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548305

Cytogenetic abnormalities and fragile-x syndrome in Autism Spectrum Disorder

Background Autism is a behavioral disorder with impaired social interaction, communication, and repetitive and stereotypic behaviors. About 5–10 % of individuals with autism have 'secondary' autism in which an environmental agent, chromosome abnormality, or single gene disorder can be identified. Ninety percent have idiopathic autism and a major gene has not yet been identified. We have assessed the incidence of chromosome abnormalities and Fragile X syndrome in a population of autistic patients referred to our laboratory. Methods Data was analyzed from 433 patients with autistic traits tested using chromosome analysis and/or fluorescence in situ hybridization (FISH) and/or molecular testing for fragile X syndrome by Southern and PCR methods. Results The median age was 4 years. Sex ratio was 4.5 males to 1 female [354:79]. A chromosome (cs) abnormality was found in 14/421 [3.33 %] cases. The aberrations were: 4/14 [28%] supernumerary markers; 4/14 [28%] deletions; 1/14 [7%] duplication; 3/14 [21%] inversions; 2/14 [14%] translocations. FISH was performed on 23 cases for reasons other than to characterize a previously identified cytogenetic abnormality. All 23 cases were negative. Fragile-X testing by Southern blots and PCR analysis found 7/316 [2.2 %] with an abnormal result. The mutations detected were: a full mutation (fM) and abnormal methylation in 3 [43 %], mosaic mutations with partial methylation of variable clinical significance in 3 [43%] and a permutation carrier [14%]. The frequency of chromosome and fragile-X abnormalities appears to be within the range in reported surveys (cs 4.8-1.7%, FRAX 2–4%). Limitations of our retrospective study include paucity of behavioral diagnostic information, and a specific clinical criterion for testing. Conclusions Twenty-eight percent of chromosome abnormalities detected in our study were subtle; therefore a high resolution cytogenetic study with a scrutiny of 15q11.2q13, 2q37 and Xp23.3 region should be standard practice when the indication is autism. The higher incidence of mosaic fragile-X mutations with partial methylation compared to FRAXA positive population [50% vs 15–40%] suggests that faint bands and variations in the Southern band pattern may occur in autistic patients.

Background In 1943 Kanner coined "infantile autism" (autism derived from Greek autos , or self) after observing 11 children, mostly boys, on the basis of their social isolation. Autism, often referred to as autistic disorder or infantile autism, is a complex behavioral disorder, which, by definition, develops prior to age three. Autism is defined completely on the basis of impairments in social interaction and communication, repetitive and stereotypic behaviors. Recent research has examined autistic traits in a population of twins and found that social impairment actually follows a unimodal distribution without a clear demarcation to separate cases of the disorder [ 1 ]. For this reason, future discussion of autism may be referred to as autism spectrum disorder (ASD) [ 2 ]. For most children, the onset of autism is gradual; however, about 30% have a "regressive" onset. Fifty to seventy percent of children with autism are defined as mentally retarded by nonverbal IQ testing. Seizures develop in about 25% of children with autism. About 25% of children who fit the diagnostic criteria for autism at age two or three years subsequently begin to talk and communicate, and by six or seven years blend to varying degrees into the regular school population. The remaining 75% continue to have a life-long disability requiring intensive parental, school, and societal support. There are no biologic markers, therefore the standard criteria, compiled by the American Psychiatric Association Manual of Psychiatric Diseases, 4th edition (DSM-IV), is the primary diagnostic reference used in the United States for autism. The causes of autism can be divided into "idiopathic," which comprises the majority of cases, and "secondary," in which an environmental agent, chromosome abnormality, or single gene disorder can be identified. About 5–10% of individuals with autism can be diagnosed with secondary autism; the remaining 90–95% have idiopathic autism. About 30% of children with idiopathic autism have complex autism, defined by the presence of dysmorphic features or microcephaly or a structural brain malformation [ 3 ]. About 70% of children with idiopathic autism have essential autism, defined as the absence of physical abnormalities. A recent CDC case-finding study in Brick Township, New Jersey reported prevalence at 40 per 10,000 for autism [ 4 ]. While the latest epidemiologic study from the United Kingdom utilizing specialized visiting nurses who monitored child health and development at seven months, 18 to 24 months, and three years of age reported a prevalence rate of 16.8 per 10,000 for autism [ 5 ]. The sex ratio for autism has been estimated at 4 males:1 female [ 6 , 7 ]. Social cognition and communication in autism may be related to dysfunction in the amygdala, hippocampus, and related limbic and cortical structures. The cerebellum may also form part of a distributed neuronal network responsible for social cognition and communication. Serotonin is the neurotransmitter implicated in autism [ 8 ] The single gene disorders in which secondary autism is observed include fragile X syndrome, tuberous sclerosis, phenylketonuria, Rett syndrome [ 3 , 9 ], Sotos, Neurofibromatosis I, Joubert syndrome [ 10 ], and Smith-Lemli-Opitz syndrome [ 11 ]. Risk to sibs of idiopathic cases is 75 times greater than the prevalence in the general population [ 7 ] and higher concordance for autism among monozygotic (60–90%) than dizygotic (0–10%) twins [ 12 ] argue for a genetic predisposition to idiopathic autism. Multiple independent whole genome scans and chromosomal abnormalities studies have pointed out several candidate regions on chromosomes 2q, 3q, 7q, 6, 13q, 15q, 16p, 17q and sex chromosomes. These regions possess candidate genes that have been screened for mutations or association with autism [ 13 ]. However, a clear involvement of a major susceptibility gene (or genes) in autism remains far from clear. The results from linkage studies and the drop in the concordance rates between monozygotic and dizygotic twins suggests that the genetic etiology of autism is certainly heterogeneous (different genes in different families), polygenic (more than one affected gene per individual) with epigenetic influences and allelic heterogeneity (different variants in the same gene may lead to different patterns of genetic disease) [ 13 - 15 ]. To gain further insight into secondary autism we have compiled data on patients with autistic traits tested for fragile-X syndrome using molecular methods and chromosome abnormalities using cytogenetic analysis and FISH. Methods A search was initiated using the key word autism in the indication field of the Genzyme Genetics, Orange laboratory database. For each case the electronic data and/or files were reviewed. The referral center, age, sex, karyotype, fluorescence in situ hybridization (FISH) and fragile X results were extracted and tabulated. G-banded metaphases were prepared using standard procedures and FISH was performed using the protocol provided by the manufacturer of the commercial probes (Vysis Inc., Illinois, MI). Fragile X test: Isolated DNA was tested by both Southern blot analysis and Polymerase chain reaction (PCR) for the size and methylation status of the CGG repeat expansion within the FMR-1 gene. Southern blot analysis was performed with the probe StB12.3 on EcoR1 and Eag1 digested DNA. PCR products were separated by acrylamide gel electrophoresis and detected with a CGG repeat probe. Results Patients with autistic traits were referred by physicians to our laboratory for genetic testing. The clinical diagnostic criteria applied were not specified on the test request forms. A total of 433 patients with an indication of autism were sent to our laboratory for genetic diagnosis. The median age was 4 years. Sex ratio was 4.5 males to 1 female [354:79] (Table 1 ). A chromosome abnormality was found in 14/421 [3.33 %] cases. A Fragile-Xq27.3 was diagnosed in 7/316 [2.2 %]. Chromosome abnormalities are summarized in Table 2 : The aberrations were: 4/14 [28%] supernumerary markers from cs 15 [ 3 ] and cs 2 [ 1 ] (Fig 1a ); 4/14 [28%] deletions of 2q37.3, 3p25, 12q21.2q23.3 and 13q13.2q14.1(Fig 1b ); 1/14 [7%] duplication of 15q11.2q13; 3/14 [21%] inversions of 10p11.2q21.2, 17q23q25 (de novo) and 14q11.2q33 (mosaic) (Fig 1c ); 2/14 [14%] translocations, one balanced t(1;14) and one unbalanced der(14;18) (Fig 1d ). The 3/4 marker/ring had a 15 centromere signal by FISH and had either nil, one or two signals for D15S11, that demarcates the involvement of the critical region of Prader Willi/Angelman syndrome (Table 2 ). Fluorescence in situ hybridization was performed on 23 of 433 patients for reasons other than to characterize a cytogenetic abnormality, 3 to rule out Williams-Beuren Syndrome, 5 for DiGeorge syndrome, 7 for proximal duplication of D15S11, 6 for subtelomere rearrangements, 1-for subtelomere rearrangement & proximal duplication D15S11 and 1 for duplication D15S11, Smith-Magenis Syndome, and DiGeorge Syndrome. All 23 FISH tests were negative (Table 3 ). Fragile X (Table 4 ): The mutations detected were: A full mutation (fM) and abnormal methylation in 3 [43 %] and mosaic mutations with partial methylation of variable clinical significance in 3 [43%]. The mosaic size mutations were: an fM [200–900 repeats(r)] / deletion mutation [30 r] with partial methylation. The faint bands for the full mutation and deletion mutation may be a reflection of the sample quality (Fig 2 , Lane 5). Two size mosaics had permutation (pM) [150 r]/fM [400 r] (Fig 2 ), and pM [155 r]/fM [800 r] with normal and abnormal methylation. A premutation mosaic female carrier with an atypical EcoR1 and Eag1 pattern and a typical BssH1 pattern gave 2.8, 3.0, 5.2–5.4 Kb bands, the Eag1 pattern suggested a DNA sequence change [14%]. PCR gave reproducible bands corresponding to 29, 65, 80 repeats and a faint band for 39 repeats. Intermediate mutation (45–54 r) was found in 5 males and 2 females. Discussion Chromosomal causes of secondary Autism Spectrum Disorder (ASD) About 1.7 % to 4.8 % of individuals with ASD have chromosome abnormalities, including unbalanced translocations, inversions, rings, and interstitial deletions and duplications (Table 5 ). The chromosome abnormalities that have been reported on more than one occasion are duplication of 15q, deletions of 18q, Xp, 2q and the sex chromosome aneuploidies 47,XYY and 45,X [ 16 ]. A recent FISH subtelomere study found one out of ten unselected patients with ASD had a subtelomeric 2qter deletion [ 17 ]. In our experience 7/7 ASD patients were negative for subtelomeric rearrangements. Children with Down syndrome have autism more commonly than expected. The incidence was at least 7 % in one study [ 18 ]. This finding suggests that chromosome abnormalities may lower the threshold for the expression of autism. In our study (4/14 = 28 %) and in other surveys, the common recurring chromosomal abnormality was duplication of the proximal 15q region (Table 5 ). About 1% of individuals with ASD have a chromosomal duplication in the Prader-Willi/Angelman region of proximal 15q [[ 19 , 20 ], present study 4/421[0.95%]]. The duplicated region q11.2q13 is on the maternal chromosome 15 in autistic patients [ 21 - 23 ]. Most commonly, this is a supernumerary isodicentric 15q chromosome detectable by routine cytogenetic studies or, less commonly, an interstitial duplication of the region detected by FISH analysis for the SNRPN gene region. These two chromosome abnormalities have only subtle effects on the physical phenotype. Supernumerary isodicentric 15q chromosomes are de novo occurrences. Duplication of proximal 15q may result from segregation of a parental chromosome translocation or an interstitial 15q duplication. An abnormal gene dosage within 15q11.2-q13 might cause susceptibility to autism. The 15q11-q13 region is shown schematically in Figures 3 & 4 . Chromosome 15q11.2-q13 has gained support as an autism candidate region on the basis of the association of maternally derived chromosomal duplications of this region with an autistic phenotype [ 20 , 24 - 27 ] and genetic evidence for linkage and allelic association in the same interval in chromosomally normal autism families [ 28 - 34 ]. The maternal specificity of chromosome 15 duplications in autism suggests a genomic imprinting effect. There are multiple imprinted genes in 15q11.2-q13, and two neurodevelopmental disorders exhibiting opposite patterns of genomic imprinting have been mapped to this region [ 35 - 37 ]. Interstitial deletion of 15q11.2-q13 specific for the paternal chromosome is the most frequent cause of Prader-Willi syndrome (PWS; MIM 176270), whereas maternal-specific deletion of the same common interval results in Angelman syndrome (AS; MIM 105830). The converse of Angelman Syndrome is observed in autism, that is a maternal duplication. The four causes of Angelman syndrome are 1) maternal deletion of 15q11.2q13, 2) paternal UPD15 3) mutations in UBE3A 4) mutations leading to imprinting errors of this region. A population-based study showed a high rate of ASD in AS [ 38 ]. But, a mutation was not identified in the UBE3A putative promoter or coding region in 10 idiopathic ASD patients [ 39 ]. Lack of expression of the maternally expressed UBE3A gene in the brain is thought to be the cause of AS. Since patients with deletions compared to other types of AS mutations have a more severe phenotype, suggests the involvement of additional gene losses, such as GABAA receptor gene cluster [ 40 ]. Transcripts increased in patients with duplications 15q11.2q13 are maternal UBE3A [ 41 ], maternal ATP10C [ 40 , 42 ] and other transcripts including antisense transcripts that could regulate gene expression [ 43 ] and may contribute to the duplication phenotype. Therefore, over expression of genes in 15q11-q13 probably confers ASD risk. Region proximal to D15S11 is considered to have no phenotypic effect. However, one of our patients had a marker 15 negative for D15S11, therefore, some duplications of 15, proximal to D15S11 and the autism candidate region may also influence susceptibility to autistic traits. Initial studies to characterize the phenotype of 15q11.2q13 duplication patients have found variation among affected people including mental retardation, motor coordination problems, seizure disorder, and impairments in attention, communication, and social function (some but not all with ASD or attention deficit hyperactivity disorder (ADHD)) [ 44 , 45 ]. It appears there may be a parent-of-origin effect on the linkage and association signals in this region of UBE3A and ATP10C [ 46 , 32 , 33 ]. Further studies across data sets, and rigorous evaluation of potential functional effects of associated alleles, and a thorough assessment of haplotype transmission within ATP10C and neighboring genes would be conclusive. The majority of linkage and association data point to the GABRB3 gene, which is one of a cluster of γ-aminobutyric acid (GABA) receptor subunits that map to the distal, apparently nonimprinted segment of the duplicated region (Fig. 3 ). Although a number of groups have detected genetic effects at GABRB3 in independent autism populations [ 29 - 31 ], other studies have failed to replicate these observations [ 47 - 49 ]. Yardin et al 2002 [ 50 ] recommended a systematic screening by FISH, of chromosome region 15q11.2q13 in cases with autistic-like syndrome A deletion of 2q37.3 was identified by FISH in a patient with autism and macrocephaly in our study. Wolff et al 2002 [ 51 ] also reported an autistic patient with a 2q37.3 deletion detected using subtelomere probes. Whole genome screens have suggested several chromosomal regions that are potentially associated with a susceptibility gene for autism. [ 52 - 57 ]. Three studies revealed positive linkage to 2q [ 52 , 53 , 57 ] and a third study demonstrated linkage to distal 2q in a subset of patients with autism and delayed onset of phrase speech [ 53 ]. Genomic scans are limited by the number of loci that are assessed; therefore, not all areas may be equally represented. It is important to note that telomeric regions may have increased meiotic recombination and may be under-represented in these types of analyses. Thus, the FISH approach is an important correlative study in the search for susceptibility genes. Macrocephaly in ASD: most children with autism are born with normal head circumference and about 20% meet the criteria for macrocephaly [ 58 ]. The increased rate of growth in head circumference appears to be most dramatic in the first year of life and corresponds to increased growth of the cerebral cortex as measured by MRI [ 59 ]. A de novo deletion of chromosome 3, del(3)(p25), was found in one case with ASD and development delay in our patient pool. A deletion of 3q region was found by Konstantareas & Homatidis 1999 [ 60 ]. Genome wide scan found a major susceptibility locus at 3q25-27 and there was also allelic association in families with autism spectrum disorder originating from a subisolate of Finland [ 61 , 62 ]. Animal models and linkage data from genome screens implicate the oxytocin receptor at 3p25-p26 [ 61 , 62 ]. To date, there have been no reports of 12q deletions in patients with ASD, to the best of our knowledge. For the first time we report an interstitial deletion 46,XY,del(12)(q21.2q23.3) in a patient with ASD, development delay, mental retardation, multiple congenital abnormality, and family history of Down syndrome. 46,XY,del(13)(q13.2q14.1)de novo was found in one of our patients' with ASD. Hyperserotonemia in autism is one of the longest-standing biochemical findings. The serotonin 2A receptor gene (HTR2A) on chromosome 13q14q21 is a primary candidate gene in autism. Converging data from recent genome screens also implicates the genomic region containing HTR2A [ 63 - 66 ]. Correlation of HTR2A disruption or deletion in our case with a 13q13.2q14.1 deletion and other 13q deletion in ASD patients would complement genome screen data. The recent physical mapping of the serotonin 5-HT(7) receptor gene (HTR7) to 10q23 [ 67 ] raises the question if the inversion inv(10)(p11.2q21.2) present in our patient, which is considered a normal familial variant could have long range influence on HTR7 and susceptibility to autism in some cases. So far there has been no observed association or link between chromosome 14 and ASD. Also, the mosaic inversion inv(14)(q11.2q33) [3/20] found in one of our patients is considered a cultural artifact when seen in 1or 2 cells. In this study, a patient with inv(17)(q23q25)de novo, had ASD, hypotonia and developmental delay. Chromosome 17q shows association with autism by genome wide scans and in linkage studies [ 57 , 66 , 68 ]. There is also interest in 17q since serotonin transporter gene (SERT) has been mapped to17q11-q12 [ 69 ]. We report a patient with 18p deletion due to an unbalanced translocation between 14 and 18. Majority of the reported cases with autism involve deletion of 18q [ 70 - 72 ]. A deletion of the 18p-arm (at band 11.3) in about 50% cells and 50% of the cells with a duplication of the long arm in peripheral blood was described in a mildly obese girl with DSM-III-R autistic disorder and moderate mental retardation [ 73 ]. Another preschool girl with selective autism and a deletion of Chromosome 18p11.l has also been described [ 74 ]. She had communication problems consistent with a diagnosis of autism. However, in the area of reciprocal social interaction she was a little less deviant than most children and had no major behavior problems typical of autistic disorder. Linkage and association studies have suggested at least 2 candidate loci, one on the short and the other on the long arms of chromosome 18 [ 75 ]. One of the breakpoints in the balanced translocation, 46,XX,t(1;14)(q25;q31.2) was 1q25, in the present study. A recent report, links D1S1675 that maps to chromosome 1q24 with autism [ 76 ] using obsessive-compulsive behaviors as a restricting criterion for the analysis. The proximity of our breakpoint and D1S1675 may be coincidental or causal. Although no cases with X- rearrangements were identified, in this study, the literature on abnormal X chromosome and autism is discussed as it has been cited in multiple cases. An autistic (ICD-10) woman had a translocation, t(X;8)(p22.13;q22.1) [ 77 ], a boy with "autistic disorder" had duplication of Xp22 [ 78 ], and a de novo Xp22.3 deletion was observed in 3 autistic females [ 79 ]. Further, mutations in cell adhesion genes NLGN4 on Xp22.3 and NLGN3 on Xq13 are reported in patients with autism [ 80 ]. Therefore, subtle Xp rearrangements have to be considered in the cytogenetic assessment of ASD patients Fragile X syndrome The typical clinical picture in FRAXA includes mental retardation, macro-orchidism, large ears, and prominent jaw. Within neurons, the FMR protein (FMRP) interacts with mRNA and ribosomes, suggesting a role in regulating protein synthesis [ 81 ]. FMRP is heavily synthesized in dendritic spines in response to synaptic activity, and abnormal dendritic spine size and shape have been noted in FRAXA patients and fmr1 knockout mice [ 82 ]. These abnormalities may correspond to an abnormal postsynaptic response that weakens synaptic connections [ 83 ]. A multicenter study in Sweden [ 84 ] found fragile X in 13 of 83 boys (16%) with infantile autism but in none of 19 girls with infantile autism. Klauck et al. (1997) [ 85 ] concluded from molecular genetic studies of 141 patients from 105 simplex and 18 multiplex families that an association of autism with fragile X is nonexistent and that the Xq27.3 region is not a candidate for autism. Stoll (2001) [ 86 ] presented 11 children under the age of 8 years and the difficulties in diagnosis of fragile X syndrome at this age. The author concluded on the importance of fragile X DNA test for all children with mental retardation, autism, or significant developmental delay without a clear etiology. Whereas only a few percent of children with autism have fragile X syndrome, at least half of children with fragile X syndrome have autistic behaviors, including avoidance of eye contact, language delays, repetitive behaviors, sleep disturbances, tantrums, self-injurious behaviors, hyperactivity, impulsiveness, inattention, and sound sensitivities. The frequency of the fragile X syndrome among individuals with autism was ascertained up to 1993 using cytogenetic method. The incidence ranged from 12.7%–1.6%. However, these studies had different criteria for classifying positive cases. The differences were: the number of metaphases analyzed ranged from 20 to 100 and the cut-off range from 1% to 4% metaphases with a fragile X [ 84 , 87 - 94 ]. Using molecular analysis the incidence of fragile X syndrome was 5% (1/20) [ 95 ], 3.3%(1/30) [ 96 ], 12% (3/25) [ 97 ] and the present study 2.2 % (7/316). The difference in incidence between the present (2.2%) and previous studies (3.3% – 12%) may be due to small sample size in the other studies or clinical criteria for selection of patients or both. Mosaicism for FRAX mutation: Forty-three percent of FRAX patients had a mosaic mutation in our study. The prevalence of males who carry a full mutation and a permutation is 15–20% [ 98 - 102 ] among the affected individuals. Nolin et al 1994 [ 103 ] analyzed a group of affected fragile X males by Southern blotting and found 41% (61/148) to be mosaic. This observation of 41% is significantly higher than previous reports 15–20%. The difference could be technical modifications, which permitted the identification of faint premutation bands in some patients. The higher percentage (43 %) of affected males with mosaicism in our study suggests that the occurrence of such individuals may be frequent in patients with ASD. The degree of mental retardation seemed not to be influenced by the presence of premutation alleles in some of the cells and a full mutation in the rest of the cells [ 104 ]. While Merenstein et al 1996 [ 105 ] study suggests there may be some variation of clinical expression in fragile X males with a full mutation and permutation. It has been hypothesized that these mosaic cases should show higher levels of functioning than those who have only the inactive full mutation gene, but previous studies have provided negative or equivocal results. In one study, the cross-sectional development of communication, self-care, socialization, and motor skills was studied in 46 males with fragile X syndrome under age 20 years as a function of two variables: age and the presence or absence of mosaicism. The rate of adaptive skills development was 2–4 times greater in mosaic cases versus full mutation cases. FMR1 protein (FMRP) levels was shown to correlate with IQ, even in mosaic males for 38% of the IQ variance [ 106 , 107 ]. There was also a trend for cases with autism to be more prevalent in the full-mutation group [ 108 ]. However, we found a high incidence (43 %) of mosaic FRAX mutation in autistic patients, which requires confirmation in other FRAX studies of large cohorts of ASD patients. Deletion mutation The molecular mechanism of the FRAX is based on the expansion of a CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. The instability of this CGG repeats containing region is not restricted to the CGG repeat itself but expands to the flanking region as well. de Graaff et al 1996 [ 109 ] described four unrelated fragile X patients mosaic for both a full mutation and a small deletion in the CGG repeat containing region. Sequence analysis of the regions surrounding the deletions showed that both the (CGG)n repeat and some flanking sequences were missing in all four patients. The 5' breakpoints of the deletions were found to be located between 75–53 bp proximal to the CGG repeat. This suggests the presence of a hot spots for deletions in the CGG repeat region of the FMR1 gene and emphasizes the instability of this region in the presence of an expanded CGG repeat [ 110 - 113 ]. All reported cases had fragile X phenotype (which may be an ascertainment bias), and the deletion was usually a faint band suggesting a recent mutation in a small population of cells. Our case had an unusual Southern band pattern, consistent with the presence of both a full mutation (3.7 and 5.8–7.9 kb; 200–900 r)and a deleted (2.8 kb; 30 r) mutation with partial methylation. Since immunohistochemical staining or Western blot analysis was not performed to assess the FMRP production it precludes clinical correlation. A 6-year old mosaic permutation female carrier had 29,65,80,39 repeats. The autism spectrum disorder in our patient may be due to diminished translational efficiency in FMRP production. Tassone et al (2000) [ 114 ] [have shown FMRP in 61% and 70% lymphocytes in two females 91/2 years and 33 years of age with 103/33, 180/30 repeats and IQ of 49 and 90, respectively. The first patient has physical, cognitive, and behavioral features of the fragile X phenotype but FMRP was in the normal range, while the second patient also had FMRP level in the normal range was treated for depression and has a history of ovarian cyst, premature menopause at 27 years, and a hysterectomy at 31 years. She also experienced social anxiety, panic attacks, mood swings, and mild obsessive compulsive behaviors. Johnston et al. (2001) [115] recently observed that emotional problems, including depression and interpersonal sensitivity, were more likely to occur in carrier females with >100 repeats. Austism spectrum disorder has been observed mostly in males with permutations [ 114 , 116 , 117 ]. The mosaic permutation in our female patient was 80 repeats as the largest expansion which is still below 100 repeats found in the affected cases in literature. Clinical correlation in our permutation mosaic case is hindered by the lack of FMRP studies and even so, the tissue distribution of mosaicism and FMRP profile would be the necessary phenotype determinant. The significance of intermediate alleles found in 7/316 (Average age 4.36 years) of our patients requires further exploration on larger independent samples as they may raise the threshold for important developmental disabilities and/or physical features [ 116 ]. The limitations of our retrospective study were a lack of uniform clinical criterion for inclusion and limited behavioral diagnostic information for purposes of dissecting the phenotype. However, for autism diagnosis it may be reliable, as shown in a recent study [ 118 ] which examined the UK General Practitioner Research Database (GPRD) and found the diagnosis of autism among general practitioners in the UK had a high positive predictive value. Conclusions In our experience, the incidence of chromosome (3.33%) and fragile-X (2.2%) abnormalities totals to 5.53% in a population of patients with an indication of autism sent for genetic testing. Since, 28% percent of chromosome abnormalities detected in our study were subtle; a high resolution cytogenetic study for ASD patients with a scrutiny of 15q11.2q13, 2q37 and Xp23.3 region should be standard practice. The higher incidence of mosaic fragile-X mutations with partial methylation in our ASD population vs incidence of mosaicism in reported populations with fragile X syndrome [50% vs 15–40%], suggests that faint bands and variations in the Southern band pattern may occur rather frequently in fragile X positive autistic patients. The mosaic FRAXA mutation with normal and abnormal or partial methylation may also enhance the threshold for autism spectrum disorder. Careful analysis and high quality gels are required to rule out the type of mutation in patients with autistic traits. FRMP estimates in positive cases would be an extremely useful adjunct especially in mosaic cases. Future studies are necessary to corroborate the high incidence of mosaicism and their role in ASD. Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here:

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517827

A Richer Map for Searching Scientific Literature

Anyone who has used a search engine quickly becomes familiar with both their power and limitations. A key-word search for “bush kerry butterfly county” turns up almost twenty-eight thousand documents, but a scant few of them are of any interest to the botanist studying the butterfly bush in County Kerry. The problem is more circumscribed but no less significant within specialized databases, such as the fourteen-plus million medical journal articles catalogued by PubMed. It is not so much that the literature is too vast, but that the search strategies are too weak. A simple list of key words used to tag and retrieve a document cannot begin to capture the richness of the information within, especially when that wealth is expressed in syntactically complex sentences like “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” But there is another way. Rather than simply extracting a limited list of key words from an article's abstract, the entire text of the document can be categorized into classes: some words represent entities (e.g., gene, cellular component, molecular function) and others relationships (e.g., physical association, purpose, comparison, regulation). The entire set of entities and relationships can be linked to create a map of the information within the document, which, like a physical map, captures some of the complexity of the territory it describes. Humans excel at this type of concept mapping, but their labors are slow and expensive. In this issue, Paul Sternberg and colleagues at the California Institute of Technology (Caltech) describe a computer-based system that performs the same task, and show that it is almost as good as humans at mapping out the scientific literature concerning the laboratory nematode, Caenorhabditis elegans . Sternberg's system, called Textpresso, includes 33 categories of terms, both of entities and relationships, and a full list of all possible examples for each entity (for genes, for example, this would be specific gene names) and relationship (for physical association, this would include bind, adhere, link, etc.). This collection, called an ontology, is then applied to sentences within the text of a document to map out the relationships within—for instance, the mention of two genes within a sentence along with any form of the word stem “regulat-” indicates that one gene probably regulates the other, and the sentence is marked accordingly. With scores of tags applied, the full markup of a sentence is typically much longer than the sentence itself. Currently, Textpresso has marked up almost 4,000 full-text articles on C. elegans , representing 60% of the entire literature. Identifying terms in scientific prose The final result is a document that can be queried in subtle ways impossible with mere key words. For instance, to find entities (whether they be transcription factors, small molecules, or anything else described to date) that interact with the aging-related gene daf-16 , one enters the terms “daf-16” and “association.” Textpresso returns 125 publications, with citations and links to the articles. (Textpresso is available at www.textpresso.org .) The results can be further refined by adding other entities or relationships, as well as by specifying author, journal, or year of publication. Textpresso's ability to find relevant documents, and ignore irrelevant ones, is still not as great as an expert human curator of the same literature. But the system can be constantly tweaked to get better and better. This process requires human intervention, and the Caltech team does not think this is likely to be automated anytime soon. On the other hand, the structure of Textpresso, and to some extent the ontological lists from C. elegans , can be used for literature analysis of other model organisms. Finally, the fully annotated literature within a field is not only a repository of scientific facts, but also a data mine of human communication, which can be queried for patterns having little to do with model organisms and much to do with how scientists communicate with each other.

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548304

Steroid-refractory ulcerative colitis treated with corticosteroids, metronidazole and vancomycin: a case report

Background Increasing evidence elucidating the pathogenic mechanisms of ulcerative colitis (UC) has accumulated and the disease is widely assumed to be the consequence of genetic susceptibility and an abnormal immune response to commensal bacteria. However evidence regarding an infectious etiology in UC remains elusive. Case presentation We report a provocative case of UC with profound rheumatologic involvement directly preceded by Clostridium difficile infection and accompanying fever, vomiting, bloody diarrhea, and arthritis. Colonic biopsy revealed a histopathology suggestive of UC. Antibiotic treatment eliminated detectable levels of enteric pathogens but did not abate symptoms. Resolution of symptoms was procurable with oral prednisone, but tapering of corticosteroids was only achievable in combination therapy with vancomycin and metronidazole. Conclusions An infectious pathogen may have both precipitated and exacerbated autoimmune disease attributes in UC, symptoms of which could be resolved only with a combination of corticosteroids, vancomycin and metronidazole. This may warrant the need for more perceptive scrutiny of C. difficile and the like in patients with UC.

