Information on the extent of postpartum maternal morbidity in developing countries is extremely limited. In many settings, data from hospital-based studies is hard to interpret because of the small proportion of women that have access to medical care. However, in those areas with good uptake of health care, the measurement of the type and incidence of complications severe enough to require hospitalisation may provide useful baseline information on the acute and severe morbidity that women experience in the early weeks following childbirth. An analysis of health services data from Lusaka, Zambia, is presented.Six-month retrospective review of hospital registers and 4-week cross-sectional study with prospective identification of postpartum admissions.Both parts of the study identified puerperal sepsis and malaria as, respectively, the leading direct and indirect causes of postpartum morbidity requiring hospital admission. Puerperal sepsis accounted for 34.8% of 365 postpartum admissions in the 6-month period. Malaria and pneumonia together accounted for one-fifth of all postpartum admissions (14.5% & 6% respectively). At least 1.7% of the postpartum population in Lusaka will require hospital-level care for a maternal morbidity.In developing country urban settings with high public health care usage, meticulous review of hospital registers can provide baseline information on the burden of moderate-to-severe postpartum morbidity. Maternal morbidity refers to complications that have arisen during the pregnancy, delivery or postpartum period. Every year an estimated 50 million women are affected by maternal morbidity. Defining, interpreting and measuring maternal morbidity, however, is recognised to be difficult and the prevalence of such morbidity  has been poorly described  dFortney & Smith  have desA network of 23 public sector clinics and a single referral hospital, the University Teaching Hospital (UTH), comprise the public health care system for Lusaka's population of approximately 1.5 million. There is also a small private sector (2% of deliveries)  and therAt the University Teaching Hospital, which acts as the district referral hospital for the city, admission and discharge registers are kept in each ward and department. In an earlier retrospective study, analysing referrals for all pregnancy-related complications, undertaken at UTH ,13, 4% oData collection was carried out by LV between July and September 2000. Ethical clearance for the study was obtained from the University of Zambia Research Ethics Committee.For the purpose of this study, the WHO definition of the postpartum period (from delivery until 6 weeks after delivery) was used as the time period inclusion criteria . All womRelevant admission and discharge registers at UTH were reviewed for the six-month period July-December 1999. Dependent on the type, timing and severity of a postpartum problem, women may be referred or may self-refer to one of three different units within the hospital: (i) Women with early postpartum complications, defined as problems occurring within 24 hours of delivery, are admitted through the labour ward admission room; (ii) Women with postpartum problems occurring more than 24 hours after delivery are referred or may present themselves to the gynaecology filter clinic, from where they may then be referred on to the emergency admission ward; (iii) Cases of breast abscess are generally admitted though the surgical unit. From any of the units women may be then admitted onto a longer stay gynaecology ward for further management and treatment. Women admitted to hospital irrespective of the length of stay were included in the data capture.Table Cases were crosschecked by name and age between the emergency admission and gynaecology wards and between the short and longer stay surgical wards, to prevent double counting. Cases of breast abscess were also crosschecked between the surgical unit and the gynaecology department to ensure cases had not been referred. Data was entered on Epi. Inf. 6.04 for analysis.Routine health service statistics for the same six-month study period (July-December 1999) were also collated. There were 19,691 deliveries within UTH and the satellite clinics , of which 1,021 were by caesarean section (an average of 39 per week). Women delivering either at UTH or the clinics were instructed to attend the local satellite clinic for postnatal follow-up visits at week 1 after delivery and again at 6 weeks. Some women were seen at UTH for a postnatal visit at week 1 to follow-up on some complication while those who had a caesarean section were reviewed at 6 weeks. Recorded postnatal check-up at the clinics at 1-week was 40% and 21% at 6 weeks; however, this data was incomplete for some clinics. At UTH, an average of 42 women per week attend the postnatal clinic – most of them after a caesarean section. During the same six-month period, there were 93 maternal deaths at UTH. Of these maternal deaths 8 (8.5%) were known to be due to puerperal sepsis. A much larger number of the women who died had sepsis and stigmata of AIDS .Due to the limitations of the routine data sources used in the retrospective review, a small cross sectional study was also conducted using prospective identification of cases in order to verify the findings. Over a 4-week period, from 14 August to 10 September 2000, all early postpartum admissions to the maternity unit at UTH were identified through the labour ward admission register. Obstetric case notes were sought and reviewed. Late postpartum cases (>24 hours and up to 6 weeks after delivery) for the same time-period were also identified and recorded by the same means as described for the retrospective study. On the gynaecology ward, cases were identified through daily review of the ward round books, to identify new admissions, and through consultation with the senior ward sister and the ward clerk.After crosschecking of data to prevent double counting, 365 maternal postpartum admissions to the hospital were positively identified for the 6-month study period July-December 1999. Cases of retained placenta (n = 55), removed in theatre, were not included in the final analysis as it was not possible to differentiate between clinic referrals and UTH deliveries.Of the 365 admissions, 236 (65%) cases were identified through the emergency admission ward, 120 were identified on the gynaecology ward and the remaining 9 cases were identified on the surgical wards. More than half of the emergency admission ward cases were referred from the satellite MCH clinics , more than half were aged between 20–29 years, a reflection of the fact that the largest number of births takes place within this age group.There were 39 different recorded diagnoses across the 365 cases Table . PuerperMalaria was the second most common diagnosis accounting for 14.5% of all cases. Hypertensive disorders including hypertension, pre-eclampsia and eclampsia accounted for 10.9%.at least 1.7% . Because this incidence refers only to women admitted live to hospital in the postpartum period, the actual incidence will be somewhat higher, with the addition of postpartum morbidities that occurred in women who were already hospital in-patients, any maternal deaths that occurred outside of the hospital, and any missed late postpartum admissions to medical wards.Using MacKeith et al.'s  estimateOver the one-month period August-September 2000, 64 admissions to the hospital for postpartum maternal morbidity were identified. The routine hospital procedures differentiate between "early" (up to 24 hours postpartum) and "late" (after 24 hours) postpartum admissions. The former are admitted through the labour ward admissions and the latter through the emergency gynaecology ward. Twenty women (31%) were "early" referrals and 44 (69%) were "late". This categorisation of the data therefore concerns time elapsed between delivery and admission to hospital, and not between time of delivery and onset of the condition.Just over one-third (7/20) of the maternal referrals in the first 24 hours after delivery were for retained placenta and just over one-third were for pregnancy- related hypertension or eclampsia (7/20). Three referrals were for postpartum haemorrhage. There were no maternal deaths in this group.Referrals from the satellite clinics accounted for 73% of all "late" (>24 hours after delivery) postpartum admissions to UTH; the remaining cases were mainly self-referral. The diagnoses of the 44 cases of late-postpartum admissions in the one-month period are shown in Table Twenty-one cases (47%) were admitted to the longer stay gynaecology ward. More than half of all cases (52%) were in the 20–29 years age group. Among the 44 women admitted to hospital at more than 24 hours postpartum, 4 died; 2 of these deaths occurred on the longer stay ward. Of the 4 deaths, one could be directly linked to obstetric events (anaemia); the others were related to non-obstetric causes .This hospital study used time-period based inclusion criteria in its identification of cases of maternal morbidity sufficiently severe to require hospital admission. The majority of morbidity thus identified was directly linked to obstetric causes e.g. puerperal sepsis, infected wounds, and pregnancy-induced hypertension. However, non-obstetric conditions, including malaria and pneumonia were found to have accounted for at least one-fifth of all postpartum admissions in the retrospective review. This mirrors the increased role that indirect causes have been found to be playing in maternal mortality rates in countries such as Zambia .We have already outlined some of the practical difficulties with using routine hospital data sources such as admission registers. Admission rates estimated from the small prospective part of the study do not suggest, however, that many cases were lost in the identification process in the larger retrospective part. The former identified an average of 11 late-postpartum maternal admissions to hospital per week in the month observed, and the latter, an average of 14 late-postpartum maternal admissions per week over the six months reviewed. In both parts of the study, puerperal sepsis and malaria were identified as leading causes of postpartum morbidity requiring hospital admission. In the prospective identification of postpartum morbidity requiring hospital admission, puerperal sepsis, malaria and hypertensive disease each accounted for the same number of admission (5 in each) but the numbers are too small to draw any conclusions. Some inaccuracy in the classification of certain morbidities may be expected due to the use of admission diagnoses. It is also a limitation in the design of this element of our study that data collection did not extend to the medical wards. It is therefore not possible for us to ascertain whether, or to what extent, there are medical ward admissions of women in the late puerperium with non-obstetric conditions such as malaria.Puerperal sepsis was the leading cause of postpartum hospital admissions in this population, accounting for 34.8% of all identified postpartum cases in the retrospective part of this study. Other hospital-based studies as well as surveys of women's self-reports of postpartum morbidity report puerperal sepsis as a leading cause of postpartum morbidity in developing countries ,6,15-18.Puerperal sepsis cases identified through the retrospective data collection part of the study accounted for more than twice as many cases as the second commonest postpartum morbidity requiring hospital admission, malaria. For the retrospective study the overall rate of puerperal sepsis cases requiring hospital-level care and admission was 0.64% of all supervised deliveries in the public sector services, a rate that falls