Plasmodium infections, but its role in pathogenesis or protection remains unclear. However, protective properties of natural antibodies have previously been demonstrated in other contexts.Polyclonal B-cell activation is well known to occur in Plasmodium-infected subjects locally detected in a survey study in the Brazilian Amazon, and from unexposed and exposed but presently uninfected control subjects, were assayed by a standardized quantitative immunoblot method allowing simultaneous detection of IgG or IgM reactivity to a large number of parasite-unrelated proteins.Sera from asymptomatic and symptomatic Escherichia coli proteins was strikingly enhanced in asymptomatic Plasmodium-infected individuals when compared to such with clinical malaria symptoms, or to uninfected control subjects. This difference was most characteristic for limited exposure times . It was more significant than a similar trend found for IgG to Plasmodium falciparum antigens, and unrelated to parasitaemia levels. Asymptomatic subjects with comparatively short exposure characteristically showed relatively elevated IgG versus IgM reactivity. Polyclonal IgG reactivity appears triggered by previous P. falciparum but not Plasmodium vivax malaria.In subjects free of coinfection with hepatitis B virus, IgG reactivity to human brain antigens and The observed difference in polyclonal antibody production seems related to intrinsic activation states of infected individuals, rather than to parasite-antigen specific immune responses. However, it appears influenced by preceding stimuli. This supports the idea that acquired clinical immunity may not exclusively depend on antigen-specific responses, but also on the individual polyclonal reaction. Plasmodium falciparum [Leishmania [Plasmodium parasites, either naturally or selected during the long co-evolution of the human species with these parasites. An example may be autoantibodies to 'band 3', a host-encoded target on cell membranes, involved in protection against malaria [Malaria remains an important health problem in sub-Saharan Africa and in some parts of Asia and South America. Resistance to therapeutic drugs and to insecticides, as well as social and environmental changes, are important factors in this situation. Increasing importance is currently given to antibodies in protection against human malaria, especially directed at erythrocytic stages of lciparum . Howeverlciparum -4 as welishmania  infectio malaria ,7 as wel malaria .Natural antibodies, immunoglobulins circulating in the absence of particular immunogenic stimuli, emerge from continuous autonomous activity of the immune system which appears largely independent of any external priming, as demonstrated e. g. in germ-free and antigen-free mice ,10. TheyThe aim of this study was to investigate whether likely natural antibody reactivity patterns measured toward targets not related to parasites, but rather derived from autologous tissue or intestinal flora, could differentiate between asymptomatic and symptomatic forms of malarial infection. Asymptomatic infection is frequent in the Brazilian Amazon , even wiP. falciparum or Plasmodium vivax, with clinical symptoms of variable degrees of intensity but low reported mortality [Asymptomatic and symptomatic subjects who are studied here were occasionally detected in a survey study of endemic Brazilian populations. They mainly consisted of migrants who lived in endemic areas for individually different time periods, and may have experienced sequential infections by ortality . ResultsP. vivax or P. falciparum clinical malaria. The malaria-exposed subjects comprised three distinct groups [see Additional file 1]. The principal study group was derived from a screening of 531 miners living in gold-mining areas in the municipality of Apiacás (AP), Mato Grosso, among whom 99 had been found parasitaemic, presenting with positive Giemsa-stained thick-blood smears [th, 1994) and to the Universidade Federal de Minas Gerais Ethics Committee (April 15th, 1998). Venous blood samples (20 ml per subject) were drawn in Vacutainer™ (Becton Dickinson, Oxnard, CA) heparinized tubes. Giemsa-stained thick-blood smears were examined at this point for blood parasitaemia. Finally, a third control group consisted of 10 healthy adult volunteers from Belo Horizonte (Minas Gerais) who had never been exposed to malaria transmission or visited endemic regions (Aparasitaemic/Not exposed). IgG and IgM to human brain proteins were assessed in the subjects described above. When measuring IgG reactivity to E. coli proteins, two of the Terra Nova and seven of the Apiacas subjects were not included, but replaced by one Terra Nova and eight Apiacas subjects not assayed for reactivity to brain proteins.78 sera analysed here originated from subjects exposed to transmission in the Brazilian Amazon endemic area, who had variable numbers of reported previous episodes of d smears . Out of P. falciparum and MSP1-19 in these populations have been analysed and described previously [The parasite density was quantified after examination of 200 microscopic fields at 1.000× magnification under oil-immersion. All slides were examined by three well-trained microscopists. Blood parasitaemia was expressed as the number of parasites per 200 leukocytes. Parasitaemia and reactivity to eviously .E. coli were run in a discontinuous SDS-PAGE 10% gel . After eletrophoresis, proteins were transferred onto a nitrocellulose membrane (Schleicher & Schuell, Germany) in a semi-dry system for one hour at 0.8 mA/cm2. After overnight blocking of free binding sites in PBS-Tween 0.2%, membranes were incubated for four hours with sera, diluted 1/20, in incubation units fixing membranes in cassettes with 28 independent channels (Miniblot System C-Shell, Immunetics Inc., Cambridge, MA). Whole membranes were then washed and incubated for 90 minutes at room temperature with secondary anti-human IgM or IgG conjugated with alkaline phosphatase (Southern Biotech, Birmingham, AL). Reactivies were revealed with NBT/BCIP (nitroblue-tetrazolium/bromo-chloro-indolyl-phosphate) substrate (Promega, Madison, WI) for three to five minutes, and membranes scanned in 8-bit grayscale and with 600 dpi resolution in a domestic scanner (Apple Color OneScanner). Thereafter, membranes were stained overnight with colloidal gold (Biorad, Hercules, CA) to reveal total protein and again scanned as described.The assay was done as described . BrieflyThe method of data processing has been described . BrieflyThese vectors were then analysed by Principal Component Analysis (PCA). PCA is a classic method of multivariate analysis designed to describe multidimensional data with high dimensionality through projection onto characteristic subspaces with lower dimensionality. Principal components are defined in a mathematically strict manner as orthogonal axes fitting maximal information in terms of total variance with decreasing proportion and uncorrelated among each other. This includes no information on experimental groups or other particularities, but provides a completely neutral and unbiased description of the data set as such.The number of bands detected on a respective extract was quantified as the number of sections showing reactivity values two-fold above the average reactivity of the standard used for adjustment. Total reactivity for a given extract was assessed as the average OD over the entire migration scale, from the first to the last section. Only distribution-independent statistics were used: Mann-Whitney rank sum test and Spearman rank correlation. P-values below 0.05 were considered significant.P. falciparum: p = 0.034; IgG anti-MSP1-19: nonsignificant) or all parasitaemic individuals (p = 0.017 and p = 0.014, respectively).As an example, Fig. The first principal component (PCA factor 1) is, by definition, the linear combination of single reactivity measurements which represents a maximum of information about a multivariate dataset in terms of total variance, here 34%. Since this is the most systematic quantitative representation, the analysis will be continued based on principal components. In PCA, factor loads, i. e., coefficients indicating the relative contribution of measured parameters to the respective factor score, can be interpreted according to whether this factor represents a general level difference or a pattern-related property. Here, the factor-1 score contained only positive loads and, thus, represents a modified measure of total reactivity. Factor-1 scores indeed showed similar groupwise distributions as band number or total reactivity and were most discriminating. Among the first few principal components, factor 1 was the only one with interpretable properties.Further properties of factor-1 scores derived from IgG anti-brain reactivities are shown in Fig. P. falciparum , or to PfMSP1-19 . Nevertheless, as can be seen in Fig. P. falciparum, these correlations are mainly due to the fact that all parameters were relatively enhanced in asymptomatic subjects. Considering asymptomatic and symptomatic individuals separately, correlations are no longer evident. Namely, some HBV-free symptomatics had elevated IgG anti-P. falciparum, but without a parallel increase in factor-1 scores.Fig. P. falciparum infection in their last clinically diagnosed episode were higher than for those who had had P. vivax malaria (Mann-Whitney test: p < 0.01). Qualitatively, this appeared to be the case for parasite carriers as for aparasitaemic subjects with reported previous malaria , but not in the presence of HBV coinfection. Also similarly to IgG anti-brain, this significance increased when only subjects living less than 10 years in Apiacas were considered (p = 0.002, not shown). As can be seen in Fig. E. coli IgG reactivities indeed correlated highly with anti-brain-IgG-derived factor-1 scores . Finally, IgG anti-E. coli-derived factor-1 scores were higher in subjects reporting previous infection with P. falciparum, compared to P. vivax infection , and also with times spent in Apiacas , but not within aparasitaemic subjects. As for IgG anti-brain, there was no significant correlation with blood parasitaemia levels, but with specific IgG anti-P. falciparum .IgM reactivities to brain proteins were measured using the same extract as described for IgG (data not shown). Generally, a smaller number of bands was detected. The IgM-derived factor-1 included only positive loads like the IgG-derived one. The scores of both were correlated , and, as for IgG, IgM-derived factor-1 scores were higher in asymptomatic than in symptomatic parasitaemic individuals free of HBV. However, IgM-derived factor-1 did not significantly discriminate between them, except when only individuals living less than 10 years in Apiacas were considered (p = 0.013). No significant difference was found either in respect to previously or presently detected E. coli reactivities. Instead, the first two principal components (factor-1 