Background Clostridium difficile infection may be a compounding factor in ulcerative colitis (UC) with data suggestive of a role in disease exacerbation or initiation by the organism in a subset of patients [ 1 ]. Adjunctive antibiotic therapy, either broad-spectrum or gram positive-specific, has shown limited efficacy in UC [ 2 , 3 ]. However, sporadic reports in which significant therapeutic potential was achieved [ 4 - 6 ] suggest that a distinct subclass of patients with an infectious-associated phenotype may exist. Although there have been an increasing number of case reports on ulcerative colitis complicated by cytomegalovirus infection [ 7 - 9 ], there are few with conclusive evidence of infection with Clostridium difficile . Herein, we report a case of steroid-resistant UC with profound rheumatologic involvement immediately preceded by C. difficile infection in which remission of UC symptoms was twice achieved in response to adjunct metronidazole and vancomycin therapy. The patient remained symptom-free without supportive therapy for a four-year period. Case presentation A 32-year-old white male presented with nausea, vomiting, crampy lower abdominal pain and diarrhea. His initial lab values were hemoglobin 15.6 g/dl (14–18 g/dl), hematocrit 45.8% (40–54%), sodium 131 mEq/l (135–146 mEq/l), potassium 3.9 mEq/l (3.5–5.5 mEq/l), bicarbonate 24 mEq/l (22–24 mEq/l), chloride 91 mEq/l (95–112 mEq/l), BUN 37 mg/dl (7–25 mg/dl), serum creatinine 1.6 mg/dl (0.7–1.4 mg/dl), and normal liver function tests. He was treated with intravenous fluids, Ciprofloxacin and anti-emetics, to which the diarrhea resolved in 2–3 days. After one month he had recurrent watery diarrhea accompanied with rectal pain, bleeding and vomiting. He also developed fever of 101.6 F. The physical exam was remarkable for decreased bowel sounds and moderate lower abdominal tenderness. There were no masses or organomegalies palpable. The rectal exam was remarkable for tenderness and heme-positive mucus. The remainder of the physical exam was uneventful. Stool samples from the initial visit were positive for Clostridium difficile cytotoxin, and depicted many WBCs (neutrophils 99% and eosinophils 1%), suggesting an infectious etiology. Stool sample was negative for giardia lamblia, salmonella, shigella, campylobacter, aeromonas and plesiomonas. There was no predominance of staphylococcus, yeast, or pseudomonas. The patient was initially started on Ciprofloxacin (500 mg bid.) for 3 days without symptom resolution. Metronidazole was added (250 mg tid.), but intolerance of the patient to metronidazole with exacerbations of nausea prompted the replacement to Vancomycin (250 mg qid) in conjunction with Ciprofloxacin for 7 days. During this initial treatment period, the patient characteristically developed migratory polyarticular joint pain and swelling involving the shoulders, elbows, fingers, hips and knees. Lack of response to antibiotics suggested an autoimmune disease as a contributing factor. At this time a sigmoidoscopy and pinch biopsy revealed diffuse acute and chronic inflammation with cryptitis, mucin depletion, and glandular foreshortening and branching suggestive of UC (Fig. 1 ). The patient was treated with Asacol (400 mg bid.) in conjunction with metronidazole (250 mg qid). After 7 days of combined therapy with continued weight loss and no improvement, prednisone (60 mg qid.) was added, resulting in cessation of GI symptoms and moderate improvement in joint pain and swelling. After 7 days of continued improvement, the antibiotic and Asacol therapy were stopped. The patient was maintained on high dose steroids for 14 days. However, fever, gastrointestinal, and joint symptoms recurred upon tapering of prednisone to 20 mg a day. When symptoms recurred, a stool culture was negative for infectious agents including Clostridium difficile antigen, confirming an autoimmune component to disease pathology in this patient. Prednisone was increased to 30 mg per day at which point gastrointestinal symptoms resolved but joint swelling and pain continued. Over the course of 3 months prednisone tapering was attempted three additional times, with each resulting in symptom recurrence. After 3 months of treatment, the patient experienced an acute dental abscess and was prescribed a combination vancomycin (250 mg tid) and metronidazole (250 mg tid). During this period, the patient was able to gradually reduce prednisone dosage without symptom recurrence. Antibiotics were continued for 30 days during which time prednisone was tapered completely. Symptoms did not recur and the patient remained symptom-free without further therapeutic intervention for 4.5 years. After this prolonged period of remission of UC symptoms, a second episode similar to the first occurred. The patient once again presented with bloody diarrhea, nausea, vomiting, and joint pain. Stool cultures were Clostridium difficile positive by culture and cytotoxin. Once again the patient was treated with 60 mg daily of prednisone and metronidazole (250 mg tid). Symptoms did not resolve, and vancomycin (125 mg tid) was added, resulting in a significant improvement in symptoms. After 2 weeks of combined therapy metronidazole treatment was stopped and symptoms recurred within a week. Metronidazole was once again added, leading to resolution of symptoms. Combined antibiotic and steroid therapy continued for 2 months during which time prednisone was tapered completely and a second remission of UC symptoms was induced. Discussion The patient was remarkable in several regards. The onset of UC and spondyloarthropathy coincided with C. difficile infection. While various therapeutic regimens of prednisone, vancomycin and metronidazole preceded a negative C. difficile toxin test, remission of UC symptoms could only be induced by the combination of these three medications. C. difficile toxin tests have shown varying efficacy in diagnosing pseudomembranous colitis. It has been demonstrated that toxin-negative patients with C. difficile positive stool culture may still have symptomatic pseudomembranous colitis [ 10 ]. Lynch et al showed that 46% of stool specimens from patients test negative by cytotoxin but positive by culture [ 11 ]. Likewise, others have shown that some cases of C. difficile infection cannot be detected by the cytotoxin assay and suggested that the organism has the ability to vary toxin production [ 12 - 16 ]. The bacterium also tends to persist as an antibiotic-resistant vegetative spore, which explains the high frequency at which the symptoms recur following treatment. Since toxin-negative patients have presented with C. difficile related diarrhea, it is reasonable to assume that the organism may exist undetectably in a symptomatic patient, but especially in a patient with inflammatory bowel disease (IBD). The elusiveness of the bacterium in diagnosis of C. difficile infected IBD patients was confirmed in 2001 by Markowitz et al. They demonstrated that single-toxin assays failed to identify C. difficile infection in approximately 40% of pediatric IBD patients and in a case report where a toxin A positive, toxin B negative variant was detected [ 14 , 17 ]. It has also been shown that IBD patients have a higher incidence of C. difficile infection than in healthy controls [ 18 ]. When considered together with the case reported here, this information leads us to the hypothesis that a small subclass of UC pathology may actually have an infectious etiology resulting from chronic C. difficile infection. IBD has been shown to be more prevalent in industrialized nations, where antibiotic treatment has its longest history [ 19 ]. The increase in IBD prevalence parallels rising antibiotic use over the last 50 years [ 20 ]. If an undetected antibiotic-resistant enteric pathogen is responsible for inducing or exacerbating IBD, then widespread antibiotic use may have contributed to the increased prevalence of IBD in these industrialized nations. This case is especially interesting considering H. pylori and its recently discovered tendency to adhere to glycoconjugates expressed in inflamed gastric mucosa [ 21 ]. In light of this new information, it is tempting to speculate that C. difficile could similarly bind to inflamed colonic tissue in this subclass of UC patients. Further research in this area could yield valuable new data. The potential utility of combined therapy is further suggested by the fact that antimicrobial resistance among C. difficile strains to metronidazole and with intermediate resistance to vancomycin is emerging in countries like Hong Kong (where one of 100 C. difficile isolates was resistant to metronidazole) and Spain (where 9% of 469 clinically significant C. difficile isolates were resistant to metronidazole, particularly in isolates recovered from HIV-positive patients and few patients also had intermediate resistance to vancomycin) [ 22 - 24 ] It is interesting to note that no resistance was found to metronidazole or vancomycin among C. difficile strains from isolates in patients from UK, Germany, Brazil, Poland or Kuwait [ 25 - 29 ]. The Public Health Laboratory Service (PHLS) Anaerobe Reference Unit (ARU) has also not been successful in detection of metronidazole resistance in any of their over 1000 C. difficile isolates tested [ 24 ]. The impact of drug resistance should be considered if long-term treatment is utilized in patients. However, a complicating factor in this context is that for metronidazole and vancomycin, minimum inhibitory concentration susceptibility breakpoints are usually set for isolates causing systemic infections that are based upon antimicrobial levels in blood (serum) and not on the levels in the intraluminal area, where higher drug concentrations can be achieved [ 30 ]. Conclusions Recurrent relapses could only be suppressed in this patient by the combination therapy of corticosteroids, metronidazole and vancomycin. Our observations suggest that this combination therapy may be effective to confront and induce remissions of UC symptoms in patients with C. difficile toxin-positive refractory autoimmune symptomatologic UC as opposed to the use of a single agent. An opportunistic C. difficile infection commonly results from the use of broad-spectrum antibiotics like quinolones. The frequent use of these antibiotics in treating IBD suggests that these patients could develop C. difficile infection during treatment, thereby exacerbating symptoms and preventing remission of UC symptoms. It may therefore be practical to probe for C. difficile infections more meticulously in patients with IBD. Competing interests The author(s) declare they have no competing interests. Authors' contributions All authors contributed equally to this work. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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535338

Size control in growing yeast and mammalian cells

Background In a recent publication it was claimed that cultured mammalian cells, in contrast to yeasts, maintain a constant size distribution in the population without a size checkpoint. This inference may be challengeable. Results (1) It is argued that "weak" size control implies the existence of a checkpoint, and unfortunately the technique used by Conlon and Raff might obscure such a weak mechanism. (2) Previous investigations of size control in yeasts have shown that individual cell data, rather than means and variances of cell populations, are prerequisites for reliable interpretation. (3) No experimental data so far obtained suggest that in any cell culture a linear growth pattern in cell mass can maintain size homeostasis on its own without size control. (4) Studies on fission yeast mutants indicate that the molecular mechanisms of size control vary with genetic background, implying that no single mechanism is likely to apply to any cell type, including cultured mammalian cells, under all conditions. Conclusion The claim that cultured mammalian cells maintain size homeostasis without a checkpoint needs to be re-evaluated by measurements on individual cells.

Introduction Conlon and Raff [ 1 ] recently stated that constant size distribution is maintained in a culture of proliferating rat Schwann cells without the need for any size checkpoint, in contrast to the situation in yeast. Before discussing their conclusion, it is important to consider their techniques and nomenclature. They used an electronic (Coulter) counter to measure the mean and variation of cell volume at intervals in whole cell populations. In fission yeast, the most detailed study to date involved the measurement of lengths (proportional to volume) of single growing cells [ 2 ]. This is a vital matter that is not fully appreciated. There is a long history of size measurements on fission yeast and the involvement of size in the control of the cell cycle, see e.g. [ 3 - 5 ], but only recently has the term "size control" come into use [ 2 , 6 , 7 ]. Although convenient, the phrase is something of a cover-all, since the nature of "size control" varies in fission yeast and the term is unlikely to denote exactly the same phenomena in mammalian cells. Moreover, mammalian cells normally form tissues, where greater emphasis has to be placed on external factors. However, size control operates in all systems where it has been possible to examine individual cells through their cell cycle. Its nature is likely to be different in each, although this will only become clear when its molecular basis is understood [ 6 ]. Discussion The "evidence against size checkpoints" in cultured Schwann cells: an assessment One of the two cases cited by Conlon and Raff as evidence for the lack of a size checkpoint is the slow change in size when cells are stimulated by fresh medium [ 1 ]. The reason we do not regard this as strong evidence comes from data on fission yeast. When either cycle time or total length extension is plotted as a function of birth length, the negative slope of the regression line is large in wild-type cells, implying that size control is "strong" and deviations from the average will be corrected within a single cycle [ 2 ]. But the slope can be less, as for example in diploids, and then the control becomes "weak", since deviations will not be corrected within one cycle. Without the support of single cell data, it is nevertheless possible to imagine the presence of a weak size control in mammalian cells, homologous to that in yeast, exerting a slow but important action over several cycles. But can such a weak size control be considered a checkpoint? This entails another semantic or philosophical problem, since "checkpoint" is as ambiguous as "size control". Those who regard very sharp responses (e.g. all or none) as a criterion would argue that a weak mechanism is not a checkpoint. However, since it fulfils its function by ensuring homeostasis in a cell population, we believe that a weak size control should also be considered a checkpoint. Another problem in [ 1 ] is the increasing size of quiescent cells blocked in S phase by aphidicolin and stimulated into growth. The mean cell volume increased linearly five-fold over 100 h. A similar if less marked increase was also found in total protein. In addition, the valuable observation was made that the rates of protein synthesis and degradation increased with size. It is almost certainly true that size-dependent synthesis and degradation would not result in a size-independent pattern of protein accumulation. In the simplest case, if the rates of both protein synthesis and degradation are linear functions of total protein (mass), protein content will show a size-dependent exponential dependence on time. Therefore, further measurements and analyses need to be carried out to resolve this discrepancy. Moreover, it follows from the hypothesis of Brooks [ 8 ] that, in the absence of size control, an exponential growth of cell volume results in a continuous increase in the dispersion of cell size at division; in contrast, linear growth would not increase the dispersion in consecutive cycles, and individual cell sizes would converge towards the mean after perturbations. To date, however, no experimental evidence has been published to show that a cell culture can maintain homeostasis by linear growth without a size checkpoint. It is plausible that this might happen because of the general need for co-ordination between the cytoplasmic and chromosome cycles, but we are not convinced that linear growth on its own would meet this requirement. In the case of fission yeast, there is evidence that linear growth without a size checkpoint cannot maintain homeostasis in the culture (see below). Results of similar experiments with fission yeast Related observations from fission yeast show that normal size control is abolished in the type of block experiment used in [ 1 ]; however this might be apparent only after release. This was first shown by Fantes [ 3 ] and was later expanded by us [ 2 ]. The time-scale, however, was shorter than in mammalian cells. It is also true, as Conlon and Raff [ 1 ] point out, that the pattern of total protein and RNA content in several yeast cdc mutants was approximately exponential over a longer period after this type of block. This result comes from an early paper and depends on measurements of absolute amounts of protein and RNA/ml [ 5 ]. There is not much evidence from the far more accurate method of measuring synthesis rates by pulses of radioactive precursor, but such evidence as exists fails to support the conclusion. In the widely used mutant cdc2-33 , the rate of protein synthesis reaches a plateau after 4 h [ 9 ]. Also, only fairly minor changes in protein and RNA synthesis rates occur over 7 h in the mutant cdc13 [ 10 ]. Because linear growth was measured in mammalian cells during a long S phase block [ 1 ], we have another objection to make here. The fission yeast cell cycle can be considered as linear segments of volume growth with points (called rate change-points) where there are changes in growth rate [ 11 ], which may be partly associated with a gene-dosage effect [ 2 ]. If a similar mechanism operates in mammalian cells during the normal cycle, it could never be observed when replication is arrested. Genetic background and size control in fission yeast There are other points about the fission yeast experiments that are relevant to size control, which is almost certainly affected by genetic background. The main rate change point (an effect of gene-dosage, see above) is shifted in position in wee mutants (S phase is also shifted), but this size control is a strong one, albeit weaker in diploids [ 2 ]. There appears to be a different mechanism in the strange double mutant wee1-50 cdc25Δ [ 12 ]. It seems improbable that all these variations involve the same molecular mechanisms; rather, they suggest a variety of options in the nature and positions of size controls. But since we do not understand the molecular mechanisms involved, these matters remain to be resolved. Another point is that the normal rules of the cycle can be broken temporarily, but not for long. For example, mammalian cells can certainly grow without entering S phase [ 6 ]; and the very large cells of early embryos only develop a size control after a number of cycles [ 13 ]. Block and release protocols can be used to induce synchrony in cdc mutants of fission yeast, and size control is lost for some cycles (but not forever), as indicated by the lack of negative correlation between length extension and birth length [ 2 , 3 ]. However, if size control has been permanently lost, as in the case of the double mutant wee1-50 rum1Δ , the cells lose viability after a few generations [ 14 ] despite the near-linearity of their length growth pattern [ 2 ]. According to the hypothesis of Brooks [ 8 ], cell size should be convergent in this case after some generations, leading to size homeostasis. However, the mathematical solution seems not to be applicable for the yeast cells, which rather die. Conclusions To return to mammalian cells: our view is that the best way forward is to try to devise ways of following individual cells through the cycle. It is easy for us to say this, since we have had the great advantage of working with regular shaped yeast cells that stay still and grow steadily on agar. The mammalian cells situation is far more difficult, but there is reasonable hope that it can be solved by methods of tracking individual cells such as fibroblasts by a semi-automatic method and measuring their dry mass by interferometry [ 15 ]. The main problem with the technique of Conlon and Raff is that it cannot distinguish between a weak (but existing!) size control and its total lack. Very recently, similar experiments have been done with erythroblasts and fibroblasts from different vertebrates, and the results seem to suggest the existence of a strong size control in every case [ 16 ]. Our present knowledge that the existence of size control (either weak or strong) seems to be general, suggests that rat Schwann cells are probably not exceptions above the rule, but they rather have a weak size control in spite of the conclusions of Conlon and Raff [ 1 ]. To choose the correct interpretation between the two possible ones is a challenge for the future.

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522877

Improving identification of differentially expressed genes in microarray studies using information from public databases

The process of identifying differentially expressed genes in miroarray studies with small sample sizes can be improved substantially by extracting information from a large number of datasets accumulated in public databases.

Background Microarray experiments are often used to identify potentially relevant genes in biological processes. By determining which genes are differentially expressed between different states, for example, hypotheses can be developed as to the role of those genes in the underlying biological mechanism [ 1 - 4 ]. However, the fact that microarrays simultaneously assess the expression of tens of thousands of genes makes it difficult to extract pertinent information from background noise. With a multitude of variables, it is easy to generate a high percentage of false positives, and validation is expensive and time-consuming. This issue is aggravated by the high cost of microarrays and often by the difficulty of obtaining enough biological or clinical samples, causing microarray experiments to be performed on a smaller scale than desirable in almost all cases. For exploratory analysis in particular, very few biological or technical replicates are run at present. For a two-class comparison, three-by-three or smaller experiments are not uncommon. For brevity, we will use the notation 'NvN' or 'N by N' to denote a two-group comparison with N arrays vs N arrays. Overall, the need for a large sample size is acute for expression profiling studies. The number of arrays needed in a study depends on many factors, including the study design, the magnitude of biological variation in the samples, technical variability introduced in the experiment, and the desired level of sensitivity and specificity for differential expression. Several studies have examined this issue. A model with additive and multiplicative noise was used to derive the number of samples necessary for detecting fold changes of given magnitude when false-positive and false-negative rates are specified [ 5 ]. The difficulty, however, is that parameters describing technical and biological variations must be estimated for the model, which is not an easy task. When 16 public datasets, mostly from cancer studies, were examined using a repeated sampling approach [ 6 ], it was observed that stable results for differentially expressed genes are not obtained until at least five biological replicates are used and that 10-15 replicates are needed for sufficient stability. This is consistent with the results obtained in [ 7 ]. According to these criteria at least, many microarray studies are vastly underpowered. From the perspective of analysis, it is always desirable to have sufficient data. Some data analysts may even insist on a minimum number of samples before starting statistical analysis. However, when practical considerations limit the sample size, it is important to work with the given data in an optimal manner to extract as much information as possible. In the context of finding differentially expressed genes, the null hypothesis for each gene is that it is not differentially expressed between two groups, usually against the two-sided alternative hypothesis that the gene is up- or downregulated. The most commonly used statistical test in this setting has been the two-sample t -test, although other similar statistics such as the signal-to-noise ratios [ 1 ] have often been used as described below. There are a variety of statistical issues involved with identifying differential expressed genes, such as the adjustment of p -values for multiple testing [ 8 ] and the use of the false-discovery rate [ 9 ]. Ideally, the joint distribution of the test statistics should be considered, in order to account for correlation among the genes [ 10 ], but in practice, because of the difficulties associated with the number of genes being many times that of the samples, most testing procedures are carried out in a univariate manner for each gene [ 11 ]. The method we introduce here also performs a test independently for each gene and ignores correlation among genes. A fundamental difficulty in drawing reliable conclusions from a small number of samples lies in accurate estimation of the gene-specific variances, or the variance of a difference in mean expression levels per gene, with which to determine the statistical significance of observed changes in expression. Because variances based on a very small number of samples tend to fluctuate wildly as a result of randomness in sampling from a population, our ability to assess differential expression is drastically impacted. A naive application of standard methods used for larger sample sizes can result in a large number of false positives for differential expression. For example, with a small sample size, the list of significant genes identified by the t -test or variations thereof is crowded by a large fraction of genes for which large t -statistics are due to underestimation of variance by chance. Many methods have been devised to address this problem. A popular approach has been some type of regularization of the t -test. A Bayesian framework for combining the variance estimate with a background variance associated with neighboring genes was developed in [ 12 ]; a method of pooling errors among genes in which expression values are similar is presented in [ 13 ]. In the popular significance analysis of microarrays (SAM) method, a small constant is added to the variance estimate to prevent it from getting too small [ 14 ]; empirical Bayes methods compensate for the lack of enough replicates by combining information across the arrays [ 15 - 17 ]. Nonparametric methods [ 18 ], analysis of variance approach [ 19 ], and Bayesian hierarchical models [ 20 , 21 ] are also available. Some of these methods are compared in [ 22 ]. Whereas all the available methods attempt to improve the identification of differentially expressed genes essentially by gathering information across similar genes, we suggest another solution. We propose estimating the natural variance of individual genes using a large number of experiments performed previously. This provides a different and potentially more stable and accurate estimate of the variance for each gene than by simply looking at the variance of a small number of expression levels, especially in studies with very small sample sizes. Using these variances as the basis for determining differential expression offers an alternative method that can reduce the false-positive rate significantly. As the most effective method, we propose a hybrid method in which we combine the variance estimate from the current dataset with the estimate from previous experiments. This approach can also be incorporated in other settings, especially in a Bayesian framework with prior distribution for variance derived from the database. It can be applied more generally to other testing procedures such as ANOVA that benefit from more accurate estimation of gene-specific variances, and can be easily extended to the estimation of the covariance matrix in multivariate analysis. More reliable calculations of such variances based on many chips is becoming increasingly possible through large public databases of previous experiments. Public databases such as the Gene Expression Omnibus (GEO) [ 23 ] contain data from many chips, with the goal of gaining information from pooling data. GEO currently has thousands of chips, with a heavy skewing towards Affymetrix MG-U74Av2 and HG-U95 chips. Specifically, there are about a thousand HG-U95A chips and another thousand MG-U74Av2 chips, and these numbers are growing steadily (our gene-specific variances were calculated when the database held only 865 chips). Other large public databases include ArrayExpress [ 24 ], Yale Microarray Database [ 25 ], and Stanford Microarray Database [ 26 ]. GEO was selected as our primary source of reference because it had the largest compilation of single-channel microarray chips. We chose to analyze Affymetrix chips because the standardization of single-channel chips allows for easier cross-experiment comparison than dual-channel chips. The dual-channel chips are often custom-made and lack consistency in the genes represented; more important, different experiments use different reference channels, which makes it difficult to compare across experiments. Results Comparing various methods We compared the performance of four methods in accurately assessing differential expression of genes: the standard t -test, the new GEO-adjusted method, the regularized t -test, and a hybrid method combining the GEO method and the regularized t -test. The primary difference between these methods lies in the denominator of each method's t -statistic. The GEO-adjusted method replaces the sample variance estimate in the denominator with the gene-specific variance calculated from the GEO database (details for the calculation of the variance, which can be either global or pooled, are described in Materials and methods). Hence, the genes are sorted using the modified t -statistic: where μ 1 i , μ 2 i are the means for the groups 1 and 2, respectively, for the i th gene, n 1 and n 2 are the sample sizes in the groups 1 and 2, and σ 2 GEO ,1 is the gene-specific variance from the GEO database. The regularized method we used added a small constant to the denominator of the t -test, ranking genes based on the modified t -statistic: where σ 0 is the fifth percentile of all variances ( σ 0 can also be calculated to minimize the coefficient of variation of the statistic [ 14 ]). The regularized t -test smoothes out the effects of underestimated variances and therefore returns a more reliable assessment of differentially expressed genes in small samples than the standard t -test. Finally, we introduce a hybrid algorithm that combines the GEO method with others through a voting mechanism. This provides a portable solution that can be combined with a variety of other testing procedures and could potentially improve the performance of any other algorithms designed to determine differential expression in experiments with small sample sizes. Testing procedure and dataset To compare the effectiveness of the methods, we determined lists of differentially expressed genes in order of significance by applying each procedure to a large number of subsets of arrays of a given size. These genes were then compared with the 'master' list of differentially expressed genes to assess the accuracy of the method. Because we generally do not know the correct ordering of genes with differential expression, we substituted the list obtained by the t -test analysis for the full dataset as the master list; with a sufficiently large dataset, this master list is a close approximation to the true list. Thus, we used the large dataset to compute a true t -statistic for each gene and then treated random small subsamples of the arrays from the dataset as simulated observed datasets from which we could compute estimated ranks for small subsamples. Because exploring all realizations of possible subsets for a large full dataset would be prohibitively time-consuming (for example, more than 10 8 combinations of 3v3 subsets for our first dataset), we sampled repeatedly for subsets until we obtained convergent results. We then compared these lists of differentially expressed genes with the master list for overlaps or correlations in the orderings. Once the size of the subsets approaches the size of the full dataset, there can be a substantial overestimation in the overlap of genes, owing to the fact that the master list is generated using the dataset from which the subsamples are derived. However, this effect appears to be negligible in our simulations because of the large size of our full dataset and the small subsample sizes that are of our interest. The dataset primarily used for testing was a prostate cancer dataset [ 27 ] that had 50 normal samples and 52 tumor samples, with follow-up tests performed on a smaller Duchenne muscular dystrophy dataset [ 28 ] to confirm results. In our subsampling process, a small number of patients are randomly selected from each group and a variety of methods were used to determine a list of differentially expressed genes. We are mainly interested in very small sizes of one to three samples per group. For concreteness, we focus on the results for 2v2 comparisons first, but we also describe 1v1 and 3v3 comparisons. Note that large datasets are utilized here solely for the purpose of evaluating the method and that the method is designed to be used for studies with small samples. Numerical results with a GEO-adjusted t -test The first measure that was used to assess the effectiveness of the GEO-adjusted method was the correlation between the rank of the top genes returned by various methods and the true rank of those genes. This method was also used in [ 6 ]. In this measure, the standard t -test was compared to the GEO-adjusted method. The behavior of the correlation coefficient was tracked as the number of genes being analyzed was increased and the averaged values over many simulations are shown in Figure 1 . If the method were perfectly effective and ranked genes in the same order as their true ranking according to the master list, the correlation should be 1. However, the correlation coefficients were surprisingly low. This reflects the great difficulty of obtaining accurate or stable lists of differentially expressed genes from small sample sizes. Nonetheless, Figure 1 reveals that the correlation improves for the t -test as the sample size increases, and that the results of GEO tend to correlate better with the master list than the results of 2v2, 3v3, 4v4, or 5v5 t -test. To further assess the reliability of the results, tests were conducted to determine the number of top 50 genes from the master list that were accurately returned using various methods. In Figure 2 , this is plotted as a function of the list length generated by these methods, at 10, 50, 100, 150, 200, 250 and 300. For example, a list of 100 genes from the 2v2 GEO method contains just over 10 genes from the top 50 genes from the master list. Again, the low overlap clearly illustrates the difficulty of obtaining an accurate list of significant genes. We believe the low numbers to be partly due to the nature of heterogeneous samples in our test datasets (see Discussion); therefore, we focus more on the trend among the various methods here. This metric indicated that the GEO method is considerably more reliable than the t -test at determining differentially expressed genes in small sample sizes. Compared to a simple t -test, the GEO method performs substantially better, returning results from a 2v2 test that are comparable to the results returned by a 5v5 experiment using t -test. Using GEO variances on a 2v2 test returns 231% more of the top 50 genes than the unadjusted t -test. While we are not suggesting that a simple t -test is a recommended method of assessing differential expression in such small sample sizes, it illustrates the potential of this method. Using gene-specific variances developed from GEO databases is clearly more accurate than the variances that an uninformed t -test derives from small samples. We do not plot the error bars for each measurement in the figures owing to space constraints, but we have verified in the important cases that the separation between the curves is significant. The GEO-adjusted method also compared favorably to a regularized t -test. By smoothing out the variance estimates, the regularized t -test returns a more accurate assessment of differentially expressed genes than the standard t -test. Thus the gains from the GEO method over the regularized t -test were less substantial than over the standard t -test but still significant, especially for shorter gene lists. Improving our ability to reliably detect the differentially expressed genes with a short list is generally more valuable than doing so with a longer list simply because these genes at the top are the ones that an investigator examines most closely. As shown in Figure 3 , the average gain of the 2v2 GEO sample versus the 2v2 regularized t -test in those three areas (50, 100 and 150 genes) was more than 30%. The performance of GEO on a 2v2 analysis seems roughly comparable to the performance of the regularized t -test on a 3v3 sample analysis. Superior performance of a hybrid method One of the greatest advantages of the GEO method is that it can be combined with other methods. Because the regularized t -test and the GEO method both use different, yet effective, techniques to smooth out variance, they can both contribute to the differential expression analysis. By using a voting method that weights and averages the results returned from the regularized t -test and the GEO method, the performance improves further (see Materials and methods for details). The results of a 2v2 chip analysis using our voting method nearly match the performance of a 4v4 regularized t -test analysis, which is quite promising (Figure 3 ). As before, our incidence of the top 50 genes in the top 10 listed, top 50 listed and top 100 listed are improved. The voting method returns 88% more of the top 50 genes than the regularized t -test alone. We also see the greatest improvement in the larger sets of genes, thus negating one of the weaknesses of the GEO-adjusted method. By combining the advantages of the regularized t -test and the additional information from the gene-specific variances, we are able effectively to pare the required number of chips in this case and to elicit better results from the chips we do have. Further details are provided in the Materials and methods section. Tests were also performed on other sample sizes, namely 1v1 and 3v3. Although we view the first case especially as an inadequate design and do not recommend it, we have found that investigators are sometimes forced to perform analysis on such a small number of samples. We are therefore interested in improving the effectiveness of such exploratory analysis, the results of which must be verified using other techniques such as quantitative reverse transcription PCR (QRT-PCR). In our 1v1 analysis, we compared the GEO method to three methods of ordering genes on the basis of differential expression: fold ratio, y / x ; percent changes relative to the mean expression levels, ( x - y )/(( x + y )/2); and z-score based on local variance correction (using locally weighted polynomial regression) across signal intensity, as implemented in SNOMAD [ 29 ]. Basic filtering of low expression was performed at the beginning. In the example dataset, the z-scores give slightly better results than the percent changes, which in turn were better than simple fold ratios. However, as shown in Figure 4 , the GEO method returns 60% more of the top 50 genes than the best of the first two standard methods and generally returns superior results, almost on the same scale as a 3v3 regularized t -test. The method based on the z-scores performs slightly better than either of the standard methods, but GEO still returns 57% more of the top 50 genes. In the 1v1 case, the voting method proves useful, improving the results of both methods. By combining the z-score method and GEO's rankings, the results are superior to a 3v3 regularized t -test analysis. The voting method captures 83% more of the top 50 genes than the best of the standard methods. These results reflect the success of the voting method in combining GEO's rankings with a variety of other methods to significantly improve the overall performance. The results from the regularized t -test and GEO method were also compared on 3v3 comparisons. Whereas GEO still returns more reliable estimates than the regularized t -test, the improvement is smaller than in the case of the smaller sample size comparisons. In the 3v3 case, the GEO-method results are comparable to those of a 4v4 regularized t -test, returning 17% more of the top 50 genes than the 3v3 regularized t -test. However, we do find that the voting method again improves the results, returning very similar numbers of correct genes as a 5v5 regularized t -test (Figure 5 ). The voting method returns 41% more of the top 50 genes than the 3v3 regularized t -test. The performance of the GEO method does not seem to be influenced greatly by the number of samples in each group. This is because the gene-specific variance estimates are fixed and adding additional samples only impacts the mean estimates for each group. In contrast, in the regularized t -test method, adding additional samples to each group refines the estimates of both the means and the variances of each group. This factor is the fundamental reason that the regularized t -test improves quickly as the number of samples is increased whereas the GEO method does not. However, GEO performs strongly even with only one sample in each population and generates results that are comparable to a 3v3 regularized t -test analysis. This indicates that the greater weakness in the small-sample t -test lies in inaccurate variance estimates, and that stable, accurate estimates of gene-specific variance can greatly improve analysis. These results are summarized in Figure 6 , which compares the performance of the standard t -test, the regularized t -test, the GEO method, and the voting method across sample sizes. The voting method is substantially better in all cases. Looking at the Duchenne muscular dystrophy dataset also provides us with corroboration of the usefulness of this method. In this situation, the dataset is much smaller (11 normals vs 12 DMD). Because two samples capture a much higher percentage of the data in 11 chips than in 50 chips, we expect the usual tests on subsamples to naturally provide results more similar to the master list. Therefore, we expect to see less of an improvement from GEO than in our cancer dataset. As before, we see the GEO results consistently providing better results in the smaller sets of genes than the standard t -test, returning 33% more of the top 50 genes in the 2v2 case (Figure 7 ) and 40-170% more of the top 10, 50, 100 and 150 genes in the 1v1 case (Figure 8 ). While the regularized t -test seems to outperform the GEO method, combining the results of both using our voting method again returns superior results. For example, averaging the ranks in the 2v2 case returns us 134% more of the top 50 genes than the regularized t -test alone and 240% more than the standard t -test (Figure 7 ). In the 1v1 case, the voting method clearly outperforms either the GEO method or the local z-score method (as implemented in SNOMAD) alone, providing results that seem roughly similar to those returned by a 2v2 regularized t -test analysis. These results indicate that, as shown in the cancer dataset, improved results can definitely be attained through incorporating gene-specific variance in differential expression analysis. Most important, because the GEO method can be combined with regularization methods through a voting procedure, it can be used to improve results regardless of how it individually performs on a dataset. Discussion Number of chips For this method to be successful, a significant number of previously run chips must be available. As public databases grow in size and number, this limitation will gradually diminish, but not all chip types currently have enough available chips to use this method. Whereas the most popular chip types (such as Affymetrix HG-U95A) have hundreds of previously run chips available, it is more difficult to find databases of the less popular ones. In an attempt to test for the number of chips sufficient to utilize this method, variance analysis was performed. In Figure 9 , we plot the variance estimate as the number of chips used in the estimation increases, for one realization of the chip ordering. Because genes at different intensity levels may behave differently, we sorted the genes by their expression levels and selected four genes, one from the middle of each quartile. As seen in each case, the variance calculated from many chips tends to converge as the number of chips grows. Generally, the variances seemed to settle near their final values once 250-300 chips are gathered. After averaging over a large number of realizations in the chip order, we find that the variance settles near its final value at 250 chips, deviating less than 5% in either direction as more chips are gathered. While it is difficult at this time to find 300 chips of similar type and tissue, it should become easier to find datasets that are more specifically correlated with the experimental set as more data are accumulated in public databases. This would allow for more useful baselines to be established in calculating gene-specific variance, and would probably substantially improve the results. Comparing across multiple tissue types When trying to estimate the gene-specific variances for a particular experiment, the best approximation would come from a database of similar experiments. Because gene expression profiles have the potential to vary widely in cell and tissue type, examining many other chips of the same tissue type should provide the best indication of the baseline variance. For example, it would make most sense to draw on a large database of cancer chips to derive relevant information for a cancer dataset. Unfortunately, because of the dearth of large datasets that match each other in tissue type, chip type, and post-processing, it is difficult at this time to test this theory. Because our public databases do not yet contain enough chips sorted by tissue type to perform this procedure, we are forced to mix all the chips of any given type together. Yet, even with only a database of totally unrelated chips, we still saw a significant increase in performance, even over already improved methods such as the regularized t -test. If our gene-specific variances were based on even more reliable estimates (such as samples from the same tissue or same disease), the performance of the GEO method would probably be increased. As public databases grow in size and organization, this should become increasingly possible. Comparing across multiple chip types We have shown here how to derive more stable variances based on chips of the same type. The problem is much more complicated when multiple chip types are involved. In fact, we have observed that even different generations within the same platform do not give concordant results. When the same tissue samples were hybridized on both HG-U95A and HG-U133A chips, the dominant feature in the data was the chip type rather than the sample characteristic, and the lists of differentially expressed genes differed substantially between the two cases. Standardizing across these two types can be done for a portion of the genes but it is an involved process (Hwang KB, Kong SW, Greenberg SA, P.J.P., unpublished work). Efficiently combining data from single-channel and double-channel arrays is even more difficult. A more comprehensive database with a larger number of arrays spanning a greater variety of experiments would alleviate the problem to some extent, but methodologies for integrating data from multiple platforms will be essential, not only for better estimation procedures for differential expression but for other purposes as well. Need for standardization in public databases Public databases are an important resource for investigators to consider. With the stores of chips accessible online, valuable information concerning genes can be compiled and used to supplement new studies and avoid duplication of effort. This methodology would be improved by modification to the databases. Mainly, it is very important to start gathering the raw files instead of processed files. With Affymetrix chips, for example, .cel files should be stored, so that they can be processed using the latest methodology and maintain their usefulness. Already, many of the chips in the GEO database are less useful because they only report values processed through MAS 4.0, an outdated methodology, and comparing these with values generated through MAS 5.0 introduces another source of variation. In addition, the chips should be categorized and sorted according to tissue type, to further facilitate grouping and analysis. These modifications would improve the ability to use previously run chips, thus countering the high costs associated with microarray experiments and enabling the sharing of information to accelerate progress. Thinking about how to take advantage of these databases could provide further improvements to methodologies and enable more tools to be used to study gene functions. Conclusions This work proposes that value lies in pooling information from previous studies. Specifically, gene-specific information can be collected from public databases housing many chips, supplementing new studies and ensuring more reliable results. We show that compiling information from databases provides us with a different and potentially more accurate estimate of gene-specific variance, improving our differential expression analysis in small samples. In addition, because this improvement seems largely independent of the method of analysis, we are able to combine it with regularization in a voting method, leading to superior results. There were particularly strong improvements in the identification of the smallest groups of most differentially expressed genes, which would probably be deemed most important by an investigator as they are easiest to validate. Overall, the scale of the improvement is significant, as it allows investigators to halve their costs in some cases and still retain similar accuracy. The same approach can also be formulated in other settings, especially in the Bayesian framework in which priors for the gene variances may be estimated from previous datasets. Furthermore, as public databases are steadily growing in size, we expect refinement of this method to deliver greater success in the future. Regardless of what method an investigator might use, public databases are clearly a useful source of information and should prove useful in supplementing microarray studies. Materials and methods Because the very nature of public datasets implies that many of the chips have been generated and processed in different manners, standardization of the data is paramount. To maintain comparability, the chips were filtered to remove any chips processed with an algorithm other than MAS 5.0. After removing other unusable chips (such as duplicates and abnormally processed chips) 471 HG-U95A chips remained. Normalization of all of these chips is crucial, in order to guarantee that scales are similar. In an effort to preserve the general characteristics of each chip, conforming their scales while allowing for some chip-by-chip variability, experiments with multiple methods of normalization were carried out. The two major types included normalizing the trimmed mean and trimmed variance of each chip and using percentage ranks instead of numerical expression levels. In the first case, all of the data points were adjusted to align the mean and variance of the middle 90% of values. In the percentage ranks method, the values were assigned percentiles, removing most normalization effects. In addition, a scale was generated that related the percentile with the average rank change of that percentile. The average rank change for a gene in the middle of the scale was significantly larger than the average rank change at either extreme. This scale was used to adjust the variances on the basis of the rank. Because the results from both normalization methods were fairly similar, only the results of the trimmed mean, trimmed variance experiments are reported here. After all of the chips were normalized, the gene-specific variance was calculated. These variances were calculated in two separate ways, using a global variance and a pooled variance: where, for each gene, x ij is the expression level of array i in experiment set j ; the mean in the experimental set j ; is the mean in all arrays; D and D j contain the indices for the experimental sets and the arrays in the j th set, respectively. The global variance tends to reflect the degree that a gene may vary between different tissue types and diseases while the pooled variance reflects the degree that a gene tends to vary within each experiment. The global variance proved slightly more effective in the cancer dataset, while the pooled variance was more effective in the muscular dystrophy dataset. This seemed to be correlated to the composition of our GEO background datasets. Our set of 471 GEO chips contained 210 cancer chips but only 42 muscle chips. Because a large proportion of the total chips were cancer chips, a global variance may have more accurately represented the information in the whole dataset. However, because so many of the GEO chips were non-muscle, readjusting them into a pooled variance format may have provided a better gene-specific assessment of general expression. We further filtered the variance calculations to eliminate artifacts created by improperly processed chips along with biases from experimentation (that is, if a certain experiment produced uniformly high values for a specific gene). Thus, the highest and lowest 10% of values for each gene across the full set of GEO arrays was trimmed off for the variance calculation. The 10% parameter was chosen experimentally, by tracking how stable variance calculations were as various percentages were trimmed. The statistical properties of these variance estimators are difficult to show rigorously. If the samples from the GEO datasets can be assumed to come from the same population as those in the current study, the estimators should be unbiased and the proposed test statistic should behave as N (0,1) asymptotically. Because the GEO data are an aggregate of many experiments under different conditions often processed differently, we cannot assume the same underlying distribution in general and hence we do not know if the estimators necessarily approach the true variance. However, these estimators appear to be reasonably good approximations to the 'true' variance as demonstrated by the numerical results, and they certainly perform better than estimates based only on the current data. A master list of the most differentially expressed genes in the dataset was determined by t -test analysis. Then, the various methods were compared with each other through a process of subsampling to determine how accurately the results reflect the master list. After two samples from each group were randomly selected, all the genes were filtered out that did not have an expression level above 100 in any of the samples. The goal was to lower false positives among the non-GEO methods, as their results could easily be influenced by small expression levels that by chance ended up with virtually no variance and thus were assigned large t -statistics. After processing in this way, the top genes derived using the t -test, the regularized t -test, and the GEO-adjusted method were compared with the master list to determine their effectiveness. This subsampling procedure was repeated 500 times for each experiment, and the results were averaged. These methods are outlined in the Results section. Although the GEO-adjusted method was superior to both the t -test and the regularized t -test, the greatest success was found by averaging the results of the regularized t -test method and the GEO method. By averaging the ranks that we receive using GEO and using the regularized t -test set, our results are improved concerning our most important genes. This is not seen when the results of the simple t -test and GEO are combined, because the lists produced by the simple t -test are simply too inaccurate. However, as GEO and regularized t -test produce lists that are similar in quality, yet different in nature, a boost can be obtained by averaging the lists. Because using just the GEO variances ignores some of our experimental data and using just our experimental variances ignores global data, it seems that an averaging or voting procedure is a superior way to optimize results. In particular, the system that we used averaged 75% of the value of the lower rank (nearer the top of the list) with 25% of the value of the higher rank. A final score was obtained by combining the results from each method in this way, and the genes were re-ranked on the basis of this score. By using the 75%/25% ratio, genes that have a particularly high ranking on one of the methods are given slightly more importance than genes that have average rankings in both methods. Empirical testing of a number of combinations showed that the 75%/25% combination returned superior results, although all combinations experimented with returned results that were better than either method alone.