in between those estimates from earlier hospital-based studies in Niger: 0.22% ; and in A number of other obstetric-related postpartum morbidities including anaemia, breast abscess, symphisiotomy, puerperal psychosis, after-pains, urinary tract infection, secondary postpartum haemorrhage and retained products of conception were also identified as late-postpartum referrals through the retrospective study. Anaemia in the postpartum period is not an uncommon health problem . SurveysMalaria and pneumonia together accounted for one-fifth of all the postpartum hospital admissions that we identified. This finding suggests the usefulness of an approach that employs a "time-period" definition to identify cases rather than simply a set of purely obstetrically-related diagnostic categories.In all, 14.5% of identified postpartum maternal admissions to hospital were due to malaria. While it is widely recognised that the severity and frequency of malaria is greater in pregnant, compared to non-pregnant women, until recently it was generally thought that the importance of pregnancy-related malaria ends with delivery  and malaA number of factors may modify susceptibility to malaria in the postpartum period. The age at which partial immunity to malaria is acquired is critically dependent on transmission intensity ,24. WidePneumonia and Pulmonary TB were important causes of postnatal morbidity in this study and were likely related to HIV/AIDS. The HIV serostatus was rarely available of the index postpartum cases, though unlinked anonymous testing of HIV in the antenatal population in 4 sentinel sites in Lusaka during 1998 showed a high HIV prevalence of 27.4% . HIV posReview of registers on the labour ward, for both the retrospective and prospective aspects of this study suggest that the majority of referrals in the early-postnatal period (first 24 hours) were for infant rather than maternal indications. Of the early postpartum referrals for maternal indications, retained placenta is the leading reason for referral from clinics to the hospital. This reflects the urban context of the study and the relative ease of transportation that permits a district policy of removal of placenta at hospital level rather than by the practitioner with essential obstetric care skills at the delivery clinic.The high public sector maternity care usage in this community permits the low-cost review of routine data to be reasonably meaningful. The caveats are those associated with extraction of data from health facility admission registers, which are not always complete, and which cannot take account of changes in diagnosis or subsequently arising complications. In the absence of more robust data, such reviews, if carried out meticulously, do offer the opportunity to identify the extent of moderate-to-severe postpartum morbidity and the principle causes. They thus may provide the groundwork for detailed condition-specific research to take place exploring aetiology, duration, time of onset and outcome, and the implications for health care provision.The author(s) declare that they have no competing interests.LV: Conceived of the study, conducted the field data collection and data analysis and contributed to the paper.SFM: Wrote the first draft of the paper, participated in the study design and advised on the data collection and analysis.YA: Contributed to the study design, advised on the data collection and analysis and contributed to the paper.All authors read and approved the final manuscript. The views expressed in this article are those of the authors and not of their institutions.The pre-publication history for this paper can be accessed here:


Nectria haemaotcocca, a saprophytic or pathogenic fungus with sexual reproduction, exhibits a bistable morphological modification characterized by a reduced growth rate and an intense pigmentation. Bistability is triggered by the presence or absence of σ, a cytoplasmic determinant. This determinant spreads in an infectious manner in the hyphae of the growing margin, insuring hysteresis of the differentiation.Bistability and hysteresis are increasingly recognized as major properties of regulatory networks governing numerous biological phenomena, such as differentiation and cell cycle progression. The full scope of the underlying molecular mechanisms leading to bistability and hysteresis remains elusive. s1 and s2 mutations completely abolish the generation of σ and of its morphological expression, the Secteur. The remaining five mutations promote its constitutive generation, which determines an intense pigmentation but not growth alteration. The seven mutations map at the same locus, Ses (for 'Secteur-specific'). The s2 mutant was obtained by an insertional mutagenesis strategy, which permitted the cloning of the Ses locus. Sequence and transcription analysis reveals that Ses is composed of two closely linked genes, SesA, mutated in the s1 and s2 mutant strains, and SesB, mutated in the s* mutant strains. SesB shares sequence similarity with animal and fungal putative proteins, with potential esterase/lipase/thioesterase activity, whereas SesA is similar to proteins of unknown function present only in the filamentous fungi Fusarium graminearum and Podospora anserina.Seven mutants specifically affected in the generation of σ were selected through two different screening strategies. The Ses provides evidence that a system encoded by two linked genes directs a bistable and hysteretic switch in a eukaryote. Atypical regulatory relations between the two proteins may account for the hysteresis of Secteur differentiation.The cloning of  Although it has long been known that cellular memory, or epigenetic states, can be created by emergent properties of regulatory or metabolic networks , thXenopus mos MAP kinase cascade. Other relevant models are necessary for the comprehension of these phenomena.In the light of these concepts, alternative inheritable regulatory states were designated (reviewed in ) based oPodospora anserina, their molecular nature and their roles remain unknown. HET-s is a prion [Fungi are an excellent source of well-defined bistable and multistable processes associated with hysteresis  frequently and randomly appear in the growth margin of the thallus as small brown areas in which hyphal elongation is reduced and secretion of pigments is drastically increased .Mutagenesis experiments of wild-type conidiospores demonstrated that both the Secteur and the Anneau are under the control of nuclear genes . In thesa and a*, suggested that the Anneau is under the control of a unique but complex locus. All a and a* mutations map to the same locus, Ans (for 'Anneau-specific'). Mutants carrying the a mutations are unable to generate the α factor, since they never present Anneau morphology and cannot be infected by that factor. Mutants carrying the a* mutations never show Anneaux, spontaneously or after contamination, but display a red color. Surprisingly, when used as donor in contamination with the wild type as recipient, these mutants are able to trigger Anneaux in 100% of attempts, showing that these a* mutants carry the α factor constitutively. It is noteworthy that the phenotype of a* mutants suggests that the mere presence of this factor is sufficient to promote a red color but is insufficient to alter growth.Several mutations that specifically prevent the formation of Anneaux were also selected . The twos*789 (formerly 789) mutant acts similarly to a* mutation in that it entails a red pigmentation  could be obtained. To this end, we developed novel mutagenesis procedures permitting the recovery of mutations that specifically prevent Secteur development. Most were nas mutations. However, a few s* alleles and two s alleles (s1 and s2) were obtained. Genetic analysis suggested that the σ factor, like the α factor, is under the specific control of a single complex genetic locus. Molecular analysis of the s2 allele, obtained by insertional mutagenesis, permitted the cloning and characterization of the Ses locus. As expected from the genetic data, the locus is complex and encompasses two linked genes, one of which has similarity with putative esterase/lipase/thioesterase enzymes. These data provide evidence that two linked genes could generate a bistable differentiation.Because of the similar properties of Anneaux and Secteurs, we decided to screen for more mutations that specifically prevent Secteur formation, to determine if nas mutants (97%). Eleven mutants were specifically affected in Secteur expression. Ten exhibited an s* phenotype identical to that of the s*789 strain. One mutant, s1, had a wild-type phenotype, except that it was unable to express the Secteur, spontaneously or after contamination tests . In some progeny, the s* and s1 mutations were recombined with the I4mod markers. These strains were kept for further crosses. Crossing s1 with s*789 or crossing the new s* mutants with s1 and with s*789 yielded progeny that were mostly segregants, with the parental phenotypes in a ratio 1:1 and few segregants with the wild-type phenotype. Their frequency ranged from 0.1 to 0.6% of the progeny  determined in previous studies [The simplest interpretation of these results is that  studies , one muss1 mutant allele appears recessive to the wild type in balanced heterokaryons, we tried to clone the Ses locus by complementation using the sib selection strategy [Ses. However, because the contamination tests necessary for this screen were labor-intensive and time-consuming, we preferred the 61a1 strain as a recipient for transformation, since it expressed Secteurs very early, and at high frequency, and never expressed Anneaux [nas phenotype. One was probably affected in the Ses locus, as it was unable to differentiate Secteurs, either spontaneously or after contamination tests. This transformant, called s2, had a wild-type morphology and behaved as did the s1 mutant previously isolated.Since the strategy . Althoug Anneaux . As illus2 carries integration in the Ses locus, we crossed it with the wild-type (wt) and s1 strains. When crossed with wt, the 100 tested ascospores segregated 1:1 for ability:inability to differentiate Secteurs. As expected, hygromycin B resistance always cosegregated with the inability to differentiate Secteurs. When s2 was crossed with the s1 strain, only seven ascospores among 1657 were unable to express the Secteur , suggesting that the two integrated copies were linked. The size of the larger ClaI-fragment, LI, was compatible with an entire copy of the vector, whereas the smaller, SI, could be interpreted as a truncated copy. Further analyses with other restriction enzymes and probes (data not shown) established the restriction map of this region  transformants  revealed two patterns of integration . This dominance in diploids explains the failure to clone Ses by complementation.It is noteworthy that despite several attempts, transformation experiments done with the s* mutation, cosmid XN31E6 was subcloned into pBluescript and the plasmids were assayed for complementation. This yielded psecX5, a plasmid carrying a 5.5-kb XhoI fragment sufficient to confer Secteur expression . One interesting feature of the sequenced region concerned the presence of several inverted repeats ranging from 32 to 290 bp and organized in a symmetrical fashion . This protein, NhHET-E-like, has a weak similarity (23% identity and 39% similarity over 455 amino acids) to the HET-E protein of P. anserina, which has been shown to be involved in heterokaryon incompatibility [SesA and SesB, were also identified. Functional analysis revealed that only these two ORFs were part of Ses, each having a different role.A 9-kb-long region surrounding the integration site in the SesA is a 210-codon ORF  