and factor-2) of the co-calculation both significantly distinguished between HBV-free asymptomatic and symptomatic parasite carriers , and, as above, not between those infected with HBV. Scores of both factors were enhanced in asymptomatic subjects. Since principal components in the same set are by definition uncorrelated, these two factors appear to represent separate effects associated with clinical states.Finally, principal components were calculated for IgG and IgM reactivities to human brain proteins together Fig. . SurprisP. falciparum IgG . However, like IgG-derived factor 1, scores were also elevated in respect to P. falciparum but not P. vivax pre-exposure .Factor-1, characterized by positive loads as the above described, was however positively influenced primarily by IgM reactivity, and properties of factor-1 scores were similar to those of the IgM-derived factor-1. Within parasitaemic subjects, scores increased with age , with exposure times in Apiacas and in endemic areas (+0.29 and +0.26, but not significant), and were correlated with anti-P. falciparum or with pre-exposure to Plasmodium species. Instead, scores were slightly higher in subjects presently infected with P. vivax, but not P. falciparum . Neither factor correlated significantly with parasitaemia.In contrast, factor-2 scores were positively influenced mainly by IgG reactivity, while a number of IgM bands had a negative impact. Thus, factor 2 can be said to represent a certain IgG/IgM relationship. Remarkably, among HBV-free parasite carriers, the highest scores were characteristically found in asymptomatics who had only been exposed to malaria in endemic areas for a limited time. Factor-2 scores showed no significant association with IgG anti-E. coli-derived factor-1 (not shown).Global IgG reactivity and subtype-specific reactivity have been previously assayed including some subjects examined here . Among pAutoantibodies detected in the context of malaria infection are an old observation, reviewed in ,28. TypiThe simultaneous standardized detection of multiple immunoglobulin reactivities on immunoblots, as utilized in the present study, is most likely to represent natural antibody repertoires and, therefore, a way to approach the problem of repertoire description in an unbiased way, avoiding a potentially misleading disease- or otherwise antigen- or specificity-oriented vision. Extracts of human tissue (brain) or whole bacterial cultures were used as ligands for antibodies with the aim of relating eventual changes in binding patterns to clinical status, and comparing whole reactivity patterns by multivariate analysis. The same semiquantitative immunoblot technique has previously demonstrated characteristics of natural IgM and IgG reactivity patterns ,31,32, aE. coli proteins, unrelated to parasites, could differentiate strikingly between asymptomatic and mild symptomatic malaria infection. Asymptomatic subjects generally showed elevated reactivity, which was concordant but far more characteristic when compared with anti-plasmodial IgG measures in the same sample. Thus, Plasmodium infection is able to alter reactivity patterns to target proteins without relation to the parasite particularly in asymptomatic subjects. Only co-infection with HBV, known to be frequent in our study population [E. coli. In both cases, the same major properties were represented by a single respective principal component, characterized by positive loads. Both were highly correlated and when reactivity to both extracts was analysed together, they coincided in a single PCA factor with maximal discriminatory power, allowing to separate 13/15 (87%) of the HBV-free asymptomatics from symptomatic patients. Thus, IgG reactivity patterns to brain and to E. coli proteins behaved somewhat like fragments of a hologram which, added together, show the same image with increased resolution.The present results show that reactivity patterns to human brain and pulation , heightepulation . RemarkaOther than previous data on malaria-associated autoantibodies, these observations cannot easily be attributed to specific immune responses, despite a positive correlation with anti-parasite reactivity. Instead, they probably reflect another phenomenon well known for malaria and other parasite infections, that of polyclonal lymphocyte activation. Polyclonal activation, however, is often discussed as an immune evasion mechanism, beneficial primarily for parasites. Our results show in contrast that the production of polyclonal IgG was associated with protection in asymptomatic parasite carriers, and that polyclonal reactions appeared to have at least short-term protective effects. A reason for this discrepancy may be that earlier studies of polyclonal activation  assessedRemarkably, in our study, asymptomatic and symptomatic infections were indeed best discriminated in subjects with relatively short exposure times. In contrast to previous data on autoantibodies, this indicates that the reactivity shift in asymptomatic subjects described here does not result from long-term adaptation to the parasite, but is representative of an intermediate state of adaptation. This interpretation is most clearly supported by the result of joint analysis of IgM and IgG reactivity to brain proteins, where two distinct principal components appeared. Factor-1, representing a general elevation of all reactivities with dominant IgM impact, shows an evident overall time dependency in parasite carriers and may, thus, reflect long-term adaptation. However, factor-2 discriminated asymptomatic from symptomatic infections best and, most characteristically, those asymptomatic subjects who had been least exposed. Factor-2 represented a relative dominance of IgG reactivity against some IgM. Thus, in order to remain asymptomatic, particularly infected subjects without long-term adaptation to the parasite may require a pattern of natural antibody production dominated by IgG but not IgM.P. falciparum has shown that antibodies play a crucial role in controlling blood stage parasitaemia [This is in principal agreement with known protective effects of antibodies. Classically, passive transfer of hyperimmune IgG to patients infected with sitaemia ,43. The sitaemia . Such cysitaemia . In contsitaemia -47.The targets of cytophilic antibodies include diverse and highly variable parasite-encoded proteins, but also host proteins such as 'band 3' , which iPlasmodium infection appear to depend on internal activation states which can be distinguished by different patterns of polyclonal antibody production. Particularly, asymptomatic but not symptomatic infection of HBV-free subjects without extensive pre-exposure to malaria was characterized by elevated polyclonal IgG reactivity in absolute terms and in relation to IgM, furthermore appearing to be triggered by previous P. falciparum but not P. vivax infections.This study shows that simultaneous detection of IgM and IgG reactivities to a broad range of targets can reveal remarkable properties which are not easily accessible by the usual specificity-focussed approaches. Thus, clinical states in CJFF and EMB did the field work and collected the samples. NMV designed and supervised the study. AC performed preliminary experiments. CF, LFG and ASN did the immunoblots and analysed the results. CF prepared software, performed statistics and wrote the manuscript.


Atherosclerosis lesions contain abundant immunoglobulins complexed with oxidized LDL (OxLDL) that are endocytosed by macrophages to form foam cells. While recent evidence supports a role for the macrophage scavenger receptor pathway in 75–90% of OxLDL uptake, in vitro evidence suggests another potential uptake pathway could involve autoantibody binding to IgG subclass-specific Fc receptors.To address this mechanism from an in vivo standpoint, the objective of this study was to utilize flow cytometry to prospectively determine monocyte Fcγ (FcR) I, II, and III receptor expression levels in patients with acute coronary syndrome (ACS, n = 48), diabetes mellitus (DM, n = 59), or neither (C, n = 88).Increased FcR I expression was found in the ACS versus DM groups [geometric mean, (95% CI) = 2.26 (2.07, 2.47) versus 1.83 (1.69, 1.98) (p < 0.001)] and versus C [1.90 (1.78, 2.03) (p = 0.005)]. Similar relationships were found with both the FcR II receptor [ACS mean = 4.57 (4.02, 5.19) versus DM 3.61 (3.22, 4.05) (p = 0.021) and versus C 3.86 (3.51, 4.24) (p = 0.09)] and FcR III receptor [ACS mean = 1.55 (1.44, 1.68) versus DM 1.36 (1.27, 1.46) (p = 0.038) and versus C 1.37 (1.30, 1.45) (p = 0.032)]. There was no difference between DM and C groups in FcR I, II or III expression.This in vivo data supports a possible second OxLDL-autoantibody macrophage uptake mechanism through an Fc receptor-mediated pathway and a potential relationship between atherosclerotic plaque macrophage FcR levels and ACS. Atherosclerosis is a chronic inflammatory process that results from hyperlipidemia and complex interactions involving other genetic and environmental factors. OxLDL plays a central role in the atherogenic process through generation of highly immunogenic neodeterminants for the immune system . Natural2 fragments were substituted for an intact anti-apo B antibody [Recent evidence supports the macrophage scavenger receptors SR-A and CD36 as a mechanism responsible for up to 90% of uptake of OxLDL that leads to foam cell formation with no other scavenger receptors compensating for their absence in a knockout mouse model . A seconantibody . Though antibody .Immune complexes with modified lipoproteins have recently emerged as an important coronary artery and macrovascular disease risk factor in DM ,13. EvidIn recent work our group has shown FcγRII expression to be increased in the platelets of patients experiencing an acute atherothrombotic event, or who are healthy with two or more atherosclerosis risk factors . Non-acuIn view of the controversy regarding the mechanism of cholesterol uptake by monocyte-macrophages in atherosclerosis and diabetes ,21 and tAll 195 patients were randomly chosen for study participation from a larger group who fit study inclusion criteria and gave written informed consent. Forty-eight patients in the study had heart disease (HD) and were within 2 hours of onset of an ACS , 59 were DM outpatients (both type 1 and type 2 were included) with no known history of HD, and an additional 88 outpatients without HD or DM were randomly chosen as controls (C). The number and nature of ARF's was documented for each group , anti-FcR II (IV.3), anti-FcR III (3G8), or a negative class-specific control antibody (MOPC-141, Sigma) was added. Following a 15 minute incubation, 5 μl of FITC-sheep anti-mouse antibody (Sigma) was added and a second 15 minute incubation done. The pellet was washed twice before 5 μl of phycoerythrin-conjugated anti-CD14 (Becton Dickinson) was added and incubated for 15 minutes. The solution was diluted