Until recently the most accepted technique of doing root canal treatment stresses multiple visit procedure. Most schools also concentrated upon teaching the multi-visit concept. However, it has now been reported that the procedure of single visit treatment is advocated by at least 70% of schools in all geographical areas. It was therefore the aims of the present study to find the incidence of post-obturation flare-ups following single and multiple visit endodontic treatment procedures, and to establish the relationship between pre-operative and post-obturation pain in patients referred for endodontic therapy in a Nigerian teaching Hospital.st, 7th and 30th day. The presence or absence of pain, or the appropriate degree of pain was recorded for each recall visits and the interval between visits. The compiled data were analysed using chi-square where applicable. P level ≤ 0.05 was taken as significant.Data collected included pulp vitality status, the presence or absence of pre-operative, inter-appointment and post-obturation pain. Pain was recorded as none, slight, or moderate/severe. Flare-ups were defined as either patient's report of pain not controlled with over the counter medication or as increasing swelling. The patients were recalled at three specific post-obturation periods, 1Ten endodontic flare-ups (8.1%) were recorded in the multiple visit group compared to 19 (18.3%) flare-ups for the single visit group, P = 0.02. For both single and multiple visit procedures, there were statistically significant correlations between pre-operative and post-obturation pain (P = 0.002 and P = 0.0004 respectively). Teeth with vital pulps reported the lowest frequency of post-obturation pain (48.8%), while those with nonvital pulps were found to have the highest frequency of post-obturation pain (50.3%), P = 0.9.The present study reported higher incidences of post-obturation pain and flare-ups following the single visit procedures. However, single visit endodontic therapy has been shown to be a safe and effective alternative to multiple visit treatment, especially in communities where patients default after the first appointment at which pain is relieved. Until recently the most accepted technique of doing endodontic treatment stresses multiple visit procedures. Most schools also concentrated upon teaching the multi-visit concept. However, it has now been reported that the procedure of single visit treatment is advocated by at least 70% of schools in all geographical areas .Some of the problems of root canal treatment are post-obturation pain, inter-appointment pain and swelling. Although these in most cases do not last long, but could be a source of embarrassment to the dentist and annoying for the patient, more so if the tooth was symptomless before the commencement of treatment. Literature review revealed varied opinions on the incidence and severity of post-obturation pain. Some authors reported slightly more post-obturation pain following single visit than with multiple visit procedures ,3. Otherst, 7th, and 30th post-obturation days, and to compare these results with those reported in previous studies.Endodontic treatment in Nigeria is carried out in the department of restorative dentistry of the four dental schools and in few private dental clinics located in major cities, health centres and general hospitals. Previous report revealed that few cases of root canal treatment were undertaken and that root canal treatment was completed in multiple visits, specifically three visits for about half the teeth treated . ReasonsConsenting patients referred to the department of Restorative Dentistry for root canal therapy within a period of twelve months were randomly assigned for either single visit or multiple visit procedures. For the multiple visit procedures, Patients that defaulted after the first appointment (incomplete treatment) were excluded from the study. For each tooth treated, the clinical factors and conditions existing before, during and after the completion of treatment were recorded. This data included pulp vitality status, the presence or absence of pre-operative pain, post-obturation flare-ups and degree of post-obturation pain. For patients requiring root canal treatment on more than one tooth, the treatment of each tooth was separated by a period of at least four weeks to allow for proper evaluation. The pulp vitality was determined by an electric pulp tester  in combination with the presence of pulpal haemorrhage.st, 7th and 30th day. At each post-obturation recall visit, the patients were interviewed to determine whether or not there were symptoms at the present visit and whether or not there had been symptoms during the interval between the present visit and the previous one. The presence or absence of pain, or the appropriate degree of pain was recorded for each recall visit and the interval between visits. Pain was recorded as none, slight, or moderate/severe. Slight pain was defined as any discomfort no mater how brief in duration that did not require medication and that did not impair masticatory function in any way. Moderate/severe pain was defined as pain requiring medication or other palliative treatment. Impairment of masticatory function (discomfort in chewing) was recorded as moderate/severe pain. Endodontic flare-ups were defined as either patient's report of pain not controlled with over the counter medication and or increasing swelling.The patients were recalled at three specific post-obturation periods, the 1The root canals were obturated with multiple gutta-percha cones and a zinc oxide-eugenol based sealer, using the lateral condensation technique. The compiled data were analysed using chi-square where applicable. P level ≤ 0.05 was taken as significant.st post-obturation day, of these 107 were completed in single visit and 136 were completed in multiple visit. Eighty-six (86) had vital pulps and 157 had nonvital pulp canal contents. Two hundred and twenty seven (227) reported for check-up on the 7th post-obturation day, of these 104 was completed in single visit and 123 completed in multiple visit. Two hundred and twenty two reported for check-up on the 30th post-obturation day, 102 completed in single visit and 120 in multiple visit.Two hundred and eighty three (283) teeth in 255 patients were treated in all, given a ratio of 1.11 teeth per patient. Of these 56 were excluded from the study due to non-availability of patients at post-obturation recall visit. These exclusions were randomly distributed between treatment groups, with no differential loss to follow-up (25 from the single visit group, 31 from the multiple visit group). The treatment groups were fairly comparable, with similar distribution of tooth types between treatment groups, Table 2 = 9.04, p = 0.002). For the multiple visit procedures, 88 teeth presented with pre-operative pain out of which 55 (62.5%) reported post-obturation pain. 48 teeth had no pre-operative pain out of which 6 (12.5%) had post-obturation pain (x2 = 12.5, p = 0.0004). These show that for both single and multiple visit procedures, there were statistically significant correlations between pre-operative and post-obturation pain , that is patients presenting with pain not controlled by over the counter medication and or increasing swelling, were recorded in the multiple visit group compared to 19 18.3%) flare-ups for the single visit group. This shows a significant difference (Mantel Haenszel chi-square = 5.18, p = 0.02), Table % flare-uTeeth with vital pulps reported the lowest frequency of pain 48.8%), while those with nonvital pulps were found to have the highest frequency of pain (50.3%), Table 8.8%, whist and 7th days respectively 35.5% and 16.3% were higher than those in the multiple visit group 30.2% and 9.8%. Chi square test indicated no statistically significant differences  whose pulps were non-vital.Many authorities in the field of endodontics advice against the completion of root canal treatment in single visit in order to prevent post-obturation pain, especially in cases presenting with pre-operative pain ,12.In the present study more flare ups occurred in the single visit group (18.3%) than in the multiple visit group (8.1%), showing a disadvantage for single visit treatment at a 95% confidence level, Table . This isPrevious studies have shown that there is a strong positive correlation between pre-operative and post-obturation pain ,14,15. Tth day in both groups, these present a strong indication that practitioners should not overreact to early post-obturation symptoms by immediately initiating endodontic retreatment procedures or extraction of the involved tooth. Apart from the reported higher incidence of flare-ups in the single visit group, the post-obturation pain experienced by the patients in both groups compares favourably well with each other. Therefore the higher incidence should not be taken as condemnation for the single visit endodontic therapy, it should however stress the fact that a thorough understanding of the basic endodontic principles is important in considering each case on an individual basis before making a decision as to whether or not it can be completed in one visit [Despite the high percentages of post-obturation pain reported on the first post-obturation day in both groups Table , seven dne visit . As it iThe present study reported higher incidences of post-obturation pain and flare-ups following the single visit procedures. However, single visit endodontic therapy has been shown to be a safe and effective alternative to multiple visit treatment, especially in communities where patients default after the first appointment at which pain is relieved.The author(s) declare that they have no competing interests.AOO conceived of the study, participated in its design, performed the statistics, and participated in the final write-up of the manuscript. CIU participated in the design, collected the data, drafted the initial manuscript, and participated in the final write-up of the manuscript.The pre-publication history for this paper can be accessed here:


Although percutaneous coronary intervention (PCI) is becoming the standard therapy in ST-segment elevation myocardial infarction (STEMI), to date most patients, even in developed countries, are reperfused with intravenous thrombolysis or do not receive a reperfusion therapy at all. In the post-lysis period these patients are at high risk for recurrent ischemic events. Early identification of these patients is mandatory as this subgroup could possibly benefit from an angioplasty of the infarct-related artery.Since viability seems to be related to ischemic adverse events, we initiated a clinical trial to investigate the benefits of PCI with stenting of the infarct-related artery in patients with viability detected early after acute myocardial infarction.The VIAMI-study is designed as a prospective, multicenter, randomized, controlled clinical trial. Patients who are hospitalized with an acute myocardial infarction and who did not have primary or rescue PCI, undergo viability testing by low-dose dobutamine echocardiography (LDDE) within 3 days of admission. Consequently, patients with demonstrated viability are randomized to an invasive or conservative strategy. In the invasive strategy patients undergo coronary angiography with the intention to perform PCI with stenting of the infarct-related coronary artery and concomitant use of abciximab. In the conservative group an ischemia-guided approach is adopted .The primary end point is the composite of death from any cause, reinfarction and unstable angina during a follow-up period of three years.The primary objective of the VIAMI-trial is to demonstrate that angioplasty of the infarct-related coronary artery with stenting and concomitant use of abciximab results in a clinically important risk reduction of future cardiac events in patients with viability in the infarct-area, detected early after myocardial infarction. Management of acute myocardial infarction (AMI) has underwent considerable changes in the last two decades and the management of patients with AMI has become more established ,2. In paData from large clinical trials indicate that after successful thrombolysis more than 50% of patients have a significant residual stenosis and about 30% of patients have spontaneous or inducible ischemia -9. In thTrials have been performed to study the effect of routine angioplasty on clinical outcome early after AMI treated with thrombolysis -20. MostTo date, non-invasive risk stratification after AMI has mainly focused on exercise stress testing. The inability to exercise, the low diagnostic accuracy and resting ECG abnormalities, however, remain important limitations in the detection of ischemia .Several post-infarction observational studies investigated viability as prognosticator and showed that the presence of viability in the infarct area was highly predictive for future coronary events like recurrent ischemia, recurrent infarction, left ventricular failure, and death -31. ViabIn this context it should be noted that other post-infarction studies have shown viability to be associated with an improved prognosis -36. Two Based on the aforementioned assumptions, we initiated a clinical trial to investigate the benefits of percutaneous coronary intervention (PCI) of the infarct-related artery in patients with viability detected in the early, subacute phase of myocardial infarction. To demonstrate viability, we use low-dose dobutamine echocardiography (LDDE). This test can safely be performed 48 hours after acute MI ,27,30,31We hypothesize that, in order to prevent future cardiac events, PCI is only useful in patients with viability in the infarct zone early after myocardial infarction.Patients admitted to any of the participating centers with an acute or recent myocardial infarction, who are not treated by direct or rescue angioplasty, and who are stable during the first 48 hours after the acute event, are screened for the study. Patients < 80 years of age are considered suitable for the study when they have definite myocardial infarction, as demonstrated by an significant rise in creatine kinase-MB levels , 1 mm ST segment elevation in two or more standard leads or 2 mm ST segment elevation in two contiguous chest leads, and/or the development of Q waves.The criteria for exclusion are: viability testing technically not possible (poor echo-window), contraindications for dobutamine echocardiography (arrhythmia), and coronary angiography , serious life-threatening non-cardiac illness, ECG abnormalities making the evaluation of the ST segment impossible (left bundle branch block, pacemaker), and an unreliable follow-up.The study complies with the Declaration of Helsinki and all ethics committees of the participating centers have approved the protocol. All eligible patients provide written informed consent.The study is a prospective, multicenter, randomized, controlled clinical trial. In the VIAMI-trial, patients who are admitted to the hospital with an acute myocardial infarction and who did not undergo primary or rescue PCI, are evaluated by LDDE within 3 days of admission. Patients with unequivocal signs of viability in the infarct-area are randomized to an invasive or a conservative treatment strategy. In the invasive strategy patients undergo coronary angiography with the intention to perform PCI with stenting of the infarct-related coronary artery. In the conservative group an ischemia-guided approach is adopted with stress testing before discharge from the hospital. When the test is highly suggestive for ischemia, coronary angiography will be performed. If revascularization is performed, this will be scored as a secondary endpoint. Patients without viability will serve as a registry group with long-term follow-up Fig . These pThe primary endpoint is the composite of death from any cause, recurrent infarction, and unstable angina. The secondary endpoints are need for revascularization, the occurrence of angina pectoris (CCS classification), and the incidence of heart failure (NYHA classification). Left ventricular function is also evaluated as determined by echocardiography at 3 months, 6 months, and 1 year follow-up.A reinfarction is diagnosed if there is an increase in the total creatine kinase and MB isoenzyme activity (2 times ULN) and either a history of ischemic chest discomfort, or electrocardiographic changes indicative for transmural ischemia or necrosis.For the diagnosis of unstable angina, the patient must be hospitalized with ischemic chest pain or discomfort occurring at rest or with minimal exertion. In addition, the need for intravenous medical intervention and/or objective evidence of myocardial ischemia is required. For extensive description of the end points definitions, see Table A clinical endpoints and safety monitoring committee will continuously check and monitor all events in a blinded manner. Reinfarction after revascularization will be scored as described in Table If the inclusion criteria are met and none of the criteria for exclusion, viability testing by LDDE is performed within 3 days of admission.Preferably, beta-blockers are withdrawn 24 hours before the test ,27,30,31Dobutamine is administrated intravenously at doses of 5, 10, and 15 μg/kg/min, for 5 minutes at each dose. When a 10% increase in heart rate is not achieved with 15 μg/kg/min, a 5-minute infusion with 20 μg/kg/min can be used as the final stage of the procedure.Viability is defined as the improvement of wall motion abnormalities in two or more segments of the infarct zone. Changes from hypokinesia to normokinesia and from dyskinesia or akinesia to hypo- or normokinesia are considered an improvement in wall motion abnormality. Dyskinesia changing to akinesia is not considered as an improvement. Patients are continuously monitored by a 12 lead ECG and blood pressure is recorded at the end of each stage. All views are recorded at rest and during dobutamine on VHS videotape. All videotapes are sent to the core-lab  and will be analyzed by 2 experienced observers. A third observer is used in case of disagreement to reach consensus.® by Philips). The images will be displayed side-by-side in a quadscreen format to facilitate the comparison of images.For subsequent off-line analysis, the echocardiographic images will be digitized from VHS videotape and transferred to a working station (EnconcertAngiography and angioplasty will be performed as soon as possible after randomization. Coronary angiography will be performed according to standard protocols. To determine the severity of culprit lesions, quantitative coronary arteriography (QCA) will be performed to measure percent diameter stenosis, reference diameter and cross-sectional area stenosis. The degree of stenosis will be determined in the view in which the stenosis is most severe.When feasible, PCI will be performed (with or without etc) when there is a significant (≥ 50%) stenosis or occlusion of the infarct-related coronary artery, with the intention to perform primary stenting of the infarct-related artery, with concomitant use of abciximab, according to the EPILOG protocol  After stPatients will be followed for a period of three years. Follow-up data will be obtained of all patients during visits at the outpatient clinic in the first year, and by telephone interview in the second and third year of follow-up. Left ventricular function (volumes and ejection fraction) is determined by echocardiography at 3 months, 6 months, and 1 year of follow-up.The VIAMI-trial is conducted to investigate the differences in clinical outcome between an invasive and a conservative strategy in patients with demonstrated viability in the infarct-area. The expected event rate in the viability positive group is estimated to be 35 percent. To demonstrate with a power of 80% (α = 0.05, two-sided) that PCI leads to a 50% reduction in event rate in the invasive group compared to the conservative group, 200 patients will be needed in each group. Therefore, we intend to randomize a total of 400 patients in this trial, with interim analysis after 200 included patients.The formal stopping rules for the study are the following: If one of the treatment strategies appears significant superior at interim analysis (P ≤ 0.01), the study will be stopped.t test. Differences between proportions will be assessed by chi-square analysis; a Fisher's exact test will be used when appropriate. Event-free survival curves are computed with the Kaplan-Meier method, and the differences between these curves are tested with a Mantel-Cox log-rank test. A primary endpoint analysis is planned at 30 days, 6 months, and 1 year of follow-up. Subgroup analyses are planned to determine whether the treatment effect is more or less pronounced in certain subgroups. The data will be subgrouped by sex, age, diabetes, anterior infarction, time from onset of symptoms to treatment, and the use of thrombolytics. All analyses will be performed on an intention-to-treat basis. Also, the outcome per-protocol will be evaluated. Such an analysis seems worthwhile, as it will reflect the true influence of PCI on post-thrombolytic ischemic events.Baseline descriptive data will be presented as mean ± standard deviations (SD). Differences in clinical and echocardiographic variables will be assessed by unpaired Student's Enrollment of patients started April 1, 2001. Recently, an interim analysis was performed after the inclusion of 200 patients, having a 30 day follow-up. The Clinical Event Committee recommended continuation of the trial.The VIAMI-trial is the first multicenter, randomized, controlled clinical trial, investigating the clinical benefits of percutaneous catheter intervention of the infarct-related artery in patients with demonstrated viability in the early, subacute phase of myocardial infarction.The authors declare that they have no competing interests.RBvL recruited and analysed all data and drafted the manuscript. GV have been involved in drafting the article and revised it critically. OK and JGFB revised the manuscript critically. CAV and FCV have given final approval of the version to be publiced.


Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci.To investigate whether there is a difference in the efficiency of heterodimer formation when two proviruses have the same or different chromosomal locations, we introduced two different Mo-MuLV-based retroviral vectors into the packaging cell line using either the cotransfection or sequential transfection procedure. The comparative study has shown that the frequency of recombination increased about four-fold when the cotransfection procedure was used. This difference was not associated with possible recombination of retroviral vectors during or after cotransfection and the ratios of retroviral virion RNAs were the same for two variants of transfection.The results of this study indicate that a mechanism exists to enable the preferential copackaging of Mo-MuLV genomic RNA molecules that are transcribed on the same DNA template. The properties of Mo-MuLV genomic RNAs transport, processing or dimerization might be responsible for this preference. The data presented in this report can be useful when designing methods to study different aspects of replication and recombination of a diploid retroviral genome. Retroviruses are a family of RNA viruses which replicate through a DNA intermediate . The uniReverse transcription entails two DNA strand transfers during minus and plus DNA synthesis. Since the retroviral virion contains two molecules of the viral RNA, the first DNA transfer might be either intramolecular, transferring to the same template, or intermolecular, transferring to the other template. In the model of Spleen necrosis virus (SNV) it was found that the minus-strand DNA transfer is exclusively intermolecular , while aA reason why there are differences in the rates of recombination between HIV-1 and gammaretroviruses (SNV and Mo-MuLV) is not known. It has been suggested that these differences may be associated with the differences in the template switching frequencies of retroviral reverse transcriptases . A recenIn this study, we have investigated whether there is a preference in the formation of homodiploid virions during the mixed retroviral infection. To explore this possibility, we have used the forced recombination system which included two Mo-MuLV-based retroviral vectors containing different selectable markers and one of the vectors having a deletion of the PBS region. These vectors were introduced into the packaging cell line using two different methods, cotransfection, to provide tandem integration, or sequential transfection, and the frequencies of recombination for the vectors have been compared.To study whether there was a preference for the formation of homodiploid virions in the mixed retroviral infection we have used two different methods, cotransfection and sequential transfection, to introduce genetically different retroviral vectors into the host cells. Since plasmid DNA transfected into eucariotic cells is usually tandemly integrated in a chromosome -19, it iIn this study Mo-MuLV-based retroviral vectors were used as partners for recombination. These vectors contained the Mo-MuLV sequences as follows: the 5' and 3' LTRs, ψ region, a part of gag-sequences before XhoI site position 1560 ), and 14560 , andSince pDΔpbsSVpuro RNA is impaired at the initiation of reverse transcription, this function can be restored when the cDNA initiated on the copackaged pDHEneo RNA is transferred to the puro RNA template during the first jump; minus-strand synthesis continues through the puro gene, and the template shift occurs within the leader region. Thus, the restoration of retroviral vector containing the puro gene is possible via homologous recombination with the neo-containing construct at the sequence identity in the leader region of the genome.The experimental scheme employed in this study is outlined in Figure It was expected that plasmid DNA of retroviral vectors pDHEneo and pDΔpbsSVpuro cotransfected into the packaging cell line would be tandemly integrated into the host genome. To study the integration of plasmid DNA, the PCR analysis was performed with the primers hybridizing to the 3' end of neo gene (T1, direct, for pDHEneo) and to the SV40 early promoter region (T2, reverse, for pDΔpbsSVpuro). Using these primers, the specific PCR products could be obtained if the pDHEneo and pDΔpbsSVpuro are located in the same chromosomal site. On the other hand, PCR products could be generated with only one of the primers when identical molecules of plasmid DNA were integrated in the opposite orientation. However, the efficiency of amplification in this case seems to be very low because such sequences will contain inverted repeats.The PCR analysis was performed using chromosomal DNA prepared from different cell clones generated after cotransfection or sequential transfection of vectors. PCR products were separated by gel electrophoresis, transferred onto nylon membrane and hybridized with 3' neo specific probe. An example is presented in Figure We also used RT-PCR-based assay to examine the ratios of retroviral virion RNA molecules for cell clones generated by different methods of transfection. Since retroviral vectors differed by localization of EcoRI sites in the leader regions, these restriction sites were used as markers to distinguish the two coamplified PCR products obtained with primers specific to this region Figure . EcoRI d3 to 6.3 × 104 CFU/ml and puromycin titers from 5.1 × 101 to 8.0 × 102 CFU/ml. In the cotransfection experiment, the G418 titers varied from 3.1 × 104 to 1.1 × 105 CFU/ml and puromycin titers from 1.4 × 103 to 3.6 × 103 CFU/ml. The frequency of recombination was calculated from the puromycin- and G418-drug-resistant colony titers .The restriction enzyme marker differences in the leader regions of vectors provided a means to analyze the nature of recombinants in NIH 3T3 cells examined by PCR assay. Cellular DNA was analyzed from eight PuroThese experiments demonstrated that the frequency of recombination between vectors localized in the same chromosomal site was about four-fold higher than that of vectors with different chromosomal locations. These data suggest that there might be a preference for the formation of diploid retroviral genome from RNA molecules that are transcribed on the same DNA template. On the other hand, it could not be completely excluded that the high frequency of recombination for retroviral vectors in the cotransfection experiments occurred during or after transfection procedure.To study the possibility of recombination between cotransfected vectors during or after transfection, we used the defective vector in which the 5' LTR promoter was deleted. This vector, pDΔpSVpuro, is almost completely homologous to pDΔpbsSVpuro with the exception of 194 bp in the U3 region . The amplified region of pLTRΔpbs was analyzed by sequencing. The resulting construct, pDΔpbsSVpuro, was generated by exchanging the KpnI-PstI (nucleotide positions 32 to 564) fragment of pDSVpuro with the corresponding fragment of pLTRΔpbs.pMOV9 containing the complete copy of Mo-MuLV provirus and retroviral vectors pDneo and pDSVpuro have been described earlier and were used as the progenitor for all the constructions ,30. pDneequences , neomyciequences . To genepDHEneo is identical to pDneo except the point mutations in the sequences flanking the DLS region. These mutations converted the Mo-MuLV sequences in this region into new restriction sites for HindIII and EcoRI. A description of the cloning steps performed to generate this vector is available upon request.To produce pDΔpSVpuro, the enhancer-promoter sequences of U3 region in pDΔpbsSVpuro were deleted. For this purpose the 3.4 kb SacI-BamHI fragment containing 36 bp of 5' U3 region starting from SacI site and including all other vector sequences of pDΔpbsSVpuro was inserted into the SacI and BamHI sites of pTZ18 plasmid.All DNA manipulations were performed by standard procedures .2, 200 μM each dNTP, 1 % DMSO, 100 nM primers for 20 cycles (94°C 1 min, 50°C 1 min, 72°C 8 min). The products were separated on 0.8 % agarose gel, transferred onto nylon membrane (Hybond-N, Amersham), and hybridized with neo-specific probe . Probes were generated by the random-primer method with [α32P] dATP [Genomic DNA purification and hybridization were performed by standard molecular techniques . DNA preod with αP dATP [3Virion RNA was purified from filtered culture medium from transfected cells and used in RT-PCR assays . BrieflyNIH3T3 (murine cell line) and GP+envAM12 (amphotropic 3T3-based packaging cell line with MLV Gag + Pol and Env genes)  were groThe cell clones producing transfected vectors were established by transfecting GP+envAM12 cells with vector plasmids using the dimethyl sulfoxide-polybrene method . PuromycViral infection was performed immediately after harvesting the virus. The supernatants were harvested from 90 % confluent stable producer cell clones after 16 hour intervals and filtered through the 0.45 μm filters. Infections were performed in the presence of 8 μg/ml polybrene (Sigma) for two hours at 37°C. Puromycin- and G418-resistant cfu titers were determined using the infection of NIH3T3 cells by end-point dilution.The authors declare that they have no competing interests.SAK carried out most experiments and made substantial contributions to conception and design. AIK and ABO carried out analysis of integrated plasmid DNA by hybridization and participated in the works with cell cultures. IKF conceived of the study, participated in the design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.


Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive neoplasm. The cytological diagnosis of these tumors can be difficult because they show morphological features quite similar to other small round blue cells tumors. We described four cases of DSRCT with cytological sampling: one obtained by fine needle aspiration biopsy (FNAB) and three from serous effusions. The corresponding immunocytochemical panel was also reviewed.Papanicolaou stained samples from FNAB and effusions were morphologically described. Immunoreaction with WT1 antibody was performed in all cytological samples. An immunohistochemical panel including the following antibodies was performed in the corresponding biopsies: 34BE12, AE1/AE3, Chromogranin A, CK20, CK7, CK8, Desmin, EMA, NSE, Vimentin and WT1.The smears showed high cellularity with minor size alteration. Nuclei were round to oval, some of them with inconspicuous nucleoli. Tumor cells are clustered, showing rosette-like feature. Tumor cells in effusions and FNA were positive to WT1 in 3 of 4 cytology specimens (2 out 3 effusions and one FNA). Immunohistochemical reactions for vimentin, NSE, AE1/AE3 and WT1 were positive in all cases in tissue sections.The use of an adjunct immunocytochemical panel coupled with the cytomorphological characteristics allows the diagnosis of DSRCT in cytological specimens. Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive neoplasm described as a distinct clinicopathologic entity in 1989 by Gerald and Rosai . Other pHistologically, a typical feature of DSRCT is the presence of clusters of tumor cells distributed within a cellular stroma. The shape of clusters varies from round to elongate. Tumor cells are small to medium-sized with round to oval hyperchromatic nuclei, with inconspicuous nucleoli. Necrotic cells and mitosis are common features. Cytoplasm is usually scanty, and cell borders are indistinct. Intracytoplasmic eosinophilic rhabdoid inclusions may be found in larger cells with nuclear pleomorphism . The immThe cytological smears of DSRCT obtained by FNAB are moderately cellular. Tumor cells show round to oval nuclei with fine chromatin and inconspicuous nucleoli. Cytoplasm is scanty to moderate, with variable number of vacuoles. Tumor cells are arranged in loose clusters. Occasionally, spindle fibroblast-like cells are observed. Stromal fragments may be detected . EffusioWe retrieved from the cytological files of Hospital do Cancer – A. C. Camargo four cytological specimens from 3 patients diagnosed with DSRCT, including one fine-needle aspiration sample and 3 fluid samples, during the last five years (2000–2004). FNA was performed on an inguinal mass of one patient. Alcohol-fixed smears were stained with Papanicolaou technique. Serous effusions were prepared with Cytospin (Shandon, Pittsburgh, Pennsylvania, USA). We evaluated two peritoneal fluid samples and one pleural fluid sample. One case  had a cellblock available. All cases were confirmed by histological analysis and immunohistochemical reactions. The histological sections were cut in sections of 4 μm and stained with H&E and immunohistochemistry. Immunocytochemical study was also performed on all cases.Immunohistochemical and immunocytochemical reactions were performed using streptavidin-biotin peroxidase technique with positive and negative controls. Diaminobenzidine was the