to the N terminus of a large putative protein of P. anserina of unknown function but with 46% identity and 60% similarity with the Het-E protein in its C terminus  procedure. The sequence of this product revealed that a polyadenylation signal existed 141 bp downstream from the SesA stop codon.F Figure  with no s Figure . It is ay Figure . SesA is Figures . SesA waSesB, a 386-codon ORF  and animals . It does not show similarity with hemiascomycetous yeast ORFs , basidiomycete ORFs , or Arabidopsis thaliana ORFs. All the proteins similar to SesB show a significant PROSITE (PS50187) esterase/lipase/thioesterase active site serine domain. The sequence of SesB in s1,s*789,s*4,s*27, and s*18 revealed that in the four s* mutant alleles, SesB contains a single point mutation , encoding a putative peptide of 157 amino acids with no homologue in the databanks. To show that ORF-C is not an exon of SesB, we cloned into pBluescript the 2.4-kb-long SmaI fragment carrying only SesB, i.e., without ORF-C or SesA. This yielded plasmid psecSm24 , supporting the hypothesis that SesB alone is per se a functional gene involved in Secteur development.F Figure , is locaTE PS5018 esterase Figures , but no TE PS5018 esteraseSesA and SesB are present in the genomes of F. graminearum and P. anserina, we investigated whether the gene arrangement was conserved in these two species. In P. anserina, for which a single SesA homologue is found, the gene arrangement is not conserved, since SesA and SesB homologues are on two different contigs. In F. graminearum, the putative 201-amino-acid homologue of SesA that displays the lowest percentage of identity is not associated with any of the SesB homologues. On the contrary, the 214-amino-acid homologue of SesA that displays the highest identity is associated with a SesB homologue in the same order  whereas the other F. graminearum homologues are less conserved (40% identity and 63% similarity over 273 amino acids for the next-best homologue). In F. graminearum, the inverted repeats 1, 2, and 3 are absent but the inverted repeat 4 is present. Overall, these data suggest an evolutionary conservation of Ses between N. haematococca and F. graminearum, both of which are in the genus Fusarium, but with a lack of conservation of the gene organization in more distant species, including the loss of SesA. There are no data concerning the potential development of Secteur in the F. graminearum strain used by the Fungal Genome Initiative for sequencing.Since homologues of both r Figure . This hos1, we co-transformed the wild-type strain with pSec3-s1 and pBC-Hygro. pSec3-s1 was obtained by cloning into 'pGEM-T easy' , a PCR amplification product constructed with oligonucleotides im317 and ip2393c , these transformants could be interpreted as bearing a co-integration of pBC-Hygro and pSecs1 at an ectopic position. Sequencing of the Ses locus in these two transformants that do not display Secteurs confirmed the presence of both alleles, i.e., the wild-type allele at the resident locus and the mutated allele s1 at an ectopic position. This clearly demonstrated the dominance of the s1 mutation, in contrast to the s* mutations, which are recessive.To investigate the dominance of Ses, the primary locus controlling the development of Secteurs.Although the Secteur and Anneau phenomena were described about 30 years ago , the molN-methyl-N'-nitro-N-nitrosoguanidine). A vast collection of mutants affected in the Secteur expression was easily obtained. One mutant with an s phenotype was isolated, along with 11 s* mutants. Apparently, NG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis was more efficient than UV mutagenesis in recovering these specific mutants . Genetic analysis showed that the structure of the Ses locus mirrors the structure of the Ans locus, indicating that α and σ could be generated through similar mechanisms, in agreement with the fact that both factors require the same set of nas genes for propagation.The experimental design, based on the differences in the growth rate between modified and normal cells, was very efficient in finding mutations that block the propagation of Secteurs after the action of both UV and NG , the recovery of the s2 mutant was an unexpected outcome. Although we may have been lucky, the recovery of this transformant may reflect a preference for plasmid integration in the region of the Ses locus, which is compatible with the high recombination frequency observed at Ses. Cloning of Ses permitted the construction by transformation of a partial diploid containing both the wild-type and s1 mutant alleles. The fact that this partial diploid has a s phenotype indicates that s1 allele is indeed dominant over the wild-type allele in partial diploids. Similar contradictions have been observed in A. nidulans by comparing diploids and heterokaryons for complementation between mutations in the regulatory and the cognate structural genes. These data were interpreted as being due to a limitation of regulatory gene products to the nucleus or by a stringent dose effect when combined with a nonrandom distribution of nuclei in heterokaryons [N. haematococca, as reported earlier [SesA products expressed from the wild-type and s1 nuclei in different portions of the thallus. Because s1 is dominant and corresponds to a missense mutation, it is likely that the product expressed from SesA in s1 is a dominant negative. If its concentration is insufficient, especially in hyphae homokaryotic for wild-type SesA nuclei, the propagation of σ and Secteur formation is favored. In contrast, in diploids an even ratio of the two products is always achieved, preventing propagation of σ and Secteur formation.Dominance/recessivity tests using balanced heterokaryons showed that us fungi . With th species . Among tokaryons ,27. In o earlier , only 1–Ses shows that it is composed of two linked and expressed genes, SesA and SesB, both of which are necessary for Secteur expression. Each has a different role. The s1 and s2 mutants map to SesA. Because s2 is an integration of a large segment of DNA at the beginning of SesA, the phenotype of the s2 mutant probably results from a complete loss of function of SesA. The s1 mutant carries a dominant missense mutation and therefore synthesizes the SesA mRNA, and probably a dominant negative protein. The SesAp protein has no evident homologue in the databanks yielding a clue to its function. As s2 does not produce the σ determinant, cannot be contaminated by mycelia carrying σ, and never differentiates the Secteur morphology, SesA is probably necessary for the production and transmission of σ.The molecular characterization of s* mutations map to SesB and determine a red-pigmented phenotype associated with the generation of the σ determinant throughout the thallus, but without any growth alteration. It is noteworthy that all the s* mutations are probably not simply preventing the production of a functional mRNA, but are also not preventing the production of an active protein, as they map outside the evolutionarily conserved region of the protein. The function of SesB and its homologue remains unknown in fungi; however the inactivation by RNA interference of the most similar gene in C. elegans is lethal during embryogenesis, suggesting that the protein produced by SesB could be essential . Based SesA and SesB display weak similarity only with proteins of unknown function. However, from our data we can infer that SesA most likely encodes a regulatory factor for SesB and that the regulation probably takes place at the protein level and probably not at the RNA level, since s1 and all s* mutation are point mutations that are not likely to affect RNA transcription and stability. Two formal models could explain how the regulation might work.To date, this situation has not been described for any of the previously reported systems responsible for bistable or multistable switches in fungi. Previous systems implicated prions ,29, chroSesA (SesAp) could exist in two states: one, AN, characterizing the Normal morphology and the other, AS, determining the Secteur morphology . In addition, the product of SesB (SesBp) can adopt the normal BN state, which can be transformed into a BS state in the presence of AS. BS would be responsible for the growth alterations observed in the Secteur and would follow the propagation of AS .Firstly, the product of y Figure . The tras mutants, SesAp would never be present, thus making the presence of SesBp constitutive . In the s* mutant, the interactions between the two proteins would be abolished, permitting the activity of both SesAp and SesBp . This model could explain the dynamic equilibrium found in the ZiS area, as well as the dose effect for SesAp, explaining the difference of heterokaryons and partial diploids.In the second model, SesAp may negatively regulate SesBp but also be negatively regulated by SesBp Figure . In the N. haematococca, is controlled by a complex, two-gene locus. Each gene has a distinct role in defining bistability, and atypical regulatory relations between the two proteins coded by the two genes may account for the hysteresis of Secteur differentiation. The data exemplify the diversity of mechanisms that generate differentiation in fungi, and, more generally, in eukaryotes.Secteur, one of the two bistable and hysteretic differentiations exhibited by the filamentous fungus N. haematococca strain wt  was used as the standard wild-type strain in this study. The origin and relevant characteristics of all strains used in this study are listed in Table The homothallic 1s1I4mod and 61a1 strains used as recipients in transformation experiments were constructed by crossing the s1I4mod strain with a1 mutant and the 61 mutant with a1I4mod strain, respectively (unpublished data). The marker segregation was recorded after three weeks. This included (1) mycelium and perithecium pigmentation, (2) fertility, and (3) the ability to express the Secteur and the Anneau as tested by transferring the progeny onto potato dextrose agar (PDA) and by inoculating 3-day cultures with s*789 (for Secteurs) and a*58 (for Anneaux).The aGeneral culture conditions and manipulations were as described by Daboussi-Bareyre . StrainsN. haematococca have been described elsewhere [4mod. This strain developed white, self-sterile perithecia whereas wild-type and fertile mutants developed self-fertile, red perithecia [The general methods for crossing the homothallic lsewhere ,32. SincThe progeny of crosses was analyzed in accordance with the following procedure: two to nine white, fertile perithecia from each cross were freed from mycelium and conidia. Each was opened to spread the ascospores on one Petri dish containing water plus 3% agar. To control viability, 35 ascospores from each perithecium were transferred to PDA and incubated for 4 days at 26°C, while the water-agar stock dishes were kept at 2°C to prevent ascospores from germinating. Then, the water-agar dishes containing many viable ascospores  were returned to 26°C to allow germination overnight. A sample of about 400 germinating ascospores from each dish was transferred to PDA. The phenotypes were recorded after a week at 26°C in the dark, except for the perithecium color, which was evaluated after an additional period of two weeks.1, s*84, and s*18 were crossed with the two strains carrying either of the markers, arg154 and lys255, that promote auxotrophy for arginine and lysine, respectively. Auxotrophic marked strains, which were recovered in the progeny, were paired on a cellophane membrane in the combinations appropriate to restore prototrophy. After 48 hours of growth on complete medium, the membrane was transferred to minimal medium. Widely inclusive inocula from prototrophic mycelia