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Calcineurin activation influences muscle phenotype in a muscle-specific fashion

Background The calcium activated protein phosphatase 2B, also known as calcineurin, has been implicated as a cell signaling molecule involved with transduction of physiological signals (free cytosolic Ca 2+ ) into molecular signals that influence the expression of phenotype-specific genes in skeletal muscle. In the present study we address the role of calcineurin in mediating adaptations in myosin heavy chain (MHC) isoform expression and muscle mass using 3-month old wild-type (WT) and transgenic mice displaying high-level expression of a constitutively active form of calcineurin (MCK-CN* mice). Results Slow muscles, e.g., soleus, were significantly larger (by ~24%), whereas fast muscles, e.g., medial gastrocnemius (MG) and tibialis anterior were significantly smaller (by ~26 and ~16%, respectively) in MCK-CN* mice compared to WT. The masses of mixed phenotype muscles, such as the plantaris and the extensor digitorum longus, were not significantly changed from WT. The soleus, plantaris, MG and diaphragm displayed shifts toward slower MHC isoforms, e.g., soleus from WT mice contained ~52% MHC-I, ~39% MHC-IIa, and ~9% MHC-IIx, whereas MCK-CN* mice had ~67% MHC-I, ~26% MHC-IIa, and ~7% MHC-IIx. The specific isoforms that were either up or down-regulated were muscle-specific. For instance, the proportion of MHC-IIa was decreased in the soleus and diaphragm, but increased in the plantaris and MG of MCK-CN* mice. Also, the proportion of MHC-IIx was unchanged in the soleus, decreased in the diaphragm and increased in the plantaris and MG of MCK-CN* relative to WT mice. Fast to slow shifts in fiber type proportions were evident for the plantaris, but not the soleus. Fast, but not slow, plantaris fibers of MCK-CN* mice had higher oxidative and lower glycolytic properties than WT. Conclusion These data suggest that calcineurin activation can influence muscle phenotype and that the specific influence of calcineurin activation on the phenotypic and mass characteristics of a muscle is dependent upon the original phenotypic state of the muscle.

Background Skeletal muscle adapts to neural and use-dependent signals by altering its mass and phenotypic properties, including the proportion of slow (I) and fast (IIa, IIx, and IIb) myosin heavy chain (MHC) isoforms [ 1 , 2 ]. Calcineurin has been implicated as a regulatory molecule involved in the transduction of contractile activity-based signals to molecular signals involved in the regulation of muscle growth and phenotypic gene expression in skeletal muscle [ 3 - 14 ]. Calcineurin is a phosphatase (protein phosphatase 2B) that is activated by calmodulin upon binding Ca 2+ and has been shown to play an important role in the regulation of cytokine gene expression in lymphocytes [ 15 , 16 ]. Substrates for calcineurin include the phosphorylated isoforms of a transcription factor known as the nuclear factor of activated T cells (NFAT) [ 17 ]. Upon dephosphorylation, NFAT isoforms translocate into the nucleus where they can bind to a conserved DNA binding site known as the NFAT response element (NRE) and alter transcription, leading to enhanced expression of specific genes [ 15 - 17 ]. It has been proposed that in skeletal muscle nuclear NFAT can activate the expression of slow muscle phenotypic genes [ 3 - 5 , 8 , 11 , 14 ]. In addition, MEF2 and NFAT appear to act synergistically in turning on slow muscle fiber specific genes [ 18 ]. Thus, increased muscle contractile activity and the resulting sustained elevations in cytosolic Ca 2+ could potentially result in altered phenotype-specific gene expression via calcineurin [ 4 ]. To date, however, no studies have reported the influence of chronically elevated calcineurin activity on the MHC isoform composition and fiber cross-sectional areas (CSAs) of limb and respiratory skeletal muscles. Therefore, in the present study, transgenic mice containing a highly expressed transgene consisting of a constitutively active form of calcineurin (CN*) driven by the muscle creatine kinase (MCK) enhancer were used to assess the influence of chronic calcineurin activation on MHC isoform protein and muscle mass and fiber cross-sectional area (CSA). Previous studies using the same line of transgenic mice demonstrated that calcineurin activation elevates the expression of some slow phenotypic genes [ 11 ], the percentage of EDL fibers with MHC-IIa [ 19 ], and proteins related to insulin-stimulated glucose uptake [ 20 ]. Results The MCK-CN* transgenic mice expressed the transgene in all muscles tested as shown in Figure 1 . As expected, due to transgene expression being driven by a fast muscle-specific enhancer (MCK enhancer), the fast MG muscle displayed the highest level of expression. The soleus (a mixed fast and slow muscle in mice), diaphragm and plantaris showed relatively similar levels of transgene expression. Figure 1 A) SYBR ® Green I stained agarose gel of PCR products following RT-PCR for the MCK-CN* mRNA product. No specific product (~900 bp, arrow) is observed in the DNase-treated RNA samples (first 4 lanes) of either wild-type (WT) or MCK-CN* transgenic (CN*) mouse gastrocnemius. Therefore, there is no contaminating genomic DNA in the RNA samples used for RT-PCR. As expected, RT-PCR of the cDNA (second four lanes) reveals that expression of the transgene is only observed in the MCK-CN* mice. A positive (+) control of tail DNA from an MCK-CN* mouse is shown on the right. B) Western blot of CN* and WT mouse soleus (SOL), diaphragm (DIA), plantaris (PLANT) and gastrocnemius (GAST) muscles for calcineurin. The polyclonal MAb recognized both the endogenous (CN, ~60 kDa) and constitutively active (CN*, ~45 kDa) forms. As expected, muscle from WT muscles did not contain the CN* protein. C) Relative semi-quantitative analysis of CN* mRNA (via RT-PCR) and protein (western blotting) in muscles of MCK-CN* mice. All values were normalized to the same soleus muscle (n = 3 – 6 per group). The asterisk (*) denotes significantly different (p ≤ 0.05) from other muscles. As shown in Figure 2 , the muscles from MCK-CN* mice showed significant shifts towards slower MHC isoforms compared to WT. However, the specific isoforms that were either up- or down-regulated were muscle specific. For instance, the soleus of MCK-CN* mice showed an elevation in MHC-I at the expense of MHC-IIa. The MG showed a reduction in MHC-IIb, the fastest of the MHC isoforms expressed in mice, and elevations in MHCs-I, -IIa, and -IIx. The diaphragm showed an elevation in MHC-I and reductions in MHCs-IIa and -IIx. The diaphragm of the MCK-CN* mice also showed a complete absence of MHC-IIb, which was present in small proportions in WT mice. The plantaris of MCK-CN* mice showed similar directional adaptations in MHC isoforms as did the MG, i.e., higher proportions of MHCs-I, -IIa, and -IIx, and reduced MHC-IIb relative to WT mice. Thus, in MCK-CN* mice, MHC-I was consistently elevated and MHC-IIb reduced (if it was present in the WT muscle). In contrast, MHC-IIa was increased in the MG and plantaris, but decreased in the soleus and diaphragm of MCK-CN* mice. Also, MHC-IIx was increased in the MG and plantaris, decreased in the diaphragm, and unchanged in the soleus of MCK-CN* mice relative to WT. Figure 2 Adult myosin heavy chain (MHC) isoform proportions of the soleus (A), medial gastrocnemius (B), diaphragm (C), and plantaris (D) muscles of wild-type (dark bars) and MCK-CN* transgenic (gray bars) mice as determined by SDS-PAGE of whole muscle extracts. The * denotes significantly different from wild-type at p ≤ 0.05. Note the elevated proportions of slower MHC isoforms in the transgenic mice compared to wild-type. The plantaris muscle of MCK-CN* mice showed a significant shift in fiber type proportions from fast to slow as measured by the proportions of fast and slow isoforms of MHC, sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA), and troponin I (Figure 3 ). Plantaris fibers that stained positively for the slow isoform of one protein stained positively for the slow isoforms of the other proteins and likewise for fast protein isoforms. In contrast, immunohistochemical techniques did not detect significant differences in the proportions of fibers containing specific MHC isoforms in the soleus of MCK-CN* mice (Figure 4 ). However, there was a non-statistically significant tendency for increased proportions of fibers with slower MHC isoforms. Figure 3 Proportions of plantaris fibers staining positively for slow and fast isoforms of MHC, sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA), and troponin I (Tn I) in wild-type and MCK-CN* transgenic mice. MHC-I, SERCA2, and TN I slow are the slow muscle isoforms and MHC-II, SERCA1 and Tn I fast are the fast muscle isoforms. The * denotes significantly different from wild-type at p ≤ 0.05. Note the elevated proportions of fibers staining positively for the slow isoforms in the transgenic mice compared to wild-type. All fibers that stained positively for the slow MHC (MHC-I) were positive for the slow isoforms of SERCA and Tn I and all fibers that stained positively for the fast MHC (MHC-II) were positive for the fast isoforms of SERCA and Tn I. Figure 4 Proportions of soleus fibers staining positively for specific fast and slow MHC isoforms in wild-type and MCK* transgenic mice. No fibers stained positively for MHC-IIb with mAb BF-F3, thus antibody RT-D9 which is specific for MHC-IIx and MHC-IIb indicated the presence of MHC-IIx. No significant differences in fiber proportions of the soleus were found between wild-type and MCK-CN* mice. To ascertain if other phenotypic properties of muscle fibers were influenced by calcineurin activation, plantaris fibers of MCK-CN* and WT mice were analyzed for marker enzymes of oxidative (succinate dehydrogenase, SDH) and glycolytic (α-glycerophosphate dehydrogenase, GPD) capacity. Fast plantaris fibers of MCK-CN* mice showed a significant increase in SDH staining intensity, indicative of elevated mitochondrial content, and a decrease in glycolytic staining intensity, indicative of reduced glycolytic capacity (Figure 5 ). In contrast, slow plantaris fibers of MCK-CN* mice were not different from WT (Figure 5 ). Figure 5 Oxidative and glycolytic enzymatic profiles of individual plantaris fibers of wild-type and MCK-CN* transgenic mice. The * denotes significantly different from wild-type at p ≤ 0.05. A) The specific activity of succinate dehydrogenase (SDH, oxidative marker) was increased in fast (type II) plantaris fibers, but unchanged in slow (type I) fibers. The specific activity of α-glycerophosphate dehydrogenase (GPD, glycolytic marker) was significantly decreased in fast plantaris fibers, but not slow fibers. B) The integrated activity (see methods for explanation of integrated activity) of succinate dehydrogenase (SDH, oxidative marker) was increased in fast (type II) plantaris fibers, but unchanged in slow (type I) fibers. The integrated activity of α-glycerophosphate dehydrogenase (GPD, glycolytic marker) was significantly decreased in fast plantaris fibers, but not slow fibers. C) The GPD/SDH ratio (glycolytic/oxidative ratio) was significantly decreased in fast fibers, but unchanged in slow fibers of the plantaris of MCK-CN* mice relative to wild-type. As shown in Table 1 , the mass of a characteristically slow muscle, i.e., the soleus, was greater; whereas, the mass of characteristically fast muscles, i.e., the MG and tibialis anterior were lower in MCK-CN* vs. WT mice. The masses of muscles that characteristically display a more intermediate phenotype, with relatively high proportions of fast oxidative (i.e., IIa and IIx) fibers, i.e., the plantaris and the extensor digitorum longus, were unchanged. Table 1 Body and absolute and relative muscle masses. Wild-type (n = 6) MCK-CN* (n = 10) Body Mass (g) 23.3 ± 0.9 20.7 ± 0.4* Absolute Muscle Mass (mg) Soleus 8.2 ± 0.6 10.2 ± 0.6* MG 46.7 ± 1.8 34.5 ± 1.9* PLT 16.8 ± 0.8 16.2 ± 0.7 TA 43.0 ± 1.2 36.0 ± 1.3* EDL 8.3 ± 1.3 7.8 ± 0.5 Relative Muscle Mass (mg/g body mass) Soleus 0.35 ± 0.02 0.48 ± 0.02* MG 2.01 ± 0.04 1.66 ± 0.09* PLT 0.72 ± 0.03 0.78 ± 0.04 TA 1.85 ± 0.04 1.74 ± 0.06 EDL 0.36 ± 0.05 0.38 ± 0.03 Abbreviations: MG, medial gastrocnemius; PLT, plantaris; TA, tibialis anterior; EDL, extensor digitorum longus. Values with asterisks are significantly different from wild-type at p ≤ 0.05. As expected, based on the overall differences in muscle mass between MCK-CN* and WT mice, the soleus contained fibers with CSAs greater than WT, whereas the MG contained fibers with CSAs smaller than WT (Figure 6A ). The plantaris, which showed no change in mass, also showed no change in fiber CSA (Figure 6B ). In the soleus, both slow and fast fibers were larger (Figure 6C ). However, slow fibers were increased in CSA by 49% and fast fibers by only 22%. This resulted in a ~20% increase in the proportion of the muscle CSA occupied by slow fibers in MCK-CN* mice (Figure 6D ). Thus, the changes in overall phenotype of the soleus can be partially accounted for by changes in the size of specific types of fibers. Figure 6 Individual fiber cross sectional areas (CSAs) in muscles of wild-type and MCK-CN* mice. A) Mean fiber CSAs in soleus and medial gastrocnemius (MG) muscle regardless of fiber type. B) Mean fiber CSAs of slow (type I) and fast (type II) fibers of the plantaris muscle. C) Mean fiber CSAs of slow (type I) and fast (type II) fibers of the soleus muscle. D) The percent of the entire soleus muscle cross-section occupied by slow (type I) and fast (type II) fibers. The * denotes significantly different from wild-type at p ≤ 0.05. Discussion Elevated calcineurin activity altered the phenotypic profile and mass of both fast and slow muscles and muscle fibers. The phenotypic changes were indicated by the general fast to slow adaptation in MHC isoform expression in MCK-CN* versus WT mice. The changes in phenotype were apparently not restricted to MHC isoforms since the plantaris of MCK-CN* mice had higher proportions of fibers that stained positive for the slow isoforms of MHC, SERCA and troponin I, suggesting a coordinated increase in the expression of slow phenotypic proteins upon calcineurin activation. Fast plantaris fibers also had elevated oxidative and reduced glycolytic profiles, suggesting that calcineurin activation influenced the metabolic profile of fibers promoting a more oxidative phenotype. These data suggest that fibers that did not show adaptations in MHC, SERCA or troponin I isoforms and remained fast were still influenced by calcineurin activation to acquire an oxidative phenotype. These changes are consistent with the proposed role of calcineurin as a sensor of contractile activity and transducer of contractile signals into molecular signals that induce the expression of a slow oxidative phenotype [ 4 ]. The overall changes in MHC isoform composition for some muscles, such as the soleus, were apparently aided by fiber type-specific changes in the CSA of the fibers, such that type I fibers were increased and type II fibers decreased (or increased only slightly in the soleus) in CSA as a result of elevated calcineurin activity. These data are consistent with the hypothesis that elevated calcineurin activity induces increased expression of slow muscle phenotypic genes resulting in elevated mass of slow muscles and muscle fibers. In addition, fibers from fast muscles may attain a smaller CSA in MCK-CN* mice relative to WT as a result of the change in phenotype from a fast glycolytic type of fiber (typically the largest fibers) towards a slow or fast oxidative type of fiber (typically the smallest fibers), see Rivero et al. [ 21 , 22 ]. Although the general adaptations in phenotype were in the direction of faster to slower isoforms, the actual transitions in MHC isoform content were muscle-specific. This is reminiscent of the muscle-specific influence of thyroid hormone treatment on muscle phenotype [ 23 ]. Of the muscles examined, the diaphragm of MCK-CN* mice showed the greatest absolute elevation in MHC-I content. Also, in the soleus and diaphragm of MCK-CN* mice, MHC-I was up-regulated at the expense of MHC-IIa. However, in the MG, MHCs-I, -IIa, and -IIx were up-regulated at the expense of MHC-IIb. The mechanisms by which some muscles (MG and plantaris) respond to calcineurin activation by up-regulating MHC-IIa and IIx whereas others (soleus and diaphragm) down-regulate these MHCs is currently unknown. In vitro data suggest that MHC-IIa is highly (100 fold) and MHC-IIx and -IIb moderately (~5 – 10 fold) stimulated by calcineurin activation in mouse C 2 C 12 cells [ 24 ]. Because the adaptations were muscle-specific it is likely that additional mechanisms must be invoked in the in vivo state to modulate the ultimate response to calcineurin activation. Since the diaphragm showed the greatest response to calcineurin activation and the diaphragm is a chronically active muscle it is tempting to speculate that the chronic motor activity of this muscle results in an up-regulation of parallel signaling pathways that may support a more pronounced calcineurin activity-induced shift in fiber phenotype. The observation that MHC-IIa and MHC-IIx were up-regulated in fast muscles and MHC-I was up-regulated in slow muscles of MCK-CN* mice is in agreement with the idea of a limited adaptive range for muscles of varying phenotype. The limited adaptive range theory essentially states that muscles from a slow developmental lineage have the capacity to adapt in the range of I ↔ IIa ↔ IIx, whereas muscles from a fast developmental lineage can adapt in the range of IIa ↔ IIx ↔ IIb [ 25 ]. Thus, MG and plantaris fibers may be restricted to express MHC-IIa as the 'slowest' MHC isoform. In contrast, soleus and diaphragm fibers have the capacity to express MHC-I as the slowest MHC. However, recent data demonstrate that the suggested limited adaptive range for slow muscle is not strict. For example, combined treatment of rats with thyroid hormone and hindlimb suspension results in substantial expression of MHC-IIb by the soleus [ 26 ]. Another potential explanation for the muscle-specific response to chronic calcineurin activation is a possible obligatory step-wise transition from MHC-IIb ↔ IIx ↔ IIa ↔ I [ 27 ]. Following this scheme a fast muscle fiber would initially express MHC-IIx and then IIa as it transitioned towards a slower phenotype. However, this is a temporal scheme and the animals in this study were 3 months of age, therefore, sufficient time was available for a complete transition of fast muscles to MHC-I. In addition, previous studies from our [ 28 - 31 ] and other [ 26 , 32 ] laboratories have demonstrated that the obligatory step-wise transition scheme is not always followed. For instance, some rat soleus fibers can transition from MHC-I to MHC-IIx without apparent expression of MHC-IIa protein following hindlimb suspension, spaceflight, or paralysis induced by either spinal cord transection or spinal cord isolation [ 28 - 31 ]. The molecular mechanisms involved in directing the muscle-specific response are unknown, but could involve parallel signaling pathways, perhaps involving CAMKinase, MEF2, PGC-1, or altered histone acetylation at specific sites. It is also possible that variability in inherent motor-neuron activity influences the expression of specific sets of parallel signaling pathways which in turn allows for alteration of only specific sets of phenotypic proteins in muscles of differing phenotype. Our data showing that the diaphragm, a chronically active muscle, had the greatest absolute increase in MHC-I protein lends support to this idea. Ultimately, these data support the idea that calcineurin activity plays a role in the regulation of muscle phenotype. Since the expression level of the transgene in these mice results in basal calcineurin activity levels that are ~10 times the activity in WT mice [ 33 ], and since the phenotypic adaptations were somewhat modest, this suggests that parallel pathways likely need to be invoked to induce a more complete fiber type shift. In addition, the activation of calcineurin did not completely supersede the developmental-based phenotypic lineage. For instance, in the MCK-CN* mice the soleus retained ~40% fast (type II) fibers and the MG was predominantly composed of fast MHC isoforms. However, it must be noted that the transgene is active in the MCK-CN* mice during all stages of development. Thus, negative feedback mechanisms that may reduce the down-stream effectiveness of calcineurin activation may be enhanced or invoked during the development and maturation of the skeletal musculature. It is also possible that parallel pathways must be invoked with calcineurin to fully activate the slow muscle specific phenotype, as suggested by Dunn et al., [ 7 ] and demonstrated by Wu et al., [ 18 ]. Presence of the transgene will only directly influence the basal calcineurin activity and it is possible that higher sustained levels of calcineurin activity, such as may occur with sustained contractile activity-induced elevations in myoplasmic calcium, would support a more complete fiber transformation. Since the Ca 2+ -calmodulin stimulation of calcineurin can be as great as 100-fold [ 15 ], it is possible that normal in vivo calcium-induced activation of endogenous calcineurin would result in a much greater elevation in calcineurin activity and a greater degree of fiber type transformation. Several studies have suggested that calcineurin activation does not influence muscle mass [ 7 , 34 - 36 ] or phenotype [ 37 ]. One of these studies, Dunn et al., [ 7 ], made use of a similar transgenic mouse model as that used in this study. However, the level of basal calcineurin activity in the mice used by Dunn et al., [ 7 ] was reported to be several times lower (~same as WT) than that reported for the mice used in our study, ~10× greater than WT, i.e., transgenic mouse line #1 from reference [ 33 ]. Thus, the lack of effect in some of the previous studies may be related to the level of transgene expression and the lower basal calcineurin activity. It is interesting to note that the transgenic mice used by Dunn et al. [ 7 ], also did not display differences in fiber phenotype compared to WT. In contrast, other studies have demonstrated that calcineurin activation and or the accumulation of nuclear NFAT may be involved in the mediation of growth inducing factors, such as IGF-1 [ 10 , 13 , 38 ]. Curiously, our data suggest that chronic calcineurin activation results in elevated mass of slow muscle and reduced mass of fast muscle. This suggests that the changes in mass and fiber CSA may be secondary to the changes in phenotype. The differential effect of chronic calcineurin activation on slow vs. fast muscle mass may be explained as follows. Elevated expression of slow phenotypic genes in a slow muscle could theoretically result in an increased accretion of slow phenotypic proteins and elevated size of slow fibers. Fast muscle fibers showed an elevation in oxidative enzyme activity, i.e., transitioned from a fast glycolytic (FG) to a fast oxidative glycolytic (FOG) phenotype. Typically, FG fibers are larger than FOG fibers in mammalian muscle. A strong inverse correlation exists between oxidative capacity and fiber CSA [ 39 , 40 ]. Thus, as fast fibers transitioned from FG towards FOG in response to calcineurin activation, they would achieve a smaller CSA. Conclusions Collectively, the data support the hypothesis that calcineurin activation plays an important role in the modulation of skeletal muscle phenotype. The responses to calcineurin activation were muscle specific, suggesting that inherent differences in signaling mechanisms within distinct muscles likely modulate the response to calcineurin activation. The observation that a 10-fold increase in basal calcineurin activity results in alterations in the expression of fast versus slow phenotypic proteins and alterations in the metabolic capacities of fibers that do not show a transition to slow fiber type isoforms suggests that the activation of this pathway can modulate multiple aspects of fiber phenotype. However, parallel pathways likely need to be invoked to completely alter the phenotype of all fibers in a muscle. Methods Animals Breeding pairs of MCK-CN* transgenic mice were obtained from the primary colony at the University of Texas Southwestern Medical Center [ 11 ] and housed at the Animal Care Facility at Virginia Polytechnic Institute and State University. This specific line of transgenic mice has been used previously to study the influence of calcineurin activation on the expression of utrophin [ 19 ], and insulin-sensitive glucose uptake [ 20 ]. Female B6C3F1 mice expressing a transgene consisting of 4800 bp of the MCK enhancer, the coding sequence of the activated form of calcineurin, which codes for amino acids 1 to 398 and lacks the autoinhibitory and calmodulin binding domains [ 41 ], and 620 bp of the human growth hormone polyadenylation signal (MCK-CN* mice) were crossed with the C57Bl6J strain. Transgenic offspring were identified by PCR amplification of tail DNA using primers specific for sequences within the calcineurin coding region (5'-GAA CCA GCA GTT CCT GTG TGT ACA CG-3') and the hGH polyadenylation signal (5'-CAC TCC AGC TTG GTT CCC GAA TAG AC-3'). Since the transgenic mice were generated in the B6C3F1 strain and were then crossed with the C57Bl6J line, all comparisons were made to wild-type (WT) littermate controls, which would share any strain dependent variables with the MCK-CN* transgenic mice. All animal procedures were approved by the Virginia Polytechnic Institute and State University and California State Polytechnic University Institutional Animal Care and Use Committees and conform to the American Physiological Society's guiding principles for the care and use of animals. MCK-CN* (n = 10) and WT littermate controls (n = 6) were sacrificed by pentobarbital injection at 3 months of age. Animals from two independent litters were used. The diaphragm, soleus, medial gastrocnemius (MG), plantaris, tibialis anterior, and extensor digitorum longus muscles were dissected free, trimmed of excess connective tissue, and weighed (diaphragm weights were not obtained). The muscles (except tibialis anterior and extensor digitorum longus) were then rapidly frozen in melting isopentane cooled with liquid nitrogen and stored at -70°C. Muscle RNA analyses Total RNA was isolated from frozen muscle (soleus, MG, plantaris, and diaphragm) samples using Trizol reagent according to the manufacturer's instructions (Invitrogen Corp., Carlsbad, CA, USA). The final RNA pellet was dissolved in nuclease-free water, treated with DNA-free™ (Ambion Inc, Austin, TX, USA) to remove potential contaminating genomic DNA, and then quantified by UV (A 260 ) light absorbance. All RNA samples had A 260 :A 280 ratios of greater than 1.8. Two μg of the isolated RNA were reverse transcribed to cDNA with Superscript II (Invitrogen Corp.) in a final volume of 20 μl. The same PCR primers used for genotyping were used to amplify the specific cDNA fragment from transgenic mice expressing the MCK-CN* transgene. The PCR reaction included: 1 μl of cDNA, 1X Platinum Taq PCR buffer (Invitrogen Corp.), 1.5 mM MgCl 2 , 0.2 mM dNTPs, 1 μM each primer, and 1 unit Platinum Taq (Invitrogen Corp.) in a final volume of 25 μl. The PCR cycle conditions were 94°C for 4 min and then 40 cycles of 94°C for 30 sec, 61°C for 30 sec, and 72°C for 30 sec, followed by a final step at 72°C for 5 min. The PCR products were separated on 1.5% agarose gels, stained with SYBR ® green I (Molecular Probes Inc., Eugene, OR, USA) and quantified using a ChemiImager 5500 (Alpha Innotech, San Leandro, CA, USA). Muscle protein analyses One MG, plantaris and soleus muscle from each animal were serially sectioned perpendicular to the fiber axis using a Microm cryostat, stained imuunohistochemically for fast or slow myosin heavy chain (MHC) isoforms using either anti-fast or anti-slow MHC monoclonal antibodies (mAb, Novocastra Laboratories, Inc.) according to Talmadge et al., [ 30 ]. Soleus tissue sections were also analyzed for the presence of MHC-IIa, MHC-IIx and MHC-IIb using mAbs specific for MHC-IIa (mAb SC-71), MHCs-IIx & -IIb (mAb RT-D9) and MHC-IIb (mAb BF-F3). Serial sections of the plantaris were also immunohistochemically stained for the presence of fast and slow isoforms of troponin I (Research Diagnostics Inc.) and sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA, Novocastra Laboratories, Inc.) isoforms using similar techniques. The sections treated for immunohistochemistry were used to measure the cross-sectional area (CSA) of fifty individual muscle fibers per muscle using a calibrated microscopy-based image analysis system consisting of a GMS-300 grayscale microscopy system (Scion, Frederick, MD) and a Nikon Eclipse E400 microscope. The MG, plantaris and soleus from the opposite limb and the whole diaphragm (including costal and crural portions) were subjected to high resolution SDS-PAGE for analysis of myosin heavy chain isoforms as detailed in Talmadge and Roy [ 42 ]. These muscles were also analyzed for constitutively active and endogenous calcineurin protein levels by semi-quantitative western blotting as described in detail previously by Spangenburg et al. [ 43 ]. The primary antibody used for western blotting was a rabbit anti-calcineurin Pan A polyclonal (AB1695) from Chemicon International (Temecula, CA, USA) which recognized both the constitutively active (CN*) and endogenous forms of calcineurin. The western blot results obtained using this primary antibody were verified (data not shown) with other antibodies that showed similar patterns of reactivity, including antibodies CN-A1 (Sigma Chemical Co., St. Louis, Mo, USA), SC-9070 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and AB1696 (Chemicon International). Quantitative histochemistry of individual muscle fibers Quantitative histochemical analyses of oxidative and glycolytic enzyme activities were measured on 50 plantaris fibers per muscle that were immunohistochemically typed for fast or slow MHC isoform content in serial sections. According to Rivero et al., [ 21 , 22 ], succinate dehydrogenase (SDH; EC 1.3.5.1) was used as a marker for oxidative capacity and α-glycerophosphate dehydrogenase (GPD) was used as a marker for glycolytic capacity of individual muscle fibers. Histochemical staining and image analysis were performed as previously described in detail [ 21 , 22 ]. The specific enzymatic activity per unit fiber volume (i.e., enzyme concentration) is expressed as change in OD per minute of reaction. The integrated enzymatic activities (ISDH and IGPD) were calculated as the product of specific enzymatic activity (OD/min for a given fiber) and CSA of that fiber which reflects the enzyme content per fiber and is expressed as OD/min × microns 2 . Statistical analyses Student's t-tests were used for comparing mean data between transgenic and WT mice at each age. Statistical differences were significant at p ≤ 0.05. Authors' contributions RJT: conception, design, data collection and analysis, manuscript preparation, research funds collection. JSO: quantitative histochemical analyses, genotyping, statistical analysis, figure preparation. MRR: electrophoretic analyses, statistical analysis, figure preparation. NDG: mRNA and electrophoretic analyses, figure preparation. SRS: western blotting, figure preparation. SJL: muscle sample collection. FJN: supplied transgenic mice, provided genotyping expertise.