chromogen. Table 22-year-old white female, with abdominal pain. Video-laparoscopy showed a liver mass and multiple peritoneal implants diagnosed as DSRCT. Six months after the diagnosis, she started chemotherapy for four months, and reduction of tumor mass was observed. One month after the end of chemotherapy, the tumor was removed. Macroscopically, tumor mass measured 5.0 × 4.0 × 3.8 cm and was involving uterus, pericolic tissue, and vagina. Histological analysis shows also involvement of both ovaries and large bowel wall. Ten out of 13 lymph nodes showed metastasis of DSRCT. The peritoneal fluid colleted during surgery was negative for neoplastic cells. Eight months after the first surgery, she presented with a recurrence in the abdominal cavity and a new resection of the tumor mass showed involvement of cecal appendix. Peritoneal fluid sample collected at that time was positive for malignant cells. In the follow up examination, seven months after the second surgery, it was found an inguinal tumor mass of 15 mm. FNA was performed and showed DSRCT metastasis. After the diagnosis, this patient was transferred to another institution.Seven year-old male with back pain and fever. CT scan showed pleural effusion and a mediastinal mass measuring 16.0 × 9.0 cm. Tumour mass showed involvement of soft tissues. Surgical biopsy and pleural drainage were performed. The patient was treated with radiotherapy and chemotherapy, but died 8 months after the diagnosis.Male, 15-year-old had acute abdominal pain and was submitted to an exploratory laparotomy that disclosed a large pelvic mass, involving epiplon and sigmoid, cecum, liver and peri-aortic lymph nodes. This patient had multiple nodules on peritoneal surface. The biopsy of tumor was performed. One month after the diagnosis, chemotherapy was initiated. The patient was submitted to chemotherapy during 8 months, with reduction of more than 50% of tumor mass. A second laparotomy was done to excise retroperitoneal and retrovesical mass. At this time peritoneal fluid sample was collected. After surgery, chemotherapy was continued. The patient is alive, with residual disease.The smears showed high cellularity. The tumor cells exhibited a slight variation in size. Nuclei were round to oval, some of them with small nucleoli. The cytoplasm was scanty. Tumor cells are clustered, with rare clusters showing rosette-like features. The background of the smears showed lymphocytes. Figure .All fluid samples showed similar features. The samples showed high cellularity. Tumor cells were more frequently arranged in tridimentional clusters, but occasionally, isolated cells are also seen. Additionally, clusters showing rosette-like features are rarely observed. Nuclei were round to oval, some of them with small nucleoli. The cytoplasm was scanty Figure .The distribution of immunoreactivity in histological and cytological samples from the patients are summarized in Table the histological sample collected before chemotherapy, exhibited immunohistochemical positivity for vimentin, epithelial membrane antigen (EMA), neuron specific enolase (NSE), chomogranin A, and desmin in dot-like perinuclear pattern. Cytokeratin expression was observed with anti-cytokeratin cocktail (AE1/AE3) and Cytokeratin 8. Tumor cells also expressed WT1 protein.tumor cells exhibited positivity for vimentin, EMA, NSE, chomogranin A, AE1/AE3 and WT1. Desmin and cytokeratins 7, 20 and 34BE12 were negative.tumor cells exhibited positivity for vimentin, NSE, AE1/AE3 and WT1. The immunohistochemical study was performed before chemotherapy.DSRCT is a rare neoplasm that affects young patients. It may present a problem in the differential diagnosis with other small round cell tumors. The diagnosis of DSRCT however can be established with correlation of clinical, cytological and immunocytochemical features. The cytological features that we found in the smears obtained by FNA are similar to other descriptions in the literature. Similar to reports of Zeppa et al , we did One of the characteristics of DSRCTs is its dissemination along serous surfaces. Due to this fact, development of serous effusions is a common clinical finding in DSRCTs patients, with detection of tumor cells in the fluid. In effusions, tumor cells may be present in aggregates but no obviously architectural arrangement is seem.Demonstration of a divergent phenotype and the reciprocal translocation characteristic of DSRCT are critical to the diagnosis.In a reported series of 32 cases of DSRCTs , 88% of The establishment of a specific reciprocal translocation t  as diagnostic in DSRCT was based on the results of Sawyer et al . Shen et22, 23. In addiction to consistent WT1 expression, the typical serosal involvement in DSRCT has raised the possibility that this tumor might be a blastematous tumour derived of primitive mesothelium [EWS gene encodes a protein which the precise function and normal role has not yet been elucidated. Recently, Thomas et al  proposedothelium . Mesotheothelium .We detected WT1 immunoreactivity in all tumors tissues and in 2 out of 3 serous effusions with malignant cells, as well as on FNAB smears. The high frequency of DSRCTs with WT1 protein expression suggests that in consensus with clinical tomographic and cytological findings, this antibody may be used to confirm the diagnosis of DSRCT in cytological samples. We observed a negative WT1 reaction in the cytological sample of patient 3. This sample was collected 10 months after the end of chemotherapy protocol. We can hypothesize if chemotherapy hampered a different antigenic pattern in malignant cells, and influenced this result.Among other small round cell tumors, most of cases of rhabdomyosarcomas and neuroblastomas do not disclose nuclear WT1 staining ,27. CompIn conclusion, cytological and immunophenotypical findings in an appropriate clinical context is sufficient to suggest DRSTC, what sounds highly contributive for us, considering the high aggressiveness of this tumor.


Australian burden of disease estimates appeared inconsistent with the reported repetitive and ubiquitous nature of dental problems. The aims of the study were to measure the nature, severity and duration of symptoms for specific oral conditions, and calculate disability weights from these measures.Data were collected in 2001–02 from a random sample of South Australian dentists using mailed self-complete questionnaires. Dentists recorded the diagnosis of dental problems and provided patients with self-complete questionnaires to record the nature, severity and duration of symptoms using the EuroQol instrument. Data were available from 378 dentists (response rate = 60%).Disability weights were highest for pulpal infection (0.069), caries (0.044) and dentinal sensitivity (0.040), followed by denture problems (0.026), periodontal disease (0.023), failed restorations (0.019), tooth fractures (0.014) and tooth wear (0.011). Aesthetic problems had a low disability weight (0.002), and both recall/maintenance care and oral hygiene had adjusted weights of zero.Disability weights for caries (0.044), periodontal disease (0.023) and denture problems (0.026) in this study were higher than comparable oral health conditions in the Australian Burden of Disease and Injury Study . A range of common problems such as pulpal infection, failed restorations and tooth fracture that were not included in the Australian Burden of Disease and Injury Study had relatively high disability weights. The inclusion of a fuller range of oral health problems along with revised disability weights would result in oral health accounting for a larger amount of disability than originally estimated. Although dental problems are widespread in number and impose a large burden on society in terms of lost production, pain and suffering, and health expenditure there is a tendency to underestimate their importance due to the generally non-fatal nature of most oral diseases and complacency arising from acknowledged improvements in oral health, such as trends toward lower caries levels among children and decreased edentulism in adults. Australians spend $2.6 billion on dental services, some 5.4% of recurrent health expenditure for 1998–99 , and perThe disability-adjusted life year or DALY ,6 providEstimates of DALYs are limited by inadequate information on the distribution of severity of disease and the course of a disease. Due to limitations in the data many of the disease models are necessarily simple and approximate, with their precision reflecting the source and nature of the data underlying the model. Also, the lack of Australian disease weights may mean that they are not completely representative of Australian societal preferences . Hence tThe DALY estimates for oral health in the Australian Burden of Disease and Injury Study seemed inconsistently low with other reports of the high prevalence and incidence of oral health conditions such as dental caries and periodontal disease . There aThe broad aims of the project were to evaluate methods used to measure the burden of disease associated with oral conditions in Australia. The specific aims were to obtain measures of burden of disease related to specific oral conditions, measure these in terms of the nature, severity and duration of symptoms, and calculate disability weights from these measures.The study was conducted using a 2-stage sampling design whereby dentists were randomly sampled from the South Australian Dental Register, randomised into one of seven equal-sized study groups and sent a mailed self-complete dentist questionnaire along with up to five self-complete patient questionnaires depending on the study group. Dentists were provided with a practitioner logbook in which to record for the first 1 to 5 adult patients (depending on study group assignment of dentist) of a random clinical day the treatment they performed and diagnosis of the oral disease or condition treated. At the conclusion of treatment the practitioner passed on a survey kit to their sampled patient(s) containing a patient questionnaire, cover letter and explanation sheet. Sampled patients completing the patient questionnaire recording basic socio-demographic characteristics and data concerning the nature, severity and duration of their symptoms. The patient questionnaires were identified using the practitioner identification number allowing linkage between the practitioner logbook data and patient questionnaire data, but maintaining the anonymity of each patient to the investigators.The emphasis of the project was to obtain precise estimates of the component measures of the burden of oral disease. These are typically expressed as percentages, such as the percentage of persons or percentage of time experiencing symptoms of a given degree of severity. Taking a parameter size of 10% as a reference estimate for any given measure, in order to achieve a level of precision of 20% or less relative standard error, a minimum target sample of 225 patients was required. This would provide an acceptable minimum level of precision for estimates as low as 10% in size, and better precision for any estimates larger than 10% in size.Data were collected during 2001–2 with a primary approach letter sent initially to each dentist, followed a week later by the survey materials, with a reminder card two weeks later, and up to four follow-up mailings of survey materials to dentists who had not yet responded in order to ensure higher response rates .Dentists recorded the details of the dental conditions that patients had, and patients recorded their experience of those dental conditions. Diagnosis of dental conditions was collected from dentists using an open-ended question in the dentist questionnaire and coded using the coding scheme adopted in the Longitudinal Study of Dentists' Practice Activity . Data onFollowing descriptive analysis of response rates and characteristics of respondents, the distribution of dental conditions was examined for the 11 most common dental conditions. These dental conditions were then examined in terms of the nature, severity and duration of each condition. Disability weights were then calculated for each dental condition by using a health state valuation algorithm based on UK population data . A patieA total of 378 dentists responded to the survey (response rate = 60%). Response rates between study groups ranged from 49% to 70% and tended to be higher in study groups that required dentists to sample less patients, but the effect was not monotonic Table . Data weThe characteristics of patients are presented in Table The distribution of dental conditions is presented in Fig Dental conditions are presented in Table a) were highest for pulpal infection, dentinal sensitivity and caries, followed by denture problems, periodontal disease, tooth wear and tooth fractures (Table b) were reduced. Pulpal infection remained the highest adjusted disability weight, followed