observed at the junction of the two partners were transferred to minimal medium and examined for their pigmentation and their ability to express the two modifications, either spontaneously or after inoculation.Heterokaryons were constructed as described in . Initial2, with a UV light source (254 nm), and then kept in the dark for 72 hours. For NG mutagenesis, 0.1 ml of a 200γ/ml solution of NG was deposited under the cellophane disc. After 1 hour, the treated cultures were exposed to light for NG degradation.Plugs taken from the growing margin of a culture modified by the Secteur were inoculated on cellophane discs on PDA and grown for 4 days at 26°C. The modified slow-growing cultures (ZiS) were submitted to mutagenesis by UV or NG. For UV mutagenesis, cultures were exposed at 450 J/mnas mutants, defined by (1) the inability to differentiate both Anneaux and Secteurs, (2) an exuberant white mycelium, and (3) the inability to differentiate perithecia. Some of these mutants could express the modifications at other temperatures, as was previously observed for mutants 727 and 100 [s* mutants were recovered as red, fast-growing sectors. They displayed the same properties as the s*789 mutant described previously [s mutant was selected as a wild-type-growing sector. This mutant displayed a wild-type phenotype, except for the inability to express the Secteur spontaneously or after inoculation. Both s* and s mutants could differentiate Anneaux.Fast-growing sectors were clearly visible a week after mutagenesis. For the estimation of the number of sectors recovered from each treated thallus, only sectors harboring different phenotypes were considered. A plug of these independent sectors was inoculated on PDA and the resulting cultures were examined for pigmentation and ability to express the Secteur and/or the Anneau at 26°C. The majority of the recovered mutants corresponded to  and 100 ,34. s* meviously , i.e., rN. haematococca genome, were used to transform the double mutant strain 61a1. Both vectors carried a hygromycin resistance gene. Transformation occurred at a frequency of about 10 transformants/μg plasmid. Southern analysis on 10 independent hygromycin-resistant transformants (HygR) indicated that the transforming DNA inserted generally at one or two genomic sites, in some cases in a tandem fashion (data not shown). This pattern of integration was suitable for recovery of tagged genes.Plasmids pAN7-1 (GenBank accession number Z32698) or pBC-Hygro , which hR independent transformants were transferred to PH8 (12 per Petri dish) and grown for 15 days at 18°C, a temperature that increased the frequency of spontaneous Secteur formation. Thalli with mutant phenotypes were frequently recovered  in a lysis buffer , 10 mg/ml mutanase , and incubated for 2–3 hours at 26°C with gentle agitation. Protoplasts were separated from cell debris by addition of 5 ml of ST buffer  and centrifugation at 2000 g for 10 min. The protoplasts collected at the interface of the two solutions were harvested by centrifugation at 750 g for 5 min, washed twice in ice-cold buffers, once in ST and then in STC  at a final concentration of 5–10 × 107 protoplasts/ml and kept on ice.Protoplasts were prepared as described in , with thH = 7.5; ). They wFor transformation, 5 μg or 10 μg of DNA suspended in a maximum of 50 μl TE buffer  were mixed gently with 100 μl protoplasts and placed on ice. After 30 min, 900 μl of PTCS buffer (50% PTC containing 0.8 M sorbitol) was added and incubation continued at room temperature for 30 min. Aliquots of the protoplasts were mixed with 2.5 ml molten (37°C) top PDAS (PDA with 0.8 M sucrose and 0.3% w/v agar), and overlaid onto 30 ml of selective medium  by PCR, Southern blot hybridization, or direct cloning using a complementation screen. Each tested gene was represented by 2 to 5 independent clones, indicating that the library contained at least four N. haematococca genome equivalents. Library XN was made with SalI partially digested wt genomic DNA following the same protocol as for the XSG library. The sequence of the mutant alleles was performed on two independent PCR amplification products, obtained with oligonucleotides im317 (5'-TGATCAACCTCCACGCACAT-3') and ip2393c (5'-AAGGAGATATCGCGCAGGCT-3'). Identification of the 3' end of the SesA and SesB genes was done by 3'-RACE using the 5'/3' RACE kit  on polyA+ mRNA extracted from 2-day-old mycelium, using for SesA oligonucleotide ip238 (5'-AAGAGGCCGTGAGACAAGAG-3'), and for SesB ip974c (5'-CCAAGACGACCGATAGAAGA-3').All the nucleic acid manipulations were performed using standard methods . For SouThe genBank accession no. for cosmid XN31E6 sequences is AY572411.SG performed all the experimental work and contributed to the interpretation of the data. PS and MJD performed part of the phylogenetic analysis, contributed to the interpretation of the data, and supervised the work. The writing of the manuscript was done in teamwork. All authors read and approved the final manuscript.


Background to the debate: Umbilical cord blood—the blood that remains in the placenta after birth—can be collected and stored frozen for years. A well-accepted use of cord blood is as an alternative to bone marrow as a source of hematopoietic stem cells for allogeneic transplantation to siblings or to unrelated recipients; women can donate cord blood for unrelated recipients to public banks. However, private banks are now open that offer expectant parents the option to pay a fee for the chance to store cord blood for possible future use by that same child (autologous transplantation.) Private banks offer expectant parents the option to pay a fee for the chance to store cord blood for possible future use by the child. The practice is controversial, for scientific and ethical reasons No one disputes the merit of public cord blood banking, in which women altruistically donate umbilical cord blood (UCB) for haemopoietic stem cell (HSC) transplantation, in a way similar to bone marrow donation. Unrelated UCB transplants have good outcomes in children and are associated with less graft-versus-host disease than adult marrow or peripheral blood stem cells ,2. PubliThe validity of directed UCB storage in “low risk” families, however, has been widely challenged. After early concerns from the American Academy of Pediatrics  and AmerFirst, UCB is very unlikely ever to be used. The probability of needing an autologous transplant is less than one in 20,000 ,10, althUmbilical cord blood is very unlikely ever to be used.Second, there are important moral issues. The persuasive promotional materials of commercial UCB banks target parents at a vulnerable time, urging them to take this “once in a lifetime opportunity” to “save the key components to future medical treatment” and freeze “a spare immune system” . Even atThird, collection imposes a considerable logistic burden on the obstetrician or midwife. In addition to consent, parental blood collection, and the associated packaging and paperwork, a large volume of blood has to be collected from the umbilical vessels in utero, requiring multiple syringes under aseptic technique. This may distract professionals from their primary task of caring for the mother and baby at this risky time or, more generally, divert delivery room staff from attending others . This apFinally, individual UCB banks need to remain in business long term if cryopreserved stem cells are to be retrieved. The commercial attractiveness of a service paid years in advance is attested to by the burgeoning number of private providers, yet it seems unlikely that all will survive. Indeed, some US providers are already in trouble for infringing collection patents. There remain reservations about whether laboratories will meet national standards and be accredited. There is a further danger that misplaced enthusiasm for commercial auto-collection will undermine the proven utility of altruistic public cord blood banks.Notwithstanding the above, we accept that the utility of UCB storage in low-risk families is very different from the entirely speculative post-mortem cryonics industry. We acknowledge the possibility that autologously stored UCB stem cells may eventually be used. Indeed, recent research documenting the multi-potentiality of UCB mesenchymal lineages  and the Stem cells may potentially be used in life-saving therapies for degenerative diseases or injuries. Stem cells self-replicate and are multi-potential—they can differentiate into diverse cell types . While sTo fully realize this potential will require collection and banking of UCB cells, which are harvested without pain or trauma from placental structures that are normally discarded after birth. We realize that UCB banking (public and private) has sparked controversy. Critics of routine banking question its cost-to-benefit ratio, citing doubts about the clinical relevance of cord stem cells or the likelihood that they will ever be used . Other cThe “stemness” of UCB cells is not merely theoretical . UCB haThe real question is who should pay for umbilical cord blood collection and storage.ESCs also represent an allogeneic source of cells—they are derived from another individual whose tissue type does not match up with the recipient, resulting in immune rejection when transplanted . We knowWith millions of healthy babies born each year, there is potentially a large UCB supply that can be stored, tissue-typed, and made available at short notice. If saved for potential use by the donor, UCB cells become a source of perfectly matched, autologous stem cells (plus there is a 25% probability of being an exact match for a sibling). Yet the American Academy of Pediatrics came out against UCB banking, saying that the odds (with some exceptions) of a donor ever using a UCB sample were low, between 1/1,000 and 1/200,000. While the chance of a donor benefiting may presently be low, this does not automatically mean that another member of society could not benefit. For people with genetic diseases or cancers, the chances of finding an immune-tolerant donor match would obviously be increased by the expansion of cord blood sampling. Also, at the pace that stem cell research is moving, perhaps there will be new uses for UCB cells in the next decade, especially in the field of tissue engineering . ImportaWho should operate cord blood banks—the private or the public sector? There are around 20 private UCB banks in the United States. They charge a collection fee, typically $1,000–$1,500, which includes testing for pathogens and genotyping. Samples are maintained in a frozen state for around $100 a year. An additional $15,000–$25,000 is charged if a sample is used for transplantation . The cost of UCB cell transplantation is significantly less than bone marrow transplantation, and the risk of graft-versus-host disease is lower . The priAny exploitation by companies of the vulnerabilities of expectant parents for financial gain is clearly unacceptable. Federal legislation to establish a national cord blood stem cell bank network—free to all donors—has been introduced in the US Senate and House of Representatives that, if approved, should diminish the risk of exploitation. But unless the network is well-designed from a sociological viewpoint, it could generate a situation where not all cultural and ethnic groups are represented or where benefits are accessible only to families with health insurance or sufficient income to afford transplants. It still remains difficult to find full matches for African, Asian, and Native Americans—mostly because of an insufficient number of UCB donors and the diversity of HLA types in different ethnic groups .The real question is who should pay for UCB collection and storage—the individual donor, who currently has only a small prospect of using their cord blood, or society as a whole? We believe that it is the job of government to assure that people of all ethnic groups are informed and educated about donating UCB. Then, to facilitate UCB banking and the development of technological innovations for its storage and clinical utility, we recommend a national network that is a mixture of for profit, non-profit, and governmental organizations.Markwald and Mironov argue that commercial UCB banking is ethically justified on the grounds that UCB transplantation is effective treatment for many haematological disorders, that autologous UCB is a useful future source of stem cells for the donor, and that there is no second chance to collect these cells.We did not dispute (indeed we acknowledged) the value of UCB HSCs for the treatment of many malignant and non-malignant haematological disorders. However, evidence of their value derives from allogeneic HSCs from public UCB banks . Like maMarkwald and Mironov state that the real question is who should pay for routine UCB collection and storage. However, they take no account of the considerable logistic burden this imposes, the extremely low chance that autologous cells will ever be used , and the costs of routine UCB collection . They alThus the real questions are, first, why should society in general, or the government as a representative of at least a substantial proportion of society, pay for a service not shown to be of any real use?  