cell morphology, which may be required for Chs2p function. A second set of 25 interacting genes show that Chs1p is intimately involved in buffering yeast cell wall robustness during vegetative growth.Our synthetic network analysis reveals a deep interactional complexity underlying chitin biology. The et al. [Haploid deletion mutants Table  are avaiet al. . Arrays et al. . NourseoBNI4, CHS1, CHS3-7 or SHC1, as described [MATa spore progeny were serially diluted in sterile water and 2 μl for each dilution was spotted onto medium selecting for the query-gene mutation [SD-His/Arg + canavanine/Nourseothricin], the interacting gene mutation [SD-His/Arg + canavanine/G-418], and both the query-gene and interacting gene mutations [SD-His/Arg + canavanine/Nourseothricin/G-418] then incubated at 30°C for ~2 days. Cell growth under the three conditions was compared and double mutants were scored as synthetic sick (SS), synthetic lethal (SL) or no interaction (No) [CHS3-7 or SHC1 [CDC3,CDC11, CDC12 and CHS2) or genes whose deletion leads to systematic growth defects in our conditions (ANP1, MNN9, PHO85 and SRV2) and these genes were used in our network analysis. No synthetic interaction between GAS1 and CHS4 or CHS7 was found by the SGA. These discrepancies with other's data [CHS1 or a set of 16 non-reciprocal interactions (that is gene A found in screen for genes interacting with gene B and gene B not found in the set of genes interacting with gene A).Synthetic genetic array analysis (SGA) was used to identify genes required for the optimal growth of strains deleted for escribed ,19. From or SHC1 ,25,56,572HPO4/0.1 M citric acid, pH 6.0) was added to pellets. Extracts were then stored at -20°C until processed for chitin measurements. Samples were thawed and subjected to two digestions with 4 μl of purified Streptomyces plicatus chitinase-63 (4 μg/ul in PBS) at 37°C for 36–40 h and then for 20–24 h. The amounts of chitin were then determined by using the modified Morgan-Elson procedure as described previously [p < 0.01 in a Student's t-test, see Stationary phase cultures were diluted 1:100 into 3 ml of YEPD and grown again for 22-24 h at 30°C. Cells from 1.5 ml culture were colleted by centrifugation (20,000 × g, 2 min). Pellets were then frozen at -20°C until used for alkali-extraction. Dry weights were determined after a 2 day incubation at 37°C. Cell pellets were resuspended in 1 ml 6% KOH and heated at 80°C for 90 min with occasional mixing. Alkaline insoluble material was pelletted (20,000 × g, 20 min), neutralized with phosphate-buffered saline for 10–20 min with occasional mixing. After centrifugation (20,000 × g, 20 min), 200 μl of McIlvaine's Buffer  containing 10 μg/ml or 50 μg/ml Calcofluor white (Fluorescent Brightener 28, Sigma), and control plates. Plates were incubated at 30°C for 48 hours, photographed, and then rechecked after 72 hours. A literature search indicated that the phenotype we found agreed with that previously reported for 29 mutants (22 interacting mutants + 7 query mutants). In 4 cases however (ARC18, PDE2, PEX6 and SPF1), we found a wild type sensitivity for mutants that had previously shown altered Calcofluor white sensitivity [Sensitivity to Calcofluor white was assessed using a modified version of the method described by Ram et al. . Cells wsitivity ,58,59. TGL participated in the design of the study and its coordination, collected and analyzed data, and drafted the manuscript. JS carried out confirmations of SGA screens and Calcofluor white sensitivity assays. CAS carried out chitin determinations and data analysis. AMS, PM, SH and AHYT carried out SGA screens. HB and CB conceived the study, and oversaw its design and coordination. HB participated in data analysis and manuscript writing. All authors read and approved the final manuscript.Title: Interactions reported previously and not included in our global analysis.Click here for fileTitle: Sensitivity to Calcofluor white and chitin levels of mutants.Click here for file


The biological potential of prostate cancer is extremely variable. Particular interest is focused on markers not expressed in normal prostatic tissues. pS2 protein expression has been demonstrated in a range of malignant tissues in an oestrogen-independent pathway. Recently, it has been demonstrated that pS2, in prostate cancer, is closely associated with neuro-endocrine differentiation. In the present study, we have analyzed, the potential of Neuro-endocrine and pS2 (TFF1) expression in human prostate cancer determined by immunohistochemistry, in primary adenocarcinoma of the prostate and attempted to correlate this with the clinico-pathologic features of the patient and neuroendocrine expression.Ninety-five malignant prostatic specimens from primary adenocarcinoma, obtained from either transurethral resection of prostate or radical retropubic prostatectomy, from 84 patients between January 1991 and December 1998 were evaluated by immuno-histochemical staining using selected neuroendocrine tumor markers i.e. chromogranin A (CgA) and estrogen inducible pS2 protein. The relationship between the expressions of pS2 was studied with CgA expression, clinical stage (TNM) and tumour grade (Gleason system). Fischer exact test was used for statistical analysis.The mean age of the patients was 70 + /- 9.2 years. The pS2 expression was seen in 10% of primary prostate cancers. Worsening histological grade was associated with greater expression of pS2 (p < 0.001). The expression of CgA was noted in 31% of malignant prostatic tissue. In pS2, positive cases 2/3rd of patients were also CgA +ve. However, there was no significant correlation between pS2 expression and the stage of disease.pS2 expression in prostate cancer significantly correlates with histological grade and the neuroendocrine differentiation, as demonstrated by Chromogranin A expression but not with the clinical stage of the disease. However, the overall expression was low consequently; no definitive conclusions can be drawn. We feel further work is required in a larger series, both in primary and metastatic cancer. The biological potential of prostate cancer is extremely variable . It is pMaisakowski et al. first described the pS2 gene in the MCF-7 human breast cancer cell line . The pS2In the present study, we have investigated the expression of pS2 in malignant primary prostatic tissue in specimens obtained from transurethral, open prostatectomy, and correlated this with neuro-endocrine differentiation and clinical stage and grade. This is a preliminary report on pS2 expression in prostate cancer, a larger study will better define the correlation between stage, grade of cancer with pS2 and CgA expression.Ninety-five malignant consecutive primary prostatic specimens were obtained from 84 patients by either trans-urethral resection of prostate (n = 69 patients) for urinary obstruction or from radical retro-pubic prostatectomy (n = 15 patients) between January 1991 and December 1998. These tissue specimens were taken from the archived records of the department of pathology. The age ranged from 52–93 years (mean 70 + 9.2 years).Immuno-histochemical staining for pS2 and Chromogranin A Sections were stained for H & E as well as for pS2 (Novocastra, UK Cat. # NCL-pS2) and Chromogranin A (DAKO, Glostrup, Denmark Cat # A0430) by immunohistochemistry using indirect immunoperoxidase technique.Briefly, 3 μm thick tissue sections were cut and mounted on poly-L-lysine (sigma) coated slides. Sections were deparaffinized in xylene and re-hydrated through graded alcohol series followed by water. Antigen retrieval was done in case of pS2 with 10 mM citrate buffer, 6.0 in a microwave oven 3 × 5 seconds at 450 W, then gradually cooled down to room temperature.Sections were washed with water followed by Phosphate buffer saline (PBS) rinse.Endogenous peroxidase in the sections was blocked for 30 minutes with 0.3% H2 02 in methanol. Sections were washed with PBS. All sections were treated with Normal Swine serum (NSS) prediluted 1:10 in PBS for 5 minutes. The sections were then incubated with the primary antibody to pS2 diluted with NSS (1:100) and Chromogranin A (1:20) for 90 minutes at room temperature. Slides were washed with PBS and incubated with peroxidase-conjugated swine anti rabbit secondary antibody (DAKO) at a dilution of 1:150 for 45 minutes at room temperature. 3, 3'-diaminobenzidine (DAB) was used as a final Chromogen. Harris Haemtoxylin was used as a counter nuclear stain. Positive and negative controls were used with all batches of IHC staining. A prostatic adenocarcinoma specimen section expressing pS2 was used as a positive control. Same case exhibiting the primary antibody was used a negative control with each staining procedure. The extent of pS2 reactivity was semi quantitatively assessed by estimating the percentage of positive acini present in the whole mounted sessions. Expression was graded ++ if more than 50% of the tissue showed expression, + if between 5 and 49% showed expression and focal if <5% showed expression.Histological grading The Gleason system was used for grading of the cancer specimens; a senior histopathologist (SP) blinded of previous Gleason grading and clinical course did this. Based upon the Gleason score patients were divided into three groups i.e. well differentiated (Gleason sum 2–4), moderately differentiated (Gleason sum 5–7) and poorly differentiated (Gleason sum 8–10).To study correlation and determine the p value Student t test was applied.The cancerous lesion composed of 35% (n = 29) stage T1, 32% (n = 27) stage T2, 25% (n = 21) stage T3 and 6% (n = 5) stage T4 disease according to the TNM classification. Based upon the stage of the disease patients were divided into three groups i.e. organ confined (T1-2), locally invasive (T3-4 and N1) and metastatic (M1) cancer.In 95-cancer specimen from transurethral resection (n = 69) for urinary obstruction and radical retropubic prostatectomy for organ-confined cancers n = 15), pS2 reactivity was detected in the adjoining normal or hyperplastic acini in only 4.2%. The pS2 expression in cancer was found in 10% . The expression of pS2 figure  in varioIn the present study, we investigated the