by caries and dentinal sensitivity, followed by denture problems, periodontal disease and failed restorations. Subtracting the intercept from the unadjusted disability weight reduced all weights (DWc) by a constant amount.Unadjusted disability weights derived from the additive model (DWes Table . When add) followed a similar rank order as for the unadjusted disability weights derived from the additive model (DWa), being highest for pulpal infection with caries ranked second-highest, but with some re-ordering of the next highest conditions . When adjusted by the percent of time that dental conditions were experienced all disability weights derived from the multiplicative model (DWe) were reduced, with pulpal infection ranked highest, followed by caries, dentinal sensitivity, denture problems, tooth wear and periodontal disease.The disability weights derived from the multiplicative model are presented in Table c) and multiplicative (DWe) models were similar in rank ordering, with pulpal infection, caries, dentinal sensitivity and denture problems ranked highest. While the adjusted disability weights derived from both models were also similar in magnitude those derived from the additive model were lower for all oral conditions except aesthetics, which was identical for both models. In the remainder of the paper the final adjusted disability weights derived from the additive model (DWc) will be presented, as this provided the most conservative estimate.The final adjusted disability weights derived from the additive , with 39.6% of patients reporting symptoms on one or more of the six dimensions . ConversThe findings from this study indicate that oral health conditions may account for a considerably higher level of DALYs than previously thought, due to the lack of quantitative data on the nature of dental conditions. While Australia has not had another national oral health survey since the initial survey of 1987–88, there have been other studies that suggest that dental problems are common ,4, and aCompared to the Australian Burden of Disease and Injury Study the adjusted disability weights for oral health conditions in this study were higher for comparable oral conditions of caries (0.044 versus 0.005 for caries involving a filling and 0.014 for caries involving an extraction), periodontal disease (0.023 versus 0.007) and denture problems (0.026 versus 0.004 for edentulism). In addition there were a range of common oral health problems such as pulpal infection, failed restorations and tooth fracture that were not included in the Australian Burden of Disease and Injury Study which had relatively high disability weights. The inclusion of a fuller range of oral health conditions along with revised disability weights would result in oral health accounting for a much larger amount of disability than originally estimated.None declared.DSB and AJS were chief investigators on the grants obtained to fund the study. DSB performed data collection, analysis and drafting of the manuscript. AJS participated in the design and coordination of the study, and completion of the manuscript. All authors read and approved the manuscript.Appendix 1: algorithm used to calculate disability weights from the additive model. Appendix 2: algorithm used to calculate disability weights from the multiplicative modelClick here for file


Focal segmental glomerulosclerosis (FSGS) made a brief media splash last year when a kidney transplant forced NBA superstar Alonso Mourning into early retirement. Mourning's condition elicited a flood of calls from fans offering their kidneys, but most people with kidney disease are not so lucky. Some 56,000 patients await transplants; many have waited over five years. FSGS, which underlies about 25% of the 60,000 kidney-related deaths each year, causes inflammation and irregular scarring in the glomeruli, clusters of blood vessels in the kidney that filter toxins from the blood. These lesions, which allow protein and blood to escape into the urine, cause progressive kidney failure. FSGS commonly occurs as an outgrowth of various primary disorders, including obesity, HIV infection, diabetes, and hypertension. Though it's not clear what causes FSGS, this form of renal pathology is becoming more common. By using the genes underlying inherited forms of FSGS as probes, scientists hope to uncover the mechanisms that unleash the disease and to find ways to stem the damage.ACTN4 gene, which encodes a protein called α-actinin-4, cause an inherited form of FSGS. The protein normally remodels actin filaments, the primary structural component of muscle and cytoskeleton. Having a single mutated copy of the gene can cause FSGS in humans, though it is unclear how. In this issue of PLoS Biology, Martin Pollak and his colleagues at Brigham and Women's Hospital at Harvard Medical School use a three-pronged approach to figure out how the defective protein wreaks renal havoc and how these physiological changes lead to FSGS. Using biochemical analysis, cell-based studies, and a newly developed “knockin” mouse model, the researchers report that FSGS-related mutations cause α-actinin-4 to engage in various aberrant behaviors that ultimately rob the protein of its function and poison cells.Mutations in the ACTN4. In these individuals, the disease appeared to strike podocytes  first. While engineering mice designed to carry mutations in this gene, the researchers created mice that lacked detectable Actn4 expression. These “knockout” mice developed severely damaged podocytes and progressive glomerular disease. In the current experiments, the researchers returned to their “knockin” mice, which carry two copies of the mutation found in the families with inherited FSGS. They also generated “normal” mice and mice harboring one normal and one mutant copy of the gene.In previous experiments, Pollak's team had discovered that some families with the inherited form of FSGS carried mutations in Actn4 mutation into podocytes, using a variety of methods, to see where in the cells the expressed proteins turned up. They also introduced fluorescently labeled mutant and normal Actn4 genes into podocytes that were grown from the three mouse types: normal proteins were diffused throughout the cytoplasm in each cell type, but the mutant proteins showed an uneven distribution. Analysis of various tissues taken from the knockin mice revealed normal levels of mRNA transcripts—indicating normal gene transcription—but “markedly reduced” α-actinin-4 protein levels. The mutant proteins, it turns out, were manufactured normally but were degraded far more quickly than normal proteins. Electron microscopy showed that podocytes in the kidneys of the knockin mice had structural defects, while the mice themselves had significantly higher levels of protein in their urine than mice with one or two normal copies of the gene did.In the biochemical experiments, the researchers investigated the mutant protein's binding behavior. Typically, two α-actinin-4 proteins form a twosome without incident, but here the mutants behaved badly, assuming improper structural conformations and forming aggregates rather than pairs. Next, Pollak and colleagues introduced the genes with the Actn4 mutations produce α-actinin-4 aggregates with significantly reduced life span, the authors explain, suggests two possible mechanisms of initiating disease: aggregation and the toxic affects of aggregation could injure podocytes, or loss of α-actinin-4 function caused by rapid degradation of the protein could produce injury. Pollak and colleagues argue that both factors likely play a role: Actn4 mutations lead to both reduced α-actinin-4 activity and protein aggregation, and the loss of protein function and the toxic effects of protein aggregation produce glomerular injury. As inherited renal disease typically emerges later in life, it may be that α-actinin-4 aggregation causes incremental but cumulative podocyte damage over time. So what does this mean for patients with progressive renal disease? While these findings may not translate into clinical applications anytime soon, they do suggest that therapies aimed at repairing the structure or expression of these essential cytoskeletal proteins might return the renegade proteins to the fold.The finding that FSGS-associated


Typically, randomization software should allow users to exert control over the different aspects of randomization including block design, provision of unique identifiers and control over the format and type of program output. While some of these characteristics have been addressed by available software, none of them have all of these capabilities integrated into one package. The main objective of the Random Allocation Software project was to enhance the user's control over different aspects of randomization in parallel group trials, including output type and format, structure and ordering of generated unique identifiers and enabling users to specify group names for more than two groups.The program has different settings for: simple and blocked randomizations; length, format and ordering of generated unique identifiers; type and format of program output; and saving sessions for future use. A formatted random list generated by this program can be used directly (without further formatting) by the coordinator of the research team to prepare and encode different drugs or instruments necessary for the parallel group trial.Random Allocation Software enables users to control different attributes of the random allocation sequence and produce qualified lists for parallel group trials. An important aspect of any trial that should be clearly stated in the final report is the method used to assign treatments (or other interventions) to participants . In the Without the use of computer software or Internet services the maintenance of the whole process of randomization and allocation is difficult. In addition, in the case of any necessary restrictions on the process of randomization (i.e. block randomization ) the com1. Independent running on a local computer without any need to access the Internet2. Different types of program output: to file (html or text files), window and system clipboard3. Provisions for different block design4. Capability to deal with a larger number of groups5. Specifying a name for each group6. Control over the format, length and ordering of the generated UIs7. Control over the format of generated sequence8. Saving or loading the randomization settings9. Viewing previously generated randomized sequencesRandom Allocation Software is a program created in Microsoft Visual Basic 6, and it installs in the same way as ordinary Windows software (i.e. running setup.exe and following on screen instructions). Once installed and run, there are some controls in the main window for specifying the number of groups (2 to 16), sample size and the name of each group. It also contains menu items to determine the program output and randomization settings. The default program output is saved into either html or text files, and it may also have output to a window or to the system clipboard. A variety of randomization options can be set in the options window. The length of generated UIs (named as Code in the program) can be between 3 to 10 characters and there are options for different alphanumeric structures. In addition, these UIs can appear in sequential or random order in the generated random list. The program can generate simple or block randomization in different types, including equal size blocks, multiple block lengths with random variation among the specified block sizes and complete randomized blocks (random number and size of blocks). The generated sequence will appear in a multiple columns format and the number of columns (1 to 10) can be changed in the options window. Output to html file will be formatted in the form of one block per table. Borders of the tables may be shown or hidden. By clicking the 'Generate' button in the main window the random sequence will be generated and opened by the default viewer for the output file (e.g. Internet Explorer for html files). Previously created output files can also be viewed from inside the program. Additional options include saving the current randomization settings, loading a previous setting and enabling the program to save the last setting upon program exit.During execution, the program produces a random sequence of allocation using the Rnd function that generates a floating point random number. The Rnd function uses the linear-congruent method for pseudo-random number generation as depicted by the following formula:x1 = (x0 * a + c) MOD (2^24) [12]where:x1 = new valuex0 = previous value a = 1140671485c = 12820163The seed of the Rnd will be the Timer function, which will return the number of seconds elapsed since midnight. Although this version of the program does not produce repeatable lists, it is possible to