And second, why should commercial banks be allowed to continue to target vulnerable parents anxious to do the best for their children while making no mention of the low chance of use, of alternative sources of available stem cells , or of the risks of reducing stocks of allogeneic HSC in public UCB banks?We agree with Fisk and Roberts that exploiting the emotional vulnerabilities of expectant parents is unjustifiable—thus we support regulation of UCB banking, monitoring, certification, and informed consent. But we disagree that there is a lack of solid scientific evidence for UCB collection and that “future therapeutic possibilities are very hypothetical.” Research on stem cells is advancing rapidly, and stem cells derived from UCB are emerging as a reasonable first choice for the field of regenerative medicine.Fisk and Roberts are inconsistent in their views. They claim that stem cells collected in UCB units often are not “in sufficient numbers for use against degenerative conditions” in adult life but then acknowledge that “the in vitro expandability of cord HSC numbers is sufficient for transplantation into an adult.” They argue that private and public UCB collections create dramatically different “logistic burdens,” but in our experience, the syringes, paperwork, and level of personal distraction are generally the same for public or private banking.We strongly disagree that private UCB banking has no future. While we anticipate a consolidation phase for this industry, surviving companies should be eager to acquire UCB units collected from competitors. If all stem cell sources were under a state monopoly—without private sector contribution—there would be less incentive or opportunity for fostering innovation in long-term storage, expansion, or phenotype characterization of UCB stem cells. The growth of new biotech companies focused on regenerative medicine would be discouraged, compromised, or undermined by the absence of competition, inadequate access to venture capital, and the typical resistance of state health-care systems and their affiliated medical professionals to innovation.Fisk and Roberts are creating obfuscations by mixing “speculative cryobionic companies” that promise “immortality and eternity” with serious biotech companies and private UCB banks that focus on a realistic commercialisation of UCB stem cells as a platform for promoting new biotech initiatives.The collection and storage of UCB stem cells is an opportunity for society to build a representative collection of UCB units that can improve the chances of identifying suitably matched donors for transplantation. Human ESCs are mired in ethical concerns and concerns about immunological intolerance. Autologous cells from the bone marrow or elsewhere lose their attractiveness if there is a genetic mutation or a progressive loss of “stemness” due to normal aging . UCB cel


IBD).Cytokine mediated induction of the mucosal addressin cell adhesion molecule-1(MAdCAM-1) expression is associated with the onset and progression of inflammatory bowel disease , widely used in the treatment of diabetes, has as well recently been highlighted as protective in models of inflammation and cancer. We found that troglitazone (10–40 μM), significantly reduced the TNF-α (1 ng/ml) mediated induction of endothelial MAdCAM-1 in a dose-dependent manner, achieving a 34.7% to 98.4% reduction in induced MAdCAM-1. Trogliazone (20μM) reduced TNF-α induced VCAM-1, ICAM-1 and E-selectin expression. Moreover, troglitazone significantly reduced α4β7-integrin dependent lymphocyte adhesion to TNF-α cultured endothelial cells.These results suggest that PPAR-γ agonists like troglitazone may be useful in the clinical treatment of IBD. Endothelial cell adhesion molecules or 'ECAMs' play essential roles in the development of chronic inflammation by recruiting leukocytes, especially lymphocytes through their ability to promote leukocyte rolling, firm adhesion and extravasation . Tissue Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of transcription factors, whose activities are regulated through the high affinity binding of small lipophilic ligands that include steroid hormones . A new cAlthough PPAR-γ is expressed at high levels in adipose tissues, PPAR-γ has also been described in many other kinds of cells, including those in the vasculature like endothelial cells, vascular smooth muscle cells and monocytes and macrophages . AlthougPPAR-α, rather than PPAR-γ activation is responsible for blocking cytokine induced ECAM expression [MAdCAM-1, one of the more important regulators of gut inflammation in IBD. However, this has not yet been investigated.However, the literature does not uniformly support protective roles for all PPARs. For example, it has been suggested that activation of pression ,41 and ttroglitazone, to limit cytokine induction of MAdCAM-1 and also VCAM-1, ICAM-1 and E-selectin, and decrease MAdCAM-1 dependent lymphocyte endothelial adhesion in vitro.In the present study we have examined the ability of a candidate glitazone PPAR-γ ligand, To confirm the presence of PPAR-γ on SVEC endothelial cells, western blotting for PPAR-γ was performed using an antibody generated to a synthetic peptide corresponding to amino acid residues 284–298 of murine PPAR-γ; importantly, this antibody exhibits no significant homology to PPAR-α. This antibody recognized a PPAR-γ specific band at 55 kD by western blotting in SVEC cells Fig. .data not shown). Troglitazone was not overtly toxic, and did not affect cell protein content (i.e. protein content per well), or change the expression of either actin or vimentin, two cell structural proteins  (data not shown).To examine the effect of PPAR-γ activation on endothelial cell inflammatory responses, SVEC endothelial cells were treated with TNF-α either with or without a pre- and co-treatment with troglitazone. TNF-α 1 ng/ml, 24 h) increased MAdCAM-1 expression to 13.4 times that of the untreated control level. This TNF-α induced MAdCAM-1 was dose dependently attenuated by pre- and co-treatment with troglitazone (i.e. from 69% to 7% of TNF-α stimulated levels) at concentrations of 10 to 40 μM  enhanced VCAM-1 expression to 8.5 times that of untreated controls. This elevated expression was significantly reduced (to only 80% of the TNF-α stimulated level) by troglitazone at 20 μM Fig. . Alone, TNF-α  increased ICAM-1 expression to a level 4.7 times greater than untreated control levels. This TNF-α enhanced ICAM-1 expression was strongly attenuated (to 32% of the TNF-α stimulated level) by troglitazone at 20 μM  increased the expression of E-selectin 4 times over untreated control levels; this increase in E-selectin was also blocked by troglitazone (to 22% of the TNF-α stimulated level) at 20 μM Fig. . Alone, Having established that troglitazone exerts a significantly protective effect against TNF-α stimulated endothelial MAdCAM-1 induction, we examined the effects of troglitazone on the adhesion of α4β7 expressing mouse lymphocytes (using the cell line TK-1) to endothelial monolayers following TNF-α treatment. TNF-stimulation (24 h) significantly increased the adhesion of TK-1 lymphocytes to SVEC monolayers. Troglitazone (20 μM) significantly reduced TK-1 adhesion in response to TNF-α stimulation at 24 h Fig. . TroglitMechanistically, the expression of all of these adhesion molecules is known to depend on the activation of NF-kB following TNF-α stimulation. Therefore, we examined whether troglitazone-mediated protection against TNF-α induced MAdCAM-1 expression and lymphocyte adhesion were related to the activation and/or inhibition of NF-kB. In this study, phosphorylation of the p65 subunit of NF-kB was used as an index of NF-kB activation. TNF-α  induced the phosphorylation of NF-kB p65; this activation was significantly reduced by the PPAR-γ ligand, troglitazone at 20 μM Fig. . Alone, MAdCAM-1 is a 60 KD endothelial cell surface molecule that is strongly expressed by mucosal endothelial cells, particularly following exposure of these cells to pro-inflammatory cytokines such as TNF-α. Expression of MAdCAM-1 has as well been reported in the brain, and in the heart, ,47, and With respect to inflammatory bowel disease, MAdCAM-1 appears to be necessary for lymphocyte homing to mucosa associated lymphoid tissue -5,50. SiTNF-α is thought to be perhaps the most important cytokine responsible for driving the onset and progression of IBD. Because of this primary role of TNF-α in IBD, anti-TNF-α antibody therapy has been successfully used in IBD to reduce both colonic injury and expression of ECAMs in IBD . While TThis is the first study to demonstrate that a PPAR-γ ligand, troglitazone, can significantly reduce the expression of MAdCAM-1, an endothelial cell adhesion molecule which is closely linked to chronic gut inflammation. Troglitazone significantly reduced TNF-α induced expression of several other ECAMs as well ,38,41,57NF-kB is a member of the Rel family of dimeric transcription factor complexes key transcription factor that modulates expression of MAdCAM-1 in inflammation, and is governs the expression of several other endothelial adhesion molecules in response to cytokines ,35,40,52PPAR-γ ligands like troglitazone apparently suppress activation of NF-kB, and in our hands reduce both basal and TNF-stimulated NF-kB p65 phosphorylation figure . ConsequPPAR-γ ligands like glitazones should not only attenuate MAdCAM-1, but also diminish the expression of other ECAMs like ICAM-1, VCAM-1 and E-selectin. This reflects a decrease in the synthesis of some, but not all proteins, since densitometry of troglitazone treated monolayers shows no difference in total protein content between wells following troglitazone, but western blotting or surface expression assay find a significant decrease in the expression of ECAMs.inhibition of ECAMs such as ICAM-1 in response to TNF-α [endothelial' in many cases,  [While studies with glitazones in endothelial models for the most part demonstrate an to TNF-α ,38, Chento TNF-α ,7 have rstances) , those rindependent pathways. Some described PPAR-γ ligands, like the cyclopentanone prostanoids, exert anti-inflammatory effects through a PPAR-γ-independent pathways [It is also possible that PPAR-γ activators might well affect inflammation