expression of pS2 protein in the adenocarcinoma of prostate and in the surrounding normal prostate tissue. We used a standard immunohistochemical method to assess pS2 expression in tissue sections of adenocarcinoma prostate instead of instead of biochemical or immuno-radiometric assay. The immuno-histochemical method for detection of pS2 expression has drawbacks in comparison to biochemical and immunoradiometric assay on tissue extracts. Both of the later methods allow precise quantification of levels of expression for a better correlation with other parameters studied.However, as we are interested in the clinical utility of pS2 expression in our prostate cancer population, we used immunohistochemistry, which allows appreciation of intra-tumoral heterogeneity of expression and of both cancerous and non-cancerous cells. pS2 protein expression has been demonstrated in a range of malignant and benign pathologies. It is highly expressed in receptor positive human breast cancer  but exprA significant implication of pS2 in prostate cancer is the close association of this marker with Neuroendocrine (NE) differentiation. There is increasing evidence that focal NE differentiation frequently occurs in prostatic adenocarcinoma and it may have significant prognostic implications -14. NE dOur results demonstrate that although the expression of pS2 protein was noted in only 1/10th of prostate cancers, it significantly correlates with the histological grade and NE differentiation; both have independent and interdependent prognostic value. There is dearth of data exploring the correlation of pS2 expression and aggressiveness of prostate cancer cell behavior. Limited literature available at present show significant association of pS2 expression with prognosis in prostate cancer, however more work is required to explore the utility of this marker in defining the biological potential of prostate cancer.The author(s) declare that they have no competing interests.MHA, conceived of the idea, wrote the manuscript and conducted clinical part of the study. FA, helped in designing the study and reviewed the draft of the manuscriptNF, helped in conducting study, helped in data collection and analysis. MI, conducted the pathological part of the study. SP, conducted and supervised the pathological aspects of the study and reviewed the manuscript and wrote methods and results related to the pathology. All authors' have read and approve of the final manuscript.The pre-publication history for this paper can be accessed here:


Due to differences in current socio-economical situation and historically shaped values, different societies have their own concepts of high-quality life. This diversity of concepts interferes with quality of life (Qol) research in health sciences. Before deciding to apply a Qol assessment tool designed in and for another society, a researcher should answer the question: how will this instrument work under the specific circumstances of my research. Our study represents an example of the utilization of qualitative research methods to investigate the appropriateness of the Rheumatoid Arthritis Quality of Life Scale (RAQol) for the assessment of Qol in Estonian patients.Semi-structured interviews were conducted with the rheumatoid arthritis (RA) patients of Tartu University Hospital and these were analyzed using the principles of the grounded theory.We described the significance of the questionnaire's items for our patients and also identified topics that were important for the Qol of Estonian RA patients, but that were not assessed by the RAQol. We concluded that the RAQol can be successfully adapted for Estonia; the aspects of Qol not captured by the questionnaire but revealed during our study should be taken into account in future research.Our results show that qualitative research can successfully be used for pre-adaptation assessment of a Qol instrument's appropriateness. With this article we are introducing our experience of how qualitative research can be utilized in the process of adapting of a quality of life (Qol) assessment instrument. We will argue the unique benefits of the qualitative method for assuring the validity of an adapted measure.Qol cannot be treated as something uniform and stable. One reason for its variability in time and space is the social nature of the "quality". Different societies with their current socio-economic situation, values and traditions, carry their own, to certain degree dissimilar understandings of high-quality life. Therefore Qol can be seen as a social construct. For further discussion on Qol domains see Schalock, 2004 .Inclusion of Qol assessment into a set of health outcome measures is, regarding the achievements in improving the survival of chronically ill patients, well justified. But the social complexity of the construct means that its evaluation is also complex compared to traditional outcome measures e.g. symptoms or results of lab tests. Restricting Qol assessment to condition-bound groups (disease-specific Qol) permits potential influences on everyday life to be homogenized; problems with the various individual significances of these condition-characteristic impacts remain. Different Qol concepts in health sciences try to improve assessment's generalizability. Functionalistically oriented health-related Qol concentrates on restrictions in performance of everyday activities . AlthougBefore deciding to use an existing instrument for Qol assessment, a researcher should answer the question: how will this questionnaire work in the particular circumstances of the research? Even an instrument that has performed excellently in the country of origin can loose some validity when applied in a different social context. There are three categories of topics that should be recognized for every Qol instrument: first, those important for the patients being investigated, and already assessed by the instrument ; second, those unimportant (or not so important) for the patients being investigated, but still assessed by the instrument; and third, those important the for the patients being investigated, but not assessed by the instrument. It is evident that an appropriate questionnaire consists mostly of the first category items, the content of the second category items is minimized and all topics important for patients are included.The items belonging to the two first categories can be determined during a standard adaptation process. The drawback of this approach is the amount of adaptation work that usually has to be done before receiving evidence about the merits of the questionnaire. Delimiting of the third category requires a deeper insight into the society-specific determinants of Qol.Because of the smallness of Estonia (population about 1.4 million) studies involving only our patients may demonstrate a lack of statistical power; also some restrictions concerning scientific potential and funding should be admitted. Therefore a promising choice for Estonian clinical and health sciences can be seen in cooperative research projects. Reasoned selection, judicious adaptation and application of internationally approved assessment instruments could be crucial for success. As a part of the Soviet Union, Estonia stayed isolated from Europe for years and quite strong ideological pressure was brought to bear on the citizens. After establishing independence in the 1990s, Estonian society has undergone abrupt ideological and economic changes. A researcher working in the Qol assessment field should take into account the suspected impact of these factors on understanding life quality among the local population.Our work offers an example of the application of qualitative interviews for exploring the essence of quality of everyday life for Estonian rheumatoid arthritis (RA) patients. We will illustrate the evaluation of the three topic categories using an example of RA specific Qol assessment scale.Rheumatoid Arthritis Quality of Life Scale (RAQol) was developed in the 1990s as an outcome measure to assess the impact of RA and its treatment on QoL. The content was derived from interviews with 50 RA patients conducted simultaneously in Great Britain and the Netherlands ,5. The t• food, drink, sleep, activity, sex, pain avoidance• warmth, shelter, security, safety, freedom from fear, stability• affection, love, physical contact, intimacy, attachment, communication, sharing experiences, sharing goals, affiliation• curiosity, exploration, play, stimulation, enjoyment, creativity, meaningfulness• identity, status, recognition, approval, appreciation, usefulness to others, respect, competence, self esteem, mastery, achievement, power, independence, freedom• time structure• self actualizationAs a RA specific instrument, the RAQol was designed to assess the fulfillment of only those needs whose importance for RA patients emerged during the interviews. The original wording from the interviews was retained as much as possible. The final RAQoL is a 30-item measure where each item is in the form of a simple statement to which patients indicate whether or not it is true for them at that moment. The following example of the RAQol items demonstrates the wide range of everyday areas covered by the instrument: self-care, different indoor and outdoor activities, emotions and conditions, and interpersonal relations.• Item 18. I have problems taking a bath/shower• Item 7. Jobs about the house take me a long time• Item 6. I find it difficult to walk to the shops• Item 16. I often get depressed• Item 12. I find it hard to concentrate• Item 13. Sometimes I just want to be left alone• Item 29. I avoid physical contactet al. 1997 [An affirmed item indicates an adverse quality of life. Each item on the RAQoL is scored '1' for the affirmed statement or '0' for the disaffirm statement. All item scores were summed to form a total score ranging from 0 (good QoL) to 30 (poor QoL). The original RAQol exists in two versions – UK English and Dutch; currently seven language adaptations of the RAQoL are available for use. Excellent test-retest reliability, internal consistency and construct validity of the original instrument and its versions has been demonstrated -10. The al. 1997 .The lack of an instrument for systematic assessment of the Qol of RA patients in Estonia raised the question of the feasibility of adapting the RAQol. Our task was to assess the value of application of the qualitative method for describing the appropriateness of the questionnaire before the adaptation. For studying the three above-described topic categories in connection with the RAQol, we formulated the following two research questions:• Are the RAQol's topics important for our patients?