revise the program in subsequent versions to save the value of the seed to reproduce the same random list. The output consisted of shuffled allocations each of which is a UI, group name pair. The program checks for the uniqueness of the UIs and generates an error message if the specified UI length is insufficient to hold the entire sample size.Runs test was used to check randomness of the output list with sample sizes from 10 to 190 (10, 30, 50, ..., 190) and from 200 to 3000 (200, 600, 1000, ..., 3000). Each runs test was carried out for the group number of 2, 3, 5 and 6. SPSS 10 software was used to perform the runs test.The program starts running with the default settings. Users may run the program with the default settings or set the number of groups, the name of each group and the sample size. Clicking the 'Generate' button figure  producesFigure In block randomization the final sample size is usually larger than the specified one.A total of 18 runs test were performed to check the randomness of the program output, which resulted in P values of 0.22 to 0.81.The main use of Random Allocation Software is to produce simple or block randomized sequences for parallel group trials. Its use is restricted to parallel group randomized trials. Compared with similar software, it enables the user to control the length, order and format of the UIs; and the type and format of the output. It allows specifying up to 16 groups for parallel trials.Random Allocation Software has been designed to produce random sequences consisting of UI, group name pairs with additional control over the output format and type. Available randomization software generally has limitations in the number of groups, naming each group, generating UIs and control over the output. Many of these problems have been addressed in the present software. As has been stated in previous sections, the main use of this software is for randomization in parallel group trials. The software can be revised to support crossover and other types of randomized trials. The experienced user may test the randomness of the program output by selecting numeric labels for group names and then exporting the generated list into a statistical software such as SPSS to execute a runs test on the exported data.Project name: Random Allocation SoftwarePublic use access: (Latest version)Operating systems: Windows 98, Me, 2000, XP. It should be noted that on some Windows operating systems  during installation of the program an error message like "Setup Cannot Continue... System Files Are Out of Date" may be displayed. If this happens, click OK and restart the system. Then run the setup.exe again. This is due to a known bug in the installation programs of Microsoft Visual Basic . This prProgramming Language: Visual Basic 6Other requirements: Internet Browser (Internet Explorer 5 or higher is recommended)License: Free for academic use.JIT = Just in TimeUI = Unique IdentifierThe author declares that he has no competing interests.The pre-publication history for this paper can be accessed here:


The goal of much care in chronic childhood illness is to improve quality of life (QOL). However, surveys suggest QOL measures are not routinely included. In addition, there is little consensus about the quality of many QOL measures.To determine the extent to which quality of life (QOL) measures are used in paediatric clinical trials and evaluate the quality of measures used.Systematic literature review.Included paediatric trials published in English between 1994 and 2003 involving children and adolescents up to the age of 20 years, and use of a standardised QOL measure. Data Sources included MEDLINE, CINAHL, EMB Reviews, AMED, BNI, PSYCHINFO, the Cochrane library, Internet, and reference lists from review articles.We identified 18 trials including assessment of QOL . In three trials, parents rated their own QOL but not their child's. Fourteen different QOL measures were used but only two fulfilled our minimal defined criteria for quality.This review confirms previous reports of limited use of QOL measures in paediatric clinical trials. Our review provides information about availability and quality of measures which will be of especial value to trial developers. Chronic disease affects approximately 18% of children  and cystEfforts to measure child QOL have proved complex but a number of generic and disease-specific measures have been reported . GenericThe quality of measures must be evaluated according to performance characteristics. Guidelines suggest good measures of QOL are reliable and valid for the group of patients for whom they are used, include a form for self-report wherever possible, are brief and developmentally appropriate, and allow completion by proxy .There is little evidence that QOL measures are routinely used in clinic practice  or cliniWe report a systematic review drawing on established methodologies  to deterrd quarter 1993, AMED (Allied & Contemporary Medicine) 1985 to December 2003, British Nursing Index (BNI) 1985 – October 2003, EMBASE, PSYCHINFO 1872–2003.The following databases were searched: MEDLINE 1966 to Nov Week 2 2003, CINAHL 1982 to December Week 1 2003, EMB Reviews: Cochrane Central Register of Controlled Trials, Your Journals at OVID, EMB reviews: ACP Journal Club 1991 to July / August 2003, EMB reviews: Database of Abstracts and Reviews of Effects 3Text word and thesaurus searches were used to minimise the chance of missing relevant articles. The following keywords were searched:• child, childhood, children, adolescent, infant, pediatric, paediatric,• quality of life, QOL,• clinical trial, randomised controlled trialSearches were restricted to English language papers.Search engines were used to search the Internet with keywords and Boolean logic. Additional references from articles identified through these searches were also pursued.These included:1) Children and adolescents up to the age of 20 years,2) RCT, formal cross-over trial, or studies evaluating one or more active drug treatment with or without placebo,3) Standardised QOL measure (For these purposes we drew on a previous review  and defi4) Articles published in English between January 1994 and December 2003.1) Samples including both adults and children.2) Comparison of surgical treatment, pain control, palliative medication, or psychological/homeopathic intervention.3) Outcomes evaluated in terms of medical data only, non-standardised measures of QOL or standardised psychological measures including symptom checklists, measures of self-esteem, or coping.Abstracts were reviewed for relevance and full articles obtained where appropriate. A summary sheet was developed and both authors independently reviewed papers to ensure reliability. Data extracted by reviewers was second coded and compared and any discrepancies were resolved through discussion.Of the 917 records retrieved from the databases, initial inspection suggested that 27 abstracts met the inclusion criteria. On reading the full articles, nine failed to meet inclusion criteria. The resulting 18 articles were included in the review -29.Disease: QOL was most frequently included in trials in atopic diseases (Asthma = 4, Rhinitis = 4, Dermatitis = 2 and Eczema = 1). Single studies were identified in Cystic fibrosis, Otis media, Amblyopia, Diabetes, Obesity associated with a brain tumour, Idiopathic short stature, and Congenital agranulocytosis.• Location: Seven studies were conducted in the U.S.A, 4 in the U.K, 3 in the Netherlands, one in Taiwan, and one in Israel. Two studies were multi-national.• Child's age: Three studies recruited children across a broad age range (1 to 18 years), 2 focused on pre-school children (1–5 years), 4 on pre-school and middle childhood (2–10 years), 2 on middle childhood (6–12 years), 6 on middle childhood and adolescence (5–18 years), and one on adolescents alone (12–17 years).• Sample size: Sample size ranged from 19 [•  from 19  to 689 [ from 19 . Power cDesign and trial aim: We identified 11 RCTs, 2 cross-over studies, and 5 studies comparing two or more active treatments without placebo or control group. Of the 11 RCTs, 1 was multi-national, 7 multi centre, and 3 single centre studies. Of the 7 non RCTs, 1 was multi-national, 2 were multi centre, and 4 single centre. Nine articles involved comparisons of two or more treatment and the remainder involved comparison of treatments with placebo.• Blinding: Seven RCTs reported blinding procedures.• Parent and caregiver QOL: Fifteen studies measured the impact of the disease on the child's QOL. Three included assessment of the caregivers QOL.• Respondent for child QOL: Of the 15 studies focusing on child QOL, 10 were based on child, and 3 on parent reports. In two studies both children and parents reported the child's QOL and in one of these clinicians also rated child QOL [• hild QOL .Generic or disease specific: In total, 12 disease specific and two generic measures were used [• ere used -38. The ere used ,29.Quality of measure: We assessed quality of measures based on minimal accepted criteria [• criteria  whereby criteria ,38. ThreDespite extensive searches we identified only 18 published reports of paediatric trials including standardised QOL measures. This undoubtedly represents a very small percentage of paediatric trials and supports previous findings that QOL data is seldom reported in paediatric clinical trials . FurtherIn considering why there are relatively few trials including QOL measures, it is important to take into account the aims and purpose of the trial ,41. The Where QOL was measured, disease specific measures were most often used (N = 12) as is normally recommended for use in clinical trials. Only two trials included measures that satisfactorily fulfilled accepted criteria . TypicalSelection of a measure of QOL is dependent on the psychometric properties of the instrument, as well as clinical and demographic variables characteristic of the sample. However, psychometric properties depend upon samples for which the scale has been validated. Hence it is important to ensure measures are used with clinical populations where psychometric data are available.There are some grounds for assuming that QOL changes during childhood, and therefore satisfactory measures target specific age groups . There aIt is also generally recommended that ratings of QOL should be made by children themselves whenever possible . In caseCONSORT  guidelinObjections to inclusion of QOL measures in trials involve anticipated increased costs, extra time needed to gain patient and parent consent, and lack of sophistication of currently available measures . A majorA second problem is that disease specific measures may simply not be available for rare conditions. Attempts to develop such measures are promising and in this review instruments for ambylopia  and agraOther methodological limitations in current work include the lack of power calculations. Where the aim of the trial includes QOL assessment, power calculations must be performed and are an essential element of clinical trial design. In cases where measurement of QOL is a secondary endpoint, sample size calculations are rare and difficult to establish. However attempts should be made to hypothesise expected changes in QOL scores in relation to the agreed sample size prior to the trial .This review supports previous findings of limited use of QOL measures in paediatric cancer trials  and exteThe measurement of QOL provides valuable information about the psychological and social impact of treatment on children especially where no differences in survival rates are anticipated. For this reason, the inclusion of QOL measurement in paediatric trials is becoming increasingly valued and mandatory ,48. TherBoth authors were responsible for planning, conducting and reporting this work and approved the final manuscript.Table 1: Study characteristicsClick here for fileTable 2: Quality of life measuresClick here for file