through NF-kB pathways  specificpathways  and blocpathways  which coSince high levels of PPAR-γ protein are expressed within the colon,  it is poOur results indicate that troglitazone and other glitazones may provide an effective means of treating forms of chronic inflammation including inflammatory bowel disease through their ability to interfere with steps in the activation of NF-kB, effectively blocking the expression of adhesion molecules like MAdCAM-1 which increase infiltration of tissues by leukocytes.Recombinant mouse TNF-α was purchased from ENDOGEN , and troglitazone was provided as a generous donation from Sankyo corp., Japan.2, and cultures were used immediately upon reaching confluence .The SVEC4-10 line is an endothelial cell line derived by SV40 (strain 4A) transformation of murine small vessel endothelial cells, originally isolated from the axillary lymph node vessels of an adult male C3H/Hej mouse . These c+ T cell lymphoma TK-1 cells  at a 1: 500 dilution overnight (4°C). Membranes were washed 2× with wash buffer. Secondary goat anti-rabbit horseradish peroxidase conjugated secondary antibody (Sigma) was added at a 1:2,000 dilution for 1 h. Membranes were washed 3 times and developed using the ECL detection system .Monolayers were either pretreated (1 h) with blockers, and then incubated with cytokines (24 h), or treated without test agents and then treated with cytokines (24 h). All cell samples were harvested at 24 hours. Equal quantities of protein (75 μg) from each sample were electrophoretically separated on 7.5% SDS- PAGE gels. Gels were transferred to nitrocellulose membranes (Sigma) and blocked with 5% milk powder in PBS at 4°C (overnight). These membranes were washed twice for 10 min with wash buffer (0.1% milk powder in PBS). Primary rat anti-mouse MAdCAM-1 mAb  was added at a concentration of 10 μg/ml and incubated at room temperature for 2 h. In p65 NF-kB phosphorylation studies, membranes were incubated with anti-phospho p65 antibody  diluted 1:1000 overnight (4°C). These membranes were washed twice with wash buffer. Secondary goat anti-rat horseradish peroxidase conjugated secondary antibody (Sigma) was added at a 1:2000 dilution for 2 h for MAdCAM-1, while goat anti-rabbit antibody was used for detecting phospho-p65 NF-kB. Lastly, membranes were washed 3 times and developed using the enhanced chemiluminescence (ECL) detection system. The density of MAdCAM-1 staining was measured by scanning the 60 KD band, using a HP ScanJet™ flatbed scanner. Images were analyzed for density using Image Pro Plus™ image analysis software . The data are expressed as a percentage of TNF-α-induced level of density.2SO4, and the samples were transferred to 96-well plates. Plates were read on a Titertek MCC340 plate reader  at 450 nm. Blanking (i.e. background) was performed on monolayers stained only with second antibody and reacted as above.Surface expressions of ECAMs were measured using the method of Khan et al. . SVEC mo6 cells/ml with 0.02 mg fluorescein diacetate (FDA) (Sigma) at 37°C for 30 min. The cells were then washed twice with ice-cold HBSS, spun at 250 g for 5 min to remove unincorporated fluorescence and suspended in HBSS. The TK-1 lymphocyte cell line used in this assay expresses high levels of the α4β7 integrin, [Briefly, TK-1 cells were suspended in culture medium and fluorescence labeled by incubating TK-1 cells at 2 × 10ntegrin, ,44 whichntegrin, . In thisntegrin, . SVEC montegrin,  and alloPPAR-γ – peroxisome proliferative activated receptor gammaMAdCAM-1 – Mucosal Addressin Cell Adhesion Molecule-1VCAM-1 – Vascular Cell Adhesion Molecule-1E-selectin – Endothelial SelectinICAM-1 – Intercellular Adhesion Molecule-1IBD – Inflammatory Bowel DiseaseTNF-α – Tumor Necrosis Factor-αDr. Sasaki accomplished the studies described in this manuscript, all authors contributed equally in the design, interpretation and execution of this article.


At just 9.8 kilobases, the HIV genome pales in comparison to the 3.2 gigabases of its human and nonhuman primate targets. The compact retrovirus encodes just 14 proteins, which play different roles in promoting viral infection and virulence. As a retrovirus, HIV uses the host's cellular machinery—including RNA polymerases, which carry out transcription—to copy its RNA genome into DNA and infiltrate human chromosomal DNA. Once the virus is integrated , its genes can be transcribed.Adept as HIV is in exploiting its host's molecular resources, the virus can't establish a foothold without the services of its skeleton crew. The HIV transcription factor Tat (“transactivator of transcription”), for example, is an essential regulator of HIV gene expression. Without Tat, HIV transcripts don't reach full length and can't effect viral replication. In a new study, Melanie Ott and colleagues identify an enzyme that regulates viral transcription by modifying Tat.trans-acting responsive element (TAR). Tat binding to TAR requires recruiting the enzyme cyclin-dependent kinase 9 (CDK-9) to the HIV promoter (where transcription begins). CDK-9 chemically modifies the RNA polymerase and enhances its transcribing efficiency.The regulation of HIV genes depends on a complex interplay between proviral DNA, cellular proteins and transcription factors, and Tat. Unlike most transcription factors, Tat activates transcription by binding to RNA, specifically to a bulging “stem-loop” structure that forms at one end of all viral transcripts called the The transcription process—including the labyrinthine protein–protein and protein–DNA  interactions—is highly regulated. One process that figures prominently in this regulation is acetylation, which adds an acetyl group  to a molecule or protein. Histone acetylation was long thought to influence transcription by regulating the structure and function of chromatin, which is an assembly of proteins (mostly histones) and DNA. Another, more widely accepted model proposes that histone acetylation controls transcription by recruiting cofactors required for transcription.Acetylation and deacetylation enzymes can also target other proteins. Of the three classes of deacetylases known to modify human histones, the sirtuins (SIRT1–7) appear to preferentially target a number of nonhistone proteins. Ott and colleagues first tested the ability of all seven SIRT proteins to deacetylate Tat by placing them in a test tube with Tat proteins. Though three SIRT enzymes caused Tat acetylation, only one, SIRT1, is a nuclear enzyme, like Tat, suggesting that SIRT1 might work similarly in living cells.Ott and colleagues went on to show that transcription via Tat occurs in the presence of SIRT1, but not when SIRT1's catalytic center is removed. Experiments using cells taken from transgenic mice lacking SIRT1 demonstrated that introducing human SIRT1 enzymes increased Tat's transcriptional effects in a dose-dependent manner, while treating cells with the small molecule HR73, a derivative of a molecule that inhibits the yeast version of the SIRT1 protein, caused a 5-fold reduction in HIV transcription.10.1371/journal.pbio.0030076]).The authors propose a cycle of transcriptional transactivation in which SIRT1 deacetylates Tat at the HIV promoter. Deacetylated Tat associates with CyclinT1 and TAR, and leads to transcription. Tat acetylation dissociates Tat from CyclinT1 and TAR, and transfers Tat to the elongating polymerase complex. Since acetylated Tat can't recruit CyclinT1 and CDK-9, the authors explain, a new round of transcription requires that new, unacetylated Tats are produced or existing Tats are deacetylated. Thus, efficient viral replication depends on adequate Tat supplies. And since HIV gene expression relies on SIRT1's enzymatic activity, inhibiting SIRT1 could prove to be a promising anti-HIV therapy. Future study will have to verify whether inhibiting SIRT1 can successfully put the brakes on HIV transcription and control the virus (See also “A New Paradigm in Eukaryotic Biology: HIV Tat and the Control of Transcriptional Elongation” [DOI:


From the moment its life begins, the fate of a multicellular organism depends on how well its cells communicate. Proteins act as molecular switchboard operators to keep the lines of communication open and the flow of cellular messages on track. But charting the protein interactions, signaling pathways, and other elements that regulate these networks is no small feat. Previous efforts have been hampered by the lack of quantitative data—measurements of signal duration, amplitude, and fluctuation—on these regulatory pathways.Hoping to fill in some of the quantitative gaps, Marc Kirschner of Harvard Medical School, Reinhart Heinrich of Humboldt University Berlin, and colleagues developed a mathematical model as a framework for understanding the quantitative relationships among signaling proteins. To do this, they focused on a well-studied signaling pathway, the Wnt pathway, which plays a role both in various stages of embryonic development and in carcinogenesis. The researchers chose the Wnt pathway in part because a lot is known about it and in part because they could collect enough of the additional measurements they needed to build a solid model from experiments. And like most signaling pathways, Wnt is highly conserved. Consequently, developing tools that elucidate the Wnt pathway will not only provide insights into this important pathway, but have implications for understanding other communication pathways in animals from jellyfish to humans.To get the additional measurements needed to build their model, the researchers reproduced aspects of the Wnt pathway in the cytoplasm of unfertilized frog eggs. Among the new data collected from these experiments were measurements of the concentrations of scaffold proteins, which bring other components in a pathway together by providing an interaction surface. Strikingly, they found that the principal scaffold proteins involved in the pathway, axin and adenomatous polyposis coli (APC), occur in dramatically different concentrations and perform their jobs in different ways. After a series of refinements based on additional experiments, the model could not only simulate the behavior of the main players in the pathway—both in the absence and presence of a Wnt signal—it also suggested why the two scaffold proteins are present in different concentrations. Axin occurs at very low concentrations relative to the other proteins in the pathway and is likely to bind with them randomly, while APC occurs in similar concentrations and probably binds with the other components in an ordered manner. Because the proteins axin interacts with are also involved in other signaling pathways, the authors propose that the low level of axin here may help the pathways retain their modularity, preventing the Wnt pathway from interfering with the other pathways.These findings demonstrate that modeling can offer powerful new insights into the workings of complex signaling systems, cutting through the static to pick up important signals even in those pathways that are well understood. The results have important implications for developmental biology and human disease: The Wnt pathway is often activated during carcinogenesis—and mutations in several of these signaling proteins have been linked to colon cancer—suggesting that cancer can develop when signals in the Wnt circuitry somehow get crossed. By predicting how quantitative factors may influence the behavior of signaling networks, mathematical models such as this could shed light on the role that breakdowns in cellular communication play in carcinogenesis. The researchers argue that future attempts to characterize these complex networks must incorporate quantification measurements, and their modeling efforts suggest ways to do that.