• What else do our patients find to be significant in connection with their everyday life quality?We decided to apply thematic analysis following the principles of the grounded theory. Our choice was determined by the second research question. To answer it, the analysis had to be guided by the data itself, to enable new motifs and theories to spring up. The grounded theory, evolved by Barney G Glaser and Anselm L Strauss in the 1960s  and deveFor data collection we decided to apply individual interviews, which we considered to be the method of choice for investigating RA patients' perceptions of quality of their everyday lives. An alternative focus-group method was rejected because of the possible intimacy of some topics, which might have been difficult to discuss openly in a group setting.Considering the formulated research questions we agreed that the semi structured interview format should be preferred.We decided not to acquaint our research subjects with the original version of the questionnaire. We thought that familiarization with the instrument might restrain the respondents from disclosing their everyday life problems in full, especially those problems not captured by the RAQol.The RAQol items were arranged into groups according to the dimensions of everyday life they reflect. Four groups emerged (note some overlapping): self-care and indoor activities (items 1, 3, 5, 7, 8, 10, 11, 18, 21, 26, 30), outdoor activities (items 2, 4, 6, 10, 14, 17, 20, 21, 25), emotions and conditions (items 9, 12, 13, 16, 19, 21, 22, 23, 24, 28) and relations (items 2, 4, 13, 15, 17, 20, 22, 25, 27, 29). We formed four open-ended interview questions to cover these dimensions. The questions were intended to introduce informal conversation during which different Qol aspects connected with the everyday life could be revealed.1. How does your disease influence your coping with everyday indoor activities including self-care?2. How does your disease influence your coping with outdoor activities?3. What emotions can you describe in connection with your disease?4. How has your disease influenced your relations with other people?We prepared three to four additional secondary questions for each of the four interview questions for cases when some guidance of the interviewee is necessary.The fifth interview question was added in order to give the interviewees a possibility to speak freely about their everyday life problems not assessed by the RAQol. The sixth question was intended to elucidate the hierarchical importance of everyday problems/restrictions for our patients.5. What other impacts does your disease have on your life?6. Which disease impacts on your life do you consider to be the most important?We assumed the structure of the interview – from more detailed to general – to be appropriate for our patients; for most of them it would be the first time to be interviewed.We were determined to include patients who met ARA 1987 diagnostic criteria for RA . We deciBy choosing interviewees from among the inpatients of the Rheumatology department of Tartu University Hospital, one of two specialized rheumatology centers in Estonia, we had access to sufficient medical data to follow the established inclusion and exclusion criteria. In Estonian rheumatology inpatient care is the dominating approach and it often also comprises some traditionally outpatient procedures. Recruiting inpatients gave us the opportunity to sample the whole spectrum of RA patients in circumstances where participation in the research would not interfere with their everyday routine. Also, conducting interviews under hospital conditions allowed us to create similar settings, free of major distractors, for each conversation.Our sampling strategy derived from the wish to collect as multifaceted data as possible. We applied principles of theoretical sampling where information gathered during previous interviews determined the selection of subsequent respondents in order to get views from different positions. The final list of characteristics, which we considered to be important for guaranteeing versatility of the sample, included: age, gender, duration of RA, severity of RA , education, working status, marital status, members of family unit, and living conditions. The characteristics of the patients are presented in Table The interviewing took place from February to June 2002.We consulted medical records to determine inclusion and exclusion criteria, patients' demographics and disease characteristics. No patients were contacted earlier than on the third day in hospital in order to allow them some time for adjustment. A day before the planned interview the goal and expected course of the interview were explained to the patient, the patient's were asked for their agreement to be interviewed. One of the patients we contacted refused to participate on account of being due to leave hospital the next afternoon. All recruited patients gave their informed consent.Interviewing was conducted in a private room in the rheumatology department. All interviews were conducted by one researcher (MT) and were audio taped. Eight interviews were carried out in Estonian; two respondents (3 and 5) were interviewed in Russian, the respondents' native language. Interviews lasted from one and a half hours to three hours.For most of our respondents it was their first chance to discuss their everyday life problems with somebody outside the family. But all of the interviewees were very willing to share their experiences after overcoming some diffidence at the beginning of the interview, and talked openly about their lives with the disease. For the interviewer, growing knowledge with every subsequent interview allowed her to move away from strict adherence to the interview questions, towards more informal conversation and, if necessary, the examination of some topics in depth.Every interview was transcribed word-for-word and discussed before recruiting the next participant. Those interviews conducted in Russian were translated from the tape by the interviewer and transcribed in Estonian.The tenth interview was exceptional. This patient was not hospitalized in the rheumatology department at that time, but we decided to invite her to our study due to the exceptionally long duration of RA recorded in her medical documentation – 50 years. The patient was contacted by phone and her agreement to participate was reached. We met at the patient's home and she was asked to tell her life story with stress on everyday problems. Thus we got an exiting narrative history of Estonian rheumatology from a patient's perspective and collected valuable information for our current research.No new topics relevant to our research questions came forth after the seventh interview. Therefore we decided to stop at the tenth. This decision agrees with the theoretical sampling idea that one should stop recruitment of respondents when the researchers decide that the study has reached its saturation ,16. The As the first step we read and discussed the interviews' transcriptions extensively. Tentative code families were chalked out as a notional framework for subsequent coding.Open coding of the transcripts was performed independently by the two of us, MT and JS. The codes adhering to the previously identified code families were ascribed to expressions composed mostly of one or two sentences in order to distinguish their leading ideas; to some expressions several codes were attached Due to the different structure and extent of the tenth interview, selective coding was applied and only those parts relevant for our research questions were coded.There were no disagreements between researchers at the level of code families; some inter-coder discrepancy appeared in appointing particular codes. Still, full consensus on open codes was reached in discussion. We analyzed the differences in coding and concluded that they could be ascribed to the coders' different – medical and sociological – backgrounds. We illustrate our conclusion with the example of coding a patient's expression: can't even go help my sister in the country with potato planting, a bit sad (2). It was coded as 'inability to offer physical help' by MT and 'alterations in traditional family relations' by JS; coders agreed on the 'relations with close ones' code family. During discussion the consensus code 'inability to perform in family roles' was established, which introduced a deeper exploration of the topic of role performance.We agreed on the coding discrepancies being a benefit rather than a drawback of our research process. They allowed us to highlight and subsequently integrate different aspects of applied codes on the boundaries of marked tentative code families. Hence we decided not to perform formal inter-coder reliability analysis due to its diminished informative potential for this particular research.Axial codes were created through grouping and condensing of open codes by MT. Axial coding was discussed and approved by all researchers. Side by side examination of affined axial codes of different interviews formed the basis for creating core categories – composition of a syllabus of motifs that emerged from interviews. Core categories were formed together by MT and JS, and were discussed and acknowledged by the whole research group.In analysis we used the selective sampling of core categories to meet our two research objectives. First, to assess the importance of the RAQol topics for our patients, we compared every single item of the RAQol with the coded data of our interviews. Our assessment of importance was born in discussion and took account of the closeness of the meanings of the item and the relevant expressions of the