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539264

Dissemination of information to General Practitioners: a questionnaire survey

Background Early identification of permanent hearing impairment in children enables appropriate intervention which reduces adverse developmental outcomes. The UK Government has introduced a universal hearing screening programme for neonates. All involved health professionals, including those in Primary Care, need to be aware of the service to enable them to offer appropriate support to their patients. A programme of information dissemination within Primary Care was therefore undertaken. The aim of the current study was to determine the extent to which the information had reached General Practitioners (GPs), the GPs' preferred mode of dissemination and the sources from which GPs accessed information Methods Postal questionnaire survey of a randomised sample of 1000 GPs in the Phase I pilot sites of the Neonatal Hearing Screening Programme (NHSP). Results Responses were received from 54.2% of the sample. Just under 50% of those responding had received information, 62.2% of respondents said they would like to receive more information and the preferred methods of dissemination were the written word and web-sites to allow access when needed. Few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject. Many are keen to delegate detail to a third party, usually the health visitor, who has traditionally had responsibility for hearing screening. Conclusions Dissemination efforts for service developments of relevance to GPs should concentrate on advertising a website address via brief but memorable posted literature and/or articles in relevant journals and magazines. The website should be GP-friendly, and have a dedicated area for GPs including information of specific relevance and downloadable information sheets.

Background Approximately 1.65 per 1000 babies born in the UK have a significant permanent hearing impairment (>40 dB HL) detectable at birth [ 1 ]. Early recognition and appropriate intervention can reduce the proven negative impact on the development of communication skills [ 2 ]. Thus, based on robust evidence [ 3 ], in June 2000 the UK Government announced an initiative to introduce effective and appropriate screening for women and children by 2004 [ 4 ] which included the implementation of a novel screen for hearing impairment for all newborns in England. Although targeted hearing screening has been in place in some areas for several years, the introduction of a universal service at birth and the proposal to abandon the health visitor distraction screen at the age of 8 months, has implications for the knowledge General Practitioners (GPs) are now expected to have. Thus a crucial part of the implementation of the new service was to provide information to GPs and other primary care staff. Previous studies addressing the issue of the most appropriate method of dissemination of information to GPs are inconclusive [ 5 ]. Rapidly developing technology is adding further methods [ 6 ], the ease of which may adversely contribute to the overload. This study uses the introduction of the Newborn Hearing Screening Programme (NHSP) to illustrate the issues around dissemination to GPs [ 6 , 7 ]. Introduction of a novel screening service is a good example because a screen is only the beginning of a long process that integrates testing, diagnosis, intervention and monitoring [ 8 ], and in the case of permanent hearing impairment, long-term follow up. The National Screening Committee recognises information dissemination to be key to effective delivery of screening programmes stating that, "There is a need for all those involved in service delivery to understand the objectives and principles of the relevant screening programme(s) and be able to adequately interpret and explain the results and implications of the screening tests to patients as appropriate." [ 4 ] Primary care professionals are often the most accessible people with whom patients, in this case parents, can raise their questions. The phased implementation of NHSP in the UK began in 2001 in 23 pilot sites and was accompanied by a campaign of information dissemination including distribution of leaflets and posters to all general practices, the development of a dedicated website and appointment of local co-ordinators. The aim of the current study was to determine the extent to which the dissemination information had reached the GPs, the GPs' preferred mode of dissemination and the sources from which GPs accessed information. Methods We designed and piloted a one-page questionnaire [see Additional file 1 ] and used it to conduct a postal questionnaire survey early in 2003 of 1000 general practitioners in ten purposively sampled primary care trusts implementing the NHSP in the pilot sites. Five of the sites were those implementing the screen as a community based service and the remaining five were implementing the screen as a hospital based service sampled to match the community sites for annual birth rate. In each site 100 GPs were randomly selected to receive the questionnaire incorporating the restriction of only one questionnaire per practice. Identification of non-respondents was not possible as all information on returned questionnaires was anonymous. A reminder would have involved a total mailing. This option was rejected for reasons of inappropriate mailings to those GPs who had responded and cost. Results Response rate Of the 960 questionnaires not returned by the Post Office and therefore assumed to have reached their destination, 520 (54.2%) were returned at least partially completed. The answers from the questionnaire are summarised in Table 1 . Table 1 Positive response to questionnaire items Total number of responses Positive response N % Information received: Received any information 506 252 49.8 Seen leaflets 508 125 24.6 Visited website 505 12 2.4 Got poster 495 64 12.9 Knows contact details of local co-ordinator 503 78 15.5 Information requested: 429 267 62.2 Via written information 104 39.0 Via website 82 30.7 Via seminars 39 14.6 Via open day 16 6.0 Via meetings 15 5.6 Via e-mail 4 1.5 Information sources: Meetings: Existence of local GP forum 436 268 61.5 Attend local GP forum 39 14.6 Attend local child health seminar 437 84 19.2 Journals read: British Medical Journal 457 363 79.4 British Journal of General Practice 457 142 31.1 The Lancet 457 4 0.9 Update 457 194 42.5 BMA News 457 165 36.1 PCT Newsletter 457 144 31.5 Practitioner 457 130 28.4 Pulse 457 32 7.0 GP 457 13 2.8 Doctor 457 6 1.3 Information received The responses indicate that just less than half of our sample had received any information about NHSP. Of those, half had seen (but not necessarily read) the leaflets but only 2.4% of responders (4.8% of those receiving information) had visited the website. Information requested When asked if they would like more information about NHSP 62.2% said yes. Of those, the route most commonly cited was via the written word (39.0%) presumably leaflets and letters but 82 (30.7%) requested information via a website. Meetings and e-mail notification were not popular preferences. Information sources To ensure that information reaches the target audience we should consider from which sources they get their information. Here it was clear that attending meetings is not common, less than 20% of respondents attended the local GP forum or child health seminar but GPs do read journals and magazines, notably the British Medical Journal (79.4%), Update (42.5%) and the British Journal of General Practice (31.1%). Open comments In open comments (N = 84 respondents, 16.6%) it appeared that many of those that had received information had actually heard about the programme from the media or their own patients. A key theme in the comments was the delegation of responsibility for the knowledge to a colleague. While few GPs perceive themselves to have a core role in the delivery of the NHSP and thence a need for knowledge in the subject, many are keen to delegate detail to a colleague, usually the health visitor, who has traditionally had responsibility for hearing screening. Discussion The response rate of more than 50% is acceptable for a survey of GPs. A second mailing might have improved this but its advantage was weighed against the attraction of anonymity. To minimise the number of questions and encourage response, no demographic details were requested. Analysis of any response bias by age of practitioner, size of practice etc was therefore not possible. A response might be more likely from people who knew something of the subject and our estimates for the level of awareness might therefore be high but it is likely that the views expressed concerning resources accessed and preferred channels of dissemination are representative. Although considerable effort had been expended in distributing letters, leaflets and posters to every general practice, less than half the respondents said they had seen it. It is possible that mail had been screened and intercepted by administrative staff in the practice or rejected as "junk mail" by the GP. It is important therefore to ensure such mailed information is identified as relevant. Some health service developments will not be high on the average GP's agenda. In terms of neonatal hearing screening s/he will see only 20 newborns per year on average and approximately one permanently hearing-impaired child in a working lifetime. Nearly two-thirds of respondents said they would like to receive more information even though many complained about "information overload". This is a weakness inherent in surveys of this nature where stated attitudes may reflect socially desirable responses rather than respondents' true attitudes. Further exploration of this issue using qualitative techniques of personal interview would contribute to the general issue of information dissemination to GPs. GPs cannot know everything about everything and do not necessarily need to but they do need to know where they can find the relevant information and to be able to access it easily [ 9 ]. Our survey indicates that brief written information combined with the internet would be useful routes to use. Conclusions We suggest that any dissemination effort for service developments of relevance to GPs should concentrate on advertising the website address via brief but memorable posted literature and/or articles in relevant journals and magazines. It is also crucial that any website be GP-friendly by having a dedicated area for GPs including information of specific relevance and downloadable information sheets. Competing interests The author(s) declare that they have no competing interests. Authors' contributions PM designed and carried out the survey, performed the analyses and wrote the original report. HF supervised the design and implementation of the study and drafted the manuscript. Both authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 A survey of awareness of the NHS Newborn Hearing Screening Programme (NHSP) amongst General Practitioners (GPs) Questionnaire Click here for file

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522876

Function-informed transcriptome analysis of Drosophila renal tubule

Analysis of the transcriptome of the Drosophila melanogaster Malpighian (renal) tubule gives a radically new view of the function of the tubule, emphasising solute transport rather than fluid secretion.

Background Microarrays allow the interrogation of the transcriptome, the set of genes transcribed in a particular cell type under a particular condition [ 1 ]. Arrays are particularly potent tools when their coverage is relatively comprehensive, based on a completed and well annotated genome, such as that of Drosophila [ 2 ]. Commonly, they are used in time series, for example of development, of life events such as metamorphosis [ 3 ], of rhythmic behavior [ 4 ] or of responses to environment, such as aging or starvation [ 5 , 6 ]. In Drosophila , arrays are frequently used for whole-organism studies, but in multicellular organisms the ease of experimentation must be balanced against two potential problems: sensitivity and opposing changes. In the first case, even large changes in gene expression in a small tissue will not significantly influence the overall levels in the whole organism; in the second, changes in opposite directions in roughly balanced populations of cells (for example, the sharpening of expression patterns of pair-rule genes) will cancel out at an organismal scale. It is thus vital to resolve gene expression not only over time but also over space. In practice, this means looking at gene expression in defined cell types and tissues as well as in the whole organism. Our assumption is that the expression of many putative genes will go undetected until such tissue-specific studies are performed [ 7 ] - with obvious consequences for post-genomics - and we illustrate this point in this paper. We applied Affymetrix arrays in the context of a defined tissue with extensive physiological characterization, the Malpighian (renal) tubule of Drosophila melanogaster . The tubule is a valuable model for studies of both epithelial development and function. Developmentally, the tissue is derived from two distinct origins: an ectodermal outpushing of the hindgut and subsequent invasion (late in embryogenesis) by mesodermal cells [ 8 ]. Tubule morphology is very precisely and reproducibly specified; in the tiny tissue of 150 cells, there are altogether six cell types and six regions, specified to single-cell precision [ 9 ]. The transport processes that underlie fluid production in the tubule are known in extraordinary detail for so small an organism [ 10 - 12 ]. The dual origin of the cell types is reflected by dual roles for the ectodermal principal cells and mesodermal stellate cells in the mature tubule; the principal cell is specialized for active transport of cations, whereas the stellate cell appears to control passive shunt conductance [ 11 , 13 , 14 ]. Cell signaling pathways are also understood in considerable detail: several peptide hormones that act on tubule have been identified [ 15 - 17 ], and the second messengers cyclic AMP, cyclic GMP, calcium and nitric oxide have all been shown to have distinct roles in each tubule cell type [ 10 , 18 - 20 ]. This wealth of physiological knowledge provides a framework for the analysis of the results, and thus - unusually in genetic model organisms - a reality check on the usefulness of the experiment. Results The principle of the experiment was to compare the transcriptome of 7-day adult Drosophila melanogaster Malpighian (renal) tubules, for which defined state there is a wealth of physiological data, with matched whole flies. As described in Materials and methods, data were analyzed by Affymetrix MAS 5.0 software, or by dChip, or dChip and Significance Analysis of Microarrays (SAM) software. Both methods of identifying differentially expressed genes from dChip-normalized data gave virtually the same results. Indeed, SAM analysis followed by further filtering produced 1,465 differentially expressed genes compared to 1,455 genes identified within filtering by dChip alone. Furthermore, the latter list is indeed a subset of the former one. For that reason we report only the list generated by dChip in comparison with MAS data. Both MAS and dChip/SAM gave comparable views of the data, despite the radically different approaches to analysis. It has been shown that the average absolute log ratios between replicate arrays calculated with dChip are significantly lower than one calculated with Affymetrix software (Li and Wong [ 21 ]). This bias affecting fold-change calculations is the price of the increased precision that manifests itself in reduced variance, and consequently in the increased sensitivity of identification of differentially expressed genes. Nonetheless, the rank correlation is good (Spearman's r = 0.6, p < 0.0001). Taking genes called as significant by both systems, MAS5 'up' call or dChip t -test p -value of 0.01, and narrowing the list by setting an arbitrary cutoff of twofold enrichment and minimum mean difference of 100, MAS5 reported 683 genes and dChip reported 671. Furthermore, the dChip-reported genes overlap with 77% of MAS5-reported genes and this number increases to 91% if only the top 500 MAS5-reported genes are considered. Our confidence in the quality of the dataset is thus high. For simplicity, and because the two analyses produce concordant results, further analysis is restricted to the MAS5 results. The full microarray data have been deposited in ArrayExpress [ 22 ]. The fly versus fly and tubule versus tubule samples were extremely consistent, despite the technical difficulty in obtaining the latter (30,000 tubules were dissected in total). In contrast, there was wide divergence between fly and tubule samples (Figure 1 ). Although a common set of housekeeping genes showed comparable abundance, there was a large set of genes enriched in the fly sample, and a smaller set of genes strongly enriched in the tubule sample. In detail, of 13,966 array entries, 6,613 genes were called 'present' in all five fly samples, compared with 3,873 in tubules. A total of 3,566 genes were present in both fly and tubule: 3,047 in fly only and 307 in tubule only. This illustrates the point that whole-organism views of gene expression are not necessarily helpful in reflecting gene-expression levels in individual tissues. The microarray data are summarized in Tables 1 , 2 . Validation of the microarray Four genes were selected from each of three fly tubule expression classes: very highly enriched; uniformly expressed; and very highly depleted. The expression of each gene was verified by quantitative reverse transcription PCR (RT-PCR) and the data are presented in Table 3 . The agreement between Affymetrix microarray and quantitative PCR determination is good, further increasing our confidence in the robustness of the dataset, and in the approximate correspondence between signal and RNA abundance as a population average. It should be noted that the absolute sizes of the ratios are quite variable; this is a property of dividing a large number by a very small one. Nonetheless, genes scored as enriched or depleted on the arrays are invariably similarly scored by quantitative RT-PCR (QRT-PCR). These data can also be used to validate the use of the normalized Affymetrix signal as a semi-quantitative measure of RNA abundance (Table 1 ). If the QRT-PCR dataset of Table 3 is normalized against corresponding signals for rp49 (generally taken to be a ubiquitous gene with invariant expression levels in Drosophila ), and compared with the globally normalized Affymetrix signal, the agreement is seen to be excellent (Figure 2 ), with a Spearman's r of 0.83 ( p < 0.0001). With appropriate caution, the normalized Affymetrix signal can thus be taken as a reasonable estimate of expression levels between genes. Table 1 shows the top 20 genes listed by mean Affymetrix signal intensity. Although this is only a semi-quantitative measure of transcript abundance, the identities of the known genes in the lists are illuminating, and persuade us that the approach has some informal value. Specifically, mRNAs for ribosomal proteins dominate the list, and transporters are conspicuous in the balance. For example, the V-ATPase that energizes transport by tubules is represented by one gene (other subunits are also abundant, but just below the cutoff for Table 1 ). The α-subunit of the Na + , K + ATPase is also highly abundant: this is more surprising, and is discussed below. Two organic cation transporters are also very abundant. Alcohol dehydrogenase, long known to be expressed in tubules [ 23 , 24 ], is also a major transcript. There are also surprises: the most abundant signal is for metallothionein A. This is entirely consistent with our classical understanding of tubule function: it has long been known as a route for metal sequestration and excretion [ 25 - 30 ]. However, in the entire literature on Malpighian tubules, we are not aware of a physiological investigation of the role of metallothionein, other than documentation of expression [ 31 , 32 ]. The microarray results can thus potently direct and inform future research. Table 2 lists the 53 tubule-enriched genes that are enriched at least 25-fold, in comparison with the whole fly (the full list is provided as an additional data file). The conspicuous feature of these data is the extent to which tubule transcripts differ from any previously published profile. When comparing fly with tubule, there is a large set of genes that are downregulated and another large set of genes that are upregulated in tubule. The extent of the upregulation is also remarkable: the top gene is 99-fold enriched; the top 10 at least 50-fold enriched; and the top 100 at least 16-fold enriched in tubule compared to fly. The standard errors are also extremely low, meaning that we can be very confident (by two separate statistical measures) of the genes called significantly enriched in tubule. The phenotype gap Another prominent feature of the signal data in Table 1 is the relatively large fraction of novel genes (those for which there is not even a computer prediction of function) at the top of the list. Indeed, five of the top 10 genes by signal intensity are completely novel - that is, there are no known orthologs - and should provide tantalizing insights into tubule function. The 'phenotype gap' [ 33 , 34 ] is a key problem in functional genomics; that is, the genetic models preferred for genomics are historically not the organisms selected by physiologists. This can lead to a log-jam in reverse genetics, which depends critically on a wide range of phenotypes to identify effects of the mutation of target genes [ 12 ]. It has recently become possible to quantify the phenotype gap [ 35 ]. The present dataset elegantly exposes the phenotype gap in Drosophila , and shows that the tubule phenotype may go some way to closing it. Around 20% of Drosophila genes have been studied in sufficient detail to attract names (beyond the standard 'CG' notation for computer-annotated genes). Figure 3 shows that the fraction of anonymous genes in the tubule-enriched list is far higher than would be expected. That is, previous work has tended to overlook these genes. Conversely, because it is possible to perform detailed physiological analysis in tubules, it is possible to close the phenotype gap for these genes. There is a general implication from these data: that functional genomics, in Drosophila and other species, will rely increasingly on the study of specific tissues, as it is only in this context that expression of genes will be either measurable or explicable. Reconciling array data with function Many microarray experiments merely classify enriched genes to their Gene Ontology families. However, the uniquely detailed physiological data available on the Malpighian tubule allows a much more informative approach. The dataset can be validated by inspection, based on known molecular functions in the tissue and new functions can be inferred from abundant or enriched transcripts in the dataset. As the array is relatively comprehensive (corresponding to the 13,500 genes in release 1 of the Gadfly annotation), the results are also relatively authoritative. Organic solutes The housekeeping ribosomal transcripts vanish from the enrichment list (Table 2 ), which is now dominated by transporters. Intriguingly, these are not for the V-ATPase that is considered to dominate active transport by the tubule, but for organic and inorganic solutes. There is a range of broad-specificity transporters - for organic cations, anions, monocarboxylic acids, amino acids and multivitamins. There are also multiple inorganic anion co-transporters for phosphate and iodide. Most are not only very highly enriched, but also highly abundant. In more detail, the results are remarkable (Table 4 ). Nearly every class of transporter is represented, and almost all of these have at least one representative that is both abundant and enriched, implying a very specific renal role; indeed, this table contains the genes with the highest average enrichments of any class, frequently more than 30-fold. Some transporters have been documented implicitly as having a tubule role; many of the classical Drosophila eye-color mutants also have an effect on tubule color, and have since been shown to encode genes for transport of eye-pigment precursors [ 12 , 36 ]. These genes now turn out to be both abundant and enriched; among the ABC transporters are scarlet and white , and among the monocarboxylic acid transporters is CG12286 , which we have recently argued to correspond to karmoisin, a probable kynurenine tranporter [ 37 ]. Glucose and other sugar transporters are consistently abundant and enriched, implying that sugar transport is a major (and previously unsuspected) role of the tubule. Inorganic transporters are also included in the table; there are also copper and zinc transporters, which is consistent with electron-probe X-ray microanalysis data that heavy metals accumulate in tubule concretions [ 38 , 39 ], and with the extreme abundance of metallothionein A (Table 1 ). As well as specific transporters, the tubule is enriched for several families of broad-specificity transporters (organic anion and cation transporters, multivitamin transporters, ABC multidrug transporters and an oligopeptide transporter). When combined these would be capable of excreting a huge majority of organic solutes. These results invite a substantial revision of our interpretation of the role of the tubule. Classically, it is considered to be the tissue that excretes waste material, both metabolites and xenobiotics, and provides the first stage of osmoregulation. However, nearly all work on insect tubules in the last half-century has focused on the ionic basis of fluid secretion and its control, as these are easily measured experimentally. Although there have been sporadic reports on the active transport of organic solutes such as dyes [ 40 - 42 ], the historical view was of a relatively leaky epithelium, with a paracellular default pathway for those solutes not recognized by specific transporters. While consistent with the more classical view of the tubule, our results also suggest that the insect is emulating a leaky epithelium to produce the primary urine by incorporating a vast array of broad-specificity active transporters in the plasma membranes of what is electrically rather a tight epithelium. Indeed, this interpretation is consistent with other independent data: the intercellular junctions in tubule are known to be of the pleated stellate variety, the invertebrate equivalent of tight junctions [ 43 ]; and, like salivary glands, tubule cells are known to be highly polytene [ 44 - 47 ] or even binucleate [ 48 ], adaptations that maximize the size of cells and thus maximize their area/circumference ratios. V-ATPases Physiological analysis of the tubule has concentrated on the secretion of primary urine, and the energizing transporter is a plasma membrane proton pump, the V-ATPase [ 13 , 49 - 51 ]. This is a large holoenzyme of at least 13 subunits, encoded by 31 Drosophila genes [ 52 , 53 ]. V-ATPases have two distinct roles, one carried out at low levels in endomembrane compartments of all eukaryotic cells and the other in the plasma membranes of specialized epithelial cells of both insects and vertebrates [ 54 ]. In such cells, the V-ATPases can pack the plasma membrane to such an extent that they resemble semi-crystalline arrays when observed by electron microscopy [ 55 ]. It is clearly of interest to find out which genes contribute to the plasma-membrane role of the V-ATPase, though this would normally involve difficult and tedious generation of selective antibodies capable of distinguishing between very similar proteins. However, the mRNAs for those V-ATPase subunits enriched in epithelia should also be particularly abundant; one could thus predict that at least one gene encoding each V-ATPase subunit should show enrichment in tubule compared with the rest of the fly. This is indeed the case (Table 5 ): invariably, one gene for each subunit is both significantly enriched, and far more abundant, than any other gene encoding that subunit. The reason that the enrichment is not higher is probably because the whole-fly samples contain other epithelia, each with enriched V-ATPase, as minor parts of the overall sample. The array data thus allow a rapid and authoritative prediction to be made on the subunit composition of the plasma membrane V-ATPase. It will be interesting to extend these data to other epithelia in which V-ATPase is known to be functionally significant. Na + , K + - ATPase The role of the classical Na + , K + -ATPase in tubule is enigmatic. In nearly all animal epithelia, transport is energized by a basolateral Na + , K + -ATPase, which establishes a sodium gradient that drives secondary transport processes. By contrast, insect epithelia are energized by a proton gradient from the apical V-ATPase [ 56 , 57 ] and, consistent with this, many insect tissues are paradoxically refractory to ouabain, the specific Na + , K + -ATPase inhibitor [ 58 ]. Accordingly, models of insect epithelial function tend not to include the Na + , K + -ATPase. It is thus interesting to note that both Atpalpha and Nervana 1 (encoding isoforms of the α and β subunits, respectively) are among the most abundant transcripts in tubule (Table 6 ). Both are about as enriched in tubule as the V-ATPase subunits, but are significantly more abundant (compare Table 5 ). By contrast, a novel alpha-like subunit ( CG3701 ), and both Nrv2 (the neuronal β-subunit) and other novel β-like subunits are at near-zero levels. As Na + , K + -ATPase has previously been documented as being particularly abundant in Drosophila tubule [ 59 ], it may thus be prudent to re-include the Na + , K + -ATPase as an important part of models of tubule function. Potassium channels Potassium is actively pumped across the tubule, and the main basolateral entry step is via barium-sensitive potassium channels, both in tubule [ 50 , 60 , 61 ] and in other V-ATPase-driven insect epithelia [ 62 , 63 ]. Of the ion channels, the potassium channel family is by far the most diverse in all animals: in Drosophila , there are at least 28, and in human 255, K + -channel genes [ 64 ]. Inspection of the potassium channels on the array (Table 7 ) clearly identifies just four that are expressed at appreciable levels. Irk3 , Ir , Irk2 and NCKQ are all both very abundant and highly enriched in tubule. Irk3 in particular is 80-fold enriched over the rest of the fly, implying a unique role in tubule. Three of these genes are members of the inward rectifier family of potassium channels: supporting the hypothesis that they are critical for potassium entry, these channels are known to be highly barium-sensitive [ 65 ]. An inward rectification of potassium current (meaning that potassium would pass much more easily into the cell than out) would be ideal for a basolateral entry step. Inward rectifier channels normally associate with the sulfonylurea receptor (SUR), an ABC transporter, in order to make functional channels [ 66 , 67 ]. In tubules, SUR mRNA is present at extremely low abundance (signal 6, enrichment 0.9 times). However, CG9270 , a gene with very close similarity to SUR (1 × 10 -28 by BLASTP) is very abundant in tubule (see Table 4 ), (signal 422, enrichment 21 times). A second very similar gene, CG31793 (previously also known as CG10441 and CG17338 ), is very much less abundant (signal 24, enrichment 0.5). We therefore predict that novel inward rectifiers, formed between Irk3 , Ir or Ir2 and CG9270 , may provide the major basolateral K + entry path in tubule. In contrast, the other classes of K + channel, and the Na/K/Cl co-transporter that has been documented in tubule, are all relatively low in both abundance and enrichment. Chloride and water flux In a fluid-secreting epithelium, a necessary correlate of the active transport of cations must be the provision of a shunt pathway for anions and a relatively high permeability to water. In Drosophila tubules, a hormonally regulated chloride conductance pathway has been shown to occur in the stellate cells, although the molecular correlate of the currents has not been determined. There are three ClC-type chloride channels in the Drosophila genome, and RT-PCR has shown that all three are expressed in tubule [ 12 ]. The array data present a prime candidate (Table 8 ). Although all three genes are expressed, only one ( CG6942 ) is both very abundant and enriched in tubule (signal 251, enrichment 4). It is thus an obvious candidate partner to provide a shunt pathway for the epithelial V-ATPase. Water flux through the tubule is also phenomenally fast: each cell can clear its own volume of fluid every 10 seconds [ 12 ]. Although traditionally it was thought that only a leaky epithelium could sustain such rates, the identification of aquaporins (AQP) (the predominant members of the major intrinsic protein (MIP) family) as major water channels in both animals and plants [ 68 ] provides an obvious counter-explanation. There is physiological and molecular data for the presence of aquaporins in Drosophila tubule [ 69 ], and AQP-like immunoreactivity has been demonstrated in stellate cells [ 12 ]. Table 9 shows that only four of the seven AQP/MIP genes are abundant, and only three enriched. One can thus tentatively assign an organism-wide role to CG7777 (signal 243, enrichment 0.6), but tubule-specific roles to CG4019 , CG17664 and DRIP . In particular, CG17664 , is both highly abundant and very highly enriched (signal 705, enrichment 7.9). Control of the tubule The hormonal control of fluid secretion is well understood. The major urine-producinig region of the tubule is the main segment [ 70 ], and is composed of two major cell types, principal and stellate cells [ 9 , 13 , 71 ]. Active cation transport in the principal cell is stimulated by the hormones calcitonin-like peptide and corticotrophin releasing factor (CRF)-like peptide, both of which act through cyclic AMP (cAMP). Another peptide family, the CAPA peptides, act through intracellular calcium to stimulate nitric oxide synthase and thus raise cyclic GMP (cGMP), an unusual autocrine role for nitric oxide [ 20 , 72 ]. In the stellate cell, the chloride shunt conductance is activated by leucokinin [ 17 , 73 ], and a role for tyramine as an extracellular signal has also been proposed [ 74 ]. So far, the CAPA and leucokinin receptors have been identified [ 75 , 76 ]; both are prominent among the receptors enriched in tubule (Table 10 ). The CAPA receptor appears much more highly enriched in tubule than the leucokinin receptor, which is consistent with our understanding of each: the tubule is the only known target of CAPA, whereas leucokinin receptors are widely distributed in the adult gut, gonad and nervous system [ 75 ]. There are many other receptors that are reasonably abundant and enriched in tubule. As well as candidate receptors for calcitonin-like and other neuropeptides, there are two glycine/GABA-like receptors that might be expected to form ligand-gated chloride channels, together with good matches to vascular endothelial growth factor-like, insulin-like and bombesin-like receptors. The localization of, ligands for, and functional roles of these receptors will be of great interest. It should be noted in this context that all hormones characterized so far act on one of the two main cell types in the principal section of the tubule. There are, however, six genetically defined cell types and six regions in the adult tubule [ 9 ], and it is likely that there will at least be ligands acting on the initial segment to stimulate calcium excretion, and others acting to regulate reabsorption by the lower tubule. If any of these receptors maps to these regions, they would be prime candidates for such roles. Overall, the main surprise from these data is the sheer range of candidate ligands that could be inferred; this more than doubles the size of the endocrine repertoire so far postulated for insect tubules. On a more general level, it is possible to trace out the key genes in all three intracellular signaling pathways that have been studied in detail in Drosophila tubule (Table 11 ). The results for signaling genes tend not to be as clear-cut as for transporters, as many are rather widely distributed, and so do not show enrichment, and many do not require high standing levels of protein (and implicitly mRNA) to achieve their effects. Nonetheless, it is possible to identify genes that are at least present, and frequently enriched, in tubule. For the cAMP pathway, it is possible to identify adenylate cyclases, protein kinase A catalytic and regulatory subunits, and a phosphodiesterase ( dunce ). For cGMP, there are both soluble and membrane guanylate cyclases, implying that the tubules may produce cGMP directly in response to novel ligands, as has recently been suggested [ 77 ]. Both Drosophila genes encoding protein kinase G are expressed in tubule, and one is highly enriched. This is consistent with the renal phenotype observed both in foraging mutants [ 78 ], and in tubules in which protein kinase G is overexpressed [ 79 ]. There is also a PDE11-like phosphodiesterase. For calcium, two genes for phospholipase C, one for calmodulin, and one for protein kinase C and for calcium/calmodulin-dependent protein kinase are apparent. There are also a number of interesting modulatory or anchoring proteins, such as 14-3-3 zeta, A-kinase anchoring proteins, and receptors for activated C-kinase ( Rack1 ). How is the tubule specified? The developmental origin of the tubule has been reviewed in detail [ 80 - 82 ]. Briefly, four unique 'tip cells', specified by a cascade of neurogenic genes, control cell division in four outpushings (anlagen) of the hindgut, to form the Malpighian tubules. Late in embryogenesis the tubule is invaded by mesodermal cells, which intercalate between the future principal cells, and which then differentiate to form stellate cells [ 8 ]. In the adult, there are known to be at least six cell types and six tubule regions [ 9 ]. These regions are specified to great precision, and it is clear that each cell in the tubule has a precise positional identity. How does this identity persist throughout the lifetime of the animal? Presumably, combinations of transcription factors interact to provide both regional and cell-type coordinates and, after early establishment, these combinations must persist into adulthood. The microarray data allow the identification of transcription factors that are either highly abundant or highly enriched in tubule. Although this is by no means a complete list of transcription factors that are of importance to tubules, it is a good starting point. Furthermore, there are enhancer trap or reporter gene constructs available for many transcription factors. Accordingly, the top transcription factors and DNA-binding proteins were identified from the array dataset (Table 12 ). Some of these transcription factors are already known to be present in tubule, and their presence is confirmed: cut , which is known to be required for development of, and expressed in adult Malpighian tubules [ 83 ]; and forkhead and homothorax , both implicated by expression or mutational analysis to be involved in tubule development [ 84 , 85 ]. Teashirt , which has recently been shown to be stellate-cell specific in the late embryo [ 8 ], is also present in the adult, with fairly high enrichment (4.6 times). The array results also implicate a further set of transcription factor genes ( ETS21C , CG4548, bowl , sequoia , tap , CG1162 , pnt , shaven , forkhead domain 59A , sloppy paired 2 , lim3 ) as important in adult. Significantly, these mainly encode transcription factors implicated in development of the nervous system (another ectodermal tissue), so their reuse in the adult tubule is not too surprising. Once the binding sites for these factors are known, it will be interesting to model gene expression in different tubule regions. As transcription factors have been studied experimentally in some detail, they are relatively well represented by enhancer trap and other in vivo construct lines. Although individual lines do not necessarily represent the complete expression pattern of their cognate genes, a collection of such lines can provide a rapid first validation of a gene list (Table 12 ). Accordingly, representative reporter gene lines were ordered from the Bloomington Stock Center [ 86 ], and their adult staining patterns in tubule and gut are shown in Figure 4 . The results are exciting: most lines showed patterned staining in tubule that is consistent with our original genetically derived map of the tubule [ 9 ]. For example, homothorax marks out the initial, main and transitional segments of the tubule, whereas CG7417 marks the complementary lower tubule domain. The latter line is widely used as a highly specific mushroom body GAL4 driver line in brain, and it is interesting that the two known lower tubule GAL4 driver lines (c507 and c232) are both insertions in alkaline phosphatase 4 , a gene which is only expressed in lower tubule and the ellipsoid bodies of brain (next to the mushroom bodies) [ 87 ]. There is also a cell-type-specific transcription factor: corto is found only in stellate cells. Several other transcription factors show ubiquitous, rather than patterned, expression in the tubule, but this is nonetheless consistent with their identification in the microarray dataset. Another interesting aspect of the data in Table 12 is the number of anonymous CG genes implicated in tubule function. These genes have been annotated as transcription factors because of DNA-binding domains, for example, but have not been characterized functionally. The epithelial phenotype gap is thus evident even in this most intensely studied group of genes. Exceptions to the rule The whole premise of microarray work is that an abundant or enriched signal indicates the importance of a gene product in a particular context. This hypothesis is normally both untested and unchallenged. The unusual depth of functional understanding of the tubule allows a more rigorous appraisal. In fact, the majority of the genes implicated in tubule function are found well up the list. There are, however, several conspicuous exceptions (Table 13 ). The calcium channels trp and trpl are normally considered to be eye-specific, and have an essential role in phototransduction [ 88 - 90 ]. It is thus not surprising to find both genes almost at the bottom of the gene list. We have shown, however, that fluid secretion is severely compromised by mutations in either gene. Similarly, nitric oxide synthase (NOS) is a major signal transducer in tubule [ 20 , 72 ]. Nonetheless, all three genes are within the 'bottom' 20 of the whole array, with signals that are barely detectable and significant depletion compared with the whole fly. This is a cautionary example: while abundant or enriched signals can be taken as reliable indicators of functional significance, the converse is not necessarily true. The tubule and human disease Consequent to the demonstration of the phenotype gap, there are some intriguing, abundant and enriched genes which by virtue of their non-uniform expression, are likely to be important in (and best studied in) tubule. A systematic approach was taken by combining the tubule-enriched gene list with the homophila database of Drosophila genes with known human disease homologs. The results (Table 14 ) show the 50 human diseases with Drosophila homologs that are upregulated at least threefold in tubules. Intriguingly, several of these genes have human kidney phenotypes. Some are extremely well studied: for example, rosy (one of the first Drosophila mutations recorded) encodes xanthine oxidase, and mutation in either human or fly produces severe nephrolithiasis with concomitant distortion of tubules (reviewed in [ 12 ]). The distension of tubules is remarkable (Figure 5 ). In both species, lethal effects can be ameliorated by a high-water, low-purine diet. Other diseases, although less well documented, have plausible renal phenotypes: for example, antenatal Bartter syndrome, a severe salt-wasting renal disease, associated with mutations in the ROMK channel (homolog ir ); Dent disease, caused by mutation in ClC5 (homolog CG5284 ); proximal renal tubular acidosis, caused by mutation in the NDAE co-transport (homolog ndae1 ); nephrophatic cystinosis, caused by mutation in a lysosomal cystine transporter (homolog CG17119 ); mucopolysaccharidosis type IV, caused by mutation in galactosamine-6-sulphatase, an enzyme enriched in both human and fly kidney (homolog CG7402 ). Overall, there is a clear message that human and fly renal function may be relatively similar over quite a wide range of properties. The tubule phenotype may also prove highly informative for other genes implicated in disease. Recently, a small 10 kDa protein, bc10, was shown to be downregulated in the transition from early-stage to invasive bladder carcinoma [ 91 ]. The normal function of this protein is not yet established, but its homolog ( bc10 ) is highly abundant (893 ± 50) and moderately enriched (1.9 ± 0.09) in tubule, and a P-element insertion within the gene P{GT1}BG02443, is available from stock centers. This comparative approach can be extended to non-human species. For example, CG4928 represents an abundant and enriched transcript (3,778, 13 times enriched), that is highly similar (1.9 × 10 -75 ) to the C. elegans gene unc-93 [ 92 ]. This is associated with a 'rubber-band' phenotype, in which motor co-ordination is sluggish; it is thus taken to be a myogenic or neuromuscular gene. The discovery that a close homolog is highly enriched in renal tissue opens new lines of investigation for this gene. Discussion These data have value at two distinct levels: specific and general. Specifically, we have found out more about the operation of the Malpighian tubule than in any single published piece of work since the very first pioneering days: a summary is given in Figure 6 . This tissue is of great interest, both for developmental studies and for integrative physiological study of epithelial function. Despite 990 papers on Malpighian tubules since the start of the twentieth century, and a really rather good understanding of ion and water transport, the microarray data provide strong indications that these are only minor properties of the tubule. Whole families of transporters are represented by abundant mRNAs and transport solutes that have yet to be studied in the context of tubule. Some datasets implicate particular genes in processes that have been studied in great physiological detail, and the presence of known genes with the novel can only increase our confidence in the result. In this context, the demonstrated abundance of transporters for almost every class of organic and inorganic solute dramatically diminishes the number of solutes for which a nonspecific paracellular pathway need be invoked. The data thus allow the conceptual view of the epithelium to alter from leaky to tight in a physiological-transport sense: this is consistent with electrophysiological data [ 93 ]. There are two areas where microarray data deserve comment. Firstly, more than 300 genes are expressed in tubule but called as absent in whole-fly samples. Although there is an obvious convenience and consistency in employing whole-organism samples for array studies, it is important to recognize that the approach is very likely to suppress the detection of those interesting genes that are not widely expressed. Secondly, the premise that abundance on an array (or more generally, abundance of an RNA species) necessarily correlates with functional significance can be spectacularly refuted by three examples, the trp and trpl channels and NOS. It is, however, probably significant that these are cell-signaling molecules, where a relatively small number of molecules can have a disproportionate influence on cell behavior. By contrast, the transport genes for which the tubule is so enriched are much more likely to exert effects proportional to their abundance. Conclusions Reverse genetics is a vital tool in functional genomics, but the 'phenotype gap' has hampered widespread implementation of this approach [ 35 ]. As the tubule presents a range of easily assayed phenotypes [ 12 ], this work specifically identifies those genes that are likely to be best studied in tubule by virtue of their very high enrichment. In addition to the obvious transport genes, it is interesting that many transcription factors and human disease gene homologs fall into this category. This work thus stresses the importance of systematic, fine-grained, tissue-specific microarray analysis in closing the phenotype gap for multicellular model organisms. Materials and methods Flies Drosophila melanogaster were kept on standard diet at 25°C and 55% relative humidity on a 12:12 h photoperiod. Malpighian tubules were dissected from 7-day-old adults, for compatibility with the extensive physiological literature on the tubule [ 10 , 11 , 13 , 15 , 17 , 19 , 20 , 39 , 70 , 75 , 94 - 96 ]. At this stage, the tubules are in a relatively stable state after adult emergence, and their secretion parameters do not change detectably between 3 and 14 days post-emergence. Microarrays Tubules were dissected in batches of 1,000 by a group of eight experimenters. Tubules were aggregated into Trizol every 15 min to minimize the distortion of the transcriptome by the trauma of dissection and in vitro incubation. Care was taken to sever the tubules from the gut at the lower ureter so that no other tissue was included in the sample. For each experimental point, whole flies from the same culture were homogenized in Trizol in batches of 100, to permit a matched pair comparison. Six repeats were performed. RNA was extracted according to standard protocols, and quality was assessed with an Agilent RNA Bioanalyzer. Samples of 20 μg total RNA were reverse-transcribed, then in vitro transcribed, according to Affymetrix standard protocols. The quality of the ccomplementary RNA (cRNA) was also checked on an Agilent RNA Bioanalyzer, with a sample in which the broad cRNA peak exceeded the height of the low molecular weight degradation peak taken to be satisfactory. Samples were then run on the Affymetrix Drosophila genome array under standard conditions. Quality control was at several levels: the Affymetrix MAS 5.0 software provided evidence of successful sample preparation, with test genes providing a 3':5' signal ratio of less than 3. dChip [ 97 ] provided an alternative view, with a direct oligo-by-oligo view on the success of hybridization across the array surface; slides with both single-probe and probe-set outlier rates of less than 5% were taken as satisfactory. Only arrays in which both results were in range were accepted. In this case, 11 of 12 arrays were satisfactory; the first tubule array failed both MAS and dChip criteria, and so the first experimental pair was discarded to leave a five-sample paired design. As will be seen from the results, this design was sufficient to identify tubule-enriched genes with a high level of confidence. As sample collection extended over the whole day, array results from morning versus afternoon samples were compared (data not shown), but no difference was found between the two groups at this very broad time resolution. Bioinformatics Microarray samples were analyzed by two independent routes. The first was low-level analysis with the Affymetrix MAS 5.0 suite and identification of differentially expressed genes using the Affymetrix Data Mining Tool. The second was low-level analysis using dChip software [ 97 ] followed by assessment of significance using SAM software [ 98 ] followed by post-analysis by dChip. The MAS5 low-level analysis consisted of background subtraction followed by robust conversion of probe-level perfect match-mismatch (PM-MM) expression values into probe-set-level signals followed by linear multi-chip normalization (scaling). Tubule enrichment was based on an Affymetrix 'up' call, and a critical level of p < 0.05. In this analysis method, tubule and fly samples were taken as matched pairs, reflecting their biological origin. The dChip-based low-level analysis consisted of background correction followed by the multi-chip, 'invariant-set' nonlinear normalization at probe level followed by the calculation of model-based expression indices using PM expression values only. Differentially expressed genes between two groups of five replicates were identified within dChip by filtering data using the following criteria: lower 90% confidence bound of fold-change [ 21 ] > 2; difference between group means on antilog scale > 100 and p -value for t -test of equal group means < 0.01. Alternatively, the differentially expressed genes were identified using SAM software with 1,000 sample permutations and false-discovery rate cutoff of 1%. These were then post-filtered using two first criteria from the dChip analysis mentioned above. Fold change was calculated as a ratio of group means. Outputs were saved as Excel files, and parsed by hand-coded Perl scripts. Additional data file A list of genes (Additional data file 1 ) called as upregulated in tubule by Affymetrix SAM 5 software, and with more than two-fold enrichment is available with the online version of this article. Supplementary Material Additional data file 1 A list of genes called as upregulated in tubule by Affymetrix SAM 5 software, and with more than two-fold enrichment Click here for additional data file