PLoS Medicine editors argue that all trial registries, reports, and databases should be freely available onlineThe  PLoS Medicine, Mike Clarke, the director of the United Kingdom Cochrane Centre, lays down a challenge to clinical researchers and journal editors [In a compelling essay in this issue of  editors . He arguFirst, we would argue that by the time a paper is sent to a journal, it is surely too late in the process to be insisting on systematic reviews. If a clinical trial report meets our criteria for originality, importance, and quality, it makes little sense for us to reject it just because the authors failed to systematically review the literature when designing their study. The time to mandate that researchers do a review is much earlier—when they apply for funding, register their trial, or seek ethics committee approval.There is no doubt that the best research builds on previous knowledge. But unfortunately, the current medical publishing system hides much of this knowledge behind subscription or “pay per view” charges, which discriminates against researchers who do not work for well-funded institutions. A group of researchers in Indonesia, for example, recently told a depressingly familiar story of trying to search the medical literature in preparation for a research project ; access Many clinical trials, especially negative ones, remain unpublished, which prevents researchers from reviewing all the data on an important health issue. There are two main reasons why certain trials are not published: one is that the pharmaceutical industry has a long history of suppressing data that are commercially unfavorable and the second is that medical journals and the popular media favor publication of positive over negative trials . While we support the recent announcement on trial registration by the International Committee of Medical Journal Editors—as a condition of considering a trial for publication, member journals will require registration of the trial in a public trials registry —we beliePLoS Medicine is committed to publishing high-quality negative trials. In this issue, for example, we publish an important randomized controlled trial of a malaria vaccine in 372 Gambian men, which found that the vaccine was ineffective at reducing the natural infection rate.The scientific literature will remain biased unless the publishing industry changes its practices and provides a place where the results from all registered trials can be published. The internet makes it possible for every single clinical trial to be publicly and seamlessly tracked through three tiers. The first tier is registration in a publicly available database. The second is the publication of a peer-reviewed summary of every trial, regardless of its outcome, in a traditional journal format, with annotations and critiques that help readers understand the trial's implications. The third is the deposition of detailed trial data in a structured, computable format that allows sophisticated searching and analyses across trials. This format will allow the development of better tools to help clinicians apply trial results to their practice. For trial data to be as useful as possible, all three tiers must be publicly accessible. Assessment of each trial's validity is critical, but should not stop crucial information about all trials being placed in the public domain.http://rctbank.ucsf.edu) are under development. And as a publisher committed to open access, PLoS will provide the second, essential tier—journals capable of peer reviewing and publishing an annotated report of every trial. Traditional medical journals, with their subscription-based model, are unlikely to be able to provide this service, because in order to attract subscribers they need to publish only the highest-profile trials. We believe that an open-access model—in which the research funder pays a publication fee to recover the costs of peer review and for hosting the report on a secure server—is the best mechanism for creating such venues. We are working to make that happen.Trial registries exist, such as ClinicalTrials.gov and the International Standard Randomised Controlled Trial Number registry. Moreover, many trials are registered in a semi-public database maintained by the United States Food and Drug Administration, and there are compelling arguments (which Turner articulates in an essay published online ahead of our December issue ) for makReturning to Clarke's challenge, our final response is to say that we have a bold vision of a freely accessible online world of clinical trials—from registration to annotated summaries to trial databases. That world would be even richer if every systematic review were made freely available. We challenge the Cochrane Collaboration to put the full text of all of its reviews into the public domain. We hope the Cochrane Collaboration will join us in the open-access revolution.


Bioinformatics and microarrays have given scientists powerful new tools to investigate the structure and activity of genes on a global scale. Rather than studying just a few genes, scientists can analyze tens of thousands within and across species. Microarrays flag which genes are expressed under particular cellular conditions in an organism, while genome sequencing offers clues to gene function and regulation. By comparing the genomic properties of different species, scientists can spot patterns that help them identify functional and regulatory elements, learn about genome structure and organization, and gain a better understanding of the evolutionary forces that shape life on Earth.The potential of these technologies to reveal insights into the fundamental structure and function of biological systems continues to grow along with the wealth of gene sequence and expression data—but the ability to interpret and merge these datasets lags behind the ability to collect them. In an effort to overcome these limitations, Sven Bergmann, Jan Ihmels, and Naama Barkai developed a comparative model that integrates gene expression data with genomic sequence information.Because functionally related genes are expected to be coexpressed in different organisms and because the sequence of some of these functionally related genes may also be conserved between organisms, Bergmann and colleagues hypothesized that “conserved coexpression” could serve as an indicator of gene function on a genomic level.  But first they had to determine whether coexpression was conserved among species. Analyzing the gene expression profiles of six distantly related organisms—bacteria, yeast, plant, worm, fruitfly, and human—the researchers found that functionally related genes were indeed coexpressed in each species. The most strongly conserved sets of coexpressed genes are associated with core cellular processes or organelles. These results indicate that conserved coexpression can improve the interpretation of genome sequence data by providing another functional indicator for homologous sequences.Since functionally related genes are expressed together in different organisms, it would be reasonable to think their regulatory networks are also conserved. To explore this idea, the researchers grouped coexpressed genes and their regulatory elements into “transcription modules” for each organism. They found significant variation in the number, organization, and relative importance of these modular components. Which components contributed most to an organism's global transcription program, for example, depended on the organism. But they also found that the transcription networks are highly clustered—meaning that genes connected to a specific gene are also connected to each other. This finding indicates that gene expression programs, regardless of their size or individual components, are highly modular. Each transcriptome contains modules that have been conserved over time along with “add-on” modules that reflect the needs of a particular species. This modularity supports the notion that variation between and among species arises from the diversity of gene expression programs.Although the regulatory details of individual gene groups varied, the researchers found common ground in the overall landscape of the expression data. The transcription programs exhibit properties typical of dynamically evolving “real-world” networks that are designed to perform in uncertain environments and to maintain connections between elements independent of scale. These properties were originally identified in studies of social networks and the World Wide Web, but they aptly describe the real-world challenges of the cell. Studies of dynamically evolving networks show that nodes  added at an early stage (much like highly conserved genes) are more likely to develop many connections, acting as a hub. Following these organizational principles, transcription networks would have a relatively small number of highly connected “hub genes”—though a much higher number than one would expect in a random network.And that is what the authors observed: the networks they constructed from the expression data had the expected number of highly connected hub genes, which tend to be essential and conserved among organisms. Since these highly connected genes are likely to have homologues in other organisms, they can serve as powerful and efficient tools for assigning function to the thousands of uncharacterized sequences found in sequence databases. This model presents a framework to explore the underlying properties that govern the design and function of the cell and provides important clues—in the form of conserved transcription modules—to the evolutionary building blocks that generate diversity.


Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34 A primate model of gene transfer into hematopoietic stem cells demonstrated MLV integration around transcription start sites whereas SIV integrated into gene-dense regions, indicating distinct safety implications for each Integration of proviral DNA into the host cell genome is an essential step in the life cycle of retroviruses. The process begins after a retrovirus enters the cell and the RNA genome is reverse transcribed into double-stranded DNA. Preintegration complexes (PICs) containing linear proviral DNA associated with several viral and cellular proteins  either e+ cells for treatment of SCID-X1, in association with proviral activation of the LMO2 transcription factor gene . Retroviral gene therapy targeting HSCs has been aggressively pursued because of its potential to treat many congenital and acquired human diseases. Its therapeutic promise was convincingly demonstrated in children with X-linked severe combined immunodeficiency (SCID-X1) and adenosine deaminase deficiency . Unfortutor gene . These sFor many years, researchers have been aware that retroviral insertional activation of proto-oncogenes can result in tumors. Administration of replication-competent oncoretroviruses to susceptible mouse strains led to tumor development, the result of a high number of repetitive insertion events in vivo during rapid cell proliferation, with outgrowth of a clone containing one or more proviruses activating growth control genes . While tWith the readily accessible human genome sequence, mapping studies of retroviral integration sites in cell lines have uncovered nonrandom integration patterns, when studied using wild-type HIV, HIV-derived, or murine leukemia virus (MLV)-derived vectors . HoweverIn order to evaluate the integrating vectors currently being developed for gene therapy applications, we compared the integration patterns of MLV and simian immunodeficiency virus (SIV) vectors. MLV vectors have been utilized for over a decade in clinical trials. However, they have a number of limitations, including inefficient transduction of quiescent cells and difficulty in maintaining stable high-level expression from tissue-specific internal genetic control elements. Thus, lentiviral vectors based on HIV or SIV “backbones” have been pursued and shown to overcome these limitations, and are now moving into clinical trials.