interviewee, the frequency of their occurrence, and the significance for the respondents. Second, to describe the Qol topics that were significant for our patients but were not evaluated by the questionnaire, we compared the coded data without a counterpart among the items with the list of needs offered by the needs-based Qol model.We will present our results as answers to the research questions. The descriptions of two of the three topic categories are included in the first, and the description of the third category in the second answer.Three groups of items can be highlighted: items whose importance was demonstrated by the data; items that could conditionally be considered to be important; items whose significance could not be shown on the basis of the interview data.Most topics assessed by the instrument were essential for our interviewed patients. For 22 of 30 items, the interview data provided sufficient evidence to consider them applicable for the evaluation of the Estonian RA patients' Qol. Examples of patients' utterances supporting the significance of the content and also the appropriateness of the format of each of these items can be given from two or more interviews.In some cases a remarkable diversity of utterances connected with one particular item was noted. We will illustrate this finding using item 17 as an example: I'm unable to join in activities with my family or friends. The following responses represent six different reasons to agree with the proposition included in the item. We have given word for word translations of the quotes into English, the IDs of patients are given in brackets.• physically difficult to walk anywhere (1)• a whole fuss with moving, don't want to torture myself (2)• I used to ride the bus a lot before, now it's so expensive, I can't afford to visit anyone very often (9)• Whenever you're sitting somewhere, are somewhere, it's hard to stay in one position all of the time, you have to make yourself move, go somewhere, or whatever (1)• when your feet are ill and you sit for a very long time, then you can't even get up and move (4)• what's the use of going if you're no good anyway (2)• of course I didn't go anywhere, only peeped from the car window (5)• not going to the pub, don't know what to do there, can't handle dancing, and drinking doesn't work out either, no point in just sitting there the whole night (8)• when my joints were so tender and painful that I had to talk about my disease all the time then I definitely didn't look for company and I couldn't eat anyhow and I used a spoon for eating food that you ought to eat with a fork (9)• as I cause such a situation that my hosts have to help and watch me all of the time, I'd rather not go (6)• conditions of a home I go to, how cold it might be, if there's only cold water, is the toilet outside (6)Difficulties related to bathing were mentioned in eight interviews and therefore item number 18: I have problems taking a bath/shower, was included in the group of the appropriate items. Still, the interviews also offered some hints that the value of the item as a measure of ability to carry out body care could be diminished by Estonian traditional sauna culture, especially popular in rural areas I don't care much for the bath, mainly I have let my kids take me to the country and have gone to the sauna, that's much more like it (2)• sometimes I go to the sauna, we have a sauna in the cottage in the countryside, its no big deal to wash myself there, in the summer at least; a sauna might really be more convenient than this bath; there's just no hassle with getting up (7)• I can manage washing myself in the sauna but I can't get up from the bath on my own (8)Motifs related to four of the remaining eight items emerged a number of times from the interviews. However, before adding these items to the list of appropriate ones, certain aspects should be taken into consideration.Two respondents (2 and 5) burst into tears during the interview when looking back on their lives with the disease, which verified the significance of item number 19: I sometimes have a good cry because of my condition. The third interviewee spontaneously talked about crying, describing it as something bright and relieving. It can be presumed that in some cases crying may be interpreted as a way of coping, which is not unambiguously related to the level of Qol.• if you cry, you should cry thoroughly and then you'll feel better, but if you keep something inside you it'll start eating at you, if you get a chance to have a good cry it'll make you feel good /.../ crying is self-purification, later you feel light; that's a feeling a healthy person doesn't get (9)A single word or expression matching English 'frustration' cannot easily be found in Estonian. The foreign word 'frustratsioon' has been introduced into Estonian quite lately but remains unknown to the majority of lay people. Although there are a number of expressions in the interview data corresponding to item number 9: I often get frustrated, formulation of an Estonian version of the item suitable for embracing them all will be complicated.• this is the feeling that you just are, the disease has already got a hold, the sequence of events just keeps going on and on (2)• feelings of injustice that someone else is well but I'm not (5)• it depresses me, and what the mind has built up is ruined by some moment and then the emotional breakdown comes again (6)• often my thoughts run ahead of me and I'm feeling rested and would like to do something, but when I get down to it, then that's it, I end up hindering the work and not doing anything myself (8)• I can't forgive my disease for ruining my life structure that had been carefully and arduously built over a long time (6)In four interviews the insecurity connected with the disease progression and its unpredictability was one of the main topics. In Estonia's rapidly changing society, securing ones future cannot be an easy task and disabled people do not have much of a chance for success. Therefore the likening of capability to determine their future to ability to control their disease by patients is expected, and item number 28: I feel that I'm unable to control my condition, can be considered to be important. But again, difficulties in the formulation of the Estonian wording of the item will arise. Though quite common in everyday spoken Estonian the equivalent to the expression 'to control a condition' was never used by the interviewees when talking about their concerns.• afraid of planning, you never know when it strikes back again (1)• not yet [unable to cope] but it might come in the future; I don't know what will happen next year /.../ you're afraid that if you really get so poorly that you can't take care of yourself who will take care of you (4)• I still try to be like a human being but don't know how long I can manage (5)• there's really nothing for granted in this world of course, but I am preparing a back-up solution in case things get worse (8)No patients reported that they were disturbed by continuously thinking about the disease. Three interviewees with a disease duration of over 10 years talked about not thinking of their condition as a positive phenomenon connected with adaptation to the disease. We concluded that item number 23: My condition is always on my mind, can be treated as significant, although our interviews revealed no evidence that the problem is recognized when present.• I don't think that I'm an ill person at the moment, just when these hands are painful or I just can't cope with everything; I don't think that I'm ill, that I'm so miserable, I don't think about it (4)• I have to change and re-adjust my basic values, but I don't think about it all of the time, maybe I have adjusted them subconsciously /.../ I then, subconsciously, not thinking about it, eat something softer or don't go biting on a big apple if my jaw joint is painful (7)• I don't' even think about my disease at this point, it's like a husband now, day and night, its there in everything I do and I know that as long as I live I will have it and I cant get rid of it and I don't make it a problem anymore (9)To this group we included four items.In one interview, the impact of pain on attention was described. We did not find this evidence sufficient for designating item number 12: I find it hard to concentrate, as significant. In our opinion, these expressions describe switching of attention to another stimulus and do not refer to concentration difficulties. One reason why the interview data did not support the importance of this item can be the lack of currently active intellectual workers in our sample.• You can't think about anything but pain; crossing the road you might get hit because you're only thinking about the pain and forget that you have to keep an eye on the road (6)No data corresponding to item number 1: I have to go to bed earlier than I would like, emerged from the interviews. Because tiredness is a characteristic feature of RA, one explanation for the inability of our data to show this item's importance is associated with the paucity of absorbing nighttime activities, especially in Estonian rural areas.Item number 24: I often get angry with myself, had no matches in our interviews. It is difficult to find any obvious explanations for this particularity but we can suggest that in some cases anger was rechanneled against the medical system – a phenomenon that we will discuss later.Our data failed to demonstrate the importance of item number 28: I avoid physical contact. We believe that intimacy connected with this topic could be the reason our respondents avoided openly discussing it. If so, due to the greater impersonality of a questionnaire format, the item can retain its significance as a part of the instrument.As a result of the analysis, three groups of topics connected with satisfaction of the needs included in the list of those crucial for Qol and important for our patients, but not assessed by the questionnaire, were described.Next we will name these needs and present the evidence of the limitations in their fulfillment. Our deeper inquiry into the reasons for these peculiarities will be presented in the discussion section.Adaptation to a disease is a difficult process comparable to passing through phases of grief. An inevitable and hurtful part of it is abandoning of old roles and the recognition of new ones.Changing role functioning was a common motif in the interviews. Regret and anxiety due to inability to perform in the roles of a healthy person were expressed by the majority of respondents.