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522876.xml

493266

Characterization of the GATC regulatory network in E. coli

Background The tetranucleotide GATC is methylated in Escherichia. coli by the DNA methyltransferase (Dam) and is known to be implicated in numerous cellular processes. Mutants lacking Dam are characterized by a pleiotropic phenotype. The existence of a GATC regulated network, thought to be involved in cold and oxygen shift, had been proposed and its existence has recently been confirmed. The aim of this article is to describe the components of the GATC regulated network of E. coli in detail and propose a role of this network in the light of an evolutionary advantage for the organism. Results We have classified the genes of the GATC network according to the EcoCyc functional classes. Comparisons with all of E. coli 's genes and the genes involved in the SOS and stress response show that the GATC network forms a group apart. The functional classes that characterize the network are the Energy metabolism (in particular respiration), Fatty acid/ Phospholipid metabolism and Nucleotide metabolism. Conclusions The network is thought to come into play when the cell undergoes coldshock and is likely to enter stationary phase. The respiration is almost completely under GATC control and according to our hypothesis it will be blocked at the moment of coldshock; this might give the cell a selective advantage as it increases its chances for survival when entering stationary phase under coldshock. We predict the accumulation of formate and possibly succinate, which might increase the cell's resistance, in this case to antimicrobial agents, when entering stationary phase.

Background The tetranucleotide GATC is methylated in Escherichia. coli by the DNA methyltransferase (Dam); this enzyme methylates the adenine residue within 5'-GATC-3' sequences in double stranded DNA. GATC motifs and their methylation by Dam play an important role in E. coli ; they are involved in mismatch repair (see [ 1 ] for a review on the subject of mismatch repair and [ 2 ] for a review concerning E. coli only) and the control of chromosome replication (see [ 3 ] for a concise overview on the subject). The methylation state of GATC is also involved in the expression of the pap operon; this operon codes for the Pap pili, which are of great importance in the pathogenicity of uropathogenic E. coli [ 4 ]. Mutants that lack Dam are characterized by a pleiotropic phenotype; they show for example an increased sensitivity to DNA-damaging agents, have a higher mutability and increased hyper-recombination [ 5 ]. Recent transcriptome analyses on Dam mutants show that nearly 10% of E. coli 's genes are affected [ 6 ]. When we sort these genes according to their EcoCyc functional classes and compare their distribution with all of E. coli 's genes, one can observe that the two distributions are different (p-value = 1.7 × 10 -7 ) and that the class Energy metabolism is particularly overrepresented in the genes sensitive to the dam genotype (see Figure 1 ). Figure 1 Comparison of the distribution of the "Oshima genes" with all of E. coli 's genes. The genes of E. coli have been classified according to the 15 classes used by EcoCyc. In this figure we compare the distribution of the genes sensitive to the dam + / dam - background according to a transcriptome analysis carried out by [6] ("Oshima genes") with all of E. coli 's genes. The distributions are different (p-value = 1.7 × 10 -7 ): the "Oshima genes" contain a much smaller proportion of "Hypothetical" genes, whilst the group "Energy metabolism" is particularly overrepresented. In 1996, Hénaut et al. [ 7 ] suggested the existence of a GATC regulated network thought to be involved in cold and oxygen shift. Its existence has been recently confirmed by Riva et al. [ 8 ], who have worked with a virtual chromosome and used Salmonella -a close relative of E. coli - as a control organism. The network consists of a number of genes which contain clusters of GATC within their coding sequences. In fast growing cells (for example in the intestine of their warm blooded host) Dam is a limiting factor and the DNA will be undermethylated [ 9 , 10 ]; this undermethylated DNA possesses an increased melting temperature and thus an increased stability [ 11 ]. According to the hypothesis of [ 7 ] and [ 8 ], the increased stability of the DNA comes into play when the bacteria undergo coldshock (and an oxygen shift), caused by the passage from the intestine to the external environment. At that moment, the transcription of genes containing a GATC cluster will be blocked at the level of the cluster because of the high stability of the hemi-methylated DNA. The aim of this article is to describe the GATC regulated network of E. coli in detail. In order to obtain more information about the characteristics of the network, we compare it with the genes known to be involved in the stress and SOS response. By examining the functions of the genes belonging to the GATC regulated network, we try to address the question about the possible evolutionary advantage of developing a regulatory network controlled by GATC clusters present within the coding regions. Results Description of the network and its conservation Firstly we assigned each gene in E. coli to one of fifteen functional classes according to EcoCyc [ 12 ]. This allowed us to examine whether the "GATC genes" (those genes in E. coli containing a GATC cluster, listed in Table 1 [see additional file 1 ]) follow the distribution of all of E. coli 's genes or not. The results are displayed in Table 2 and Figure 2 and show that the distribution (in percentage) of the "GATC genes" in the fifteen classes is significantly different from all of E. coli 's genes (p-value = 6 × 10 -15 ). As the GATC network is thought to come into play when the cell suffers from stress, namely coldshock (coupled to an oxygen shift), we compared the "GATC genes" with genes induced under various stress conditions according to EcoCyc ("EcoCyc genes") as the two groups might overlap. The results of this second comparison are displayed in Table 2 and Figure 3 and show that the distribution (in percentage) of the genes in the fifteen classes is clearly different in the two groups (p-value = 5 × 10 -9 ). A third comparison was made, between the "GATC genes" and the group of genes, whose expression is strongly affected by mitomycin C ("Mitomycin C genes") according to a recent transcriptome analysis [ 13 ]. Mitomycin C is an antitumor drug and antibiotic which has a strong ability to cause interstrand DNA cross-links [ 14 ]. Addition of mitomycin C to a culture of E. coli provokes a general stress in the bacteria, affecting almost 30 % of E. coli 's genes; 7% of E. coli 's genes show particularly strong changes in expression levels, including the genes in the SOS response and genes belonging to other stress response pathways [ 13 ]. We have compared this latter group of genes with the "GATC genes". Again the comparison shows the two groups of genes to be distinct (p-value = 2 × 10 -6 , see Table 2 , Figure 4 ). Table 2 Distribution of the different groups of genes according to the EcoCyc functional classification Functional class "GATC genes" all of E. coli 's genes "EcoCyc genes" "Mitomycin C genes" Amino acid metabolism 2 134 0 5 Biosynthesis of cofactors, prosthetic groups, carriers 2 127 2 1 Cell envelope 2 194 5 10 Cellular process 1 102 18 14 Central intermediary metabolism 4 149 9 15 Energy metabolism 16 363 6 29 Fatty acid/Phospholipid metabolism 8 64 1 4 Hypothetical 11 1847 12 62 Nucleotide metabolism 11 120 2 14 Other categories 3 236 30 15 Regulatory functions 1 104 12 5 Replication 4 89 14 19 Transcription 1 47 5 7 Translation 0 150 2 61 Transport/binding protein 10 369 28 42 Total 76 4095 146 303 The table shows the distributions of four groups of genes discussed in this paper, (classified according to the EcoCyc functional classification): the "GATC genes" (genes containing a GATC cluster), all of E. coli 's genes, the "EcoCyc genes" (genes induced under various stress conditions according to EcoCyc) and the "Mitomycin C genes" (genes induced by the stress caused by the antibiotic mitomycin C). This table allows the reader to perform the statistical analyses carried out in this paper. The comparison of the distribution of the "GATC genes" with each of the other three groups shows that in all three comparisons three functional classes are overrepresented in the "GATC genes": Energy metabolism, Fatty acid/ Phospholipid metabolism and Nucleotide metabolism. These three classes characterize the GATC regulated network. For an easier interpretation these data are also displayed in the form of histograms in Figures 2, 3 and 4. Figure 2 Comparison of the distribution of the "GATC genes" with all of E. coli 's genes. The genes of E. coli have been classified according to the 15 classes used by EcoCyc. In this figure we compare the distribution of the genes containing a GATC cluster ("GATC genes") with all of E. coli 's genes. (See Table 2 for the corresponding numerical data.) The distributions are different (p-value = 6 × 10 -15 ): the "GATC genes" contain a much smaller proportion of "Hypothetical" genes, whilst the groups "Fatty acid/ Phospholipid metabolism", "Nucleotide metabolism" and "Energy metabolism" are overrepresented. Figure 3 Comparison of the distribution of the "GATC genes" with the "EcoCyc genes". The genes of E. coli have been classified according to the 15 classes used by EcoCyc. In this figure we compare the distribution of the genes containing a GATC cluster ("GATC genes") with the genes induced under various stress conditions according to EcoCyc ("EcoCyc genes"). (See Table 2 for the corresponding numerical data.) The distributions are different (p-value = 5 × 10 -9 ): the "GATC genes" contain a much smaller proportion of genes belonging to "Other categories", genes involved in "Cellular processes" and "Regulatory functions", whilst the groups "Fatty acid/ Phospholipid metabolism", "Nucleotide metabolism" and "Energy metabolism" are overrepresented. Figure 4 Comparison of the distribution of the "GATC genes" with the "Mitomycin C genes". The genes of E. coli have been classified according to the 15 classes used by EcoCyc. In this figure we compare the distribution of the genes containing a GATC cluster ("GATC genes") with the genes whose expression is sensitive to the stress caused by the antibiotic mitomycin C (genes involved in the SOS response and other stress response pathways) according to the recent transcriptome analysis carried out by [13] ("Mitomycin C genes"). (See Table 2 for the corresponding numerical data.) The distributions are different (p-value = 2 × 10 -6 ): the "GATC genes" contain a much smaller proportion genes involved in "Translation", whilst the groups "Fatty acid/ Phospholipid metabolism", "Nucleotide metabolism" and "Energy metabolism" are overrepresented. The three comparisons show us that the "GATC genes" form a group apart. A characteristic of the GATC network is the overrepresentation of three classes, namely Fatty acid/ Phospholipid metabolism, Nucleotide metabolism and Energy metabolism, indicating that the genes belonging to these three classes are the ones which characterize the network and therefore deserve a closer examination. Two distinct arguments corroborate the fact that the three classes (Fatty acid/ Phospholipid metabolism, Nucleotide metabolism and Energy metabolism) are at the heart of the GATC Network: The first argument comes from an analysis of some transcriptome data. Oshima et al. [ 6 ] carried out a transcriptome study on E. coli in a dam + / dam - background. We have classified the genes affected by this background according to the 15 functional classes in EcoCyc and compared the distribution of these genes with the "GATC genes". At first sight the distribution (in percentage) of the genes in the fifteen classes is different in the two groups (p-value = 1 × 10 -4 ). If, however, we do not take into consideration the class "Hypothetical", the two distributions do no longer differ from each other, with a p-value of 0.03 (Table 3 [see additional file 2 ]. If, on the other hand, we ignore the class "Hypothetical" in the three comparisons mentioned above, i.e. the comparisons of the "GATC genes" with all of E. coli 's genes, the stress-induced genes according to EcoCyc and the mitomycin C sensitive genes, the distributions continue to be significantly different. The second argument stems from the comparison of E. coli 's "GATC genes" with the "GATC genes" present in Salmonella . E. coli contains 76 "GATC genes", Salmonella 57 (and 3 pseudogenes) and they follow the same distribution in the 15 EcoCyc functional classes (p-value = 0.75). Twenty-three genes are in common to the two bacteria (i.e. they contain a GATC cluster in both organisms); even if we do not take these genes into account, the rest of the "GATC genes" in the two organisms still follow the same distribution (p-value = 0.33). We expected the network to be evolutionarily conserved, but not to be identical in the two organisms. A particularly interesting case is given when the same pathway is affected in both organisms, but through different genes, as this represents a particularly strong argument for the evolutionary conservation of the GATC regulated network, beyond the mere conservation of the sequences themselves. The following serve as examples: The first regards the propionate catabolism, where three genes contain a cluster. prpE contains a GATC cluster in both E. coli and Salmonella . prpB , which belongs to the same catabolic pathway, is affected in E. coli only. According to our hypothesis, transcription from these two genes would be halted during coldshock. prpR codes for the positive regulator of the propionate catabolism operon, to which prpE and prpB belong to. It contains a cluster in Salmonella . According to our hypothesis, transcription of prpR would be halted under coldshock, thus inhibiting transcriptional activation of the propionate catabolism operon, effectively blocking propionate catabolism. It is interesting to note that the GATC regulated transcription block acts on two different levels: the genes involved in catabolism, as well as their operon's regulator. Formate metabolism is affected by GATC clusters in both organisms; according to our hypothesis, the insertion of selenocysteine (required by all three formate dehydrogenases) is blocked in E. coli via selB , in Salmonella through cysN (see the corresponding entry in Table 1 [see additional file 1 ] for details). Furthermore, we find that in E. coli hyfR contains a cluster, whose product is required for the induction of the formate hydrogenlyase 2 [ 15 ]; by blocking hyfR , the formation of the complex would be hindered and thus formate metabolism further inhibited. In both organisms, a nitrate/ nitrite response regulator is part of the GATC network: narL in E. coli and narP in Salmonella . The two enzymes fulfil equivalent roles in respiration, both being involved in the (co-) regulation of a number of genes encoding oxidoreductases and dehydrogenases. The regulated oxidoreductases include a periplasmic nitrite reductase ( nrfABCD ) and two nitrate reductases ( narGHJI and napFDAGHBC ); the formate dehydrogenase-N ( fdnGHI ) is regulated by NarL [ 16 - 19 ]. Discussion Evolutionary advantage of GATC network regulation According to our hypothesis, the GATC network comes into play, when E. coli passes from a warm, nutrient rich environment, where it grows rapidly, to a cold, nutrient poor environment, where growth might be expected to be considerably slower, if not completely arrested. This process occurs naturally, when the cells pass from the intestine of their warm blooded host to the external environment. When growing rapidly, the DNA is undermethylated [ 9 , 10 ] and possesses an increased melting temperature and thus an increased stability [ 11 ]. We hypothesize that when the bacterium undergoes coldshock, the transcription of genes containing a GATC cluster will be blocked at the level of the cluster because of the high stability of the hemi-methylated DNA. We will now draw the logical consequences of this hypothesis: if a gene contains a GATC cluster, its transcription will be blocked when the bacterium undergoes coldshock and the product the gene codes for will no longer be formed, affecting the biological process it is involved in. We will apply this principle systematically to the genes belonging to the three functional classes characterizing the GATC network (Fatty acid/ Phospholipid metabolism, Nucleotide metabolism and Energy metabolism), in order to try answering the following question: is there one and the same selection pressure that can explain why these three particular classes are affected by the GATC network? Nucleotide metabolism Two main groups of genes can be distinguished: those involved in the synthesis of Nucleotides ("Nucleotide Synthesis", see column "Subclass", Table 1 [see additional file 1 ]) and those involved in DNA repair ("DNA repair", see column 'Subclass", Table 1 [see additional file 1 ]). The shut down of macromolecule synthesis such as DNA, RNA and ATP when undergoing coldshock might be seen as the "effort" of the cell to eliminate wasteful energy expenditure, as growth conditions in the new environment will probably be less comfortable than in the host's intestine. The halt of the DNA repair machinery, on the other hand, might have a different reason: when the cell passes from the intestine to the outside environment, it is halted in the middle of a rapid growth phase, which means that the DNA double helix will be open in certain regions and "loose ends" of newly formed DNA will be present (like, for example, Okazaki fragments). The DNA repair machinery might interpret these fragments as damaged pieces of DNA and proceed to its elimination [ 20 ]. An immediate halt of the DNA repair machinery when undergoing coldshock would prevent such "erroneous" repair. Energy metabolism Again, we will look at two classes of genes, which are particularly interesting: those involved in respiration and those involved in the metabolism of succinate ("Respiration" and "Succinate", column "Subclass", Table 1 [see additional file 1 ]). Respiration The respiratory system of E. coli has a modular character (see [ 21 ] for a comprehensive introduction to the subject). There are three types of respiratory components (see Figure 5 ): Figure 5 The respiratory components of E. coli and Salmonella . This figure gives an overview of the respiration of E. coli and Salmonella and how it is influenced by the GATC network. As can be seen, the large majority of enzymes involved in respiration are affected, directly or indirectly, by the GATC network. Listed on the left hand side are the gene complexes that make up the dehydrogenases, together with the reactions they catalyse. The resulting reducing equivalents are passed through the common quinone pool and are used by the oxidoreductases, listed on the right hand side, together with the reactions they catalyse. Also given are the means by which the various dehydrogenases and oxidoreductases are affected through the GATC cluster network. "Heme": the gene complex contains a heme, whose synthesis is blocked by a GATC cluster in gltX . "NarL/ NarP" the gene complex is under control by NarL/ NarP whose respective genes are blocked by a GATC cluster. "Selenocysteine": the gene complex contains selenocysteine whose insertion into the protein is blocked (GATC clusters in selB / cysN ). "GATC cluster": the gene complex itself contains a GATC cluster. For further details please refer to the results section." Sal .": Salmonella . 1) Substrate specific dehydrogenases which oxidize their substrates and feed electrons to the mobile quinone pool. 2) Quinones, which deliver reducing equivalents to the terminal oxidoreductases. 3) Terminal oxidoreductases which reduce the terminal electron acceptors. The heme synthesis is blocked in E. coli (and Salmonella ) at the level of the aminolevulinic acid (ALA) production. ALA is synthesized from glutamate through the action of GltX, whose corresponding gene, gltX , contains a GATC cluster (in both organism). Hemes play a fundamental role in the energy conserving electron transport chains and are also present as cofactors in a number of enzymes [ 22 ]. Thus, if heme synthesis is blocked, the repercussions are felt by the heme containing enzymes, notably by the dehydrogenases poxB , sdhABCD , fdnGHI , fdoGHI , fdhF and lldD and the oxidoreductases torCAD , torYz , nirB , nrfAB , cyoABCD , cydAB and cyxAB. Respiration is further affected through narL , containing a GATC cluster in E. coli (in Salmonella it is narP which contains a GATC cluster) which codes for a nitrate / nitrite response regulator and is involved in the (co-) regulation of a number of genes encoding oxidoreductases and dehydrogenases. The oxidoreductases regulated include a periplasmic nitrite reductase ( nrfABCD ) and two nitrate reductases ( narGHJI and napFDAGHBC ); the formate dehydrogenase-N ( fdnGHI ) is regulated by NarL [ 15 - 18 ]. As discussed in the section above, the formate dehydrogenases require selenocysteine, whose incorporation is halted. We can thus expect formate to accumulate in the cell when it undergoes coldshock, an aspect discussed further below. Two more dehydrogenases – glpA and lldD – and oxidoreductases – dmsA and nirB - are blocked in E. coli . If we now look at the respiration as a whole, at all the respiratory enzymes affected by the clusters, directly or indirectly, the following picture emerges (see Figure 5 ): the great majority of enzymes are blocked. Interestingly, Nyström [ 23 ] points out that during aerobic starving conditions or stasis the aerobic respiration in E. coli is blocked (by ArcA). Applying our hypothesis we come to the conclusion that genes involved in aerobic respiration are blocked and moreover, that almost the entire respiration, aerobic as well as anaerobic, is brought to a standstill. A possible explanation for this can be found in the work of Dukan & Nyström [ 24 ]. They point to the fact that when encountering stress (oxidative, nutritive, osmotic or thermal) the cell is likely to enter the stationary phase. Dukan & Nyström studied E. coli cultures when entering stationary phase under different conditions; they found that cell viability depends on the conditions the cell was prior to entering the stationary phase. Cells entering the stationary phase aerobically showed a very high mortality. Cells entering the stationary phase anaerobically, though, showed a much higher viability. Seen from this perspective it could be that what we predict to happen through the GATC regulation is the "attempt" of the cell to stay in an "anaerobic mode": the bacterium does not, for example, start producing enzyme complexes needed for aerobic respiration. Blocking respiration (when entering stationary phase) might be a preventive measure because all respiration, in the presence of oxygen, is a potential danger as it can lead to the production of free radicals and peroxide. Metabolism of succinate In the section above, we predict that formate probably accumulates in the cell when undergoing coldshock. A second prediction is that succinate might also be accumulating. If we look at the TCA cycle in anaerobiosis we note that the reductive branch (leading from oxaloacetate to succinyl CoA) is not affected by GATC clusters except for the last step, leading from succinate to succinyl CoA. Thus, we expect succinate to accumulate during coldshock. (In Salmonella , a recently discovered pathway leading from succinate to propionate is also blocked through ygfH (see the relevant entry in Table 1 [see additional file 1 ]), also suggesting an accumulation of succinate in the cell.) As mentioned above, Dukan & Nyström point out that a cell undergoing stress (including coldshock) is likely to enter stationary phase; this coupled with recent work carried out on Salmonella and E. coli [ 25 ] might give an explanation: the authors suggest that when the cells are in stationary phase, an accumulation of formate and succinate has a protective effect against antimicrobial agents. The mechanisms and exact nature of this protection are not yet understood. Fatty acid / Phospholipid metabolism We can make two observations regarding this group. The first is, that the synthesis of two enzymes which are essential for fatty acid synthesis are under GATC cluster control: the biosynthesis of ACP, the central coenzyme of fatty acid biosynthesis [ 26 ] is halted through acpS and ilvD (see the relevant entry in Table 1 [see additional file 1 ]); furthermore, the synthesis of biotin, an essential cofactor in fatty acid synthesis, is hindered through mioC , which contains a GATC cluster (recent work suggesting that MioC is an essential cofactor for the biotin synthase [ 27 ]). The second observation regards the metabolism of propionate. As mentioned above, propionate catabolism is halted in both E. coli and Salmonella . Propionate, a short chain fatty acid (SCFA) is present in relative abundance in the warm blooded hosts' intestine [ 28 , 29 ], and less so in the external environment. It might be, that the GATC network acts to quickly stop a catabolic process for which it will have less use once in the external environment. The (control) mechanism So far we have stressed the importance of the physical property of hemi- or unmethylated GATC motifs, especially when present in clusters: the increased stability of the DNA, which comes into play when the ambient temperature of the bacterium is suddenly lowered. We hypothesize that this increased stability hinders transcription of the gene containing a GATC cluster when the cell undergoes coldshock and is likely to enter stationary phase; we have looked at the effect this might have on the cell. A question we might ask is that of the control mechanism: apart from the control exerted by the physical property of hemi- or unmethylated GATC motifs, is there something else that recognizes these motifs? The protein SeqA, for example, recognizes hemimethylated GATC sequences placed on the same face of the DNA double helix and it does so in a cooperative, histone-like manner, forming a homotetramer [ 30 ]. It might indeed be that the GATC cluster network also requires the intervention of such a protein in order to fully exert its functions. This question, however, reaches beyond the abilities of the research in silico and would require an intervention from the wet lab. Conclusions E. coli and Salmonella possess a GATC cluster regulated network. The clusters are found within the coding sequences and their distribution is not at random. Three functional classes characterize the network: Nucleotide metabolism, Energy metabolism and Fatty acid / Phospholipid metabolism. We hypothesize that the network comes into play when the cell passes from the warm, nutrient rich environment of its warm blooded host's intestine to the external environment, i.e., when the cell undergoes coldshock and is likely to enter stationary phase. According to our theory, the transcription of genes containing a GATC cluster will be blocked at the level of the cluster when the bacterium undergoes coldshock and the product the gene codes for will no longer be formed, affecting the biological process it is involved in. We have applied this principle to the three functional classes that characterize the GATC network and come to the conclusion that respiration is almost completely under GATC control and will be blocked at the moment of coldshock; this might give the cell a selective advantage as it increases its chances for survival when entering stationary phase under coldshock. We also predict the accumulation of formate and possibly succinate, which might increase the cell's resistance, in this case to antimicrobial agents, when entering stationary phase. Methods Procedure The procedure has been described elsewhere [ 8 ], here a general outline: We have taken a classic genomic approach, analyzing the statistical distribution of GATC along the chromosome, using a realistic model of the chromosome as theoretical reference. We thus identify local enrichments, or clusters, in the real chromosome. A GATC cluster is identified in the following manner: GATC pairs separated by less than 8 bp and triplets in regions spanning less than 62 bp are kept in a preliminary screening. In a second step we retain only those regions where there are at least four GATC motifs and where the average distance between pairs is shorter than 18 bp. We confirm the presence of GATC clusters within the genes. In order to verify that the particular distribution observed in E. coli is not a statistical artefact, but has a physiological role, we have carried out the same analysis on Salmonella , making the hypothesis that the genes containing a GATC clusters should be largely the same in the two bacteria. This has been indeed observed, showing that the genes containing a GATC cluster are part of a regulation network We thus obtain a list of genes for E. coli (76) and for Salmonella (57 and three pseudo genes) containing GATC clusters, displayed in Table 1 [see additional file 1 ]. With the help of the data mining tools listed below, we try to attribute a function to each of these genes and group them into classes according to EcoCyc's system [ 12 ]. For certain genes, which have more than one function, we have chosen the class based on the particular context of the current study (see Table 1 [see additional file 1 ] for details). In order to compare the genes belonging to E. coli 's GATC network (the "GATC genes", Table 4 [see additional file 3 ], column F) with those involved in the stress and SOS response, we retrieved the genes listed under "adaptation" and "SOS response" at EcoCyc (the "EcoCyc genes", Table 4 [see additional file 3 ], column D). We also retrieved he genes whose expression is sensitive to mitomycin C, which in addition to the SOS response also includes genes belonging to other stress response pathways, according to a recent transcriptome analysis carried out by [ 12 ] (the "Mitomycin C genes", Table 4 [see additional file 3 ], column E). Again, the "EcoCyc genes" and "Mitomycin C genes" were classified according to EcoCyc's system. A summary of the results is displayed in Table 2 . Data mining tools In order to gain information about the "GATC genes" identified, we have used the following databases: • EcoCyc [ 12 ], • KEGG [ 34 ], • METAVISTA ® [ 35 ](proprietary data base of the Metabolic Explorer society), • PubMed [ 36 ], • Swiss-Prot [ 37 ] using the SRS search tool to interrogate the SWALL (SPTR) database (accessible as SWall on the SRS server). Authors' contributions AR performed the molecular genetic studies, participated in the statistical analysis and drafted the manuscript, MOD participated in the design of the study and the statistical analysis, TC, NG and CH were responsible for the data mining, AH conceived the study, participated in its design and coordination. All authors read and approved the final manuscript. Supplementary Material Additional File 1 The "GATC genes" in E. coli and Salmonella The table lists all genes that contain a GATC cluster in E. coli or in Salmonella . ORF(#ID): the ORF number and #ID of the genes according to Oshima et al. [6]. Empty cells indicate genes not analyzed in their work. The column " EcoCyc genes " denotes the genes induced under various stress conditions according to EcoCyc, " Mitomycin C genes " denotes the genes induced by the stress caused by the antibiotic mitomycin C (genes involved in the SOS response and other stress response pathways) according to the recent transcriptome analysis carried out by [13], the two columns " GATC genes " denote genes containing a GATC cluster (in E. coli or in Salmonella ). The column " Oshima genes " denotes genes in E coli sensitive to the dam + / dam - background. "EcoCyc functional class (modified)" gives the genes, classified according to the EcoCyc functional classes, with the modifications made by us (changes are justified in the last column). " Subclass " refers to the various groups of genes discussed in the results/ discussion section of this paper. "1" : the gene is affected, "0" : the gene is not affected, "N.A." : no information was available. Click here for file Additional File 2 Distribution of the different groups of genes according to the EcoCyc functional classification, without the class "Hypothetical" The table shows the distributions of the five groups of genes discussed in this paper, (classified according to the EcoCyc functional classification) after removal of the class "Hypothetical". The five groups are: the "GATC genes" (genes containing a GATC cluster), all of E. coli 's genes, the "EcoCyc genes" (genes induced under various stress conditions according to EcoCyc), the "Mitomycin C genes" (genes induced by the stress caused by the antibiotic mitomycin C) and the "Oshima genes" (genes sensitive to the dam + / dam - background). The distributions of the "GATC genes" and the "Oshima genes" do not differ from each other (p-value = 0.03). The comparisons of the "GATC genes" with all of E. coli 's, genes, the "EcoCyc genes" and the "Mitomycin C genes", however, show that the distributions continue to be significantly different, even after the removal of the class "Hypothetical". Click here for file Additional File 3 The file contains the raw data used for this article and allows the reader to re-trace all the calculations made Click here for file