with 1 ml ammonium chloride lysing solution and incubated for 10 minutes or until the solution was clear, and the pellet washed twice before flow cytometry to determine relative receptor expression levels was carried out according to the manufacturer's specifications (Becton Dickinson). Monocytes were readily identifiable from other blood cells by their forward and side scatter properties along with CD14 expression.Following establishment of saturating concentrations for each antibody, mean inter and intra-individual coefficients of variation for each of the three Fc receptors were calculated in the antibody labeling assay employing blood samples from five healthy laboratory volunteers who met control patient criteria and FcR I values found to be 3.2 and 9.7%, FcR II 11.7 and 16.1% and FcR III 4.1 and 26.9% respectively.The ratios of FcR I, FcR II, FcR III and MOPC-141 antibody expression were calculated for each patient and the logarithm of the ratios were used to analyze the results. An analysis of variance (ANOVA) was performed to compare the groups of (1) these 3 ratios with the 3 groups of patients, (2) the 3 ratios with the total number of major ARFs, (3) the 3 ratios with each ARF, such as hypertension, diabetes, and smoking, and (4) the 3 ratios with MI, and unstable angina in group 1. The overall p-values were based on the ANOVA F-test. If the overall F-test p-value < 0.05, the LSD method (least significant difference)  was usedSignificantly increased FcR I expression was found in ACS patients compared with DM patients [geometric mean FcR I expression, (95% CI) = 2.26 (2.07, 2.47) versus 1.83 (1.69, 1.98) (p < 0.001)] and compared with C [1.90 (1.78, 2.03) (p = 0.005)] , compared to absence of current cigarette smoking, 1.91 (1.82, 2.01) (p = 0.010)  ,24, the No difference was noted in expression of any Fc receptor between the diabetes and control groups. Given that the control group was the largest of the three and that there was a difference noted in the expression levels of all three receptors between the two smaller groups, it can be concluded that increasing the sample size of either of the two groups with similar expression levels could possibly lead to an increase in the difference between them, but this difference is still unlikely to be of any significance compared with that between the ACS group and the other two groups.There was an interesting trend in both the unadjusted and adjusted analyses Table  in whichAn issue pertinent to this study would be the possible effects of inflammation on monocyte FcR expression levels. In diabetes patients it would be reasonable to speculate a significant number of activated cells in the circulation would be unlikely since in most patients with atherosclerosis the inflammatory reaction is circumscribed to the vessel wall. Overexpression of monocyte Fc receptors may have been a possibility in ACS however. Figure Observational studies of this nature have certain limitations related to their design and patient selection. As an example, selection bias needs to be considered in any study involving a population of volunteers associated with an atherosclerosis prevention trial (Groups 2 and 3). Even though the ethnic composition of the groups was the same, the implication of this selection bias is that the results are not generalizable to the population at large and this is attested to by the demographic characteristics in Table Through bridging innate and adaptive immune processes, macrophages play an important role in the progression of atherosclerosis and mediating plaque disruption that is considered to be the inciting event in the majority of coronary thrombi ,28. In tThe overall effect of the humoral immune response on atherogenesis is likely to be complex. Of note, for example, is that the FcγRII receptor has an inhibitory role on B cells that are rarely seen in plaques, while it mediates phagocytosis and release of inflammatory mediators from cells of the myeloid lineage when cross-linked by immune complexes ,32. ThusThe authors declare that they have no competing interests.


In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously.Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related.Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and α-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi. Phylogenetic relationships among animals, fungi, and plants have been a controversial issue. Although fungi traditionally had been considered more closely related to plants than to animals, Whittaker and Margulis  classifiEukaryotic RNA polymerase III (RNAP III) transcribes a variety of small RNAs like tRNAs, 5S rRNA, and several viral RNAs . Short iSaccharomyces cerevisiae, a subunit of 138 kDa binds to a B-block, and its gene which is called TFC3, has been cloned [TFC3 is a single-copy gene and essential for cell viability [S. cerevisiae 138 kDa subunit, although all of them bind to similar DNA regions, suggesting a significant degree of evolutionary divergence for RNAP III factors [S. pombe TFIIIC from the S. pombe sequence database by homology searches using the S. cerevisiae TFIIIC subunits as queries; one of these subunits named Sfc3p, is similar to the S. cerevisiae B-block binding subunit. It has been thought that, like the B-block binding subunit from S. cerevisiae, Sfc3p does not share homology with the human B-block binding subunit. On the other hand, Sfc1p, Sfc4p, and Sfc6p, which are other subunits of S. pombe TFIIIC, show homologies not only to S. cerevisiae TFIIIC subunits but also to human TFIIIC subunits [in vivo, and the isolated Sfc3p complex is active in the in vitro RNAP III-mediated transcription of S. pombe tRNA genes [In TFIIIC of n cloned . The open cloned . TFC3 isiability . In humaiability ,15, and  factors . Huang e factors  identifisubunits . It has NA genes .in vitro [Arabidopsis proteins, which appear to be the homologs of both the human and yeast subunits.The lack of clear homology between the yeast and human B-block binding subunits is strange, because transcription of tRNA genes by RNAP III is initiated by the binding of these subunits to the B-block regions, and the internal promoter sequences are highly conserved between vertebrates and yeasts (see above). Interestingly, bacterial tRNA genes also have conserved RNAP III promoters Fig.  18]. Al. Al18]. S. cerevisiae are aligned using the early Clustal program, they show little sequence similarity [Chironomus tentans (GenBank identification number: 18073910) is structurally similar to the human B-block binding subunit [C. tentans protein as a query, the best hits were the human and rat B-block binding subunits with 28 % identities. A comparable level of similarity has been found also in the Drosophila hypothetical protein, but no other related proteins were identified in the database [Chironomus and Drosophila proteins were hit (23 % and 20 % identities respectively) , Mus musculus (GIs 38087408, and 21595152), Caenorhabditis briggsae (GI 39594187), and Arabidopsis thaliana (GIs 25402830, 9665127, 15218016, 25404859, and 30685327), were also hit with low E-values: E-values were 0, 0, 0, 0, 0, e-169, e-167, e-148, and 5e-87, respectively  with 0.0, S. pombe Sfc3p with e-159, the Magnaporthe grisea hypothetical protein (GI 38108450) with e-121, the Saccharomyces bayanus hypothetical protein fragment (GI 10863079) with e-100, and the Arabidopsis hypothetical protein (GI 15218016) with 0.002 (data not shown). It is noteworthy that the Arabidopsis protein of GI 15218016 was hit although the E-value was not so good. This protein was hit also in the PSI-BLAST search using the human B-block binding subunit as a query . When Iery Fig. .S. pombe Sfc3p protein as a query. Fig. Magnaporthe grisea and Neurospora crassa hypothetical proteins (GI 38108450 and GI 32412546), were hit with very good E-values (0 and e-168 respectively) . Four hypothetical proteins of Arabidopsis thaliana were hit with E-values worse than those of the two fungi proteins but well above the threshold: GI 15218016 with e-113, GI 25402830 with e-88, GI 9665127 with 2e-88, and GI 25404859 with 7e-70 ; the N-terminal 350 amino acid sequences of the rat and human subunits showed similarities to the N-terminal region of S. pombe Sfc3p protein.Next, I performed a PSI-BLAST search using the ly) Fig. . The Asp-70 Fig. . The Araery Fig. . While bes Figs.  and 2B. Arabidopsis proteins (GIs 25402830, 9665127, 15218016, and 25404859), were hit with low E-values in both of the PSI-BLAST searches using the human and S. pombe subunits as queries. Thus, I performed a PSI-BLAST search using one of these Arabidopsis proteins (GI 25402830) as a query. Fig. Arabidopsis proteins were hit with E-values of 0, and three of them were identical to the proteins which were hit in the PSI-BLAST searches using the human and S. pombe subunits as queries. In addition to these, the human and rat subunits were hit with low E-values (e-132 and e-108 respectively). The N-terminal 700 amino acids of the human and rat subunits were most similar to the Arabidopsis proteins, but the short regions of the C-terminal ends also were similar  (data not shown). However, it should be noted that these Arabidopsis proteins were hit with E-values better than threshold when a PSI-BLAST search was performed using the S. pombe B-block binding protein as a query.The four lar Fig. . The hyp830 Fig. . The B-bArabidopsis proteins were similar to the B-block binding subunits of vertebrates and yeasts, despite the fact that vertebrate and yeast subunits share no recognizable homology [Arabidopsis proteins of GIs 25402830, 9665127, 15218016, and 25404859 represent a 'missing link' between the vertebrate and yeast B-block binding subunits.It is interesting that the four homology -17. ThesDrosophila are compared, the most conserved sequences are located within the N-terminal two-thirds of the proteins, while the C-terminal one-third is much less conserved [Chironomus tentans protein, which probably binds to the B-block in the RNAP III promoter, also has similarities to the human, rat, and Drosophila proteins in the N-terminal region [Arabidopsis homologs (GIs 25402830, 9665127, and 15218016), mainly are located in the N-terminal halves of the proteins , are located in the N-terminal one-third regions of the proteins   proteins are hit with E-values of e-29, 3e-7, 6e-54, 2e-20, 2e-39, and 2e-21 respectively (data not shown). These regions contain the domains shown to have sequence similarities [S. pombe B-block binding subunit as a query, alignments consisting of its C-terminal region and each of the Magnaporthe, Neurospora, and S. cerevisiae sequences were generated, but no homology to the C-terminal regions of the Arabidopsis proteins was suggested  was hit after four iterations; aa positions 1032–1146 of the S. cerevisiae subunit aligned to positions 1673–1774 of the Arabidopsis protein (GI 9665127) with an E-value of 5e-21 (data not shown). Consequently, the