• What man isn't disturbed by the inability to take care of himself, then you're like a kid not a man (8)• My appearance isn't as attractive [as a woman] anymore as it could have been without the disease (4)• I can't even go help my sister in the country with potato planting, a bit sad (2)• just sad because of him [the son], he asks me why I can't come outside to play soccer with him, well, I really can't (7)• totally like a small kid, someone else has to help you all the time, whatever it is, meals or something else (1)• I walked with a stick, it was a catastrophe, such an old granny (3)• I want to do something, just to make something and do a job, my hands want to work; people drop by and try to rope you in – I can't, there's no doer, all the time "I can't", other days I can't at all (2)• I am afraid of going to work soon; this hand is so ugly; what a hairdresser with such a horrible hand! (4)The new role of a diseased person was generally interpreted as something deprecatory; being ill was considered as opposite to being normal.• I can't move; my movements aren't like they should be, not like healthy and normal people have (1)• before I used to wear high heel shoes like normal people (3)• you're like some prehistoric creature, everybody goes by modern means but you like going back to the stone age (6)Feeling ashamed of the disparity led to preoccupation with concealing the condition. Surrounding people were often seen as appraisers; their opinions were valued more highly than success in coping was. As a result, even the simplest aids that could be noticed by others were rejected. In some cases the fear of being labeled as different elicited the preference of social isolation.• I don't want to see things that point to my condition, don't want these to be seen, and of course, don't want to have to always hide everything (6)• I control myself so that others won't notice the way I am – I have so many acquaintances that for a long time didn't know I was ill (9)• I'm even afraid to tell anyone that this hand is so ill, I'm so quiet (4)• [a special cup with two ears] I have it at home, but I don't use it; at this point I still try to be humanlike (5)• you don't go shopping with a crutch, no way (8)• I feel very uneasy eating in company; I still want to eat like people do, with regular tableware (9)• if I still find it impossible to eat or drink in company, then I don't; I won't go into company [to eat] like this (6)A well functioning medical system should strengthen the feelings of security and stability of its clients. In the words of our respondents the medical system constituted an enemy. The system was something to blame, to vent anger on; at the same time it was described as an inevitability that had to be obeyed and against which protection was required.• the system is wrong, the sick funds and all, why can't I get procedures done that are necessary for me (3)• you sign up there [for a rheumatologist], you wait and wait, a lot changes in that time; you wait a month and a half; you get there; by that time the drugs have run out, later you're at fault for not having taken them (2)• I went to a private clinic, almost like crying at the door, there are no vacancies, there's no one I could talk to, and I didn't. I went away to the country/.../now I'm here and now I'm told that this Achilles' has been broken for at least a month (10)• [left alone with the disease] just can't be such a thing, not alone, but life is like this, don't know what to wish (2)• helplessness, no person to protect me [against the system] (3)non grata turns in the course of the disease. One of the respondents regarded physicians as co-victims of the system, incapable of defending their patients.Physicians, some of them named as saviors and supporters, were still more often seen as being an impersonal part of the system. Professional incompetence and superficiality were connected with • if I had been sent to the rheumatologist right away then maybe things wouldn't have gotten this bad; thus, when my neck was stiff at first, it was thought I had caught a cold and it would pass (1)• the troubles started when my leg trauma was labelled as radiculitis (2)• initially no action was taken and consequently the general attitude [of the doctors] became such that nobody had the guts to do anything with me, and because it was such a awkward situation because they didn't have the right doctor who would have done something, I was left a bit high and dry (10)• the kidney doctor said: not to come to me, I am out of money, go to the GP /.../you go to the GP, she can't help you, prescribes what she can, she can't prescribe rheuma-drugs (2)The ability to do necessary shopping is directly assessed by item number 6: I find it difficult to walk to the shops. Difficulties with walking to the shop were described by our respondents and the item was considered to be important. But other problems connected with shopping emerged even more often in the interviews – managing in the shop, checking out, and carrying the purchases.• I don't want to stand in a line, my feet start to ache (7)• [shopping trolley] is very big, you have to push it hard and it's narrow there [in the shop] (9)• checking out at the counter you're in a hurry and then comes this psychological moment that you get nervous and can't handle it (7)• I open my wallet, they take what they need (5)• when the bag is too heavy then my feet can't stand it; you have calculate how much you buy (6)• sometimes you would like to buy more but you can't carry it; I just put stuff that I really need in the cart (9)For our interviewees, a topic closely related to shopping was the capability to use public transport. The obstacles encountered when entering, leaving and riding a bus were described.• I can't get onto the bus, even worse with the tram; the steps are very steep, I can't manage however I try (3)• I can't get off, I ride to the next stop; the bus doesn't pull over to the sidewalk but stops further away (6)• very bad to ride, I just stagger, lose my balance, sometimes you can fall quite badly; the worst is when you have to stand (9)• sitting down and getting up [on bus] is difficult for me, it's better I hold on to something for some time (5)The lack of money was mentioned by all of our respondents. Its interference with the satisfaction of the majority of needs was described.• I wouldn't say my spending has increased a lot, but my income is, yes, notably lower; before I even paid more income tax than I get paid now; the habits I had before have become impossible for me, financially (1)• a handicapped person could also make his/her life comfortable and tolerate everything if it was possible financially (6)• the pension is so tiny, I can't do anything with this; I can't manage a family with this (7)Our results showed the high significance of the majority of the RAQol items for the interviewees. This allows us to state that the RAQol can successfully be adapted into Estonian for usage in international research projects. Our results also highlight the difficulties in translating some specific items (number 9 and 28), which should be reckoned with during the adaptation process.Three Qol aspects that were important for Estonian RA patients but were not evaluated by the RAQol – the issues concerning changes in role performance, safety and stability of communication with the medical system, as well as some issues of procuring the necessities of life-were revealed by the analysis of interviews.Next we would like to discuss the reasons for these peculiarities and suggest some additional resources for in-depth reading.homo sovieticus mentality was the placing of collective interests above personal ones; individuals were appreciated by their contribution to the common good. Successful performance in social roles approved by the regime was honored; inability to meet validated ideals was considered shameful. Although we can talk of a dramatic change of approbated values and ideals after the end of the Soviet era – an independent, successful, competitive young individual is idealized now –, but the tendency to disapprove of the inability to fit expectations has remained.Only 13 years have elapsed since the end of Soviet rule in Estonia. Our respondents spent a considerable part of their lifetimes in the Soviet ideological environment and this has undoubtedly influenced their values and beliefs. An obligatory component of the We believe that this potpourri of old and new values and attitudes could explain the high significance of themes connected with roles and role functioning in our respondents' conversations. A person disabled because of disease could not perform successfully in acknowledged Soviet-time roles ; the same is true for present-day Estonia. Being categorized as "socially uncompetitive" would alter the identity and self-esteem of the disabled individuals and concealment of the condition could be seen as defense reaction. In the light of this, topics connected with role functioning and social acceptability should be included among those observed during a Qol investigation. See also -21.The transition from one economic and ideological system to another in Estonia has caused social instability, which is reflected in an increase in people's subjective sense of insecurity and fear. The most insecure time for the Estonian population was in the early 90ties; since that time, due to stabilization of the economy and economic development, a sense of security is returning. Still, there is remarkable disaffection with different spheres of public administration; future-oriented reorganizations that do not provide immediate benefits are treated with caution. The health care system, which has undergone fundamental reforms in the post-soviet period, is the favored object of criticism