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546329

SIRT1 Regulates HIV Transcription via Tat Deacetylation

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.

Introduction The Tat protein of human immunodeficiency virus 1 (HIV-1) is essential for the transcriptional activation of the integrated HIV-1 provirus. Without Tat, HIV transcriptional elongation is inefficient and results in abortive transcripts that cannot support viral replication [ 1 , 2 ]. Tat is produced early after infection from rare full-length genomic transcripts generated despite the elongation defect. These transcripts lead to the synthesis of a few Tat molecules sufficient to stimulate HIV transcription elongation, leading to the production of additional Tat transcripts and protein. Tat activates HIV transcription through the trans -acting responsive element (TAR), an RNA stem-loop structure that forms at the 5′ end of all viral transcripts [ 3 , 4 ]. The TAR stem contains a three-nucleotide bulge structure recognized by the arginine-rich motif (ARM) in Tat (amino acids 49–57). In vivo, Tat binding to TAR requires cyclinT1, a cofactor that interacts cooperatively with both the N-terminal transactivation region of Tat (amino acid 1–48) and loop sequences at the top of the TAR stem-loop structure [ 5 ]. CyclinT1, a component of pTEFb (the positive transcription elongation factor b), recruits the cyclin-dependent kinase 9 (CDK9) to the HIV promoter. CDK9 hyperphosphorylates the C-terminal domain of RNA polymerase II, which potently enhances the processivity of the RNA polymerase II complex [ 6 ]. We and others have shown that Tat is acetylated at lysine 50 by the transcriptional coactivators p300 and human GCN5 (general control of amino acid synthesis 5) [ 7 , 8 , 9 , 10 ]. Tat acetylation is important for Tat activity and defines a critical cyclinT1-independent step in Tat transactivation [ 11 ]. Tat acetylated at lysine 50 cannot form a ternary complex with cyclinT1 and TAR RNA. It dissociates from TAR and binds instead to the p300/CREB-binding protein-associated factor (PCAF) via its bromodomain [ 12 , 13 ]. Our current working model is that Tat acetylation disrupts the Tat/TAR/cyclinT1 complex and leads to the transfer of Tat and PCAF to the elongating polymerase. According to this model, both forms of Tat, unacetylated and acetylated, play distinct roles in the HIV promoter transcriptional cycle and lead to the sequential recruitment of the cofactors cyclinT1 and PCAF. Because of the limiting amounts of Tat protein in the early stages of HIV infection and the critical role of unacetylated Tat for pTEFb recruitment to TAR, the question arises whether Tat acetylation can be reverted via a cellular Tat deacetylase. There are three distinct classes of human histone deacetylases (HDACs) based on their homology with yeast transcriptional repressors. Class I and II HDACs are homologous to the yeast proteins Rpd3p (reduced potassium dependency 3) and Hda1p (histone deacetylase A1), respectively [ 14 , 15 ]. The deacetylase activity of class I and II HDACs is efficiently inhibited by trichostatin A (TSA) and other related hydroxamate-based inhibitors. Class III HDACs, also named sirtuins (SIRTs), are homologous to the yeast transcriptional repressor silent information regulator 2p (Sir2p) [ 16 ]. Sir2p is a TSA-insensitive histone deacetylase that requires nicotinamide adenine dinucleotide (NAD + ) as a cofactor [ 17 , 18 , 19 ]. Seven homologs of Sir2p have been identified in the human genome. Called SIRT1–7, they all contain a highly conserved catalytic domain [ 20 ]. Despite their enzymatic activity on histone substrates in vitro, recent experimental evidence suggests that SIRT proteins predominantly target nonhistone proteins for deacetylation, in both the nucleus and the cytoplasm. The nuclear SIRT1 protein deacetylates p53 [ 21 , 22 , 23 ], TAF I 68 (Tata box-binding protein-associated factor I of 68 kDa) [ 24 ], PCAF and myoblast determination protein (MyoD) [ 25 ], p300 [ 26 ] and Forkhead transcription factors [ 26 , 27 ], the p65 subunit of nuclear factor kappa B (NF-κB) [ 28 ], and the Ku70 telomeric protein (also known as the thyroid autoantigen of 70 kDa or Ku antigen) [ 29 ]. The cytoplasmic SIRT2 protein is found associated with the microtubule network and deacetylates lysine 40 of α-tubulin [ 30 ]. SIRT3 is a mitochondrial matrix protein whose target has not been identified [ 31 , 32 ]. Here, we identify the class III HDAC SIRT1 as a specific Tat deacetylase and demonstrate that SIRT1 is a novel cofactor necessary for efficient Tat-mediated transactivation of the HIV promoter. Results To test the ability of SIRT1–7 to deacetylate Tat in vitro, we transfected HEK 293 cells with expression vectors for human SIRT1–7 and immunoprecipitated the FLAG-tagged proteins ( Figure 1 A). The immunoprecipitated material was incubated with a full-length synthetic Tat protein carrying an acetylated lysine at position 50 (AcTat). The extent of Tat deacetylation was determined by Western blot (WB) with antibodies specific for the acetylated ARM in Tat [ 11 ]. Incubation of AcTat with immunoprecipitated SIRT1, SIRT2, and SIRT3 resulted in deacetylation of Tat lysine 50 ( Figure 1 B). These enzymes also deacetylate histones as determined in a standard histone deacetylase assay ( Figure 1 B). All reactions contained equal amounts of AcTat as determined by immunoblotting with streptavidin-horseradish peroxidase conjugate (SA-HRP), which recognized the biotin label attached to the N terminus of AcTat (SA-HRP in Figure 1 B). SIRT enzymes in the reactions were visualized by immunoblotting with FLAG antibodies (FLAG in Figure 1 B). Figure 1 In Vitro Tat Deacetylation by Human SIRT Proteins (A) Scheme of Tat deacetylation assay with immunoprecipitated SIRT1–7 proteins. Expression vectors for FLAG-tagged SIRT proteins were transfected into HEK 293 cells, immunoprecipitated, and incubated with synthetic Tat (72 amino acids) carrying an N-terminal biotin label and an acetyl group at position 50 (AcTat) in the presence of NAD + . Immunoprecipitated material was also analyzed in a radioactive ( 3 H) histone deacetylase assay using an H3 peptide as a substrate. (B) WB analysis of deacetylation reactions with antibodies specific for acetylated lysine 50 in Tat (α-AcTat), with SA-HRP, or with α-FLAG antibodies. (C) WB of Tat deacetylation by immunoprecipitated SIRT1 in the presence or absence of NAD + , TSA, or nicotinamide (Nic). SIRT2 and SIRT3 proteins are localized primarily in the cytoplasm and the mitochondria [ 30 , 31 ], and SIRT1 resides in the cell nucleus [ 23 , 33 ]. Since Tat is a predominantly nuclear protein, we focused our efforts on SIRT1. The SIRT1-mediated deacetylation of Tat was dependent on NAD + and completely inhibited by nicotinamide, an inhibitor for class III HDACs [ 34 , 35 ]. TSA, a specific inhibitor of class I and II HDACs, had no effect ( Figure 1 C). These results demonstrate that the Tat deacetylase activity within immunoprecipitated SIRT1 material can be solely attributed to SIRT1 and not to a contaminating class I or II HDAC. To test whether Tat and SIRT1 interact, Tat/FLAG and SIRT1/influenza hemagglutinin (HA) were overexpressed in HEK 293 cells, and cellular lysates subjected to coimmunoprecipitation assays. Tat was detected with an α-FLAG antiserum in material immunoprecipitated with SIRT1 by the α-HA antibody in cells transfected with SIRT1- and Tat expression vectors, but no signal was obtained when SIRT1 or Tat alone was expressed (IP: α-HA in Figure 2 A). Conversely, SIRT1 also specifically coimmunoprecipitated with Tat/FLAG (IP: α-FLAG in Figure 2 A). The same was observed when Tat/T7 was coexpressed with SIRT1/FLAG and was immunoprecipitated with α-T7 antibodies ( Figure 2 B). No coimmunoprecipitation of Tat was observed with SIRT2 and SIRT6 ( Figure 2 B), two SIRT proteins that can also localize to the cell nucleus (BN and EV, personal communication), or any other SIRT protein (unpublished data). Furthermore, Tat coimmunoprecipitated with endogenous SIRT1 in Tat-expressing, but not in vector-transfected, HEK 293 cells ( Figure 2 C). No SIRT1- or Tat-specific signals were obtained after immunoprecipitations (IPs) in the absence of α-SIRT1 antibodies, excluding nonspecific binding of Tat to the Sepharose beads to which the antibodies were bound. Figure 2 Physical Interaction between Tat and SIRT1 (A) Immunoprecipitation (IP) and WB of FLAG-tagged Tat (Tat-FLAG) and HA-tagged SIRT1 (SIRT1-HA) after transfection of corresponding expression vectors (+) or empty vector controls (−) into HEK 293 cells. (B) The same experiments as in (A) performed with T7-tagged Tat and FLAG-tagged SIRT1, SIRT2, and SIRT6. (C) Coimmunoprecipitation of FLAG-tagged Tat with endogenous SIRT1 in HEK 293 cells transfected with the Tat expression vector or the empty vector control. IPs were performed with or without rabbit α-SIRT1 antibodies. (D) WB of recombinant SIRT1 protein after pulldown with synthetic biotinylated Tat or AcTat. Tat proteins were detected with antibodies specific for acetylated lysine 50 in the Tat ARM (α-AcTat) or SA-HRP. (E) Immunoprecipitation/WB of FLAG-tagged Tat or TatK50R and HA-tagged SIRT1. WT, wild type. To test whether Tat and SIRT1 interact directly, increasing amounts of biotinylated synthetic Tat (72 amino acids) were incubated with recombinant full-length SIRT1. After pulldown with streptavidin-conjugated agarose, SIRT1 coimmunoprecipitated with Tat in a dose-dependent manner ( Figure 2 D). Recombinant SIRT1 bound equally well to acetylated and unacetylated synthetic Tat, indicating that the interaction occurred independently of the acetylation state of Tat ( Figure 2 D). WB with AcTat antibodies showed that AcTat remained acetylated during incubation with the SIRT1 enzyme ( Figure 2 D). Re-blotting with SA-HRP detected both Tat proteins in equivalent amounts in the binding reactions ( Figure 2 D). We also tested the ability of a Tat mutant protein (termed TatK50R to indicate mutation of lysine to arginine at position 50 of the Tat protein) to interact with SIRT1. This mutation preserves the basic charge at position 50, but cannot be acetylated. After transfection into HEK 293 cells, TatK50R accumulated to lower concentrations than wild-type Tat, but was bound to SIRT1 efficiently in coimmunoprecipitation assays ( Figure 2 E). These results collectively indicate that Tat binds SIRT1 directly and independently of lysine 50. The effects of SIRT1 on Tat function were assessed after transfection into HeLa cells. SIRT1 modestly, but reproducibly, enhanced Tat-mediated transactivation of an HIV promoter luciferase construct (HIV LTR in Figure 3 A). In contrast, expression of a catalytically inactive SIRT1 protein (termed SIRT1H363Y to indicate mutation of histidine to tyrosine at position 363 of the SIRT1 protein) suppressed Tat transactivation in a dominant-negative manner, indicating that the catalytic activity of SIRT1 is necessary for Tat transactivation. Similar results were obtained when an HIV promoter reporter construct containing mutant binding sites for the transcription factor NF-κB was used (HIV LTR ΔNF-κB in Figure 3 A). This result indicates that the superinduction of Tat activity by wild-type SIRT1 and the suppression of Tat activity by catalytically inactive SIRT1 were dependent on the interaction between SIRT1 and Tat rather than on the interaction between SIRT1 and NF-κB/p65 [ 28 ]. Importantly, SIRT1 (both wild-type and SIRT1H363Y mutant) had no effect on the transcriptional activity of the Rous sarcoma virus (RSV) LTR, a promoter used to drive Tat expression in these cotransfection experiments. Figure 3 SIRT1 Is a Positive Cofactor for Tat Transactivation (A) Cotransfection of SIRT1 or the catalytically inactive SIRT1 mutant SIRT1H363Y with the HIV LTR luciferase construct and increasing amounts of a Tat expression vector (RSV-Tat: 0, 2, 20, and 200 ng), an HIV LTR luciferase construct containing mutated binding sites for the transcription factor NF-κB and RSV-Tat (20 ng), or with an RSV-luciferase construct (200 ng) in HeLa cells. The average of three experiments is shown (± standard error of the mean [SEM]). (B) WB analysis of HeLa cells 72 h after transfection of siRNAs directed against SIRT1 or GL3 control siRNAs. (C) Cotransfection of the HIV LTR luciferase construct with increasing amounts of CMV-Tat or CMV-TatK50R (0, 50, 100, 200, 400, and 800 ng) 48 h after transfection of double-stranded siRNAs directed against SIRT1 or GL3 control siRNAs in HeLa cells. Luciferase activity was measured 24 h after plasmid transfection and 72 h after siRNA transfection. Note that all luciferase reporter vectors used in this study are based on the pGL2 luciferase vector, which is not affected by GL3-specific siRNAs [ 36 ]. The average of three experiments is shown (± SEM). (D) The transcriptional activity of increasing amounts of the CMV-luciferase reporter (0, 50, 100, 200, 400, and 800 ng) was similar in SIRT1 knockdown or GL3-treated control cells. The average of two experiments performed in duplicate is shown (± SEM). (E) WB of endogenous SIRT1 or actin 72 h after transfection of siRNA directed against SIRT1 or mutated SIRT1 siRNA. (F) Cotransfection of the HIV LTR luciferase with increasing amounts of CMV-Tat (0, 2, 20, and 200 ng) in HeLa cells pretransfected with wild-type or mutant SIRT1 siRNA oligonucleotides as described in (C). WT, wild-type. The effect of SIRT1 on Tat transactivation was further examined using small interfering RNA (siRNA)-mediated knockdown of SIRT1. HeLa cells were transfected with double-stranded RNA oligonucleotides directed against SIRT1 or against firefly luciferase expressed from the pGL3 vector as a control. All luciferase reporter constructs described in this study are based on the pGL2 vector, which is not affected by GL3 siRNAs (siRNAs directed against firefly luciferase expressed from the pGL3 vector) [ 36 ]. Levels of endogenous SIRT1 were markedly reduced at 72 h after transfection of siRNAs specific for SIRT1 ( Figure 3 B). At that time, a significant decrease in Tat transactivation was noted in cells that had received the SIRT1 siRNA, but not the GL3 siRNA ( Figure 3 C). The SIRT1 siRNA slightly enhanced the basal HIV promoter activity without Tat, and had no effect on the transcriptional activity of TatK50R, the Tat mutant that cannot be acetylated ( Figure 3 C). Loss of SIRT1 had no effect on the transcriptional activity of the immediate early promoter of the cytomegalovirus (CMV) used to drive Tat expression in these experiments ( Figure 3 D). In addition, Tat levels in HeLa cells transfected with SIRT1 siRNAs were comparable to Tat levels detected in cells transfected with GL3 siRNAs as determined by WB (unpublished data). To confirm the specificity of the SIRT1 siRNA, mutant double-stranded siRNA oligonucleotides were generated which contained a two-nucleotide mismatch between the target mRNA for SIRT1 and the antisense strand of the siRNA. Transfection of mutant SIRT1 siRNA did not affect expression of endogenous SIRT1 protein in HeLa cells, indicating that the mutation abrogated SIRT1 mRNA cleavage ( Figure 3 E). SIRT1 siRNA, but not mutant siRNA, suppressed Tat transactivation of the HIV LTR luciferase construct, confirming that the observed suppression was dependent on the loss of SIRT1 protein ( Figure 3 F). Since SIRT1 only modestly enhanced Tat transactivation in HeLa cells, which already express significant levels of SIRT1, we examined the effect of SIRT1 on Tat transactivation in a SIRT1-negative background. We obtained mouse embryonic fibroblasts (MEFs) derived from SIRT1 knockout mice [ 37 ]. The HIV LTR luciferase reporter and the Tat expression vector were introduced into these cells by nuclear microinjections because of their low responsiveness to various transfection protocols. Because murine cyclinT1 does not support Tat transactivation [ 38 , 39 ], an expression vector for human cyclinT1 was included in the microinjections. A 120-fold increase in HIV promoter luciferase activity was detected in the presence of Tat and human cyclinT1 in SIRT1 +/+ MEFs ( Figure 4 A). In contrast, Tat-mediated transactivation of the HIV LTR was reduced in SIRT1 −/− MEFs ( Figure 4 A). Ectopic expression of increasing amounts of human SIRT1 resulted in a dose-dependent increase of Tat transactivation in SIRT1 −/− MEFs ( Figure 4 B). In contrast, transactivation of the 5xUAS promoter by Gal4-VP16 was reduced in response to SIRT1 (Gal4-VP16 is a fusion between the binding domain of the DNA-binding transcription factor required for the activation of the GAL genes in response to galactose in Saccharomyces cerevisiae [termed Gal4], and the activator domain of the herpes simplex virus transactivator protein [designated VP16]) ( Figure 4 C). These results collectively demonstrate that SIRT1 represents a positive factor for Tat function. Figure 4 Impaired Tat Transcriptional Activity in Murine SIRT1 −/− Cells (A) Nuclear microinjection of HIV LTR luciferase, RSV-Tat, and a human cyclinT1-expressing construct into MEFs derived from SIRT +/+ or SIRT −/− mice. In all experiments, a fixed amount of DNA was injected by adding the empty vector control. Cells were coinjected with CMV-GFP, and the luciferase activity per GFP-positive cell was calculated. An average of two injections is shown. (B) The HIV LTR luciferase construct together with RSV-Tat and the cyclinT1-expressing construct were coinjected into SIRT −/− MEFs in the presence of increasing amounts of a plasmid expressing human SIRT1. The average of three experiments is shown (± SEM). (C) Coinjection of the human SIRT1 expression vector together with the 5xUAS luciferase construct containing five Gal4 binding sites upstream of the thymidine kinase promoter and a Gal4-VP16 expression plasmid into SIRT1 −/− MEFs. The average of three experiments is shown (± SEM). This model was further tested in nuclear microinjection experiments using synthetic full-length Tat and AcTat. Microinjection of increasing amounts of either Tat or AcTat proteins into HeLa cells caused a marked transactivation of the HIV LTR luciferase reporter in a dose-dependent manner (Wt TAR in Figure 5 A). AcTat transactivated the HIV promoter approximately 1.5–3-fold better than Tat. Transactivation by Tat and AcTat was dependent on the bulge and loop regions of TAR, indicating that transactivation by both proteins required the formation of an intact Tat/TAR/cyclinT1 complex [ 4 , 5 , 40 ] (TAR ΔBulge and TAR ΔLoop in Figure 5 A). In agreement with this conclusion, transactivation by both Tat proteins was inhibited in a dose-dependent manner by 5,6-dichlorobenzimidazole riboside (DRB), a CDK9 inhibitor known to block Tat function ( Figure 5 B) [ 6 ]. Figure 5 Transcriptional Activity of AcTat Depends on Deacetylation by SIRT1 (A) AcTat functions through TAR and cyclinT1 binding. Nuclear microinjection of increasing amounts of synthetic Tat or AcTat together with wild-type (wt TAR), TAR Δbulge, or TAR Δloop mutant HIV LTR luciferase constructs into HeLa cells. Cells were coinjected with CMV-GFP, and luciferase activity was calculated per GFP-positive cell. An average of three experiments is shown (± SEM). (B) AcTat transactivation requires CDK9. HeLa cells microinjected with Tat or AcTat (each 30 ng/μl) and the HIV LTR luciferase reporter were treated with increasing amounts of DRB, a known CDK9 inhibitor, for 4 h. (C) AcTat transcriptional activity is inhibited by nicotinamide, but not TSA. HeLa cells injected with HIV LTR luciferase and increasing amounts of AcTat were treated with TSA (400 nM) or nicotinamide (5 mM) for 4 h. The average of two experiments is shown. (D) SIRT1 is necessary for AcTat, but not Tat function. HeLa cells were transfected with siRNAs specific for SIRT1 or GL3 control siRNAs 48 h before microinjection of HIV LTR luciferase and Tat or AcTat (each 30 ng/μl). The average of three experiments is shown (± SEM). According to our working model, AcTat represents a second step in the transactivation cycle [ 11 ]. Since AcTat cannot form the trimolecular complex with cyclinT1 and TAR RNA in vitro, we hypothesized that AcTat becomes partially deacetylated by the Tat deacetylase upon microinjection. This would allow the initiation of the transactivation process by unacetylated Tat binding to TAR with cyclinT1 and CDK9. To further test this hypothesis, we treated cells with deacetylase inhibitors after microinjection of AcTat and the HIV promoter construct. Treatment with TSA, an inhibitor of class I and II HDACs, enhanced the transcriptional activity of AcTat as well as the basal HIV promoter activity (TSA in Figure 5 C). In contrast, nicotinamide, an inhibitor of class III deacetylases, blocked transactivation of the HIV promoter by AcTat while stimulating basal HIV promoter activity (Nicotinamide in Figure 5 C). Similarly, knockdown of SIRT1 using siRNA inhibited transcriptional activity of AcTat, while slightly enhancing Tat-mediated or basal transcriptional activity of the HIV promoter ( Figure 5 D). These results support the model that the transcriptional activity of AcTat depends on deacetylation by SIRT1 in cells. The identification of SIRT1 as an enzyme that catalyzes an important step in HIV transcription suggests that it could be targeted therapeutically. Splitomicin was identified as a small molecule inhibitor of the S. cerevisiae Sir2p protein [ 41 ]. While splitomicin did not inhibit human SIRT1, we identified a splitomicin derivative, called HR73, which is structurally related to a previously described inhibitor of Hst1, a homolog of Sir2p in yeast [ 42 ]. HR73 effectively inhibited the histone deacetylase activity of SIRT1 in vitro with an IC 50 (concentration causing 50% inhibition) of less than 5 μM ( Figure 6 A and 6 B). Treatment of HeLa cells with HR73 suppressed Tat-dependent HIV transcription in a dose-dependent manner (3-fold at approximately 1 μM) after transfection of the Tat vector and the HIV LTR luciferase construct (HIV LTR in Figure 6 C). In separate experiments, HR73 induced hyperacetylation of another target of SIRT1, the tumor suppressor p53, at the same concentration (1 μM) (Wei Gu and EV, unpublished data). Importantly, HR73 (1 μM) did not suppress the activity of the RSV LTR, the promoter driving Tat expression in our experiments (RSV LTR in Figure 6 C). Figure 6 Inhibition of HIV Gene Expression by a Small Molecule Inhibitor of SIRT1 (A) In vitro histone deacetylation assays including recombinant SIRT1, radioactively labeled histone H3 peptide, and indicated concentrations of splitomicin or HR73. The average (± SEM) of two experiments performed in duplicate is shown for each point. (B) Chemical structures of splitomicin and its derivative HR73. (C) Inhibition of Tat transactivation by HR73. RSV-Tat (0, 20, and 200 ng) and HIV LTR luciferase (200 ng) or RSV-luciferase (200 ng) vectors were transfected into HeLa cells. Transfected cells were treated with indicated concentrations of HR73 or DMSO for 8 h. (D) Inhibition of HIV gene expression by HR73. GFP expression in Jurkat T cells infected with HIV NL4–3 containing the GFP open reading frame in place of the viral nef gene or with an HIV-based lentiviral vector expressing GFP from the heterologous EF-1α promoter. Treatment with HR73 (1 μM in DMSO) or DMSO was performed for 36 h after overnight infection. The average (± SEM) of four experiments is shown. To examine the effect of HR73 on HIV infection, we generated infectious HIV particles using a molecular clone of HIV NL4–3 that contained the green fluorescent protein (GFP) open reading frame in place of the viral nef gene (HIV NL4–3 is a specific viral isolate) [ 43 ]. To restrict analysis to a single infection cycle, this clone also contained a frameshift mutation in the viral env gene. Viral particles were produced by cotransfection with a construct expressing the glycoprotein of the vesicular stomatitis virus (VSV-G). Jurkat T cells were incubated with viral supernatant for at least 18 h, washed to remove extracellular virus, and treated with HR73 (1 μM) or dimethyl sulfoxide (DMSO), as a control. We observed that HIV gene expression was reduced 5-fold in cells treated with HR73 as measured by GFP expression (HIV GFP in Figure 6 D). In contrast, GFP expression in cells infected with an HIV-based lentiviral vector expressing GFP from the elongation factor 1α (EF-1α) promoter was not affected by HR73 treatment (HIV [EF-1α] in Figure 6 D). These data confirm the selectivity of HR73 for HIV transcription and demonstrate that other steps in the viral life cycle, including reverse transcription, nuclear import, and integration, remain unaffected by HR73. These experiments collectively show that the SIRT1 deacetylase activity is required for HIV gene expression and establish SIRT1 as a potential drug target in the treatment of HIV-1 infection. Discussion Our previous work documented that Tat acetylation by p300 at lysine 50 is a necessary step in Tat-mediated transactivation and leads to the recruitment of PCAF to the elongating polymerase. We now report the identification of SIRT1, an NAD + -dependent class III protein deacetylase, as a Tat deacetylase and regulator of Tat activity. Tat and SIRT1 coimmunoprecipitate and synergize to activate the HIV promoter. Conversely, knockdown of SIRT1 via siRNA inhibits Tat-mediated transactivation of the HIV LTR. Tat transactivation is defective in SIRT1-null MEFs and can be rescued by expression of SIRT1. Recent observations indicate that SIRT1 can regulate transcription via histone-dependent and -independent mechanisms [ 20 ]. Since SIRT1 itself does not bind to DNA directly, targeted deacetylation of histones is thought to occur through its interaction with specific DNA binding factors, such as CTIP2 (chicken ovalbumin upstream promoter-transcription factor-interacting protein 2) [ 44 ], members of the hairy-related basic helix-loop-helix repressors (HES1 [hairy and Enhancer-of-split 1] and HEY2 [hairy and Enhancer-of-split related with YRPW motif 2]) [ 45 ], the MyoD/PCAF complex [ 25 ], and the N-CoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptor) co-repressors [ 46 ]. In addition, SIRT1 binds and deacetylates histone H1 and promotes the formation of facultative heterochromatin [ 33 ]. SIRT1 also deacetylates nonhistone factors, including the tumor suppressor p53 [ 21 , 22 , 23 ]. Detailed studies of acetylation of p53 and its regulation by SIRT1 have revealed a number of intriguing parallels to Tat regulation. p53 is acetylated by p300 at multiple lysine residues, including lysine 382, leading to an increased transcriptional activity of p53 [ 47 ]. Activation of p53 up-regulates the expression of genes whose products promote cell-cycle exit or apoptosis [ 22 ]. Therefore, p300 is a positive regulator of p53 activity, while SIRT1 negatively regulates p53 functions. SIRT1 deacetylation of p53 suppresses the induction of apoptosis and promotes cell survival in response to DNA damage [ 21 , 22 ]. Knockout mice for Sir2α show increased p53 acetylation after DNA damage leading to increased thymocyte apoptosis after ionizing radiation [ 48 ]. Interestingly, both SIRT1 and p53, and the Tat cofactor cyclinT1, are localized in promyelocytic leukemia protein bodies [ 23 , 49 ]. These unique nuclear substructures represent the natural accumulation sites of promyelocytic leukemia protein and are altered or disrupted in certain tumors and in response to different cellular stresses, including Tat expression (EV, unpublished data). Interestingly, HDAC1 (a class I HDAC) has also been reported to deacetylate p53 [ 50 ], but the relative contributions of both HDAC1 and SIRT1 to p53 deacetylation remain unclear. In a previous study, an enhancement of Tat-mediated transactivation was observed in response to TSA, an inhibitor of class I and II HDACs, and was interpreted as evidence that Tat acetylation is under the control of a class I or II HDAC [ 7 ]. However, a positive effect of TSA on Tat-mediated transactivation can occur for a number of reasons that are independent of Tat acetylation levels. First, the HIV promoter can be activated by TSA in the absence of Tat, a phenomenon likely to be mediated by transcription factors responsive to TSA that bind to the HIV LTR, such as Sp1 (stimulatory protein 1), YY1 (Yin Yang 1), Eed (embryonic ectoderm development protein), and NF-κB/p65 [ 51 , 52 , 53 ]. Tat-independent activation of the HIV LTR by TSA has been documented both in vitro using chromatinized templates, and in vivo using cell lines containing integrated HIV genomes defective for Tat-mediated transactivation [ 54 , 55 , 56 ]. Second, TSA activates many promoters that are used to drive Tat expression in transient transfection experiments, making interpretations of these experiments problematic. We have observed that Tat acetylation at lysine 50 is not specifically enhanced in response to TSA [ 11 ]. These results are supported by functional experiments with Tat mutants. In cotransfection experiments, enhanced transcriptional activity of Tat in response to TSA is preserved when lysine 50 is replaced by an alanine [ 7 ] or an arginine (MO, unpublished data). Although these experiments are complicated by the significant increase in Tat expression (due to the stimulatory role of TSA on Tat expression), they provide evidence that lysine 50 in fact might not be a target for a TSA-sensitive enzyme in HIV transcription. Another system that offers intriguing parallels to our observation is the role of the SIRT1/MyoD/PCAF complex in myogenesis [ 25 ]. In this complex, PCAF acetylates both itself and MyoD. SIRT1, in contrast, deacetylates both PCAF and MyoD, leading to a retardation of myogenesis. The negative effect of SIRT1 in the regulation of muscle gene expression plays at two distinct levels. First, SIRT1 induces deacetylation of histones in the promoter regions. Second, SIRT1 deacetylates MyoD, which leads to an inhibition of its transcriptional activity [ 57 ]. Our previous experiments documented the specific interaction of acetylated Tat and PCAF via the PCAF bromodomain and the ARM region of Tat and the role of this interaction in Tat transactivation [ 13 ]. The possibility that Tat bound to the elongating polymerase and to PCAF is regulated by a PCAF-SIRT1 complex is intriguing and will be further explored. In most identified sites of action, SIRT1 showed a negative activity on transcriptional regulatory mechanisms, resulting in an inhibition of target gene expression. In contrast, we have identified a positive regulatory activity of SIRT1 on Tat-mediated transactivation. According to our working model, Tat becomes acetylated at the level of the HIV promoter after binding to TAR. The acetylation of the Tat ARM can potentially affect other biological activities associated with this domain, including RNA binding, nuclear or nucleolar import, and protein stability. Tat acetylation could also be involved in the delivery of Tat to the transcription complex, and/or might moderate interactions with cyclinT1/CDK9 prior to transcription. However, we provided evidence that Tat acetylation leads to a dissociation of Tat from cyclinT1 and TAR and to its transfer to the elongating polymerase [ 11 ]. Acetylated Tat, bound to the elongating polymerase, recruits the transcriptional coactivator PCAF via its acetyl group and the bromodomain of PCAF [ 12 , 13 ]. An unresolved question raised by this model is the fate of acetylated Tat at the end of the transcription cycle. Since acetylated Tat cannot recruit cyclinT1 to the TAR element, a new transcription cycle would require either the synthesis of a new unacetylated Tat protein or the deacetylation of Tat. Tat concentrations are limiting early in the virus life cycle since Tat expression is under the control of the basal LTR. Tat recycling is therefore likely to represent a rate-limiting step in HIV transcription early during the replicative cycle of the virus. Experiments presented in this manuscript demonstrate that SIRT1 catalyzes this rate-limiting step in HIV transcription. These observations raise a number of additional questions. The demonstration that Tat transactivating activity is controlled by SIRT1 connects HIV transcription with the metabolic state of the cell. The HDAC activity of SIRT1 requires NAD + as a cofactor. NAD + and its reduced form NADH serve as cofactors in many metabolic and stress reactions involving oxidation and reduction. Changes in the cellular NAD + /NADH ratio may regulate SIRT1 enzymatic activity, Tat transcriptional activity, and HIV replication and pathogenesis. The site of Tat deacetylation is unknown at this point. We hypothesize that the deacetylation could be triggered by molecular events linked with the end of the transcriptional elongation process. Deacetylation of Tat by SIRT1 could lead to its dissociation from the elongating polymerase and from PCAF. The previous demonstration that PCAF and SIRT1 can be coimmunoprecipitated from cells supports this model [ 25 ]. The identification of SIRT1 as an enzyme that catalyzes a rate-limiting step in HIV transcription suggests that it could be targeted therapeutically. Since our preliminary experiments indicated that splitomicin, an inhibitor of the S. cerevisiae Sir2 enzymatic activity, did not inhibit the mammalian sirtuins, we screened several compounds structurally related to splitomicin for their ability to inhibit the mammalian sirtuins. This process led to the identification of HR73, an inhibitor that suppresses SIRT1 enzymatic activity in vitro at low micromolar concentrations. This inhibitor induced p53 hyperacetylation in vivo (unpublished data) and significantly decreased HIV transcription. These results validate SIRT1 as a novel therapeutic target for HIV infection. Future efforts will analyze in additional molecular detail the role of Tat acetylation and deacetylation in HIV transcription and replication. Materials and Methods Cells and plasmids HeLa, HEK 293, and Jurkat cells (obtained from the American Type Culture Collection, Manassas, Virginia, United States), and wild-type or Sir2α knockout MEFs [ 37 ] were maintained under standard cell culture conditions. The HIV LTR luciferase plasmid [ 58 ], the RSV LTR luciferase construct [ 11 ], the 5xUAS luciferase construct [ 59 ], the LTR ΔNF-κB -luciferase, the full-length (101 amino acid) CMV-Tat/FLAG and CMV-Tat/T7 expression vectors [ 8 ], the full-length (101 amino acid) RSV-Tat expression vector [ 60 ], the full-length cyclinT1 expression vector [ 5 ], the Gal4-VP16 expression vector [ 13 ], the human SIRT1–7 expression vectors with a C-terminal FLAG tag [ 30 ], as well as wild-type and mutant human SIRT1 and SIRT1H363Y containing a C-terminal MYC (myelocytomatosis oncogene) tag [ 23 ] were previously described. The SIRT1 cDNA was subcloned to generate a C-terminal HA-tagged fusion in a derivative pcDNA 3.1 (+) backbone (HA vector) by standard PCR-based strategies. The mutant CMV-Tat/FLAG expression vector TatK50R was generated by site-directed mutagenesis with the following primers. Forward, 5′- CCTATGGCAGGAGGAAGCGGAGACAGCG-3′ and reverse, 5′- CGCTGTCTCCGCTTCCTCCTGCCATAGG-3′. The mutant HIV LTR luciferase constructs (nucleotides 1–791) were generated by site-directed mutagenesis with the following primers. TAR Δbulge (T 223 →A) forward, 5′- GGTTAGACCAGAACTGAGCCTGGGAGC-3′ and reverse, 5′- GCTCCCAGGCTCAGTTCTGGTCTAACC-3′. The TAR Δloop mutation (C 230 →A, T 231 →G, and G 234 →T) was generated with the following primers. Forward, 5′- GGTTAGACCAGATCTGAGCAGGGTAGCTCTCTGGCTAACTAGGG-3′ and reverse, 5′- CCCTAGTTAGCCAGAGAGCTACCCTGCTCAGATCTGGTCTAACC-3′. The CMV-luciferase construct was generated by cloning the firefly luciferase gene as a HindIII/BamHI fragment obtained from pGL2 Basic (Promega, Madison, Wisconsin, United States) into pcDNA3.1 (Invitrogen, Carlsbad, California, United States). The CMV-GFP expression plasmid is commercially available (Clontech, Palo Alto, California, United States). Synthesis of Tat and HR73 Synthesis of 72-amino acid Tat proteins was as described [ 11 , 13 ]. HR73 was synthesized starting from Phenylmeldrum's acid and 6-bromo-1-dimethylaminomethyl-2-naphthol as described [ 61 ]. Identity and purity were assured by mass, infrared, and NMR spectroscopy as well as by elemental analysis. In vitro Tat deacetylation assay Human SIRT1–7 FLAG-tagged plasmids were transfected in HEK 293 cells with Lipofectamine reagent (Invitrogen). Cells were lysed 24 h after transfection with lysis buffer (50 mM Tris-HCl [pH 7.5], 0.5 mM EDTA, 0.5% NP40, and 150 mM NaCl) in the presence of protease inhibitors (Roche Molecular Biochemicals, Indianapolis, Indiana, United States). Equal amounts of total proteins were immunoprecipitated with α-FLAG M2 agarose (Sigma, St Louis, Missouri, United States), for 2 h at 4 °C. Immunoprecipitated material was washed twice with IP buffer and one time with SIRT deacetylation buffer (50 mM Tris-HCl [pH 9], 4 mM MgCl 2 , and 0.2 mM DTT). The beads were resuspended in 100 μl of SIRT deacetylation buffer containing 1 μg synthetic Tat (72 amino acids) carrying an N-terminal biotin label and an acetyl group at position 50 [ 11 ]. Reactions containing TSA (400 nM; WACO Bioproducts, Richmond, Virginia, United States) or nicotinamide (5 mM; Sigma) were preincubated for 10 min at room temperature. After addition of NAD + (1 mM), reactions were incubated for 2 h at room temperature with constant agitation. Reactions were stopped by the addition of SDS loading buffer, boiled, and after brief centrifugation, analyzed by WB with rabbit α-AcTat antibodies [ 11 ] or SA-HRP (Jackson Immunoresearch Laboratories, West Grove, Pennsylvania, United States). SIRT1–7 proteins were detected with polyclonal α-FLAG antibodies (Sigma). The histone deacetylation assay with recombinant SIRT1 (1–1.3 U/reaction; Biomol, Plymouth Meeting, Pennsylvania, United States) was performed as described previously for SIRT2 [ 30 ] in 100 μl of SIRT deacetylase buffer containing NAD + (Sigma) and enzymatically [ 3 H]-acetylated histone H3 peptide (amino acids 1–24) [ 62 ]. Splitomicin (a gift from Julian Simon, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States) and HR73 in DMSO were added to the reactions at the indicated concentrations with all components of the reactions in the absence of NAD + for 10 min at room temperature prior to the initiation of the reaction by addition of NAD + (1 mM). Coimmunoprecipitation experiments HEK 293 cells were cotransfected in duplicate with expression vectors for CMV-Tat/FLAG; CMV-Tat/T7 or CMV-TatK50R/FLAG; and the SIRT1/HA or SIRT1-, SIRT2-, and SIRT6-FLAG expression vectors or the respective empty vector controls using Lipofectamine reagent (Invitrogen). Cells were lysed after 24 h in 250 mM NaCl, 0.1% NP40, 20 mM NaH 2 PO 4 (pH 7.5), 5 mM EDTA, 30 mM sodium pyrophosphate, 10 mM NaF, and protease inhibitors (Roche Molecular Biochemicals). Duplicates were pooled, and 1 mg of lysate was immunoprecipitated either with monoclonal α-HA (Roche Molecular Biochemicals) together with protein G-Sepharose (Amersham Biosciences, Piscataway, New Jersey, United States) with α-FLAG M2 agarose (Sigma) or α-T7-agarose (Amersham Biosciences) for 2 h at 4 °C. Beads were washed three times in lysis buffer, boiled in SDS loading buffer, and analyzed by WB with polyclonal α-FLAG (Sigma), monoclonal α-HA (Roche), or monoclonal α-T7 (Novagen, Madison, Wisconsin, United States) antibodies. For the IP of Tat with endogenous SIRT1, HEK 293 cells were transfected only with CMV-Tat/FLAG or the CMV-empty vector using Lipofectamine reagent. Cell lysates were immunoprecipitated with rabbit α-SIRT1 antibodies (generated against amino acids 506–747) together with protein G-Sepharose (Amersham Biosciences). Immunoprecipitated material was analyzed by WB with the M2 α-FLAG antibody (Sigma) or rabbit α-SIRT1 antibodies. For in vitro interactions, 10 U of recombinant SIRT1 (Biomol) was incubated with biotinylated synthetic Tat or acetylated Tat proteins (0, 0.25, 1, and 4 μg) together with streptavidin-Sepharose (Amersham Biosciences) in lysis buffer in the presence of 5 mM nicotinamide (Sigma) for 3 h at 4 °C. Pelleted beads were washed three times in lysis buffer, resuspended in SDS loading buffer, and analyzed by WB with polyclonal α-SIRT1 antibodies, rabbit α-AcARM, or SA-HRP (Jackson Immunoresearch Laboratories). RNAi and transfection experiments Double-stranded siRNAs directed against nucleotides 408–428 in the SIRT1 mRNA or control GL3 siRNAs (both Dharmacon Research, Lafayette, Colorado, United States) were transfected into HeLa cells plated in six-well plates with Oligofectamine reagent according to the manufacturer's guidelines (Invitrogen). The mutant SIRT1 siRNA was identical to SIRT1 siRNA except for a two-nucleotide mismatch between the target mRNA for SIRT1 and the antisense strand of siRNA at nucleotides 418 and 419. After 48 h, cells were retransfected with the HIV LTR luciferase construct (200 ng) together with increasing amounts of CMV-Tat expression vectors (0, 50, 100, 200, 400, and 800 ng in GL3/SIRT1 siRNA experiments; 0, 2, 20, and 200 ng in SIRT1/mutant SIRT1 siRNA experiments) and corresponding amounts of empty pcDNA3.1 vector (Invitrogen). In the control experiment, CMV-Tat was replaced by the CMV-luciferase construct, and HIV LTR luciferase was replaced by an HIV LTR promoter construct driving the expression of chloramphenicol acetyl transferase (CAT) [ 55 ]). Cells were harvested 24 h later and either processed for luciferase assays (Promega) or WB of total cell extracts with polyclonal α-SIRT1 or α-actin (MP Biochemicals, Aurora, Ohio, United States) antibodies. In cotransfection experiments, human CMV-SIRT1 or CMV-SIRT1H363Y (600 ng) was cotransfected into HeLa cells plated in six-well plates with the HIV LTR luciferase reporter (200 ng) or the LTR ΔNF-κB -luciferase reporter (200 ng) and increasing amounts of RSV-Tat (0, 2, 20, and 200 ng) using the Lipofectamine reagent (Invitrogen). In the control experiment, RSV-Tat was replaced by RSV-luciferase (200 ng), and the HIV LTR luciferase construct was replaced by the HIV LTR CAT reporter. In transfections with HR73, HeLa cells were cotransfected with the HIV LTR luciferase reporter (200 ng) and RSV-Tat expression vectors (0, 20, and 200 ng) or the empty vector using Lipofectamine reagent. The RSV-luciferase construct was used as described above. After 4 h incubation with the DNA/Lipofectamine mix, the culture medium was changed and supplemented with indicated concentrations of HR73 dissolved in DMSO or DMSO alone. Cells were harvested 8 h later and processed for luciferase assays. Microinjection experiments Subconfluent MEFs (70%) were grown on Cellocate coverslips (Eppendorf, Westbury, New York, United States), and nuclear microinjections were performed at room temperature with an automated injection system (Eppendorf Micromanipulator 5171 together with Eppendorf Transjector 5246). Samples were prepared as a 20 μl injection mix containing the HIV LTR luciferase reporter or 5xUAS luciferase (each 100 ng/μl), RSV-Tat (10 ng/μl) or Gal4-VP16 (50 ng/μl), CMV-cyclinT1 (100 ng/μl), CMV-SIRT1 (100 or 300 ng/μl), together with CMV-GFP (50 ng/μl) in sterile water. At 6 h after microinjection, cells were examined under a Nikon Eclipse TE300 inverted fluorescent microscope (Nikon, Tokyo, Japan) to determine the number of GFP-positive cells, washed in cold phosphate buffer, and stored at −70 °C for luciferase assays (Promega). In HeLa cells, synthetic Tat or AcTat proteins (each 30 or 100 ng/μl) were coinjected with the wild-type or mutant HIV LTR luciferase reporters (each 100 ng/μl) together with CMV-GFP (50 ng/μl), and harvested 4 h after injection. Cells were treated immediately after injection with DRB (10 or 50 μM; Sigma), TSA (400 nM), or nicotinamide (5 mM). Microinjections in siRNA-treated cells were performed 48 h after siRNA transfection. Viral infection experiments The HIV molecular clone HIV-R7/E − /GFP containing the GFP open reading frame in place of the nef gene and a frameshift mutation in the env gene, as well as the method to generate pseudotyped viral particles with VSV-G, were previously described [ 43 ]. The number of infective particles per milliliter was established by infecting 3 × 10 5 Jurkat cells with different amounts of viral suspension. The titer of the viral stock was measured by flow cytometric analysis of GFP expression 48 h after infection. The pHR′-EF-1α/GFP construct is a minimal nonreplicative HIV-1 genome containing a heterologous promoter, EF-1α, driving GFP expression [ 63 ]. Viral particles were produced by cotransfection of the VSV-G-encoding pMD.G and the HIV-based packaging vector pCMVΔR8.91 as described [ 64 ]. All vectors for the production of HIV-based lentiviral vectors were provided by Didier Trono, University of Geneva, Switzerland. Jurkat T cells were incubated overnight with HIV-R7/E − /GFP or pHR′-EF-1α/GFP viral particles at a theoretical multiplicity of infection of 0.5 in 24-well plates. Cells were repeatedly washed and resuspended in fresh medium containing HR73 (1 μM) or DMSO alone. Viral infection was monitored 36 h later by flow cytometry analysis using a Calibur FACScan (Becton Dickinson, Palo Alto, California, United States).