C-terminal sequences of the human, rat, mosquitoes, Drosophila, Arabidopsis and fungi proteins, can be aligned by Clustal W  were predicted to contain three α-helices with corresponding locations  as queries, did not significantly hit any of the plant sequences except the Arabidopsis proteins shown in this study. Therefore, I performed a tblastn search of the Viridiplantae database using the Arabidopsis protein GI 25402830 as a query. The Oryza sativa sequence GI 37990182 was hit with the best E-value (8e-85) among Viridiplantae sequences, except the Arabidopsis genes already described . The Arabidopsis sequences at aa positions of 83–144, 234–262, 698–713, and 1812–1847 showed similarities to sequences corresponding to bp positions of 539027-538842, 527862-537776, 534044-533997, and 526942-526835 in the C. reinhardtii scaffold 16, with E-values of 9.7e-5, 80.2, 80.2, and 9.7e-5 respectively   Fig. . Althoug 10 Fig. . The resS. pombe subunit as a query, limiting the search to the fungi database. The sequence hit with the best E-value among the basidiomycete sequences, was the Ustilago maydis protein GI 461005911 . Moreover, a PSI-BLAST search performed using the Arabidopsis homolog (GI 25402830) as a query of the fungi database also did not hit the Ustilago sequence of GI 46100591 with an E-value better than 10, although the S. cerevisiae and S. pombe B-block binding subunits were hit with E-values of 0.93 and 9.7 respectively (data not shown). These results indicate that animal and plant B-block binding subunits are more similar to the yeast subunits than to the Ustilago protein GI 46100591. The overall results in this section demonstrate that the greater similarity between the plant and animal B-block binding proteins extends to the green alga protein, and the greater differences in fungi go beyond the yeast taxa.Because yeasts may not be representative of all fungi, it is important to demonstrate that the greater similarity between the animal and plant B-block binding proteins extends beyond the yeast taxa. To this end, I searched for homologs of the B-block binding protein in the basidiomycete genomes. I performed a PSI-BLAST search using the 911 Fig. . The Cryngi Fig. . SubsequArabidopsis. The Arabidopsis proteins (GIs 25402830, 9665127, 15218016, and 25404859), which show strong similarity to B-block binding subunits, are the hypothetical proteins translated conceptually from the nucleotide sequences of the chromosome I [[]. The lengths of the inferred amino acid sequences of three of these Arabidopsis proteins (GIs 25402830, 9665127, and 15218016), are close to those of the amino acid sequences of the vertebrate subunits . The PSI-BLAST was limited searches of the eukaryota databases, when the amino acid sequences from the Oryza and Chlamydomonas coding regions were used as queries. The fungi database was used in a search for homologs of the S. pombe B-block binding subunit in basidiomycetes. PSI-BLAST was run three times for each of the queries. To search for homologs of the Arabidopsis protein GI25402830 in the plant sequences, the tblastn program was used [Arabidopsis protein GI25402830 in the Chlamydomonas reinhardtii sequences, the tblastn program at the Joint Genome Institute website , was used. Clustal W in the EMBL-EBI website  was used to align the multiple amino acid sequences [) [To search for similarities between the B-block binding subunits of vertebrates and yeasts, I used the PSI-BLAST program in the NCBI website . PSI-BLAwas used . To searequences . Clustalences [) .


Violent political conflict is on the front pages, in Iraq, Afghanistan, and Sudan. This provocative piece discusses lessons we can learn from past conflicts in dealing with future ones We must define better practice and promote organisational learning Violent political conflict, and its impact, is again on the front pages—in Iraq, Afghanistan, and Sudan. While the situation in Darfur is now particularly urgent (see sidebar) ,2,3,4,5,Such violent political conflicts stir us—the global health community—to discover our own humanity amidst the bloodshed. How best should we respond? Despite unique features in each setting, we must learn lessons from previous conflicts to help guide our response to current and future ones. There are six key lessons that emerge from studying health in conflict settings.Violent conflict is driven by politics and economics . Complex political emergencies (1) occur within and across state boundaries, (2) have political antecedents typically relating to competition for power and resources, (3) are protracted in duration, (4) express existing social, political, economic, and cultural structures and cleavages, and (5) are often characterised by one sector preying on other parts of the community [ommunity . Damage ommunity . A politommunity .The current crisis in Darfur reflects a devastatingly acute episode in the chronic internal conflict that has plagued Sudan since 1983. The cost of this conflict has been enormous: over 2 million lives lost, over 628,000 refugees from Sudan in neighbouring countries, and over 4 million people internally displaced [In southern Sudan, the conflict has led to widespread ill health and has severely compromised the well-being of women and children. Indicators of immunisation, nutrition, primary school completion, and antenatal care are among the worst in the world. About 95,000 children under five years old died last year, most from preventable disease .Statistics from UNICEF are chilling: ‘A girl born in southern Sudan has a better chance of dying in pregnancy or childbirth than of completing primary school….One in nine women dies in pregnancy or childbirth but only one in a hundred girls completes primary school’ .Communities in Darfur face ongoing violence from militia supported by the government of Sudan. The fighting has resulted in large-scale destruction of villages, rape, and kidnapping. About 15,000–30,000 lives are estimated to have been lost from January 2003 to June 2004 [Despite widespread concern, information gaps abound, and humanitarian agencies report having access to only a fraction of those most affected. Yet, genocide is taking place in real time.Appreciating context is crucial. The nature of the conflict—its background, history, and the different forms of violence involved—will greatly influence health outcomes. Most conflicts are today intra-national rather than international [national . Internanational . Recent national .Better care can save lives. Emergency relief efforts are increasingly based upon empirical evidence, and priority health issues are much more effectively addressed than previously. Emphasis is typically placed upon disease surveillance, immunisation, control of infectious diseases, reproductive health, water and sanitation, shelter, and nutrition [utrition . Mental utrition .We need enhanced accountability for humanitarian action. Despite a developing evidence base for health-related humanitarian action, evaluations of humanitarian activities have found ongoing problems. These include poor standards of delivery, duplication of efforts by different agencies, lack of coordination, and failing to learn from prior experience. The Sphere Project has advocated minimum standards for the delivery of humanitarian assistance, and has established a “Humanitarian Charter” (http://www.sphereproject.org). The project's objectives and achievements have been to improve the quality of humanitarian action and promote a movement concerned with the rights and dignity of those caught up in war and disaster [http://www.alnap.org) seeks to ensure that lessons are learned, distilled, and disseminated. At a meeting in Stockholm in June 2003, key international donors committed themselves to ‘good humanitarian donorship’, which recognises the importance of promoting standards in humanitarian action [disaster ,16. The n action . Howevern action .Militarization of humanitarian efforts is problematic. Multinational military forces have played a major part in recent conflicts in Kosovo, East Timor, Sierra Leone, Iraq, and Afghanistan. The military has become increasingly involved not only in waging war but also in seeking to win the peace; it is increasingly active in delivering emergency relief. It not only provides services—sometimes necessary to deliver needed relief—but also seeks to ‘win hearts and minds’ while operating within structures responsive to military and foreign policy directives. The result has seen a blurring of the separation between military and humanitarian efforts [ efforts . This ca efforts . Emergin efforts .The transition from emergency relief to development is poorly managed. The objectives of humanitarian relief activity (saving lives and livelihoods) differ from those of development (building sustainable systems, promoting equity, building systems of governance, and eradicating poverty). In each phase there are different actors, strategies, and approaches. The increasing politicisation of humanitarian intervention [rvention ,22 bringDespite the knowledge we have gained on responding to violent political conflict, many important gaps remain.We still do not hear the voices of those most affected or of the service providers seeking to assist. The reality of people's experiences is inadequately appreciated ; whateveWe know little about how communities and systems survive adversity. In most settings, the inherent ability and ingenuity of people and systems allows them to withstand instability and insecurity. Health personnel and health systems could play a valuable role in these fragile settings—assisting individuals, communities, and systems to further develop their coping strategies, adaptations, and responses. But whether health systems do so and how is unclear. Failing to support and maintain these systems may result in much greater challenges when we seek at a later stage to resuscitate them.We also know relatively little about whether the health sector can indeed make a special contribution to building the peace. While it has been forcefully argued that the health sector is uniquely placed to play a role in peace building , the eviPerhaps the most important gap of all is between observing lessons and putting them into practice. We urgently need to transform evidence and experience into sound policy. We need more sophisticated policy analyses, more sensitive policy-making, and more relevant research. Policy in these difficult areas will never be entirely evidence-based—often it will at best be ‘evidence-informed’. Our objective must be to promote organisations and systems that are able to reflect on experience, work with partners to critically analyse and learn, and thereby formulate better responses. Violent political conflict will continue to challenge the global health community. International policy-makers and funders must support more extensive documentation and reflection: the building blocks of better practice.