for the Estonian media. In the Soviet era health care was funded from the state budget and all citizens had free access to health services. Today's health care delivery system in Estonia is financed through health insurance; the private sector is growing. The introduction of family practitioners, with novel financing principles and responsibilities, in the mid 1990ties has changed previously existing relations between patients and medical specialists; access to consultants has lost its immediacy. Bureaucracy, long waiting lists, visit charges (something unthinkable for Soviet medicine) and open discussion of the financial difficulties of the health service in the Estonian media have all made their contributions to lowering confidence in the health care system in the eyes of our patients. Communication with health care deliverers constitutes a significant part of everyday life for RA patients. Therefore the impact of this communication on the everyday life of patients cannot not be ignored. In the case of a transitional society like Estonia's, the effect of health care delivery on patients' needs for stability and safety should be considered. See also -26.In health care planning, the strategic military interests of the Soviet Union were given priority; this resulted in the development of an excessive large hospital network. Habilitation and rehabilitation were upstaged and drastically under-funded. In the Soviet era the people with special needs were partly institutionalized as disabled; their problems and even their existence (with the exception of war veterans) were simply hushed up by the authorities and media. There were no adaptations for people with physical special needs in public places and in the physical environment of towns in Soviet Estonia; elementary facilities were not accessible. People in wheelchairs began to be seen on our street with arrival of western tourists in early 90ties; they were joined shortly afterwards by our own special needs persons. During the following years adaptations considering the requirements of people with special needs have little by little been introduced into the physical environment. However, due to limited resources the improvements are coming slowly and conditions have not reached the level where the comfort of special needs persons can be guaranteed. Coping with errands and chores should be taken into account as a distinct Qol topic in Estonia today. See also . the average monthly pension for the disabled in Estonia in 2003 was 1110 Estonian kroons (approximately 71 EUR). This constituted one sixth of average monthly gross wages and salaries, and was 301 kroons (19 EUR) lower than estimated minimum means of subsistence for 30 days in the same period. From these figures we can see that financial troubles can overshadow every aspect of the everyday life of our patients.According to the Statistical Office of Estonia In our opinion adding the assessment of these three aspects – changes in role performance, safety and stability of communication with the medical system, and some aspects of procuring the necessities of to life – should be considered by Estonian researchers, especially when carrying out disease-specific Qol studies at the national level.In our research we used qualitative interviews for assessing the appropriateness of a RA-specific Qol instrument, the RAQol, for adaptation for use in Estonia. We described the importance of the items to our patients and identified Qol topics that were significant for our respondents but that were not assessed by the questionnaire. We also discussed the nature of the discrepancies in significance of Qol topics for Estonian patients.Our results show that the utilization of a qualitative study as an introductory part of Qol assessment instrument adaptation provides possibilities for more thoroughly considered Qol research. By evaluating the significance of items in the particular context, it allows the mechanical acceptance of instruments just because they have performed well in other societies and cultures to be avoided. Moreover, it offers an opportunity to identify topics that are not included in the instrument but are important for local interpretation of Qol, which are otherwise often overlooked. For researchers, qualitative studies offer a deeper understanding of the instrument in question and of the research topic – Qol of the patients.The data collected during this qualitative research process also has the potential to be used for a wider analysis of the Qol of Estonian RA patients. Still, our current interests were centered on the appropriateness of the adaptation of a particular Qol instrument, and therefore the use of the gathered data was quite limited. But we believe that the knowledge gained will be beneficial for our forthcoming research projects.MT: the idea and conceptual construction of the research, recruitment of the participants and conduction of the interviews, coordination of the coding and analysis processesJS: methodological construction and supervision of the research, active participation in the coding and analysis processesKM and EH: participation in discussions and decision-making throughout the whole course of the researchAll authors have read and approved the final manuscript.


Immune deficiency, Centromeric region instability, and Facial anomalies syndrome). In normal cells the inactive X has characteristic silencing type histone modification patterns and the CpG islands of genes subject to X inactivation are hypermethylated. In ICF cells, however, genes subject to X inactivation are hypomethylated on the inactive X due to mutations in the DNA methyltransferase (DNMT3B) genes. Therefore, if DNA methylation is upstream of histone modification, the histones on the inactive X in ICF cells should not be modified to a silent form. In addition, we determined whether a specific methyl-CpG binding protein, MeCP2, is necessary for the inactive X histone modification pattern by studying Rett syndrome cells which are deficient in MeCP2 function.In mammals, there is evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with modification complexes that can deacetylate and/or methylate nucleosomes in the proximity of methylated DNA. We examined this idea for the X chromosome by studying histone modifications on the X chromosome in normal cells and in cells from patients with ICF syndrome (We show here that the inactive X in ICF cells, which appears to be hypomethylated at all CpG islands, exhibits normal histone modification patterns. In addition, in Rett cells with no functional MeCP2 methyl-CpG binding protein, the inactive X also exhibits normal histone modification patterns.These data suggest that DNA methylation and the associated methyl-DNA binding proteins may not play a critical role in determining histone modification patterns on the mammalian inactive X chromosome at the sites analyzed. Although it has been known for some time that histone modifications play a role in gene expression , it is oNeurospora, histone methylation appears to determine DNA methylation patterns [Arabidopsis, non-CpG DNA methylation also appears to be determined by histone methyltransferases, whereas CpG methylation does not [MECP2 mutations exhibit histone hyperacetylation [Mecp2 mutations [In patterns ,4. In Ardoes not ,6. In madoes not -11. Mutadoes not , supporttylation . Histoneutations . Thus, Dutations .We are especially interested in the X chromosome with respect to the question of the relationship between DNA methylation and histone modification. The mammalian X chromosome is unusual in that about a thousand gene-associated CpG islands are hypermethylated on the inactive X and hypomethylated on the active X. Except for imprinted loci, methylation patterns at most other regions of the genome are similar between homologs. Histone modification differences known to be associated with either silent or expressed chromatin also distinguish the active and inactive X chromosomes -19. ThusImmune deficiency, Centromeric region instability, and Facial anomalies". In most cases, the molecular defects result from mutations in the DNMT3B methyltransferase gene [MECP2. This suggests that this specific methyl-DNA binding protein does not have a major role in silencing the inactive X through histone modification.To examine more fully the possible relationships between DNA methylation and histone modification, we have utilized cell cultures from individuals with a human hypomethylation disease called the ICF syndrome. This disease is clinically characterized by "ase gene -22. Certase gene  and LINEase gene . If DNA We examined histone modifications known to be associated with the inactive X chromosome in two ICF cell lines and normal control cells. Specific histone modifications including histone H3 and H4 acetylation, and histone H3 methylation at K4 and K9, were examined using antibodies to stain nuclei and metaphases -17. We aThe expected patterns of macroH2A1 histone concentration and histone modification on the inactive X were found in cells derived from ICF individuals and in control normal cells  on the inactive X, and in the second case, we had one clone with the wild type MECP2 on the inactive X (1789V) [We examined histone modifications and macrohistone association in clones from two individuals heterozygous for a mutation in tivation . MECP2 mtylation ,13, as w (1789V) . For allG6PD, NEMO, MECP2, SYBL1, AR, FMR1, and PGK1) in ICF cells so that we could employ allele-specific chromatin immunoprecipitation (ChIP) analysis. We restricted our search for polymorphisms to the promoter region, as several reports have indicated that marked differences in histone modifications between active and inactive alleles are seldom detected at other regions [SYBL1  and AR (androgen receptor in Xq12).To verify our cytological histone modification results at the gene level, we searched for promoter polymorphisms at seven X-linked loci  was informative in one ICF sample (PT 4) and in several controls, thus permitting separation of the active and inactive alleles in cloned cells, and in cultures with highly skewed X inactivation. The antibodies were highly specific under the amplification