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Nutrition and cancer: A review of the evidence for an anti-cancer diet

It has been estimated that 30–40 percent of all cancers can be prevented by lifestyle and dietary measures alone. Obesity, nutrient sparse foods such as concentrated sugars and refined flour products that contribute to impaired glucose metabolism (which leads to diabetes), low fiber intake, consumption of red meat, and imbalance of omega 3 and omega 6 fats all contribute to excess cancer risk. Intake of flax seed, especially its lignan fraction, and abundant portions of fruits and vegetables will lower cancer risk. Allium and cruciferous vegetables are especially beneficial, with broccoli sprouts being the densest source of sulforophane. Protective elements in a cancer prevention diet include selenium, folic acid, vitamin B-12, vitamin D, chlorophyll, and antioxidants such as the carotenoids (α-carotene, β-carotene, lycopene, lutein, cryptoxanthin). Ascorbic acid has limited benefits orally, but could be very beneficial intravenously. Supplementary use of oral digestive enzymes and probiotics also has merit as anticancer dietary measures. When a diet is compiled according to the guidelines here it is likely that there would be at least a 60–70 percent decrease in breast, colorectal, and prostate cancers, and even a 40–50 percent decrease in lung cancer, along with similar reductions in cancers at other sites. Such a diet would be conducive to preventing cancer and would favor recovery from cancer as well.