The molecular events, following ischemia and reperfusion (I/R) of the liver during transplantation are largely unknown. There is evidence that apoptotic and necrotic events may take place, and occasionally result in primary graft dysfunction. We herein report two cases, where significant I/R injury correlated with the development of liver calcification and primary liver dysfunction.Both patients with clinical and biochemical evidence of primary graft dysfunction demonstrated calcification at light and electron microscopy levels. In addition, one patient had macroscopic evidence of calcification on cross-sectional imaging. Both patients died secondary to the sequelae of the graft dysfunction.Severe I/R-induced injury to the liver, clinically leads to graft dysfunction. This is due to advanced apoptotic and/or necrotic events at the hepatocyte level that may, on the most severe form, lead to calcification. The study of microcalcification at the early posttransplant period could provide insight in the events taking place following significant ischemia/reperfusion-induced injury to the graft. Liver transplantation (LT) remains the only treatment for end-stage liver disease. However the transplant procedure mandates cold perfusion, hypothermic storage, warm ischemia and warm reperfusion of the graft, resulting in Ischemia/Reperfusion (I/R)-induced injury to the transplanted graft. Although the introduction of the University of Wisconsin solution (UW) has improved clinical outcomes, I/R injury remains one of the major clinical problems following LT leading, in cases of marginal donor quality or prolonged cold or warm ischemic time, to the development of graft dysfunction or non function.Recently the cellular events following liver I/R during LT have been brought to sharp focus. Nevertheless, it is still controversial if the major mode of cell death during I/R is apoptosis and/or necrosis . It has In this report, using a combination of cross-sectional imaging, histochemistry, and light (LM) as well as transmission electron microscopy (TEM) we identified liver calcification, in two grafts with severe I/R injury following LT.A 65-year-old man of Asian origin underwent LT at our institution on June 2002, for decompensated Hepatitis B related cirrhosis. The patient did not have a history of abnormal calcium metabolism, or hyperparathyroidism. Upon listing his serum ionized calcium was 1.07 mmol/L. The donor was a previously healthy, 42-year-old male, who suffered intracranial bleeding following a motor vehicle crash. He did not suffer any period of hypoxia but he was hypotensive (SBP = 90 mm Hg) prior to procurement. His serum liver function tests were normal prior to harvesting (AST 32 U/L, ALT 40 U/L). The organ was procured by our institution's transplant team. During procurement the liver was found to be well perfused with no focal injuries and no macroscopic evidence of steatosis.The recipient underwent an uncomplicated conventional LT without the use of a veno-venous bypass. There were no periods of hypoxia or severe hypotension during transplantation. The cold ischemic time of the graft was 8 hours while the warm ischemic time was 45 minutes. The graft reperfused well and no biopsies were taken. Intravenous methylprednisolone (500 mg) was administered intraoperatively, and postoperatively the patient received induction with Antithymocyte Globulin, which is the protocol followed at our institution. During transplantation the patient received a total of 4 units of packed red blood cells (PRBCs) and 6 units of fresh frozen plasma (FFP).At the time of LT, the international normalization ratio (INR) was 1.53, while serum total bilirubin was 103 μmol/L Figure . On postA 55-year-old man of Greek origin underwent LT at our institution for ethanol and hepatitis B related cirrhosis, as well as a large hepatocellular carcinoma. The patient did not have a history suggestive of hyperparathyroidism or abnormal calcium metabolism, and upon listing his serum ionized calcium was 0.94 mmol/L. The donor was a previously healthy, 52 year-old male who suffered a closed head injury during a motor vehicle crash. The organ was procured by our transplant team. Prior to procurement the donor did not suffer any periods of hypoxia or hypotension. His liver function tests were normal prior to harvesting (AST 36 U/L, ALT 45 U/L). During procurement the liver was found to be well perfused, with no evidence of aberrant vascular anatomy and no evidence of trauma. Macroscopic examination of the liver did not show any evidence of steatosis. The recipient underwent a conventional LT without venovenous bypass and without intraoperative hypotension. The cold ischemic time was 10 hours while the warm ischemic time was 40 minutes. Following reperfusion, the graft appeared well perfused and again no biopsies were taken. Intraoperatively the patient received 500 mg of methylprednisolone, and postoperatively he was induced with Antithymocyte Globulin. During transplantation the recipient received 3 units of PRBCs and 6 units of FFP. In the early postoperative period the patient had to be explored once for retroperitoneal bleeding associated with hypotension (SBP< 90 mm Hg).nd and 3rd postoperative day (4700 U/L and 6598 U/L respectively). At that time, ultrasonography demonstrated uniformly patent vessels , while Computed Tomography (CT) showed areas in the right lobe of the liver isodense with the spinal column  were transplanted without any problems. Neither recipient had any evidence of calcium metabolism problems, since both had normal serum calcium upon listing. Both recipients had an anticipated intraoperative course without periods of hypotension and without massive transfusion requirements. Finally, both grafts did not demonstrate any vascular problems in the postoperative period by Ultrasonography or CT scan examination or any evidence of intrahepatic thrombosis in postmortem or explant examination.Ischemic stress has been previously reported to induce calcium accumulation at the cell level, either by impaired energy metabolism and/or plasmalemmal alterations. This elevated intracellular calcium concentration is responsible for cytoskeletal modifications, which alter cell shape, and for the activation of phospholipases, which results in perpetuation of membrane damage and finally, mitochondrial calcification .Although, the crystal shape, composition and organization of HA in our samples are similar to those observed in bone and cartilage , as wellThe observation of calcified, vacuole-like structures in hepatic cells from these two livers could be suggestive of mitochondrial calcification. In addition, the extensive presence of phagocytic structures in the pre-calcified regions of these livers suggests an intense apoptotic/necrotic process undergone after I/R injury in these regions. Further investigation, however, is required to understand the mechanism(s) and the mode of calcification in the liver.In conclusion, we believe that the described phenomenon is underreported at least in the liver transplant literature. Furthermore, it appears that there is a correlation between the development of severe I/R injury leading to apoptosis and/or necrosis and calcifications detectable even by light microscopy. We think that the development of microcalcifications should be studied more extensively in the context of I/R injury following liver transplantation. Although, such a phenomenon appears to correlate with significant I/R injury, evident by biochemical data, it has the potential to be provide further information on the pathways of severe I/R injury post transplant.None declared.GNT: conceived the study and wrote the manuscriptMA: carried out the electron microscopy studiesEC: participated in the design and analyzed the light microscopy results, critical review of the manuscriptAE: analyzed light microscopy resultsAV: carried out electron microscopy studiesPM: participated in the design of the studyAll authors read and approved the final manuscript.I/R: Ischemia and ReperfusionLM: Light MicroscopyTEM: Transmission Electron MicroscopyHRTEM: High-resolution Transmission Electron MicroscopyEDS: Energy Dispersive SpectroscopyHA: HydroxyapatiteThe pre-publication history for this paper can be accessed here:


To evaluate the efficacy of diode laser photocoagulation in patients with central serous retinopathy (CSR) and to compare it with the effects of argon green laser.Thirty patients with type 1 unilateral CSR were enrolled and evaluated on parameters like best corrected visual acuity (BCVA), direct and indirect ophthalmoscopy, amsler grid for recording scotoma and metamorphopsia, contrast sensitivity using Cambridge low contrast gratings and fluorescein angiography to determine the site of leakage.Patients were randomly assigned into 2 groups according to the statistical random table using sequence generation. In Group 1 (n = 15), diode laser (810 nm) photocoagulation was performed at the site of leakage while in Group 2 (n = 15), eyes were treated with argon green laser (514 nm) using the same laser parameters. Patients were followed up at 4, 8 and 12 weeks after laser.The mean BCVA in group 1 improved from a pre-laser decimal value of 0.29 ± 0.14 to 0.84 ± 0.23 at 4 weeks and 1.06 ± 0.09 at 12 weeks following laser. In group 2, the same improved from 0.32 ± 0.16 to 0.67 ± 0.18 at 4 weeks and 0.98 ± 0.14 at 12 weeks following laser. The improvement in BCVA was significantly better in group 1 (p < 0.0001) at 4 weeks. At 4 weeks following laser, all the patients in group1 were free of scotoma while 6 patients in group 2 had residual scotoma (p < 0.05). The mean contrast sensitivity in group 1 improved from pre-laser value of 98.4 ± 24.77 to 231.33 ± 48.97 at 4 weeks and 306.00 ± 46.57 at 12 weeks following laser. In group 2, the same improved from 130.66 ± 31.95 to 190.66 ± 23.44 at 4 weeks and 215.33 ± 23.25 at 12 weeks. On comparative evaluation, a significantly better (p < 0.001) improvement was noted in group 1.Diode laser may be a better alternative to argon green laser whenever laser treatment becomes indicated in patients with central serous retinopathy in terms of faster visual rehabilitation and better contrast sensitivity. In addition, diode laser also has the well-recognized ergonomic and economic advantages. Central serous retinopathy (CSR) is a retinal disorder affecting young adults, characterized clinically by a well-defined, translucent, circumscribed detachment of neurosensory retina at the posterior pole, usually involving the macula. The detachment results from accumulation of transparent fluid in the potential space between retinal pigment epithelial layer and the neurosensory retina. However, the source of this fluid and the exact etiopathogenesis of this disorder remain ill understood. Maumenee  describeAlthough, other treatment modalities have been suggested for central serous retinopathy, argon green (514 nm) laser photocoagulation has been the most widely accepted modality. The 810 nm-diode laser owing to deeper penetration should prove to be more efficacious in the treatment of central serous retinopathy given the current thinking that CSR may primarily be a choroidal disease. The present study was undertaken to test the efficacy of diode laser photocoagulation in central serous retinopathy in comparison to argon green laser. According to the best of our knowledge, this is the first clinical study in which a comparative evaluation between the above two groups has been done in central serous retinopathy.Thirty patients of unilateral central serous retinopathy presenting to the retina services at the Rajendra Prasad Centre for Ophthalmic Sciences were selected for