conditions chosen, so that a strong signal was seen for the pull-down experiment with antibody and little or no signal for the "no antibody" control  has reported on a ChIP study at the ive gene . Here wethylated . In the rol Fig. .AR gene were then separated on an automated sequencer according to CAG repeat number. Two major peaks are detected in the input control DNA, corresponding to the active (A) and inactive (I) X alleles, differing in CAG repeat number  also showed normal histone H3 hypoacetylation and K4 H3 hypomethylation in spite of the very low levels of DNA methylation in this CpG island region  is the sensitivity of the cytological histone modification assay too low to detect active-type histone modifications on the ICF inactive X? and (2) is the extent of methylation on the ICF inactive X greater than is suggested by CpG island and LINE-1 methylation patterns?AR, in ICF cells ([AR gene. For genes that escape X inactivation in ICF cells, we expect their histone modification patterns to be those of expressed genes, and one of us (RSH) has previously reported this to be the case for the SYBL1 gene  and harvested in trypsin:EDTA (Gibco Invitrogen Corp.) under standard conditions ,48.For analysis of interphase stages, cells were plated on alcohol-washed 22 mm square cover slips in 35 × 10 mm Petri dishes. On the following day the medium was removed and the cells were washed once with PBS followed by fixation in 95% ethanol:5% acetic acid for 1 min at room temperature. The rest of the procedure followed the "Immunocytochemistry Protocol" of Upstate (Lake Placid, NY). DAPI-stained slides mounted in antifade were examined with a Nikon Microphot FXA microscope and images were captured with a Nikon Coolpix 995 digital camera. The inactive X was recognized under DAPI staining as sex chromatin. Absence of a particular histone modification on the inactive X was seen as a hole or gap at the sex chromatin location.For analysis of metaphase cells, we plated cells in 150 × 25 mm tissue culture dishes and added colcemid (Gibco Invitrogen Corp.) 48 h later (0.1 μg/ml for 2 h). The medium was removed and the cells were washed once with Hanks' balanced salt solution, followed by trypsinization (Gibco Invitrogen Corp.) with slight agitation to collect metaphase cells. Serum was added to stop tryptic action and the cells were recovered by centrifugation, then placed in hypotonic solution (3 mg/ml KCl and 1 mg/ml sodium citrate) at 37°C for 15 min. Cells were collected on to slides using a Cytospin centrifuge and then fixed in 95% ethanol:5% acetic acid for 1 min. The rest of the procedure followed the Upstate protocol mentioned above, followed by DAPI staining, mounting in antifade, and examination with a Nikon Microphot FXA microscope.Antibodies used to detect histone modifications were obtained from Upstate and included: "Anti-acetyl-Histone H4," recognizing acetylated lysines 5, 8, 12, and 16, "Anti-acetyl-Histone H3 (Lys 9)," "Anti-dimethyl-Histone H3 (Lys 9)," "Anti-dimethyl-Histone H3 (Lys 4)," and "Anti-Histone macroH2A1."2 tissue culture flask (about 3 × 106 cells) was washed with PBS and treated with 4% formaldehyde (pH > 7) for 10 min at 37°C. Protease inhibitor cocktail (Complete) from Roche Diagnostics (Indianapolis, IN) was used in place of individual inhibitors, and Protein A-Separose 4B  was used to collect immune complexes. After elution of immune complexes, they were heated at 65°C for 4 h to reverse crosslinks, and the DNA was recovered with a "QIAquick" PCR purification kit from Qiagen Inc. .Chromatin immunoprecipitation was performed using the protocol of Upstate with slight modifications. For each experiment, a near-confluent 75 cmAR DNA was across the polymorphic 5' CAG repeat region as previously described [Primary amplification of escribed , using 2escribed , using 6escribed .SYBL1 gene was performed similarly to that of AR, except the allele-specific reaction entailed XhoI digestion of the primary amplification product followed by these products being separated by agarose gel electrophoresis. Conditions for PCR amplification and XhoI digestion were as previously described [Analysis of the 5' region of the escribed .ac = acetylatedChIP = chromatin immune precipitationDAPI = 4,6-diamidino-2-phenylidoleFB = fibroblastFAM = 5-carboxyfluoresceinFITC = fluorescein-isothiocyanateICF = immune deficiency, centromeric region instability, facial anomaliesH3 = histone 3H4 = histone 4K4 = lysine 4K9 = lysine 9LINE-1 = long interspersed nuclear element 1MeCP2 = methyl-CpG binding proteinWT = wild typeXa = active X chromosomeXi = inactive X chromosomeSMG and RSH conceived the study design, supervised and coordinated its progress, and drafted and prepared the final manuscript. KRV and PL carried out the cell culture and cytological studies. TKC carried out the ChIP analyses. JT and UF developed the cloned Rett cell cultures. All authors read and approved the manuscript.


It is a major challenge of computational biology to provide a comprehensive functional classification of all known proteins. Most existing methods seek recurrent patterns in known proteins based on manually-validated alignments of known protein families. Such methods can achieve high sensitivity, but are limited by the necessary manual labor. This makes our current view of the protein world incomplete and biased. This paper concerns ProtoNet, a automatic unsupervised global clustering system that generates a hierarchical tree of over 1,000,000 proteins, based solely on sequence similarity.In this paper we show that ProtoNet correctly captures functional and structural aspects of the protein world. Furthermore, a novel feature is an automatic procedure that reduces the tree to 12% its original size. This procedure utilizes only parameters intrinsic to the clustering process. Despite the substantial reduction in size, the system's predictive power concerning biological functions is hardly affected. We then carry out an automatic comparison with existing functional protein annotations. Consequently, 78% of the clusters in the compressed tree (5,300 clusters) get assigned a biological function with a high confidence. The clustering and compression processes are unsupervised, and robust.We present an automatically generated unbiased method that provides a hierarchical classification of all currently known proteins. The explosive growth in the number of sequenced proteins has created a glut of proteins that are sequenced but whose structure and function are as yet unknown. A common way to tackle this problem is to use database searches to find proteins similar to a newly discovered protein, thus inferring protein function. This method is generalized by protein clustering or classification where databases of proteins are organized into groups or families in a manner that attempts to capture protein similarity. Such classification into families is a critical component in structural and functional genomics -4. The nClassifying the entire protein space into families serves not only as a method for large-scale protein annotations but also to support functional and structural genomic initiatives . Some pr. For the SwissProt-based tree, there are 227,436 clusters (including the proteins as singletons). The classification provided by ProtoNet provides the full range of cluster granularity: from single proteins to huge protein clusters that carry no biological relevance (the root clusters). We test the biological validity of ProtoNet, by its examination from different perspectives, using external-defined protein keyword annotations. Four different annotation sources are used  in ordeAs mentioned, ProtoNet contains 227,436 clusters, which is obviously much more than the upper estimate of 30,000 protein families ,26. TherIn this paper we describe methods for the unsupervised compression of the ProtoNet tree, by using intrinsic tree-based parameters of the clusters that correlate well with biological validity. By preserving the unsupervised nature of the ProtoNet data, we prevent biasing towards previously discovered findings and better allow for future generalizations, in addition to maintaining the automation of the process.Finally, automatic functional annotation to proteins is of great importance. In ProtoNet, an automatic method for assignment of biological annotation to the protein clusters is used, yielding high-confidence functional assignments for a large majority of the proteins' clusters.In order to measure the correspondence between a given cluster and a specific annotation, and allow for supervised validation of the ProtoNet clusters, we define the notion of a correspondence score (CS). The CS for a certain cluster and a given keyword measures the correlation between the cluster and the keyword, using the well-known intersect-union ratio.c be the set of annotated proteins in cluster C; Let k be the set of proteins in the system annotated by keyword K; We define:Let C be a cluster in the ProtoNet tree, and K be a keyword (from a specific source) that annotates (some of) the proteins in the system; Let best cluster). The score for a given cluster on keyword K ranges from 0 (no correspondence) to 1 .The cluster receiving the maximal score for keyword K is considered the cluster that best represents K within the ProtoNet tree , GO (Molecular Function) and ENZYME (subclass, sub-subclass and entry). The results Table  show a h-100). Note that even for the SCOP fold level, which is associated with proteins that may be extremely remote in sequence, ProtoNet's relative success is extremely high .To avoid trivial correspondences between a keyword and a cluster, such as the assignment of a keyword that annotates only one protein to its singleton cluster, we tested our success only with keywords that annotated at least two proteins (for SCOP and ENZYME keywords). For InterPro and GO, we selected a threshold of 20 proteins per keyword, as the majority  of the annotations is included above this threshold, thus allowing the test to focus on the more significant keywords.In order to validate the hierarchical structure of ProtoNet, we compare it with the hierarchical structure of GO as described in Figure A total of 1577 GO terms were selected as described, with 1798 edges between them according to the GO hierarchy. 