Proteomics is rapidly evolving into a high-throughput technology, in which substantial and systematic studies are conducted on samples from a wide range of physiological, developmental, or pathological conditions. Reference maps from 2D gels are widely circulated. However, there is, as yet, no formally accepted standard representation to support the sharing of proteomics data, and little systematic dissemination of comprehensive proteomic data sets.Proteome Experimental Data Repository (PEDRo), which makes comprehensive proteomics data sets available for browsing, searching and downloading. It is also serves to extend the debate on the level of detail at which proteomics data should be captured, the sorts of facilities that should be provided by proteome data management systems, and the techniques by which such facilities can be made available.This paper describes the design, implementation and use of a The PEDRo database provides access to a collection of comprehensive descriptions of experimental data sets in proteomics. Not only are these data sets interesting in and of themselves, they also provide a useful early validation of the PEDRo data model, which has served as a starting point for the ongoing standardisation activity through the Proteome Standards Initiative of the Human Proteome Organisation. Bioinformatics tools and techniques depend directly or indirectly upon experimental data. However, interpreting experimental data often requires access to significant amounts of additional information about the sample used in the experiment, the conditions in which measurements were taken, the equipment used to take the measurements, etc. Recent proposals for models that capture such experimental descriptions alongside experimental results include MIAME for transcriptome data  and PEDRIn proteomics, which is rapidly evolving into a high-throughput experimental approach, there is (as yet) no standard representation for experimental data. As a result, limited tool support is available for disseminating, searching, comparing or analysing the results of experiments conducted using different techniques and equipment in different laboratories. Thus, while experimental results can be analysed, often in a labour-intensive manner in-house, the development of bioinformatics techniques for archiving, sharing and wider exploitation of proteomics results is still in its infancy. This paper seeks to contribute to the development of effective and systematic support for proteome data management by:1. Describing a database for storing, searching and disseminating experimental proteomics data. This material should be relevant to the developers of future proteome data management systems in that it discusses and illustrates various design and implementation decisions that have an impact on the role and maintenance of the resulting database.Proteomics Experimental Data Repository) data model [2. Making available data sets from several labs whose data have been included in the initial release of the database. These data sets themselves result from substantial experimental activities, and are representative of the sorts of information that in-house and public proteome data repositories must capture. As the database stores data in an XML format that conforms to the PEDRo  , the StaEstablishing the most appropriate kinds of data to include in a database such as PEDRo is not straightforward, as this depends on the use that is to be made of the data. In a large data repository, users may want to search for results based on widely varying criteria – for example, the proteins identified, the change in the level of a protein over time, the mechanism by which a sample was studied, etc. Furthermore, the users of a proteome data repository may themselves be diverse, and include: experimentalists with minimal direct experience of proteomics, but who are interested in proteins or organisms for which proteome studies have been conducted; proteome scientists who want to identify how successful specific techniques have been in different contexts; or mass-spectrometric analysts who want to compare their results with those of others.This wide range of potential users encourages the creation of a rich repository for proteomics data that provides detailed descriptions of many different aspects of an experiment. However, populating a database such as PEDRo is not a trivial task, as several of the different kinds of data included in PEDRo currently have to be entered manually, which is time-consuming for data providers. Even though a data entry tool has been developed to ease data entry (available from ), experiMany bioinformatics databases, such as UniProt  and PDB The PEDRo database makes extensive use of XML for capturing, transmitting, storing and searching proteomics data. In particular:1. The data-capture process uses a software tool, illustrated in Figure 2. The database stores the XML captured using the data entry tool directly, using Xindice , an open3. The data are presented to users by generating web pages from the stored XML using XSLT , which wThe software components used within PEDRo (and the role they play in data capture, storage and dissemination) are illustrated in Figure sample generation (e.g. organism, growth conditions, tagging), sample processing (e.g. gel properties, spot details), mass spectrometry (e.g. machine settings, peak lists) and in silico analyses (e.g. database search program used and results obtained). Table in silico data analyses using more than one program. Furthermore, the data captured covers a range of different organisms, including Saccharomyces cerevisiae, Candida albicans, Candida glabrata, Mus musculus, Arabidopsis thaliana and Streptomyces coelicolor.The data stored in the database for each experiment is as described in the PEDRo model , and thuS. cerevisiae and the pathogenic fungus, C. albicans [C. glabrata , whose genome sequence has only just been completed. The Streptomyces coelicolor M600 data set is the largest proteomics time course analysis of this strain in terms of numbers of proteins identified. It adds significantly to our knowledge of expression of some of the 20 gene sets annotated as being determinants of the biosynthesis of secondary metabolites, including antibiotics. Somewhat similar experiments, but differing in many aspects of their metadata, are reported on the SWICZ database [Trichinella spiralis [These PEDRo data are significant in biological terms. For example, they include the first direct comparison of proteomic responses in two fungal species, namely responses to amino-acid starvation in the baker's yeast albicans . In addidatabase . This prspiralis .browsing – interactively listing or navigating through database entries; searching – identifying database entries on the basis of simple restrictions on the values of one or more fields; visualising – presenting a visual representation of the data as a starting point for browsing; or querying – specifying a search that is to be conducted over the database using a query building interface or by providing inputs to pre-written (or "canned") queries. Functional genomics databases tend to emphasise browsing and searching. For example, the Stanford Microarray Database [Web-based interfaces to biological databases tend to support one or more of the following tasks: Database  supportsDatabase  supportsDatabase  supportsS. cerevisiae data from PEDRo will also be made available through GIMS [PEDRo also emphasises browsing and searching. Figures ugh GIMS , for exaA significant motivating factor behind the development of the PEDRo repository has been to allow informed discussion, assisted by concrete examples, into the level of detail and forms of model that are most appropriate for a proteome data repository. As the PEDRo model is being used as the starting point for the HUPO-PSI activity on models for proteome data, early validation of this model is important. The following observations have been made about the PEDRo model during the data capture process:Sample description is neither very precise nor systematic. The effective description of samples is an open issue that spans different kinds of functional genomic data. For example, work is underway on the development of an ontology for characterising microarray experiments, focusing, in particular, on samples [1.  samples . HoweverThere is only limited support for relative protein abundance data (e.g. DIGE and stable isotope labelling strategies). Thus, for example, there is no place in the model to describe an expression ratio for a protein species derived from quantitative experimental strategies, only the ability to capture the 'raw' numbers. In fact, the PEDRo model was not designed to capture expression ratios, partly because such numbers are easily derived from the captured primary data, and partly because the particular method of their derivation may be contentious. It is hoped that the HUPO-PSI model will provide generic constructs for representing relationships between certain kinds of measurement (e.g. relative protein expression readings), to which can be attached the specific detail for individual techniques. However, it also seems important to avoid the pitfalls associated with overly permissive models, as these provide a less stable foundation for the developers of analytical tools than their more proscriptive counterparts.2. The gel model is not particularly detailed. Thus, for example, there is no detailed description of the image analysis software used, the descriptions of individual spots are fairly minimal, and no details are captured on spot excision. An earlier critique of the PEDRo model for gels, and some possible extensions, is provided by [3. vided by . It seemOverall, the appropriate level of detail for a proteomics repository is somewhat subjective, but can usefully be based on guiding principles; agreement as to the principles should then avoid scope-based discussions at a very fine-grained level. The current PEDRo model essentially supports the principle that enough detail should be captured about an experiment to:i. Allow results of different experiments to be analysed/compared.ii. Allow suitability of experiment design and implementation decisions to be assessed.iii. Allow protein identifications to be re-run in the future with new databases or software.There is also an additional negative principle, to the effect that the model itself should not be designed to include dependencies on characteristics relating to the configuration or properties of an individual piece of equipment. Accordingly, we have attempted to allow experimental methods and results to be described in significant detail, but without including parameters and properties that are likely to be superseded rapidly when new models of equipment are introduced, and without including parameters that can only be understood with reference to the documentation of a particular product.in silico analysis. Such design decisions are appropriate for certain categories of user of a proteomics database, but not for others. The UAB database has been designed to provide access to processed experimental results for biomedical researchers, but does not provide enough information to allow detailed comparisons of the ways in which the results were obtained.The data stored in PEDRo is more comprehensive for each experiment than is the case for most existing proteome databases. For example, in the longest established experimental proteomics resource, SWISS-2DPAGE , the empThe ProteomeWeb  providesIn terms of quantities of data, there are fewer data sets in PEDRo than in SWISS-2DPAGE, reflecting the fact that PEDRo is a newly created resource (Release 16 of SWISS-2DPAGE contains 34 reference maps), but somewhat more than in the UAB Proteomics Database. The Open Proteomics Database (OPD) supports the browsing and downloading of comparable amounts of data to those in PEDRo, and also includes mass spectrometry data, although quite a lot of the data are in flat-file format . HoweverThe need for wider and more systematic dissemination of experimental proteomics data is widely recognised, as argued in , and atti. The nature and variety of information that should be recorded about proteomics experiments.ii. The functionality that should be provided by repositories that make large-scale proteomic data available.iii. The computational architecture that should be used to provide the functionality at (ii).iv. The nature of the tools that should be developed for use with such a repository.de jure standard for proteome data representation and sharing. This paper describes a database that includes a rich collection of representative data sets. Furthermore, the paper describes the functionality (issue ii) and architecture (issue iii) of an exploratory system for disseminating such data. In the same way as we see models for representing proteomic data evolving in the light of practical experience, we anticipate that the PEDRo repository, and the overall understanding of the data access and dissemination requirements for proteomic data, will evolve as the opportunities presented by high-throughput experimental techniques and comprehensive data sets become more fully understood.This paper has sought to address issues (i), (ii) and (iii), with a particular emphasis on (i). Following on from  we belie, by following the Database link.The database can be accessed using a web browser at KG and CG implemented the software. SJ, NM and CFT contributed to the development of earlier prototypes. TM coordinated the data capture activity. CK, CE, AW, SH, DS, ZY, AJPB, AH, KC, LH, MM, PG, JH, KSL, SJG conducted or led experimental activities that generated the data in the database, and contributed to feedback on the model. AB, SJH, SJO and NWP oversaw the database design and development activity, and the latter led the write-up.