the study. They were randomly assigned into 2 groups according to the statistical random table using sequence generation. As it was a pilot study we decided only the convenient sample size i.e. 15 in each group. The allocation of patients into 2 groups was done by a person who was not involved in the study and the sequence was concealed until interventions were assigned to prevent bias. The patient selection was based on the following inclusion criteria: age less than 50 years, type 1 CSR with a single leak on fluorescein angiography that was at least 300 microns away from fovea, presence of an indication for laser treatment  and no history of any treatment in the past. Patients with multiple leak CSR, type 2 or type 3 CSR or leak at papillomacular bundle or leak within 300 μ from the foveal centre were not included in the study. Patients with other ocular pathology such as choroidal neovascularization, choroidal inflammatory or neoplastic disorder or a congenital optic nerve pit were also excluded from the study.Informed consent was obtained from all the patients included in the study before initiating treatment and an approval of the institute body ethics committee was obtained to undertake the study. In all the patients the following parameters were evaluated before laser treatment and also during the follow-up period: best corrected visual acuity (BCVA) on Snellen's acuity chart, fundus examination by direct and indirect ophthalmoscopy, amsler grid for determining the degree of scotoma and / or metamorphopsia, contrast sensitivity using Cambridge low contrast gratings and fluorescein angiography (Canon CF-60 UD camera using 400ASA, 35-mm black and white film).Technique of scoring scotoma and metamorphopsia on the amsler grid is described below:The amsler grid consists of a 10-cm square divided into smaller 5 mm squares. When it is viewed at one-third of a meter, each small square subtends an angle of 1°. The patient was asked to look directly at the central spot with the uncovered eye and to mark out any black spot, distortion of lines, or blurred area on the grid. The number of small squares within the area delineated by the patient was counted and scotoma and metamorphopsia score was graded numerically as shown in table Patients included in the study were randomly assigned to one of the following two groups. In group 1 (n = 15), the site of leakage was treated by diode laser (810 nm). Laser was performed with Volk area centralis contact lens (Keeler Ltd, Clewer Hill Road, Windsor, Berks, SL4 4AA) and the parameters used were spot size of 100 microns, duration of 100 to 200 milliseconds and power just enough to produce minimal greying. In group 2 (n = 15), patients were treated with argon green laser (514 nm) following the same laser parameters. Patients were followed up at intervals of 4, 8 and 12 weeks after laser treatment and the results were analyzed, which included a comparative analysis of the outcome between the two groups. Descriptive statistics i.e. mean and standard deviation was calculated for each variable at each follow-up for the 2 groups. To see the significant difference between the 2 groups at each point we applied "student 't' test" (unpaired). To see the trend within the group we applied "two way analysis of variance (ANOVA)". 'P' value of 0.05 was considered as statistically significant. BMDP 7.0 statistical package was used for the statistical analysis.The average age of the patients in group 1 was 34.06 ± 2.54 years (range: 30–40 years) and in group 2, it was 34.66 ± 3.23 years (range: 30–42 years). Twelve cases (80%) in group 1 and 13 patients (86.66%) in group 2 were males. All the patients presented to us within eight days (range: 3–8 days) of onset of visual symptoms. The average number of laser spots applied to the leakage site in group 1 was 3.13 ± 0.71 (range: 2–4) and in group 2 it was 3.26 ± 0.77 (range: 2–4). All the patients came regularly for the follow-up as per the protocol during entire period of study. The study was performed during the period between January 1998 and June 2000.'t' test, showed a statistically significant difference at 4 weeks with patients treated by diode laser (group 1) having a better improvement (p < 0.001)  in decimal at the time of presentation in patients assigned to group 1 was 0.29 ± 0.14. Best corrected visual acuity four weeks after diode laser treatment had improved to 0.84 ± 0.23. The BCVA further improved to 0.97 ± 0.17 and 1.06 ± 0.09 at 8 weeks and 12 weeks respectively. In group 2, the mean BCVA at the time of presentation was 0.32 ± 0.16 and it improved to 0.67 ± 0.18 at 4 weeks follow-up after argon green laser photocoagulation. Further improvement in visual acuity to 0.91 ± 0.19 and 0.98 ± 0.14 at 8 weeks and 12 weeks follow up respectively was observed. Statistical analysis of data pertaining to improvement of BCVA in the two groups by student All the patients in both the groups had central scotoma at the time of presentation. At the first follow up at 4 weeks following laser treatment, all the patients in group 1 (n = 15) were free of scotoma while 6 patients in group 2 (n = 15) had residual scotoma of grade 1 . On comparative evaluation, this difference was found to be statistically significant (p < 0.05). At 8 weeks and 12 weeks follow up, none of the patients had residual scotoma; however, 3 patients in group 2 developed a paracentral scotoma corresponding to the site of laser scar.Four patients in group 1 and five in group 2 had complaints of metamorphopsia at the time of presentation. At the first follow up at 4 weeks, 2 cases in group 1 and 4 cases in group 2 were still having grade 1 metamorphopsia. On comparative evaluation, this difference in the two groups was not found to be significant statistically. At 8 weeks and 12 weeks follow up, all patients in both the groups were free of metamorphopsia.'t' test between the two groups showed that there was an initial rapid improvement in contrast sensitivity in both the groups but group I patients had a significantly greater improvement than group 2 patients at all the follow up visits (p < 0.001)  (unpublished data). The mean absolute value of contrast sensitivity at the time of presentation in group 1 was 98.4 ± 24.77. This improved to 231.33 ± 48.97 at the first follow up at 4 weeks following diode laser photocoagulation. It further improved to 286.00 ± 48.52 at 8 weeks follow up and 306.00 ± 46.57 at the third follow up at 12 weeks. In group 2, the mean value of contrast sensitivity at the time of presentation was 130.66 ± 31.95, which improved to 190.66 ± 23.44 at 4 weeks following argon green laser treatment. This value further improved to 210.00 ± 27.25 at 8 weeks and 215.33 ± 23.25 at 12 weeks follow up. Comparative evaluation of the improvement in contrast sensitivity by student In both the groups, the commonest site of fluorescein leakage was the area superonasal to fovea. Fluorescein angiography repeated 4 weeks later showed that in both the groups, all the patients had complete resolution of the leakage site. No recurrence was reported till completion of follow up at 12 weeks.Visual prognosis in central serous retinopathy in most cases is good as they resolve spontaneously. Klein M et al.  studied Encouraging results of laser photocoagulation has been found in various studies which conclude that laser photocoagulation could effectively decrease the time for visual recovery in patients with central serous retinopathy -10. HoweResults of treatment of the leakage site in patients with central serous retinopathy with ruby , kryptonStudies performed with indocyanine green angiography in cases of central serous retinopathy have reported that hyperpermeability of choroidal vessels could be the primary pathology in CSR and retinal pigment epithelial abnormality is only secondary to it -4.Brancato R et al  performeOur observations showed that following diode laser treatment in patients with central serous retinopathy, the recovery of visual acuity was more rapid in comparison to the argon laser treatment group. Further, all the patients treated with diode laser showed complete resolution of scotoma at the first follow up at 4 weeks and none of them developed a scotoma corresponding to the site of laser treatment. In contrast, in patients subjected to argon green laser treatment, there was a significant residual scotoma at 4 weeks and 20% of the patients developed scotoma corresponding to the laser scar.Following laser treatment, contrast sensitivity in both the groups improved rapidly in the first four weeks. However, there was significantly greater improvement in contrast sensitivity in eyes subjected to diode laser. This observation is indicative that diode laser is more beneficial with regard to conservation of contrast sensitivity following laser treatment in patients with central serous retinopathy.Development of subretinal neovascularization following laser treatment for central serous retinopathy has been described earlier in literature . HoweverThe present study suggests that diode laser photocoagulation brings about a faster quantitative and qualitative visual recovery in comparison to argon green laser in eyes with central serous retinopathy. The main aim of performing laser photocoagulation in eyes with CSR is to hasten resolution and this is better fulfilled by diode laser. Further, as argon laser can hit up to the outer retinal layers, it can seal the site of leaking retinal pigment epithelium, but it cannot reduce the fluid overload over the pigment epithelial layer caused by hyperpermeability of choriocapillaries, which can be the cause of recurrence of CSR. As diode laser hits the choroid, which is the exact site of pathology, it should reduce the fluid overload on the retinal pigment epithelium.In the present study, our observations are suggestive that diode laser may be a better alternative to argon laser whenever laser treatment is indicated in patients with central serous retinopathy. In addition, diode laser also has the well-recognized economic and ergonomic advantages of being less costly, more efficient, portable and easy to maintain ,21, whicThe author(s) declare that they have no competing interests.LV designed the study and performed laser. RS performed the data collection and wrote the manuscript. PV followed up the patients. HKT performed the angiography.All authors read and approved the final manuscript.Presented in part in the Annual Meeting of the American Academy of Ophthalmology, 1997 in San Francisco, California.The pre-publication history for this paper can be accessed here:


Postoperative administration of paracetamol or its prodrug propacetamol has been shown to decrease pain with a morphine sparing effect. However, the effect of propacetamol administered intra-operatively on post-operative pain and early postoperative morphine consumption has not been clearly evaluated. In order to evaluate the effectiveness of analgesic protocols in the management of post-operative pain, a standardized anesthesia protocol without long-acting opioids is crucial. Thus, for ethical reasons, the surgical procedure under general anesthesia with remifentanil as the only intraoperative analgesic must be associated with a moderate predictable postoperative pain.We were interested in determining the postoperative effect of propacetamol administered intraoperatively after intraoperative remifentanil. Thirty-six adult women undergoing mammoplasty with remifentanil-based anesthesia were randomly assigned to receive propacetamol 2 g or placebo one hour before the end of surgery. After remifentanil interruption and tracheal extubation in recovery room, pain was assessed and intravenous titrated morphine was given. The primary end-point was the cumulative dose of morphine administered in the recovery room. The secondary end-points were the pain score after tracheal extubation and one hour after, the delay for obtaining a Simplified Numerical Pain Scale (SNPS) less than 4, and the incidence of morphine side effects in the recovery room.p value less than 0.05 was considered as significant.For intergroup comparisons, categorical variables were compared using the chi-squared test and continuous variables were compared using the Student t test or Mann-Whitney U test, as appropriate. A In recovery room, morphine consumption was lower in the propacetamol group than in the placebo group (p = 0.01). Pain scores were similar in both groups after tracheal extubation and lower in the propacetamol group (p = 0.003) one hour after tracheal extubation. The time to reach a SNPS < 4 was significantly shorter in the propacetamol group (p = 0.02). The incidence of morphine related side effects did not differ between the two groups.Intraoperative propacetamol administration with remifentanil based-anesthesia improved significantly early postoperative pain by sparing morphine and shortening the delay to achieve pain relief. Postoperative administration of paracetamol or its prodrug propacetamol has been shown to decrease pain with a morphine sparing effect -4. The eIn order to evaluate the effectiveness of intraoperative paracetamol administration in the management of postoperative pain and morphine consumption, a standardized anesthesia protocol without long-acting opioid is necessary. Thus, for ethical reasons, the surgical procedure under general anesthesia with remifentanil as the only intraoperative analgesic must be associated with a moderate predictable postoperative pain.Therefore, the present study was designed to evaluate the effect of intraoperative administration of propacetamol during remifentanil-based anesthesia on postoperative pain in patients undergoing reduction mammoplasty.After approval by the Local Ethical Committee and written informed consent, 36 consecutive female patients who underwent elective reduction mammoplasty were included. Exclusion criteria were the preoperative use of analgesic drugs; a body mass index ≥ 35, an American Society of Anesthesiology physical status ≥ 3 and sensitivity to paracetamol. Pain evaluation using a Simplified Numerical Pain Scale (SNPS) was explained to the patients during the preoperative anesthetic visit and the day before surgery. A standardized surgical technique was used for all patients.-1 was given orally 12 h and 2 h before anesthesia as premedication. Before induction of anesthesia, patients were randomly allocated to either Placebo group or Propacetamol group. Randomization was based on computer-generated codes maintained in sequentially numbered, opaque envelopes. The preparation of the propacetamol or saline infusion was done by a nurse who was not in charge of the patient. The anesthetist, the patient and the nurse's staff caring for the patients in the recovery room were unaware of the treatment group. In both groups, anesthesia was induced with propofol 2.5 mg.kg-1 followed by a slow bolus (1 min) of remifentanil 1 mcg.kg-1. Tracheal intubation was facilitated by atracurium 0.5 mg.kg-1. Anesthesia was maintained with remifentanil 0.1 mcg.kg-1.min-1 and isoflurane  with nitrous oxide (N2O) in 50% oxygen. Remifentanil infusion rate was increased or decreased by 0.05 mcg.kg.min-1 in order to maintain an arterial systolic pressure of 20% more or less than the baseline value. One hour before the end of surgery, which corresponded to the beginning of the skin closure, patients received either propacetamol 2 g in 50 cc saline (Propacetamol group) or 50 cc saline alone (Placebo group) infused over 10 min. At the end of surgery, administration of isoflurane and N2O were withdrawn and the patient was transferred in the recovery room. The anaesthetist, the patient and the nurse's staff caring for the patients in the recovery room were unaware of the treatment group. Remifentanil infusion was interrupted when the patient arrived in recovery room. Tracheal extubation was performed within a few minutes after remifentanil discontinuation. Clinical monitoring included heart rate, blood pressure, pulse oxymetry, respiratory rate, and sedation score (0: awake, 1: drowsy and 2: asleep).Hydroxyzine 2 mg.kgFrom the time of extubation, pain was evaluated on using a SNPS (from 0, no pain to 10 the worse pain) and intravenous 2 mg morphine was administered on request every 5 min until pain relief (SNPS<4). When pain relief was reached, SNPS was subsequently evaluated every 15 min. Morphine was interrupted when the sedation score went up to 1, systemic arterial pressure < 80 mmHg or respiratory rate less than 8/min. During the data collection period, intravenous morphine titration was further administered if SNPS was up to 4. Patients did not receive antiemetic prophylaxis. If post-operative nausea and vomiting (PONV) occurred, metoclopramide 10 mg and ondansetron 4 mg if necessary were intravenously administered.Patients fulfilling Aldrete criteria  were disMorphine requirement, pain and sedation scores were measured every 5 min until pain relief was obtained. When SNPS was below 4 during 15 min, parameters were subsequently recorded every 15 min. Morphine side effects (nausea, vomiting, urinary retention, shivering and itching) and need for supplemental medications (e.g., antiemetics) were also recorded.The total dose of morphine in recovery room was the primary end point. Pain scores after extubation and one hour after tracheal extubation, delay for morphine requirement, delay for pain relief and incidence of morphine side effects were recorded.p value less than 0.05 was considered as significant. Anticipating a standard deviation of 2.49 [Data are expressed as mean (± standard deviation) for quantitative variables normally distributed, or otherwise as median (25th – 75th percentiles) when data were not normally distributed, and as percentage for categorical variables. Data were analyzed using Statview 5.0 software (SAS Institute Inc, USA). For intergroup comparisons, categorical variables were compared using the chi-squared test and continuous variables were compared using the Student t test or Mann-Whitney U test, as appropriate. A  of 2.49 , it was Thirty-six patients were included during a 12 months period: 19 in Propacetamol group and 17 in Placebo group (Table In recovery room, cumulative morphine consumption was significantly lower in the Propacetamol group than in the Placebo group Table . Five miThe incidence of morphine adverse effects was similar in both groups: 5 patients had nausea (3 in Propacetamol and 2 in Placebo group, p=NS) and 4 patients had vomiting (2 in each group, p=NS), no other side effects were observed. In one patient receiving placebo, morphine titration was interrupted because of nausea and vomiting.This study shows that propacetamol administered one hour before end of surgery reduced the morphine dose given over the first four postoperative hours and shortened the elapsed time to obtain a SNPS under 4 in patients undergoing elective reduction mammoplasty. In our study, surgical technique and anesthetic protocol were similar in both groups. Remifentanil was the only analgesic used during anesthesia period and was interrupted before morphine administration. Thus, the beneficial effect on postoperative pain observed is clearly linked to intraoperative administration of propacetamol.In a recent study , VerchèrOur results are apparently at variance with those of other previous studies. Paracetamol given rectally immediately after induction of anesthesia  or at th-1) pain score and analgesic demand were significantly reduced in the early postoperative period [Cobby and colleagues observed a morphine sparing effect when paracetamol 1.3 g was administered rectally at the end of hysterectomy . One expe period .The presently observed incidence of postoperative nausea and vomiting was of 25% and was similar to that of a previous study . DespitePropacetamol was administered at the beginning of skin closure, which corresponds to one hour before the end of surgery. This delay may be insufficient to achieve pain control immediately after tracheal extubation, as the peak effect of intravenous propacetamol was shown to occur only two hours after its administration . NeverthIn summary, intraoperative propacetamol administration in women undergoing reduction mammoplasty improved significantly early postoperative pain in recovery room, and should be recommended for postoperative pain management.The pre-publication history for this paper can be accessed here:


In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes. Despite overall similarities between genomes, initiation of DNA replication and speed of duplication in different parts of the genome differs amongst EBV strains Biochemical studies performed in higher eukaryotes have shown that DNA replication initiates at specific sites, or within initiation zones, suggesting the involvement of particular DNA sequences called replicators  genome in human cell lines. During latent replication, the EBV genome is maintained as a circular episome (∼175 kb in size), and the host cell provides both the replication machinery and the licensing apparatus that limit the genome's duplication to once per cell cycle . By utilizing a different procedure to stretch DNA molecules we were able to collect a large number of images of the EBV genome representative of different stages of duplication. This allowed us to determine how DNA replication initiates, progresses, and terminates throughout the EBV genome and to precisely measure the duplication time of specific portions of the EBV genome.These improvements allowed us to obtain important new results as well as to extend previous observations. Here we show that initiation events are not limited to a specific portion of the EBV genome (namely the initiation zone detected by 2D gel electrophoresis), but, unexpectedly, take place throughout the EBV genome. Multiple initiation events were also detected in individual EBV episomes. Hence, if the initiation sites do correspond to replicators, the latter must necessarily be highly redundant (present at a frequency of one or more every 20 kb).Our new results also indicate that, in these two EBV strains, both the frequency and the order of activation of the initiation sites vary considerably throughout the viral genome. This variation involves initiation sites such as oriP, the sequence of which is highly conserved in the two EBV strains . Hence, In order to study DNA replication, we used a procedure that we call single molecule analysis of replicated DNA (SMARD). This procedure labels the replicating DNA in a way that allows us to determine the position, the direction, and the density of the replication forks in a steady-state population of replicating molecules (in this case, EBV episomes). This in turn allows us to determine how DNA replication initiates, progresses, and terminates throughout the genomic region analyzed. SMARD presents several advantages over procedures that utilize different labeling schemes and allows us to overcome most of the limiting factors that have traditionally affected studies of replication performed on DNA fibers see .In our procedure, an asynchronous population of cells is sequentially labeled with 5′-iodo-2′-deoxyuridine (IdU) and 5′-chloro-2′-deoxyuridine (CldU) . The lenIn the experiments described in this study, agarose-embedded total DNA was prepared from cells labeled with halogenated nucleotides. The circular EBV episomes were