+ cells transduced with an MLV- (Vector-genome junction sequences were retrieved from mature granulocytes and mononuclear cells (MNCs) from rhesus macaques transplanted 6 mo to 6 y prior with mobilized peripheral blood (PB) CD34 an MLV-  or SIV-d an MLV- , both co an MLV- . This mo+ for the MLV-transduced animals, and 6–7 mo posttransplantation for the SIV-transduced animals. In our extensive prior analysis of 46 rhesus macaques, genetic marking levels and clonal integration patterns are stable by 3–4 mo posttransplantation, and remain stable for up to 6–7 y (We used a modification of the sensitive linear amplification-mediated (LAM)-PCR method  to retrito 6–7 y . This aphttp://genome.ucsc.edu), and (c) showed at least 90% identity to the July 2003 human genome assembly  juxtaposed to the vector long terminal repeat (LTR), (b) yielded a unique best hit by BLAT software .Of the 992 integration sites analyzed, 232 (23%) were distributed among the four major classes of transposable repetitive elements, and therefore could not be mapped to a unique position in the genome. These insertions accounted for 59 of 491 (12%) and 173 of 501 (34.5%) of the MLV and SIV integration events, respectively. Human transposon-derived repeats encompass at least 45% of our genome and their distribution is highly variable, with density varying from 2% to 98% depending on the location .We correlated the 760 integration sites (432 for MLV and 328 for SIV) that unequivocally mapped to a unique position in the genome to the locations of annotated genes, using the UCSC Genome Browser Reference Sequence (RefSeq) Genes track, which displays the positions of National Center for Biotechnology Information mRNA Reference Sequences . We obsep = 0.0002).We next examined whether specific regions of the transcription units were more likely sites of integration than others. We analyzed the distribution of the integration events within the transcription unit by dividing the distance of each integration site from the transcription start site by the gene length. The resulting ratio, reported as the total number of integration events in RefSeq genes for each vector, provides the percentage of integrations within ten equal sections of transcription units. While SIV targets the entire transcription unit with no noticeable preference, 42 of 212 (20%) of the MLV integration sites that land within RefSeq genes, as compared 18 of 241 (7%) for SIV, are located within the first one-tenth of the transcription unit, indicating MLV's clear predilection for the 5′ portion of transcription units (p < 0.0001). The frequency of insertions within 10 kb downstream of the 3′ end is almost identical for the MLV and the in silico-generated random sets (5.3% versus 4.8%).To further explore MLV preferential integration in the vicinity of transcription start sites, we determined the distance to the nearest 5′ and 3′ ends of a RefSeq gene for each integration site. Interestingly, whereas SIV integration events do not favor locations upstream or downstream of transcription units , 48 of 4p < 0.0001). We broadened this analysis to a 60-kb window centered on transcription start sites (p = 0.02), but their distribution is clearly nonrandom and favors a 10-kb window centered around transcription start sites. This pattern is markedly different from the distribution of SIV sites: Although there is no predilection for integration in the vicinity of transcription start sites, there is a strong preference for integration within transcription units, rather than upstream of them. Of the SIV-derived sites, 122 of 328 (37%) are within 30 kb downstream of the transcription start site, while only 30 of 328 (9%) are within 30 kb upstream (p < 0.0001). Taken together, these data show a distinct integration pattern between MLV- and SIV-derived vectors (p < 0.00001 using an omnibus contingency Chi2 test): While the latter appear to integrate predominantly within transcription units, MLV vectors strongly favor integration within a 10-kb window centered on transcription start sites.We then looked at the proviral integrations within a 2-kb window on either side of transcription start sites. This survey revealed a strong tendency for MLV vectors to integrate close to transcription start sites, with 46 of 432 (11%) of the total MLV integration events occurring within 2 kb upstream or downstream, as compared to 7 of 328 (2%) for SIV (rt sites . The ovep < 0.00001 using an omnibus contingency Chi2 test).In order to ask whether the preferential integration of SIV vectors within transcription units might be associated with physical properties of the genome such as gene density, we analyzed the overall distribution of integration sites. The highest density of SIV integration sites per Mbp are on Chromosomes 17, 19, and 22 , the three most gene-dense chromosomes, with 15, 23, and 17 genes per Mbp, respectively . Since ep < 0.005). Of the 22 SIV proviruses within this 20-Mbp ridge, ten were found clustered within a 2-Mbp, extremely gene-dense region (62 RefSeq genes per Mbp). Unexpectedly, only two out of these ten integration sites are inside transcription units, underscoring the strong tendency of SIV vectors to target gene-rich regions of the genome even if not within genes. Another feature of ridges is that they are noticeably enriched for short interspersed element (SINE), but depleted for long interspersed element (LINE) repeats , are distributed among chromosomes. These ridges are characterized by typical expression levels per gene up to seven times higher than the genomic average . This fe repeats . This co repeats  and may n = 32), three times (n = 6), five times (n = 1), and seven times (n = 1). Among these 40 genes, seven are known to be involved in oncogenic translocations: ARHGEF12, MDS1, MKL1, MSF, HMGA2, RAD51L1, and RUNX1. Seven independent integration events have been identified in MDS1, predominantly within the second intron, 20–180 kb upstream of the first intron of EVI1.Given their distinct patterns of integration, we compared identified common integration sites of MLV and SIV vectors. Using the definition of a common integration site as two or more proviruses integrated within a transcription unit , we haveA better understanding of retroviral integration patterns has evolved due to the availability of the complete murine and human genome sequences. Prior mapping studies have been performed in cell lines or in primary cells cultured short-term in vitro. However, integration site patterns may be cell type–dependent, for instance, if gene activity impacts integration site selection , or if sMLV-derived retroviruses are currently the most widely used vectors in clinical gene transfer protocols. Reports of proto-oncogene activation by replication-defective MLV vectors in mice and humans mandate more detailed evaluation of their potential for insertional mutagenesis. Separating the impact of overexpressing a growth-altering transgene from the insertional events themselves is particularly important to assess in primary repopulating HSCs. The main limitation of murine oncoretroviruses as gene therapy vectors is the requirement that cells pass through mitosis in order for the PIC to reach the nucleus and integrate. Since lentiviruses can transduce noncycling cells, lentivirus-based vectors have been actively developed and a clinical trial using these vectors has commenced. A detailed analysis of lentivector integration patterns is essential to assess the risk of insertional mutagenesis of these vectors compared to MLV vectors.Although HIV-derived vectors can enter Old World monkey cells, they encounter a block prior to reverse transcription that is mediated by the dominant repressive factor TRIM5α, a component of cytoplasmic bodies , and thuMacaca mulatta is 92.5%–95% identical to the humans at a DNA level , but no propensity toward integration in any specific region of the transcription units, in contrast to MLV vectors. Although we did not observe regional hot spots for SIV integration, as previously reported in cell lines for HIV , we inston array . Intereson array  and the on array  shows a on array , suggestThis striking tendency was not observed with the MLV-derived set of integration sites. While the ratio of MLV integration sites within transcription units was significantly higher than expected compared to in silico-generated random integration sites, the MLV proviruses displayed a unique and specific affinity for the region surrounding the transcription start site of annotated genes. The finding that among the 491 MLV integration sites, only 12% are within SINEs or LINEs may support the fact that MLV inserts into 5′ regulatory elements where insertions of transposable elements are probably strongly selected against. This also indicates tethering between some transcription factor(s) and MLV PIC protein(s).These observations, consistent with previous comparative analyses in vitro , likely MDS1 gene, whereas MDS1 was not found in the SIV dataset of integration sites. MDS1 is adjacent to the EVI1 locus, which has been implicated as a retrovirally activated proto-oncogene in a number of murine leukemogenesis studies ((MDS1/EVI1) may occur at a much higher frequency than previously expected. Studies are ongoing to better understand the causes and consequences of retroviral integration within this genomic locus.A large number of genes were identified with two or more integration events, and thus were deemed common integration sites, including ten genes that had both MLV and SIV integrations. This suggests either that these genes are particularly susceptible to integration events due to open chromatin or other factors that favor both types of viruses, or that integration events in these particular genes alter expression and favor engraftment and long-term contributions to hematopoiesis. However, the most striking finding was the occurrence of seven independent hits by MLV in the first two introns of the  studies . This un studies . This suIt is important to stress that the very long-term follow-up of a large cohort of nonhuman primates, including all animals in the current study, has revealed completely normal hematopoiesis and lack of any progression towards neoplasia . All ani+ cells were enriched from mobilized PB by immunoabsorption and transduced for 96 h with either amphotropic MLV vectors LNL6 and G1Na containing the neomycin resistance gene (n = 22) (green fluorescent protein gene (n = 3) (n = 12), or 100 ng/ml megakaryocyte growth and development factor . All animals received cells transduced on flasks coated with Retronectin . In addition, two MLV animals also received cells transduced on autologous marrow stromal cells  , or for  (n = 3) . All traal cells . Cells wal cells .LAM-PCR and cloning of insertion site vector genomic fusion sequences was performed as described  using 5′For statistical comparison to the integration site sets, we computationally generated 1,000 sets of integration sites. For MLV, we made 1,000 datasets, each containing 432 randomly selected genomic coordinates; for SIV, we made 1,000 datasets of 328 points each. All human chromosome sequences were concatenated into a single long sequence. We used the random number generator function in Perl to pick a number between 1 and the total number of nucleotides in the human genome , then identified this position in the concatenated sequence and correlated this position back to its chromosomal origin. If this coordinate fell within a sequencing gap, a new number was picked. We performed an ANOVA on the in silico-generated integration sites to demonstrate that 1,000 random sets were sufficient (unpublished data).http://research.nhgri.nih.gov/projects/Dunbar/May2004/). Two-sided p values were obtained using the Chi2 test.We used a bioinformatic pipeline  to map thttp://david.niaid.nih.gov/david/ease.htm). This software was used to rank functional clusters by statistical overrepresentation of individual genes in specific categories relative to all genes in the same category. The functional clusters used by EASE were derived from the Gene Ontology classification system (http://www.geneontology.org).Genes identified as targeted by retroviral insertion were analyzed for significant functional clusters of genes using the EASE bioinformatics software (Dataset S1(78 KB TXT).Click here for additional data file.Dataset S2(60 KB TXT).Click here for additional data file.Figure S1(38 KB PDF).Click here for additional data file.Figure S2(16 KB PDF).Click here for additional data file.Table S1(12 KB PDF).Click here for additional data file.Table S2(18 KB PDF).Click here for additional data file.Table S3(8 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) under the accession numbers AY728482 to AY728804 for SIV, and AY733679 to AY734083 for MLV.The retroviral integration site sequences larger than 50 bp discussed in this paper have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/LocusLink/) for the genes discussed in this paper are ARHGEF12 (23365), EVI1 (2122), HMGA2 (8091), LMO2 (4005), MDS1 (4197), MKL1 (57591), MSF (10801), RAD51L1 (5890), RUNX1 (861), andTRIM5α (85363).LocusLink ID numbers (