771 out of 1291 (60%) edges that were produced by the ProtoNet hierarchy appear in the GO hierarchy. This number is highly significant considering the fact that there exist over 1,200,000 possible edges between the 1577 vertices in the graph (considering it as a DAG). It should be noted that there are some terms in GO that are connected to many other vertices. These vertices may bias the results of this test. In order to confirm that ProtoNet performs well without these vertices as well, the vertices were removed manually and the test was repeated, with similarly significant results (33% of the edges were correct).In order to allow unsupervised automatic compression of the ProtoNet tree, we searched for an intrinsic parameter of the clustering process that would specify clusters of biological validity. By applying such a parameter one could dispose of clusters that do not pass a certain threshold value, remaining with clusters of high biological validity. Once we remain with a subset of biologically valid clusters, the new hierarchy amongst them can be reconstructed according to the original tree hierarchy.The agglomerative hierarchical clustering scheme defines a set of terms that are intrinsically associated with the process. In such a scheme, each cluster is created from smaller clusters, which are captured as its descendants in the clustering tree. The ProtoLevel (PL) ranges from 0-100 and is used as a standard quantitative measure of the relative height of a cluster in the merging tree. The PL of a cluster is defined as the arithmetic average of the BLAST E-score of the pairs of its proteins. The PL of the leaves of the tree is defined as 0, whereas the PL of a root equals 100. The larger the PL, the later the merging that created the cluster took place. Therefore, the PL scale is considered as an internal monotonic timer of merging, during the clustering process. As mentioned above, a cluster is said to be created when the merging of its two children clusters forms it. The PL at this point is said to be the birthtime of this cluster. The deathtime of a cluster is defined as the PL at its termination, i.e. the point at which it merges into its parent cluster (or 100 if it has no parent). The lifetime (LT) of a cluster is defined as:LT = deathtime - birthtimeTherefore, the LT of a cluster reflects its remoteness from the clusters in its "vicinity" in protein sequence space.We examined the LT distribution of the set of InterPro best clusters in comparison with the LT distribution of all clusters in ProtoNet Figure . The resIn order to search for a reasonable LT threshold value , several threshold values were examined Figure . The resThe mean best CS was examined for all four external sources Table . The resThe following scheme was used to annotate the protein clusters: For each cluster C and keyword K we define the following score:TP is the amount of true positives (proteins in C that have the keyword K), FN is the amount of false negatives (proteins not in C that have the keyword K) and FP is the amount of false positives (proteins in C that do not have the keyword K).Where For each cluster, we search against all keywords of GO and InterPro for the keyword with the highest AS. If the AS of the cluster is greater than 0.25, the cluster is assigned that keyword as its annotation. The logic behind the score and the threshold is as follows: the score is determined by two parameters, the specificity and the sensitivity; let us consider the two worst-case limit cases. In the first case, specificity>0.5 and sensitivity = 1: a majority of the proteins of the cluster share the keyword, and there exist no other known proteins that have the keyword. In the second case, specificity = 1 and sensitivity>0.25: all proteins of the cluster share the keyword and they constitute more than 1/4 of the total proteins known to have this keyword. In both cases, the keyword can be assigned to the protein cluster with a high degree of confidence. All other clusters fall in between these cases.Using this method, all 6,879 clusters that contain 20 or more proteins and that remain after the compression were tested. 5,355 (77.8%) clusters passed the high confidence threshold and were therefore given an annotation. Figure Figure The challenge of protein classification by using sequence similarity has been addressed extensively by several different methods. In order to assign function to proteins, advanced methods (such as Hidden Markov Models implemented in Pfam) have been used to learn sequence-based patterns on "seeds", manually validated alignments of known protein families. The widely-used BLAST algorithm is considered to be a reliable tool for sequence alignment, but has been shown to lack sensitivity for weak similarities that may be detected by signature detection methods. We show here that by using an unsupervised bottom-up clustering method based on BLAST E-values, we have been able to construct a global hierarchy of the SwissProt proteins that can be validated by external biological sources, merely by undertaking a global view of the protein space.The four different external sources that were used for validation reflect different aspects of the protein space: InterPro annotation is predictive and is based on various signature detection methods; GO annotation assignments are both based on prediction and from known research, while the GO hierarchy was constructed completely manually; SCOP is a semi-manual classification of structures that is not necessarily reflected in sequence; the ENZYME database supplies Enzyme Commissions, which constitute a hierarchy that is based on the enzymes' chemical reactions. ProtoNet successfully constructs clusters that correspond highly to all four of the sources. Even high levels of SCOP (such as the Fold classification), which are considered to have no detectable sequence similarity, are partially matched . NotablAfter validating the biological relevance of the ProtoNet clusters by using external sources, we examined the hierarchy of ProtoNet. The test showed that the hierarchy presented by ProtoNet significantly corresponds to the manually-constructed biological hierarchy of GO. It is important to note that the method used by ProtoNet is not expected to fully recapture the GO hierarchy due to the fact that ProtoNet is structured as a collection of trees while GO is structured as a DAG. In this sense, the approach of ProtoNet may be limited in the portrayal of evolutionary complexity (as in cases of multiple domains). However by using a domain-based clustering approach, allowing multiple entities of each protein in the hierarchy, a substantial improvement in the CS quality measure may be achieved (unpublished results).An intrinsic parameter that reflects the stability of clusters during the clustering process was used in order to compress the cluster sets, leaving 16.4% of the clusters; removing the singletons clusters as well leaves 12.2% of the clusters. As mentioned above (see Methods), the entire ProtoNet scaffold contains 227,436 clusters that are represented by 630 roots; following this condensation, there are only 27,823 clusters that are represented by 2,236 roots. We show that although a massive portion of the clusters is discarded, very little performance is lost by this condensation process. It is obvious that prior to the condensation process, ProtoNet holds within it both clusters that correctly represent biological groups and clusters that are irrelevant artifacts of the clustering process (e.g. the large root clusters that are constituted of tens of thousands of proteins). Therefore, by allowing a major reduction without significant loss of biological coherence ProtoNet seems to present a more correct view of the protein world.An automatic unsupervised method for the classification of proteins has some important advantages over supervised methods : First, an unsupervised method is unbiased in automatic assignment of function to proteins, a major goal in bioinformatics. Also, it allows high-throughput analysis of whole genomes and enhances understanding of global biological systems without the need for the manual annotation of every protein. Using an automatic annotation method, we are able to successfully annotate 77.8% of the major protein clusters (of size 20 or more) that remain after the compression of the ProtoNet tree. The annotation uses a relatively conservative threshold and therefore yields high-confidence annotations. This further suggests that the clusters remaining after the condensation process are relevant biological clusters and not mere artifacts.One aspect that we have rigorously examined is the robustness of the ProtoNet tree: given a different set of proteins to cluster or a different clustering method, would the resulting tree be significantly different, or are its properties maintained? Interestingly, changing the underlying protein databases (ranging in size from 30,000 to over 1,000,000 proteins), the substitution matrices used for the preliminary BLAST, or the merging strategy  producedProtoNet version 2.4 which was used for the analyses described in this paper is based on classification of the SwissProt database (version 40.28) that contains 114,033 proteins. The entire ProtoNet scaffold contains 227,436 clusters that are contained in 630 trees. Most trees (611) are singletons and only one contains most (>99%) of the proteins. For more details on the construction of the ProtoNet hierarchy see . ProtoNeSeveral external sources were used as a biological reference for validation of the ProtoNet tree: InterPro  is an exWe have used EBI mappings of InterPro and GO to SwissProt proteins.Annotation Score (AS), Correspondence Score (CS), Directed Acyclic Graph (DAG), Enzyme Commission (EC), Gene Ontology (GO), Lifetime (LT).ML conceived of the compression of the ProtoNet tree. NK, MF and MF participated in implementation of the condensation and in the design and implementation of the various tests. All authors participated in the analysis of the results. All authors read and approved the final manuscript.