In order to perform a 3D reconstruction of electron microscopic images of viruses, it is necessary to determine the orientation (Euler angels) of the 2D projections of the virus. The projections containing high resolution information are usually very noisy. This paper proposes a new method, based on weighted-projection matching in wavelet space for virus orientation determination. In order to speed the retrieval of the best match between projections from a model and real virus particle, a hierarchical correlation matching method is also proposed.A data set of 600 HSV-1 capsid particle images in different orientations was used to test the proposed method. An initial model of about 40 Å resolutions was used to generate projections of an HSV-1 capsid. Results show that a significant improvement, in terms of accuracy and speed, is obtained for the initial orientation estimates of noisy herpes virus images. For the bacteriophage (P22), the proposed method gave the correct reconstruction compared to the model, while the classical method failed to resolve the correct orientations of the smooth spherical P22 viruses.This paper introduces a new method for orientation determination of low contrast images and highly noisy virus particles. This method is based on weighted projection matching in wavelet space, which increases the accuracy of the orientations. A hierarchical implementation of this method increases the speed of orientation determination. The estimated number of particles needed for a higher resolution reconstruction increased exponentially. For a 6 Å resolution reconstruction of the HSV virus, 50,000 particles are necessary. The results show that the proposed method reduces the amount of data needed in a reconstruction by at least 50 %. This may result in savings 2 to 3 man-years invested in acquiring images from the microscope and data processing. Furthermore, the proposed method is able to determine orientations for some difficult particles like P22 with accuracy and consistency. Recently a low PH sindbis capsid was determined with the proposed method, where other methods based on the common line fail. Three-dimensional (3D) reconstruction of virus particles like SARS (Severe Acute Respiratory Syndrome) and HSV (Herpes Simplex Virus) using electron microscopy yields crucial information for understanding the assembly and infectivity mechanism. The structural determination begins with acquisition of projection images in an electron-microscope. A major part of data processing is aimed at determining the direction of projection for each particle image (2D projection of virus) so that a 3D reconstruction can be computed. The first step in a virus reconstruction is the detection and selection of the individual particle images from a large area of an electron micrograph.There are different criteria to determine the particle orientation. One criterion is based on the computational search of the common lines in the computed Fourier Transforms of individual or multiple particle images . An imprOne approach is to take two consecutive pictures of the same particles one close-to-focus Fig.  and anotA data set of 600 HSV-1 capsid particle images in different orientations was used to test the HWPM method. The defocus range of herpes particles was chosen to be close to focus between 1.7 μm and 0.4 μm. An initial model of about a 40 Å resolution . A grid A match of the particle into the best 3 of the 200 classes was obtained using the wavelet correlation coefficient (wccf) criterion. Next, the particle was compared to the 39 projections of the best three classes, and the correct orientation was that of the projection giving the highest wccf. The hierarchical implementation wavelet projection matching reduced the time at least by a factor of 10 compared with the classical projection matching method. In the example of 600 particles, by using HWPM it took approximately 3 hours to determine the orientations, instead the 33 hours it took with the classical matching algorithm. Both algorithms were running on the SGI Origin-2000 supercomputer using 10 processors.At this point, each particle had been assigned the orientation of the closest projection. A quality factor was assigned to each orientation, which was the wavelet correlation coefficient. Particles having high wccf coefficients were selected for reconstruction of a first 3D model of the virus.Refinement of initial orientations obtained by HWPM was realized by the same iterative refinement process used in focal pair method . This reIn order to assess the accuracy of the orientations obtained with the HWPM. A comparison with the focal pair method Fig. , which iFig. HWPM was tested on a P22 empty shell capsid which was circular and whose shell is very thin (~40 Angstrom). Twenty micrographs of the P22 empty shell capsid with defocus range [0.5 to 2 μm] were used for testing purpose. The total number of particles is 1340, each image has a size of 300 × 300 pixels, and the dimension of each pixel is 2.8 Å.Concerning the initial orientations determinations using HWPM method, an initial model of around 20 Å resolutions was used to generate projections which uniformly covered the asymmetric triangle of the icosahedrally symmetric model. A grid sampling of 2° in each direction of the asymmetric triangle of icosahedral symmetry was used to obtain an initial orientation, targeting a structure of 30 Å. The number of projections obtained with this grid was about 200 projections. A match of the particle with the projections was obtained by using the wccf criterion. The correct orientation was selected as the one of the projection giving the highest wccf. The better half of the orientations projections (650) according to wccf criterion was chosen for final reconstruction.The initial orientations for the same set of data were determined using the Improved Common Line (ICL) method, with the same input parameters for the software described in . ICL useFig. During the last thirty years the common lines methods were a great method to resolve icoshedral particles up to 7–8 Å . RecentlAt high resolution reconstructions, the number of particles needed increases drastically, and the data with a signal-to-noise ratio valid up to the targeted resolution, tend to be very noisy. For an 8.5 Å structure of HSV-1 it took about 6000 particles for a final reconstruction. For a 6.5 Å structure resolution, the estimated value was about 50,000 particles using the same electron microscope .To further increase the resolution of the HSV virus to 6.5 Å or higher (4 Å), the focal pair method would be impracticable. The focal pair method, for intermediate resolution up to (8 Å) for big viruses like HSV, works well for orientations determinations. The number of particles selected for the final reconstruction about 40% of the original number of particles (taking into account the far-focus and close-focus micrographs). It is necessary to emphasize that results from both methods are very similar in terms of visual resemblance. But, there are two advantages of HWPM over the focal pair method. First, focal pair method uses as much as double the data used for the HWPM. Second, the quality of the density maps shows that HWPM gives a better resolution for the same number of particles ; second most of the data are very close-to-focus with defocus range of 0.5 μm to 1.3 μm. The data was noisy and had a very low contrast. The ICL method was able to give 40% of good orientations for the defocus range between 1.9 and 1.2 μm, here the data was closer to focus, which reduced the percentage of good orientations to less then 22%.The application of the HWPM to the P22 empty shell capsid gave the expected structure Fig. . The wavAnother example of real data reconstruction is the VP5-VP19C recombinant. After long investigation using CL and ICL algorithms, the classical projection matching scheme was also tested in order to determine the orientations, but unfortunately all those methods failed. The wavelet filtering and matching was used during the classification step of the recombinant particle VP5-VP19C ,16, whicThe third examples for low PH sindbis: Three years of investigation using CL and ICL methods failed to obtain the correct density map of the low PH sindbis capsid which is subject to conformational changes and an alteration of the symmetry. Recently the proposed method (HWPM) was tested on low PH sindbis and the correct structure was finally observed and analyzed .Wavelet multi-resolution analysis and processing improves particle detections , classifThis paper describes the development and implementation of a new method for orientation determination for low contrast images of virus particles. This method is based on wavelet filtering, which enhances the contrast of the particles and reduces the noise, and on weighted projection matching in wavelet space. A hierarchical implementation of this method increases the speed of orientation determination. Results show that, HWPM have been able to determine accurately more than 85% of the orientations of low-contrast particles. Compared to the focal pair method (for orientation determination from low contrast data) the HWPM reduced the amount of data required in a reconstruction by at least 50 %. In addition the accuracy of the orientations obtained by the proposed method is higher than those obtained by focal pair method . This imThe choice of wavelet filter bases depends on the signal. Signals coming from different sources have different characteristics. For audio, speech, image and video signals the best choices of wavelet bases are known. The best choice for electron microscopic images is not clear. The problem is to represent typical signals with a small number of convenient computable functions.-1) relevant to data analysis.An investigation to choose the best wavelet bases for electron microscopic images was performed here. During this study, simulated and real electron microscopy images were used. The majority of the wavelets basis existing in Matlab-5 software -24 was tThe principle of the wavelet decomposition is to transform the original raw particle image into several components: one low-resolution component called "approximation" , which iThe approximation component is obtained after applying a bi-orthogonal low-pass wavelet filter in each direction  followed by a sub-sampling of each image by a factor of 2 for each dimension. The details are obtained with the application of a low-pass filter in one direction and a high-pass filter in the other, or a high-pass filter in both directions. The noise is mainly present in the detail components. A higher level of decomposition is obtained by repeating the same filtering operations on the approximation. The wavelet correlation coefficient between two wavelet-transformed images, for a given level, is :1 to Wp are weights given for each components of the wavelet correlation, p is the number of components of wavelet decomposition. A1, A2 are the approximations. ⊗ denote the correlation between two components images. D1i, D2i are the details (Fig. Where WInitial orientation determination is based on model-based projection matching approach . The levThe choice of the number of classes is optimized to give the best tradeoff between speed and accuracy. The classification gives a uniform distribution of projections into the classes. The next step is to compare each wavelet-transformed raw image with the closest projection to the center of each class, and then rank the classes in terms of wccf Fig. . The fin