converted to linear molecules by digestion with a restriction endonuclease (PacI or SwaI). After pulsed field gel electrophoresis, the EBV DNA was recovered by agarase treatment  and streIn order to define precisely how the Raji EBV genome replicates, we recovered the images of 245 PacI-linearized EBV episomes that incorporated halogenated nucleotides along their entire length (112 fully stained in red, 84 fully stained in green, and 49 stained in both red and green). The results of this experiment are shown in However, the progression of DNA replication throughout the EBV genome is better described by the replication profile of the molecules analyzed C. This pThe RRF contains the initiation sites most frequently utilized to begin the duplication of the Raji EBV episomes. More than 80% of the molecules analyzed were stained in red throughout intervals 1–7 and 31–35 , and a small portion of the RRL. We recovered 209 fully substituted 105-kb fragments (94 red, 79 green, and 36 red and green). These molecules were analyzed as described for the PacI-linearized EBV episomes . We founThe movement of the replication forks throughout the EBV genome is described by the profiles of replication fork abundance  can also be calculated . More details are provided later in the text. However, it is important to note that the highest Sd values were detected within the central portion of the Raji RRF (segments A, I, and G). This result can be explained in two ways. First, replication forks may move faster throughout the RRF. Alternatively, the RRF could contain a significant level of multiple initiation events. For the reasons mentioned below we favor the second possibility.We found that different portions of the EBV genome replicate at different speeds, with values that range from a minimum of 0.3 kb/min ; the third is discussed in the last section of Results (differences in duplication speed across segments of the RRF of different sizes).The first line of evidence is provided by the immunostaining pattern of the EBV molecules. Although discontinuities in the immunostaining make it difficult to detect multiple initiation events when the distance between converging forks is 5 kb or less, the replication patterns of some of the molecules are compatible with the presence of multiple initiation events within RRF (molecule 8 in (a–i). We also reexamined the hybridization patterns of ten different fragments analyzed in a previous study  are used at a different frequency in different EBV strains . HoweverThe most striking differences in the replication of the two EBV strains were found in the order of activation of the initiation sites. In Raji episomes, primary initiation events are restricted to an 80-kb region approximately corresponding to the RRF  could potentially explain the different location of the RRF in Raji and Mutu I episomes. However, the following considerations indicate that this is not the case. Initiation events occurring at oriP take place in the vicinity of the dyad symmetry (DS) element and produce replication forks that stall in the leftward orientation at the family of repeats (FR) element . InitiatSimilar conclusions apply to the RRF of the Raji EBV genome (∼80 kb in size), in which primary initiation events were detected at various locations . This is very similar to the range of speeds found in the Raji episomes (0.3–4.7 kb/min). Therefore, DNA replication appears to progress with similar kinetics in the two EBV strains.In the previous sections we have shown that the order of activation of the initiation sites in Raji and Mutu I EBV episomes is not conserved. Here, we wanted to determine whether the quantitative aspects of DNA replication were also different. SMARD was used to calculate Sd values in the two viral strains. Segments A′–G′ divide the Mutu I EBV genome in seven parts of identical size . Instead, the RRF contained some of the lowest Sd values (segment B′). This is probably due to the presence of strong pausing sites within this portion of the Mutu I EBV genome. Nevertheless, segment B′ replicates faster than the corresponding portion of the Raji EBV genome . This allowed us to measure the average speed of the replication forks in various portions of the EBV genomes in which these conditions are satisfied.Previous observations have suggested that in mammalian cells the speed of the replication forks can vary . Here, wH and segment J  replicates even faster , nd G see  are compSd of a genomic segment and its location within the RRFs. For example, the high Sd value for segment G′ indicates the presence of active initiation sites outside the Mutu I RRF. Therefore the factors that influence the order of activation of the initiation sites are at least partially distinct from the factors that control their frequency of activation.In this study, we determined how DNA replication initiates and progresses in EBV episomes latently replicating in two human Burkitt's lymphoma cell lines (Raji and Mutu I). Previous experiments had suggested that some variability in the utilization of oriP might exist among different EBV strains . Here, hA′ , segment C′ (molecule 34), segment D′ (molecule 8), segment F′ (molecules 5 and 15), and segment G′ (molecules 4 and 31). Similarly, in Raji episomes, replication bubbles were detected within every restriction fragment analyzed by 2D gel electrophoresis throughout a region of about 80 kb  is reduced by one unit in the Mutu I strain (not shown), while the Raji EBV genome contains two short deletions see A Polack. In prinThis study also revealed modifications in the pausing of the replication forks in the oriP region. Accumulation of replication forks is clearly present in both EBV strains within this genomic region. However, only 25% of replicating Mutu I episomes contain replication forks at this location see D, comparWe also found that some features of the episomal duplication do not vary. In both Raji and Mutu I cells, replication forks move freely throughout the EBV genome , several aspects of their duplication are not conserved between Raji and Mutu I. In mammalian cells, prereplication complexes are believed to form at the end of mitosis, when general transcription is shut off . HoweverOne of the questions we tried to answer is whether initiation of DNA replication is regulated at the level of individual initiation sites. Clues to a possible regulatory mechanism can be found in the replication profiles of the EBV episomes. The RRFs are localized in specific portions of the EBV genome that differ in the two EBV strains. These regions are tens of kilobases in size (about 80 kb in Raji episomes and 20 kb in Mutu I episomes) and encompass multiple initiation sites. The early activation of an individual initiation site could be sufficient to generate a RRF. However, our results have demonstrated that within each RRF various initiation sites have a similar tendency to be activated at the beginning of the duplication of each episome. Therefore, the order of activation of the initiation sites varies at the level of genomic regions rather than at the level of individual initiation sites and might reflect the presence of a particular chromatin organization.Recent findings have shown that histone acetylation can influence the timing of replication origin firing in yeast . In thisSd detected within the RRF compared to the RRL  encompass known common fragile sites in mammalian genomes , but none of the Raji EBV episomes showed forks paused in this orientation compare D. TherefVarious groups have suggested that different cellular proteins could participate in regulating the activity of oriP . HoweverIn this study we have shown that, while the basic features of DNA replication are conserved (i.e., the progression of the replication forks), the activity of the initiation sites (order and frequency of activation) varies significantly in different EBV strains and across different portions of the EBV genome. Importantly, using SMARD we are now beginning to detect similar modifications in the utilization of initiation sites across transcriptionally active chromosomal loci of the mouse genome (data not shown). Additional mechanisms could regulate DNA replication at transcriptionally silent loci, as suggested by the complete absence of initiation events throughout an approximately 450-kb portion of the mouse IgH locus in non–B cell lines . It is t5 and 8 × 105 cells/ml. The experiments presented in this manuscript were performed at approximately 5 × 105 Raji cells/ml, using two labeling periods (240 min each) with 25 μM IdU (first label) and 25 μM CldU (second label). IdU was added directly to the growing culture, followed by low-speed centrifugation of the cells at the end of the first labeling period and resuspension in warm medium containing CldU.Raji cells were grown in exponential phase (as described in 5 and 8 × 105 cells/ml) before the replicating DNA was labeled. The conditions used for growth and labeling were the same as those used for Raji cells, with the exception that the labeling periods were only 210 min each. This Mutu I cell line is characterized by the presence of a small fraction of cells in which EBV replicates lytically, producing molecules linearized at the terminal repeats. However, this did not affect our analysis of the latently replicating episomes because only the DNA molecules that were circular before the digestion with PacI were recovered from the agarose gels and analyzed by SMARD.Early passages of the Mutu I cells , type II (only EBNA1, the LMPs, and the EBERs expressed), and type III . Although both Raji and Mutu I are human Burkitt's lymphoma cell lines, their transcription profiles are different. The Mutu I cell line used in this study was an early passage of a type I clone isolated in the Alan B. Rickinson laboratory. Raji cells, instead, have a type III-like transcription pattern and they also carry a deletion of the EBNA3C gene (Only ten EBV genes (out of about 100) can be expressed during latency  and rat anti-CldU  were used as primary antibodies , while Alexa Fluor 568-conjugated goat anti-mouse (Molecular Probes) and Alexa Fluor 488-conjugated goat anti-rat (Molecular Probes) were used as secondary antibodies. The immunostaining has almost no background. As described previously , the speStudies performed by fiber autoradiography have previously shown that the results obtained using DNA fibers (such as the average size of the replicons) are significantly affected by the length of the labeling period utilized to label the replicating DNA  of the restriction fragments analyzed by 2D gel electrophoresis were as follows: fragment a, DraI (79202)–EcoRI (82920); fragment b, EcoRI (82920)–DraI (88865); fragment c, DraI (88865)–EcoRI (91421); fragment d, EcoRI (93162)–EcoRI (95239); fragment e, EcoRI (95239)–DraI (103226); fragment f, EcoRV (100583)–EcoRV (116863); fragment g, HindIII (110942)–EcoRI (125316); fragment h, XbaI (161383)–EcoRI (1); and fragment I, EcoRV (126415)–EcoRI (137221). The probes used to detect the restriction fragments were as follows: pHindLHI for fragments a–c, pHindE for fragments d–e, pSalF for fragment f, the pHindC fragment XbaI (121146)–BglII (120341) for fragment g, the p107.5 fragment XhoI (169423)–XhoI (167487) for fragment h, and the pHindC fragment HpaI (131959)–XbaI (133151) for fragment i. The plasmids pHindLHI, pHindE, pSalF, pHindC, and p107.5 were kindly provided by John L. Yates. Two different preparations of replication intermediates were used to study the replication patterns of fragments a–c.The procedures for the enrichment of replication intermediates, 2D gel electrophoresis, and Southern analysis were essentially as described previously . Prepara