Review Background The field of investigation of the role of nutrition in the cancer process is very broad. It is becoming clearer as research continues that nutrition plays a major role in cancer. It has been estimated by the American Institute for Cancer Research and the World Cancer Research Fund that 30–40 percent of all cancers can be prevented by appropriate diets, physical activity, and maintenance of appropriate body weight [ 1 ]. It is likely to be higher than this for some individual cancers. Most of the research on nutrition and cancer has been reductionist; that is, a particular food or a nutrient has been studied in relation to its impact on tumor formation/regression or some other end point of cancer at a particular site in the body. These studies are very helpful in seeing the details of the mechanisms of disease. However, they do not help give an overall picture of how to prevent cancer on a dietary level. Even less, they tell little of how to eat when a person already has a cancer and would like to eat a diet that is favorable to their recovery. This review will focus on those dietary factors which has been shown to be contribute to increased risk of cancer and then on those additional protective dietary factors which reduce cancer risk. Finally, some whole-diet studies will be mentioned which give a more complete picture of how these individual factors work together to reduce cancer risk. Over Consumption of Energy (Calories) Eating too much food is one of the main risk factors for cancer. This can be shown two ways: (1) by the additional risks of malignancies caused by obesity, and (2) by the protective effect of eating less food. Obesity has reached epidemic proportions in the United States. Sixty-four percent of the adult population is overweight or obese [ 2 ]. About 1 in 50 are now severely obese (BMI > 40 kg/m2) [ 3 ]. Mokdad et al [ 4 ] found that poor diet and physical inactivity was the second leading cause of death (400,000 per year in the USA), and would likely overtake tobacco as the leading cause of death. It was estimated in a recent study, from a prospective cancer prevention cohort, that overweight and obesity accounted for 14 percent of all cancer deaths in men and 20 percent of those in women [ 5 ]. Significant positive associations were found between obesity and higher death rates for the following cancers: esophagus, colon and rectum, liver, gallbladder, pancreas, kidney, stomach (in men), prostate, breast, uterus, cervix, and ovary [ 5 ]. The authors estimated that over 90,000 cancer deaths per year could be avoided if the adult population all maintained a normal weight (BMI < 25.0) [ 5 ]. Clearly, obesity is a major risk factor for cancer. On the other side, careful menu planning brings about an approach entitled CRON-Calorie Restriction with Optimal Nutrition. The basic idea is to eat a reduced amount of food (about 70–80 percent of the amount required to maintain "normal" body weight) while still consuming all of the necessary amounts of vitamins, minerals, and other necessary nutrients. The only restriction is the total amount of energy (calories) that is consumed. While being difficult to practice, this approach has a lot of scientific merit for being able to extend average life spans of many species of animals including rats, mice, fish, and possibly primates (currently being tested). Along with this life span extension is a reduction in chronic diseases that are common to mankind, reviewed in Hursting et al [ 6 ]. A recent meta-analysis of 14 experimental studies found that energy restriction resulted in a 55% reduction in spontaneous tumors in laboratory mice [ 7 ]. Calorie restriction inhibited induced mammary tumors in mice [ 8 ] and suppressed implanted tumor growth and prolonged survival in energy restricted mice [ 9 ]. Among Swedish women who had been hospitalized for anorexia nervosa (definitely lower caloric intake, but not adequate nutrition) prior to age 40, there was a 23% lower incidence of breast cancer for nulliparous women and a 76% lower incidence for parous women [ 10 ]. So, too many calories is definitely counter-productive, and slightly less than normal is very advantageous. Glucose Metabolism Refined sugar is a high energy, low nutrient food – junk food. "Unrefined" sugar (honey, evaporated cane juice, etc) is also very concentrated and is likely to contribute to the same problems as refined sugar. Refined wheat flour products are lacking the wheat germ and bran, so they have 78 percent less fiber, an average of 74 percent less of the B vitamins and vitamin E, and 69 percent less of the minerals (USDA Food database, data not shown). Concentrated sugars and refined flour products make up a large portion of the carbohydrate intake in the average American diet. One way to measure the impact of these foods on the body is through the glycemic index. The glycemic index is an indication of the blood sugar response of the body to a standardized amount of carbohydrate in a food. The glycemic load takes into account the amount of food eaten. An international table of the glycemic index and glycemic load of a wide variety of foods has been published [ 11 ]. Case-control studies and prospective population studies have tested the hypothesis that there is an association between a diet with a high glycemic load and cancer. The case control studies have found consistent increased risk of a high glycemic load with gastric [ 12 ], upper aero digestive tract [ 13 ], endometrial [ 14 ], ovarian [ 15 ], colon or colorectal cancers [ 16 , 17 ]. The prospective studies' results have been mixed. Some studies showed increased risk of cancer in the whole cohort with high glycemic load [ 18 - 20 ]; some studies found only increased risk among subgroups such as sedentary, overweight subjects [ 21 - 24 ]; other studies concluded that there was no increased risk for any of their cohort [ 25 - 28 ]. Even though there were no associations between glycemic load and colorectal, breast, or pancreatic cancer in the Nurses' Health Study there was still a strong link between diabetes and colorectal cancer [ 29 ]. Perhaps the dietary glycemic load is not consistently related to glucose disposal and insulin metabolism due to individual's different responses to the same glycemic load. Glycated hemoglobin (HbA 1c ) is a time-integrated measurement of glucose control, and indirectly, of insulin levels. Increased risk in colorectal cancer was seen in the EPIC-Norfolk study with increasing HbA 1c ; subjects with known diabetes had a three-fold increased risk of colorectal cancer [ 30 ]. In a study of a cohort in Washington county, Maryland, increased risk of colorectal cancer was seen in subjects with elevated HbA 1c , BMI > 30 kg/m 2 , or who used medications to control diabetes [ 31 ]. However, glycated hemoglobin was not found to be associated with increased risk of colorectal cancer in a small nested case-control study within the Nurses' Health Study [ 32 ]. Elevated fasting glucose, fasting insulin, 2 hour levels of glucose and insulin after an oral glucose challenge, and larger waist circumference were associated with a higher risk of colorectal cancer [ 33 ]. In multiple studies diabetes has been linked with increased risk of colorectal cancer [ 34 - 37 ], endometrial cancer [ 38 ], and pancreatic cancer [ 35 , 39 ]. It is clear that severe dysregulation of glucose metabolism is a risk factor for cancer. Foods which contribute to hyperinsulinemia, such as refined sugar, foods containing refined sugar, and refined flour products should be avoided and eliminated from a cancer protective diet. Low Fiber Unrefined plant foods typically have an abundance of fiber. Dairy products, eggs, and meat all have this in common – they contain no fiber. Refined grain products also have most of the dietary fiber removed from them. So, a diet high in animal products and refined grains (a typical diet in the USA) is low in fiber. In prospective health studies low fiber was not found to be a risk for breast cancer [ 25 ]. It is possible that fiber measurements are just a surrogate measure for unrefined plant food intake. Slattery et al [ 40 ] found an inverse correlation between vegetable, fruit and whole grain intake plant food intake and rectal cancer, while refined grains were associated with increased risk of rectal cancer. A threshold of about 5 daily servings of vegetables was needed to reduce cancer risk and the effect was stronger among older subjects [ 40 ]. Many other nutrients are co-variants with fiber, including folic acid, which is covered in detail below. Red Meat Red meat has been implicated in colon and rectal cancer. A Medline search in February 2003 uncovered 26 reports of human studies investigating the link between diet and colon or colorectal cancer. Of the 26 reports, 21 of them reported a significant positive relationship between red meat and colon or colorectal cancer [ 17 , 41 - 64 ]. A recent meta-analysis also found red meat, and processed meat, to be significantly associated with colorectal cancer [ 65 ]. Meat, and the heterocyclic amines formed in cooking, have been correlated to breast cancer in a case-control study in Uruguay as well [ 66 ]. Omega 3:6 Ratio Imbalance Omega 3 fats (alpha-linolenic acid, EPA, DHA) have been shown in animal studies to be protect from cancer, while omega 6 fats (linoleic acid, arachidonic acid) have been found to be cancer promoting fats. Now there have been several studies that have tested this hypothesis in relation to breast cancer, summarized in Table 1 . Except for the study by London et al [ 67 ], all of these studies found an association between a higher ratio of N-3 to N-6 fats and reduced risk of breast cancer. Long chain N-3 and N-6 fats have a different effect on the breast tumor suppressor genes BRCA1 and BRCA2. Treatment of breast cell cultures with N-3 fats (EPA or DHA) results in increased expression of these genes while arachadonic acid had no effect [ 68 ]. Flax seed oil and DHA (from an algae source) both can be used to increase the intake of N-3 fat, with DHA being a more efficient, sure source. Table 1 Breast Cancer and Omega 3:6 Ratio. Reference # of cases w/ breast cancer # of controls Post / pre Menopausal Measure of n-3 / n-6 fat Outcome Odds ratio (95% Confidence Interval) [183] 565 554 (population and hospital) Pre & post Diet FFQ ↑N3/N6 ratio in premenopausal women = Non-signif. ↓Breast cancer risk 0.59 (0.29–1.19) In study site with population controls, find ↑N3/N6 ratio = Signif ↓Breast Cancer risk 0.50 (0.27, 0.95) [184] EURAMIC study Nested case-control study in population study Post Adipose tissue 4 out of 5 centers showed ↑N3/N6 ratio = ↓Breast Cancer risk 0.65 (p for trend = 0.55) [185] 241 88 w/ benign breast disease Both Adipose tissue ↑DHA = ↓Breast cancer 0.31 (0.13–0.75) ↑Ratio of long chain N-3:N-6 fat = ↓Breast cancer 0.33 (0.17–0.66) [186] 73 74 w/ macromastia ? Adipose tissue N-6 fat content signif. higher in cases P = 0.02 For given level of N-6 fat, EPA and DHA had a protective effect P = 0.06 [187] 71 (within ORDET study) 142 (nested case control) Post RBC membranes ↑DHA = ↓Breast cancer 0.44 (0.21–0.92) [67] 380 397 Post Adipose tissue No associations between N-3:N-6 ratio and breast cancer [188] 314 (within Singapore Chinese Health study) Diet, FFQ ↑Intake of N-3 fat from fish / shellfish = ↓Breast cancer, for all 3 highest quartiles 0.74 (0.58–0.94) Among women in lowest quartile of N-3 fat intake, ↑N-6 fat intake = ↓Breast cancer 1.87 (1.06–3.27) Flax seed Flax seed provides all of the nutrients from this small brown or golden hard-coated seed. It is an excellent source of dietary fiber, omega 3 fat (as alpha-linolenic acid), and lignans. The lignans in flax seed are metabolized in the digestive tract to enterodiol and enterolactone, which have estrogenic activity. In fact, flax seed is a more potent source of phytoestrogens than soy products, as flax seed intake caused a bigger change in the excretion of 2-hydroxyestrone compared to soy protein [ 69 ]. Ground flax seeds have been studied for its effect on cancer, including several excellent studies by Lilian Thompson's research group at the University of Toronto. In one study the flax seed, its lignan fraction, or the oil were added to the diet of mice who had previously been administered a chemical carcinogen to induce cancer. All three treatments reduced the established tumor load; the lignan fraction containing secoisolariciresinol diglycoside (SDG) and the flax seed also reduced metastasis [ 70 ]. In another study the flax lignan SDG was fed to mice starting 1 week after treatment with the carcinogen dimethylbenzanthracene. The number of tumors per rat was reduced by 46% compared to the control in this study [ 71 ]. Flax or its lignan (SDG) were tested to see if they would prevent melanoma metastasis. The flax or lignan fraction were fed to mice two weeks before and after injection of melanoma cells. The flax treatment (at 2.5, 5, or 10% of diet intake) resulted in a 32, 54, and 63 percent reduction in the number of tumors, compared to the control [ 72 ]. The SDG, fed at amounts equivalent to the amount in 2.5, 5, or 10% flax seed, also reduced the tumor number, from a median number of 62 in the control group to 38, 36, and 29 tumors per mouse in the SDG groups, respectively [ 73 ]. More recently Thompson's research group studied mice that were injected with human breast cancer cells. After the injection the mice were fed a basal diet (lab mouse chow) for 8 weeks while the tumors grew. Then one group continued the basal diet and another was fed a 10% flax seed diet. The flax seed reduced the tumor growth rate and reduced metastasis by 45% [ 74 ]. Flax seed has been shown to enhance mammary gland morphogenesis or differentiation in mice. Nursing dams were fed the 10% flax seed diet (or an equivalent amount of SDG). After weaning the offspring mice were fed a regular mouse chow diet. Researchers then examined the female offspring and found an increased number of terminal end buds and terminal ducts in their mammary glands with more epithelial cell proliferation, all demonstrating that mammary gland differentiation was enhanced [ 75 ]. When these female offspring were challenged with a carcinogen to induce mammary gland tumors there were significantly lower incidence of tumors (31% and 42% lower in the flax seed and SDG groups, respectively), significantly lower tumor load (51% and 62% lower in the flax seed and SDG groups, respectively), significantly lower mean tumor size (44% and 68% lower in the flax seed and SDG groups, respectively), and significantly lower tumor number (47% and 45% lower in the flax seed and SDG groups, respectively) [ 76 ]. So, flax seed and its lignan were able to reduce tumor growth (both in number and size of tumors), prevent metastasis, and even cause increased differentiation of mouse mammary tissue in suckling mice, making the offspring less susceptible to carcinogenesis even when not consuming any flax products. Other researchers have tested flax seed and prostate cancer. In an animal model using mice, Lin et al [ 77 ] found that a diet supplemented with 5% flax inhibited the growth and development of prostate cancer in their experimental mouse model. A pilot study of 25 men who were scheduled for prostatectomy surgery were instructed to eat a low-fat diet (20% or less of energy intake) and to supplement with 30 g of ground flaxseed per day. During the follow-up of an average of 34 days there were significant changes in serum cholesterol, total testosterone, and the free androgen index [ 78 ]. The mean proliferation index of the experimental group was significantly lower and apoptotic indexes higher compared to historical matched controls. Ground flax seed may be a very beneficial food for men battling prostate cancer. However, a meta-analysis of nine cohort and case-control studies revealed an association between flax seed oil intake or high blood levels of alpha-linolenic acid and prostate cancer risk [ 79 ]. It is quite likely that the lignans in flax seed are a major component of flax's anti-cancer effects so that flax oil without the lignans is not very beneficial. Some brands of flax seed oil retain some of the seed particulate because of the beneficial properties of the lignans. Fruits and Vegetables One of the most important messages of modern nutrition research is that a diet rich in fruits and vegetables protects against cancer. (The greatest message is that this same diet protects against almost all other diseases, too, including cardiovascular disease and diabetes.) There are many mechanisms by which fruits and vegetables are protective, and an enormous body of research supports the recommendation for people to eat more fruits and vegetables. Block et al [ 80 ] reviewed about 200 studies of cancer and fruit and vegetable intake. A statistically significant protective effect of fruits and vegetables was found in 128 of 156 studies that gave relative risks. For most cancers, people in the lower quartile (1/4 of the population) who ate the least amount of fruits and vegetables had about twice the risk of cancer compared to those who in the upper quartile who ate the most fruits and vegetables. Even in lung cancer, after accounting for smoking, increasing fruits and vegetables reduces lung cancer; an additional 20 to 33 percent reduction in lung cancers is estimated [ 1 ]. Steinmetz and Potter reviewed the relationship between fruits, vegetables, and cancer in 206 human epidemiologic studies and 22 animal studies [ 81 ]. They found "the evidence for a protective effect of greater vegetable and fruit consumption is consistent for cancers of the stomach, esophagus, lung, oral cavity and pharynx, endometrium, pancreas, and colon." Vegetables, and particularly raw vegetables, were found to be protective; 85% of the studies that queried raw vegetable consumption found a protective effect. Allium vegetables, carrots, green vegetables, cruciferous vegetables, and tomatoes also had a fairly consistent protective effect [ 81 ]. Allium vegetables (garlic, onion, leeks, and scallions) are particularly potent and have separately been found to be protective for stomach and colorectal cancers [ 82 , 83 ] and prostate cancer [ 84 ]. There are many substances that are protective in fruits and vegetables, so that the entire effect is not very likely to be due to any single nutrient or phytochemical. Steinmetz and Potter list possible protective elements: dithiolthiones, isothiocyanates, indole-32-carbinol, allium compounds, isoflavones, protease inhibitors, saponins, phytosterols, inositol hexaphosphate, vitamin C, D-limonene, lutein, folic acid, beta carotene (and other carotenoids), lycopene, selenium, vitamin E, flavonoids, and dietary fiber [ 81 ]. A joint report by the World Cancer Research Fund and the American Institute for Cancer Research found convincing evidence that a high fruit and vegetable diet would reduce cancers of the mouth and pharynx, esophagus, lung, stomach, and colon and rectum; evidence of probable risk reduction was found for cancers of the larynx, pancreas, breast, and bladder [ 1 ]. Many of the recent reports from prospective population-based studies of diet and cancer have not found the same protective effects of fruits and vegetables that were reported earlier in the epidemiological and case-control studies [reviewed in [ 85 ]]. One explanation is that people's memory of what they ate in a case-cohort study may have been tainted by their disease state. Another problem might be that the food frequency questionnaires (FFQ) used to measure food intake might not be accurate enough to detect differences. Such a problem was noted in the EPIC study at the Norfolk, UK site. Using a food diary the researchers found a significant correlation between saturated fat intake and breast cancer, but using a FFQ there was no significant correlation [ 86 ]. So, inaccurate measurement of fruit and vegetable intake might be part of the explanation as well. It must be noted that upper intakes of fruits and vegetables in these studies are usually within the range of what people on an American omnivorous diet normally eat. In the Nurses Health Study the upper quintiles of fruit and vegetable intake were 4.5 and 6.2 servings/day, respectively [ 87 ]. Similarly, the upper quintiles of fruit and vegetable intake in the Health Professionals Follow-up Study were 4.3 and 5.4 serving/day for fruits and vegetables, respectively [ 87 ]. Intakes of fruits and vegetables on the Hallelujah Diet are much higher, with median reported intakes of six servings of fruits (646 g/day) and eleven servings of vegetables per day (971 g/day) [ 88 ] in addition to a green powder from the juice of barley leaves and alfalfa that is equivalent to approximately another 100 g/day of fresh dark greens. So, it is very possible that the range of intakes in the prospective population based studies do not have a wide enough intake on the upper end to detect the true possible impact of a very high intake of fruits and vegetables on cancer risk. Cruciferous Vegetables Cruciferous vegetables (broccoli, cauliflower, cabbage, Brussels sprouts) contain sulforophane, which has anti-cancer properties. A case-control study in China found that intake of cruciferous vegetables, measured by urinary secretion of isothiocyanates, was inversely related to the risk of breast cancer; the quartile with the highest intake only had 50% of the risk of the lowest intake group [ 89 ]. In the Nurses' Health Study a high intake of cruciferous vegetables (5 or more servings/week vs less than two servings/week) was associated with a 33% lower risk of non-Hodgkin's lymphoma [ 90 ]. In the Health Professionals Follow-up Study bladder cancer was only weakly associated with low intake of fruits and vegetables, but high intake (5 or more servings/week vs 1 or less servings/wk) of cruciferous vegetables was associated with a statistically significant 51% decrease in bladder cancer [ 91 ]. Also, prostate cancer risk was found to be reduced by cruciferous vegetable consumption in a population-based case-control study carried out in western Washington state. Three or more servings per week, compared to less than one serving of cruciferous vegetables per week resulted in a statistically significant 41% decrease in prostate cancer risk [ 92 ]. Similar protective effects of cruciferous vegetables were seen in a multi-ethnic case-control study [ 93 ]. A prospective study in Shanghai, China found that men with detectable amounts of isothiocyanates in their urine (metabolic products that come from cruciferous vegetables) had a 35% decreased risk of lung cancer. Among men that had one or two genetic polymorphisms that caused them to eliminate these isothiocyanates slower there was a 64% or 72% decreased risk of lung cancer, respectively [ 94 ]. Broccoli sprouts have a very high concentration of sulforophane since this compound originates in the seed and is not made in the plant as it grows [ 95 , 96 ]. One sprout contains all of the sulforophane that is present in a full-grown broccoli plant. So, if sulforophane is especially cancer-protective, it would seem reasonable to include some broccoli sprouts in an anti-cancer diet. Selenium Selenium is a mineral with anti-cancer properties. Many studies in the last several years have shown that selenium is a potent protective nutrient for some forms of cancer. The Arizona Cancer Center posted a selenium fact sheet listing the major functions of selenium in the body [ 97 ]. These functions are as follows: 1. Selenium is present in the active site of many enzymes, including thioredoxin reductase, which catalyze oxidation-reduction reactions. These reactions may encourage cancerous cells to under apoptosis. 2. Selenium is a component of the antioxidant enzyme glutathione peroxidase. 3. Selenium improved the immune systems' ability to respond to infections. 4. Selenium causes the formation of natural killer cells. 5. P450 enzymes in the liver may be induced by selenium, leading to detoxification of some carcinogenic molecules. 6. Selenium inhibits prostaglandins that cause inflammation. 7. Selenium enhances male fertility by increased sperm motility. 8. Selenium can decrease the rate of tumor growth. A serendipitous randomized, double-blind, controlled trial of a 200 μg/day selenium supplement in the southeastern region of the USA (where soil selenium levels are low) found that the primary endpoints of skin cancer were not improved by the selenium supplement, but that other cancer incidence rates were decreased by selenium [ 98 , 99 ]. There was a significant reduction in total cancer incidence (105 vs 137 cases, P = 0.03), prostate cancer (22 vs 42 cases, P = 0.005), a marginally significant reduction in colorectal cancer incidence (9 vs 19 cases, P = 0.057), and a reduction in cancer mortality, all cancer sites (40 vs 66 deaths, P = 0.008) (selenium versus control group cases reported, respectively) [ 98 ]. The selenium supplement was most effective in ex-smokers and for those who began the study with the lowest levels of serum selenium. Several prospective studies have also examined the role of selenium in cancer prevention, particularly for prostate cancer, summarized in Table 2 . Table 2 Prospective Nested Case Control Studies of Selenium and Prostate Cancer. Reference Study # Cases # Controls Outcomes Comment [189] Physicians Health Study 586 577 ↑Se = ↓risk of advance prostate cancer (OR = 0.52, 95% CI = 0.28–0.98) Result only in men with PSA ≥ 4 ng/mL [190] Netherlands Cohort Study 540 1,211 ↑Se = ↓risk prostate cancer (OR for quintiles of Se = 1.0, 1.05, 0.69, 0.75, 0.69; 95% CI = 0.48–0.99) Results greatest in ex-smokers [191] Baltimore Longitudinal Study of Aging 52 96 ↑Se = ↓risk prostate cancer (OR for quartiles of Se = 1.0, 0.15, 0.21, 0.24 [192] Washington County, Maryland 117 233 Top 4/5 of Se had reduction in prostate cancer risk; statistically significant result for Se only when γtocopherol levels were high Men in top quintile of serum γtocopherol had 5-fold reduced risk of prostate cancer compared to lowest quintile [193] Health Professional Follow-up Study 181 181 ↑Se = ↓risk of advanced prostate cancer Adjusted OR = 0.35 (95% CI = 0.16–0.78) [194] Prospective study ↑Se = ↓risk of gastrointestinal and prostate cancer Results not statistically significant Overall, it appears that poor selenium levels, especially for men, are a cancer risk. If a person has low selenium levels and other antioxidant defenses are also low the cancer risk is increased even further. Women do not appear to be as sensitive to selenium, as breast cancer has not been found to be influenced by selenium status in several studies [ 100 - 104 ], although both men and women were found to be protected by higher levels of selenium from colon cancer [ 100 ] and lung cancer [ 105 , 106 ]. Good vegetarian sources of selenium are whole grains and legumes grown in selenium-rich soil in the western United States, brazil nuts (by far the most dense source of selenium), nutritional yeast, brewers yeast, and sunflower seeds. Chlorophyll All green plants also contain chlorophyll, the light-collecting molecule. Chlorophyll and its derivatives are very effective at binding polycyclic aromatic hydrocarbons (carcinogens largely from incomplete combustion of fuels), heterocyclic amines (generated when grilling foods), aflatoxin (a toxin from molds in foods which causes liver cancer), and other hydrophobic molecules. The chlorophyll-carcinogen complex is much harder for the body to absorb, so most of it is swept out with the feces. The chemoprotective effect of chlorophyll and its derivatives has been tested in laboratory cell cultures and animals [ 107 , 108 ]. There is so much compelling evidence for anti-carcinogenic effects of chlorophyll that a prospective randomized controlled trial is being conducted in Qidong, China to see if chlorophyllin can reduce the amount of liver cancer cases, which arise from aflatoxin exposure in their foods (corn, peanuts, soy sauce, and fermented soy beans). A 55% reduction in aflatoxin-DNA adducts were found in the group that took 100 mg of chlorophyllin three times a day [ 109 ]. It was supposed that the chlorophyllin bound up aflatoxins, but there were chlorophyllin derivatives also detected in the sera (which had a green tint to it) of the volunteers who took the supplement, indicating a possible role in the body besides binding carcinogens in the gut [ 110 ]. Protective Vitamins Vitamin B-12 Vitamin B-12 has not been proven to be an anti-cancer agent, but there is some evidence indicating that it could be beneficial. The form of administered vitamin B-12 may be important. Some experimental cancer studies have been carried out with various forms of vitamin B-12. Methylcobalamin inhibited tumor growth of SC-3 injected into mice [ 111 ], and caused SC-3 mouse mammary tumor cells to undergo apoptosis, even when stimulated to grow by the presence of growth-inducing androgen [ 112 ]. Methylcobalamin, but not cyanocobalamin, increased the survival time of mice bearing implanted leukemia tumor cells [ 113 ]. 5'-deoxyadenosylcobalamin and methylcobalamin, but not cyanocobalamin, were shown to be effective cytotoxic agents [ 114 ]. Methylcobalamin also was able to increase survival time and reduce tumor growth in laboratory mice [ 115 ]. Laboratory mechanistic evidence for the effects of vitamin B12 were seen in a laboratory study with vitamin B-12 deficient rats. Choi et al [ 116 ] found that the colonic DNA of the B-12 deficient rats had a 35% decrease in genomic methylation and a 105% increase in uracil incorporation, both changes that could increase risk of carcinogenesis. In two prospective studies (one in Washington Country, Maryland and the Nurses' Health Study) a relation between lower vitamin B12 status (but not deficiency) and statistically significant higher risk of breast cancer was found [ 117 , 118 ]. So, there is evidence from laboratory studies, prospective cohort studies, and mechanistic studies showing that vitamin B-12 is an important nutrient for genetic stability, DNA repair, carcinogenesis, and cancer therapy. Folic Acid Folic acid is the dark green leafy vegetable vitamin. It has an integral role in DNA methylation and DNA synthesis. Folic acid works in conjunction with vitamin B-6 and vitamin B-12 in the single carbon methyl cycle. If insufficient folic acid is not available uracil is substituted for thymidine in DNA, which leads to DNA strand breakage. About 10% of the US population (and higher percentages among the poor) has low enough intakes of folic acid to make this a common problem [ 119 ]. As shown in Tables 3 and 4 , many studies have found a significant reduction in colon, rectal, and breast cancer with higher intakes of folic acid and their related nutrients (vitamin B-6 and B-12). Alcohol is an antagonist of folate, so that drinking alcoholic beverages greatly magnifies the cancer risk of a low-folate diet. Genetic polymorphisms (common single DNA base mutations resulting in a different amino acid encoded into a protein) in the methylenetetrahydrofolate reductase and the methionine synthase genes which increase the relative amount of folate available for DNA synthesis and repair also reduces the risk of colon cancer [ 120 - 123 ]. Cravo et al [ 124 ] used 5 mg of folic acid a day (a supraphysiological dose) in a prospective, controlled, cross-over study of 20 patients with colonic adenoma polyps. They found that the folic acid could reverse DNA hypomethylation in 7 of 12 patients who had only one polyp. Table 3 Folate and Colon / Rectal Cancer. Reference Study # Cases # Controls Outcomes Comment [195] Case / control USA 35 64 Folate supplementation = 62% lower incidence of neoplasia result not SS [196] Case / control NY state 800 Matched neighbor-hood controls ↑Folate = ↓rectal cancer, OR = 0.5 men, OR = 0.31, women Folate no effect for colon cancer SS [197] Case / control Majorca, Spain 286 498 Colon cancer related to total calories, cholesterol, animal protein, low fiber, low folic acid . [198] Case / Control Wash. state 424 414 ↑Alcohol = ↑cancer risk; ↑fiber = ↓risk; no relation to folate intake 2.5X risk for 30 g/day alcohol [199] Nurses' Health Study & Health Professionals Follow-up Study 564 women, 331 men ↑folate = ↓risk of colorectal adenoma: OR women = 0.66, OR men = 0.63 [200] Case / Control, Italy 1,326 2,024 hospital controls Protective trends for β-carotene, ascorbic acid, vit E, and folate (OR = 0.32, 0.40, 0.60, 0.52, respectively) Similar for colon and rectal cancer [201] US male health professional cohort 205 ↑Alcohol = ↑colon cancer (OR = 2.07 for ≥ 2 drinks/day; folate weakly protective; ↑Alcohol + ↓folate = ↑colon cancer risk (OR = 3.30) [202] α-tocopherol, β-carotene study cohort of smokers 144 276 ↑dietary folate = ↓colon cancer (OR = 1.0, 0.40, 0.34, 0.51, P-trend = 0.15); alcohol intake increased risk [203] Case control, population based Composite dietary profile (alcohol intake, methionine, folate, vit B 12 , B 6 ) trend of increasing risk for high risk group Marginal SS [204] Nurses' Health Study 442 ↑folate intake = ↓colon cancer (OR = 0.69); long-term use of multi-vitamins beneficial Folate intake includes multi-vitamins [205] NYU Women's Health Study 105 523 ↑folate = ↓colorectal cancer risk (OR = 0.52, P-trend = 0.04 Alcohol increased risk [206] NHANES I Epidemiologic Follow-up Study ↑folate = ↓colon cancer (OR men = 0.40, P-trend = 0.03; ↑alcohol, ↓folate = ↑colon cancer (OR men = 2.67 Results not stat. signif in women [207] Nurses' Health Study 535 ↑folate intake = ↓colon cancer in women with family history (OR = 0.48) Folate effect greater in women with family history [208] Canadian National Breast Screening Study 295 5,334 ↑folate = ↓colorectal cancer (OR = 0.6, P-trend = 0.25 Results not SS [209] Prospective cohort in The Netherlands 1,171 Rectal: OR, men 0.66, women no trend Trends SS only in men [210] Case / Control Italy 1,953 4,154 ↑folate = ↓colorectal cancer (OR = 0.72) Population drinks alcohol regularly [211] Iowa Women's health Study 721 ↑folate + (↑B 12 or ↑B 6 ) = ↓colon cancer (OR = 0.59, 0.65, respectively Nutrients not independent, alcohol increases risk [212] Case / Control NC state 613 996 ↑β-carotene, vit C, calcium = 40–60 % ↓risk colon cancer in whites; in African Americans ↑ vit C and E = 50–70% ↓risk colon cancer; no relation to folate to cancer risk Colon cancer rates higher in Aftrican Americans in NC; due to less UV light absorption with dark skin? [213] Wheat Bran Fiber trial, test for recurrence of adenoma polyps 1,014 men and women ↑homocysteine = ↑risk (OR = 0.69); ↑plasma folate = ↓risk (OR = 0.66) ↑folate or B 6 intake (diet + supplements) = ↓risk (OR = 0.61 SS; cut-off for highest quartile is 664 μg/day (way above RDA) SS = statistically significant Table 4 Prospective Studies of Folate and Breast Cancer. Reference Study # Cases # Controls Outcomes Comment [214] Nurses' Health Study 3,483 ↓folate intake + alcohol = ↑risk of breast cancer (OR = 0.55, P-trend = 0.001) Folate intake not associated with overall risk of breast cancer [215] Canadian National Breast Screening Study 1,336 5,382 ↓folate intake + alcohol = ↑risk of breast cancer (OR = 0.34, P-trend = 0.004) Folate intake not associated with overall risk of breast cancer [216] Prospective study in USA with postmenopausal women 1,586 Among drinkers, ↓folate intake = ↑breast cancer risk (OR = 1.59) No association in overall cohort [125] Shanghai Breast Cancer Study, China 1,321 1,382 ↑folate intake = ↓ risk (OR = 0.71, P-trend = 0.05); ↑folate, ↑methionine, ↑B 6 , ↑B 12 = ↓risk (OR = 0.47, P-trend = 0.01) No alcohol, no supplements, unprocessed, unfortified foods [217] Nurses' Health Study II, study of premenopausal women 714 Vitamin A protective (OR = 0.28); Vitamins C, E, and folate not associated with risk. [118] Nurses' Health Study 712 712 matched ↑plasma folate = ↓risk (OR = 0.73, P-trend = 0.06). For women who drank alcohol, ↑plasma folate even more protective, OR = 0.11. ↑plasma B 6 and plasma B 12 were also protective [218] Prospective study in USA with postmenopausal women 1,823, 308 with family history (FH) FH- +Alcohol = ↑risk (OR = 1.40) FH- + Alcohol + ↑folate = normal risk; FH+ ↓folate = ↑risk for drinkers (OR = 2.21) and non-drinkers (OR = 2.39); FH+ +Alcohol + ↑folate = ↑risk (OR = 1.67); FH+ + ↑folate = normal risk Women with family history of breast cancer can reduce risk by increasing folate intake and not drinking. FH = Family History Folate may be more important for rapidly dividing tissue, like the colonic mucosa. Therefore, the cancer risk associated with low folate intake is probably higher for colon cancer than for breast cancer. Most of the breast cancer studies only found a protective effect of folate among women who consumed alcohol (see Table 4 ). However, among women residents of Shanghai who consumed no alcohol, no vitamin supplements and ate unprocessed, unfortified foods there was a 29% decreased risk of breast cancer among those with the highest intake of folate [ 125 ]. So, there may be a true protective effect that is masked in the western populations by so many other risk factors. Two studies showed that the risk of cancer due to family history can be modified by high folate intake, so a prudent anti-cancer diet would be high in dark green leafy vegetables. The mean intake of folic acid on the Hallelujah Diet was 594 μg/day for men and 487 μg/day for women [ 88 ]. Vitamin D Vitamin D is produced primarily from the exposure of the skin to sunshine. Even casual exposure of the face, hands, and arms in the summer generates a large amount of vitamin D. In fact, simulated sunshine, equivalent to standing on a sunny beach until a slight pinkness of the skin was detected, was equivalent to a 20,000 IU oral dose of vitamin D 2 [ 126 ]. (Note that the RDA is 400 IU for most adults.) It has been estimated that 1,000 IU per day is the minimal amount needed to maintain adequate levels of vitamin D in the absence of sunshine [ 126 ], and that up to 4,000 IU per day can be safely used with additional benefit [ 127 ]. The concentration of the active hormonal form of vitamin D is tightly regulated in the blood by the kidneys. This active hormonal form of vitamin D has the potent anti-cancer properties. It has been discovered that various types of normal and cancerous tissues, including prostate cells [ 128 ], colon tissue [ 129 ], breast, ovarian and cervical tissue [ 130 ], pancreatic tissue [ 131 ] and a lung cancer cell line [ 132 ] all have the ability to convert the major circulating form of vitamin D, 25(OH)D, into the active hormonal form, 1,25(OH) 2 D. So, there is a local mechanism in many tissues of the body for converting the form of vitamin D in the body that is elevated by sunshine exposure into a hormone that has anticancer activity. Indeed, 25(OH)D has been shown to inhibit growth of colonic epithelial cells [ 133 ], primary prostatic epithelial cells [ 134 ], and pancreatic cells [ 131 ]. So, the laboratory work is confirming what had been seen some time ago in ecological studies of populations and sunshine exposure. The mortality rates for colon, breast, and ovary cancer in the USA show a marked north-south gradient [ 135 ]. In ecological studies of populations and sunlight exposure (no individual data) sunlight has been found to have a protective effect for prostate cancer [ 136 ], ovarian cancer [ 137 ], and breast cancer [ 138 ]. Recently Grant found that sunlight was also protective for bladder, endometrial, renal cancer, multiple myeloma, and Non-Hodgkins lymphoma in Europe [ 139 ] and bladder, esophageal, kidney, lung, pancreatic, rectal, stomach, and corpus uteri cancer in the USA [ 140 ]. Several prospective studies of vitamin D and cancer have also shown a protective effect of vitamin D (see Table 5 ). It could be that sunshine and vitamin D are protective factors for cancers of many organs that can convert 25(OH)D into 1,25(OH)D 2 . Table 5 Prospective Studies of Vitamin D and Cancer. Reference Study Vit D measure # Cases # Controls Outcomes Comment [219] 19-year cohort study of 1,954 men Diet history ↑vit D + calcium = ↓colorectal cancer (rates for lowest to highest intakes were 38.9, 24,5, 22,5 and 14.3/1000 population Significant effect even after adjustments for confounding factors; 2.7 fold reduction. [220] Washington county, Maryland cohort Serum 25(OH)D 34 67 matched ↑serum vit D = ↓colon cancer. Relative risk was 0.25 for 3 rd quintile and 0.20 for 4 th quintile. 4–5 fold reduction [221] Physicians' Health Study Serum 25(OH)D & 1,25(OH)D 2 232 414 No relation between vitamin D metabolite levels and prostate cancer [222] Nurses' Health Study Dietary and supplement intake Colon cancer RR = 0.42 (SS) for total vitamin D, comparing top and bottom quintiles Calcium not related to colon cancer risks; 2.4 fold reduction [223] Finnish clinical cohort Serum 25(OH)D & 1,25(OH)D 2 146 292 ↑serum 25(OH)D = ↓risk of rectal cancer, RR by quartile = 1.00, 0.93, 0.77, 0.37, P trend = 0.06. Serum 25(OH)D 12% lower in cases than in controls (12.2 vs 13.8 ng/l, P = 0.01; 2.7-fold reduction [224] NHANES I Follow-up Study Sunlight and diet 190 women Cohort matched Risk reductions for breast cancer for women in regions with high solar radiation (RR 0.35 – 0.75). [225] Helsinki Heart Study Serum 25(OH)D 149 596 ↑serum 25(OH)D = ↓prostate cancer. 1.7 fold greater risk for below median level compared to above median level. Young men (<52 years old) with low 25(OH)D had much higher risk of advanced prostate cancer (OR = 6.3) [226] Randomized controlled trial for colon adenoma recurrence Serum 25(OH)D & 1,25(OH)D 2 , and supplementary calcium 803 subjects total Above medium 25(OH)D and supplemental calcium reduced adenoma recurrence (RR = 0.71) Calcium and vitamin D appeared to work together to reduce colon cancer risk. [227] Norway, Finland, Sweden cohort of men Serum 25(OH)D 622 1,451 ≤ 19 nmol/l and ≥ 80 nmol/l of 25(OH)D at higher risk of prostate cancer. (40–60 nmol/l had lowest risk). Antioxidants α- and β-Carotene and other Carotenoids Carotenoids have been studied vigorously to see if these colorful compounds can decrease cancer risk. In ecological studies and early case-control studies it appeared that β-carotene was a cancer-protective agent. Randomized controlled trials of β-carotene found that the isolated nutrient was either neutral [ 141 ] or actually increased risk of lung cancer in smokers [ 142 , 143 ]. Beta-carotene may be a marker for intake of fruits and vegetables, but it does not have a powerful protective effect in isolated pharmacological doses. However, there is a large body of literature that indicates that dietary carotenoids are cancer preventative (See Table 6 ). Alpha-carotene has been found to be a stronger protective agent than its well-known isomer β-carotene. Studies tend to agree that overall intake of carotenoids is more protective than a high intake of a single carotenoid. So, a variety of fruits and vegetables is still a better anti-cancer strategy than just using a single vegetable high in a specific carotenoid. Table 6 Studies of Carotenoids and Lung Cancer. Reference Study # Cases # Controls Outcomes Comment [228] Hawaiian cohort 332 865 Dose-dependent inverse associations for dietary β-carotene, α-carotene, lutein; Subjects with highest intake of all 3 had the lowest risk Previous study showed variety of vegetables more protective than just foods rich in a particular carotenoid [229] Washington county, Maryland residents 258 515 ↑Serum/plasma levels of cryptoxanthin, β-carotene, lutein/zeaxanthin = ↓cancer (OR = 0.74, 0.83, 0.90, SS) [230] Case control, Spain 103 206, hospital No association for intake of α-carotene, β-carotene, or lutein. [231] Case control, Uruguay 541 540 ↑total carotenoids = ↓cancer (OR = 0.43, SS) Risk reduction for vit E and glutathione also seen. [232] Finland cohort 138 ↑α-carotene = ↓cancer (OR = 0.61, SS); β-carotene inversely related but not SS. 90% of α-carotene from carrots ↑Fruits and ↑root vegetables = ↓cancer (OR = 0.58, 0.56, respectively, SS) [233] Nurses' Health Study & Health Professionals Follow-Up Study 794 ↑α-carotene, lycopene, total carotenoids = ↓cancer (OR = 0.75, 0.80, 068 respectively, SS); Never smokers + ↑α-carotene = ↓cancer (OR = 0.37, SS) 4–8 year lag between diet assessment and date of diagnosis gave strongest correlations. [234] Shanghai men's cohort 209 622 ↑serum β-cryptoxanthin = ↓cancer (OR quartiles = 1, 0.72, 0.42, 0.45, P-trend = 0.02); Smokers with above median level of total carotenoids had a SS 37% reduction in cancer risk Study population had ~50% lower mean levels of serum carotenoids compared to US whites. [235] Canadian National Breast Screening Study 155 5,631 Non-significant inverse trend in risk for α-carotene and β-cryptoxanthin β-cryptoxanthin most from citrus, red peppers [236] Japan Collaborative Cohort Study 147 311 ↑α-carotene, β-carotene, canthaxanthin, total carotenoids = ↓risk (OR = 0.35, 0.21, 0.37, 0.27 respectively, SS); lycopene and β-cryptoxanthin reduce lung cancer risk, but not significantly [237] Singapore Chinese Health Study 482 ↑dietary β-cryptoxanthin = ↓cancer risk (OR = 0.73, 0.63 for smokers, SS) No significant associations of other carotenoids with lung cancer [238] Pooled analysis of 7 cohorts in USA and Europe 3,155 ↑ dietary β-cryptoxanthin = ↓lung cancer (OR = 0.76, SS) Other dietary carotenoids not significantly related to lung cancer. SS = statistically significant difference between comparison groups. The richest source of α-carotene is carrots and carrot juice, with pumpkins and winter squash as a second most-dense source. There is approximately one μg of α-carotene for every two μg of β-carotene in carrots. The most common sources of β-cryptoxanthin are citrus fruits and red sweet peppers. Lycopene Of the various carotenoids lycopene has been found to be very protective, particularly for prostate cancer. The major dietary source of lycopene is tomatoes, with the lycopene in cooked tomatoes being more bioavailable than that in raw tomatoes. Several prospective cohort studies have found associations between high intake of lycopene and reduced incidence of prostate cancer, though not all studies have produced consistent results [ 144 , 145 ]. Some studies suffer from a lack of good correlation between lycopene intake assessed by questionnaire and actual serum levels, and other studies measured intakes among a population that consumed very few tomato products. The studies with positive results will be reviewed here. In the Health Professionals Follow-up Study there was a 21% decrease in prostate cancer risk, comparing the highest quintile of lycopene intake with the lowest quintile. Combined intake of tomatoes, tomato sauce, tomato juice, and pizza (which accounted for 82% of the lycopene intake) were associated with a 35% lower risk of prostate cancer. Furthermore, lycopene was even more protective for advanced stages of prostate cancer, with a 53% decrease in risk [ 146 ]. A more recent follow-up report on this same cohort of men confirmed these original findings that lycopene or frequent tomato intake is associated with about a 30–40% decrease in risk of prostate cancer, especially advanced prostate cancer [ 147 ]. In addition to the two reports above a nested case control study from the Health Professional Follow-up Study with 450 cases and controls found an inverse relation between plasma lycopene and prostate cancer risk (OR 0.48) among older subjects (>65 years of age) without a family history of prostate cancer [ 148 ]. Among younger men high plasma β-carotene was associated with a statistically significant 64% decrease in prostate cancer risk. So, the results for lycopene have been found for dietary intakes as well as plasma levels. In a nested case-control study from the Physicians' Health Study cohort, a placebo-controlled study of aspirin and β-carotene, there was a 60% reduction in advanced prostate cancer risk (P-trend = 0.006) for those subjects in the placebo group with the highest plasma lycopene levels, compared to the lowest quintile. The β-carotene also had a protective effect, especially for those men with low lycopene levels [ 149 ]. In addition to these observational studies, two clinical trials have been conducted to supplement lycopene for a short period before radical prostatectomy. In one study 30 mg/day of lycopene were given to 15 men in the intervention group while the 11 men were in the control group were instructed to follow the National Cancer Institute's recommendations to consume at least 5 servings of fruits and vegetables daily. Results showed that the lycopene slowed the growth of prostate cancer. Prostate tissue lycopene concentration was 47% higher in the intervention group. Subjects that took the lycopene for 3 weeks had smaller tumors, less involvement of the surgical margins, and less diffuse involvement of the prostate by pre-cancerous high-grade prostatic intraepithelial neoplasia [ 150 ]. In another study before radical prostatectomy surgery 32 men were given a tomato sauce-based pasta dish every day, which supplied 30 mg of lycopene per day. After 3 weeks serum and prostate lycopene levels increaed 2-fold and 2.9-fold, respectively. PSA levels decreased 17%, as seen also by Kucuk et al [ 150 ]. Oxidative DNA damage was 21% lower in subjects' leukocytes and 28% lower in prostate tissue, compared to non-study controls. The apoptotic index was 3-fold higher in the resected prostate tissue, compared to biopsy tissue [ 151 ]. These intervention studies raise the question of what could have been done in this intervention was longer and combined synergistically with other effective intervention methods, such as flax seed, increased selenium and possibly vitamin E, in the context of a diet high in fruits and vegetable? Vitamin C Vitamin C, or ascorbic acid, has been studied in relation to health and is the most common supplement taken in the USA. Low blood levels of ascorbic acid are detrimental to health (for a recent article see Fletcher et al [ 152 ]) and vitamin C is correlated with overall good health and cancer prevention [ 153 ]. Use of vitamin C for cancer therapy was popularized by Linus Pauling. At high concentrations ascorbate is preferentially toxic to cancer cells. There is some evidence that large doses of vitamin C, either in multiple divided oral doses or intravenously, have beneficial effects in cancer therapy [ 154 - 156 ]. Oral doses, even in multiple divided doses, are not as effective as intravenous administration. Vitamin C at a dose of 1.25 g administered orally produced mean peak plasma concentrations of 135 ± 21 μmol/L compared with 885 ± 201 μmol/L for intravenous administration [ 154 ]. While vitamin C is quite possibly an effective substance, the amounts required for these therapeutic effects are obviously beyond dietary intakes. However, intravenous ascorbate may be a very beneficial adjuvant therapy for cancer with no negative side effects when administered properly. Other Antioxidants There are many more substances that will have some benefit for cancer therapy. Most of these substances are found in foods, but their effective doses for therapy are much higher than the normal concentration in the food. For example, grape seed extract contains proanthocyanidin, which shows anticarcinogenic properties (reviewed by Cos et al \ [ 157 ]. Also, green tea contains a flavanol, epigallocatechin-3-gallate (EGCG), which can inhibit metalloproteinases, among several possible other mechanisms [ 158 ]. And there are claims for various other herbal substances and extracts that might be of benefit, which are beyond the scope of this review. Probiotics The bacteria that reside in the intestinal tract generally have a symbiotic relationship with their host. Beneficial bacteria produce natural antibiotics to keep pathogenic bugs in check (preventing diarrhea and infections) and produce some B vitamins in the small intestine where they can be utilized. Beneficial bacteria help with food digestion by providing extra enzymes, such as lactase, in the small intestine. Beneficial bacteria help strengthen the immune system right in the gut where much of the interaction between the outside world and the body goes on. Beneficial bacteria can help prevent food allergies. They can help prevent cancer at various stages of development. These good bacteria can improve mineral absorption, maximizing food utilization. However, the balance of beneficial and potentially pathogenic bacteria in the gut is dependent on the diet. Vegetable fiber encourages the growth of beneficial bacteria. A group of Adventist vegetarians was found to have a higher amount of beneficial bacteria and lower amount of potentially pathogenic bacteria compared to non-vegetarians on a conventional American diet [ 159 ]. Differences in bacterial populations were seen between patients who recently had a colon polyp removed, Japanese-Hawaiians, North American Caucasians, native rural Japanese, and rural native Africans. Lactobacillus species and Eubacterium aerofaciens , both producers of lactic acid, were associated with the populations with the lower risk of colon cancer, while Bacteroides and Bifidobacterium species were associated with higher risk of colon cancer [ 160 ] There is a solid theoretical basis for why probiotics should help prevent cancer, especially colon cancer, and even reverse cancer. Probiotics produce short chain fatty acids in the colon, which acidify the environment. Lower colon pH is associated with lower incidence of colon cancer. Probiotic bacteria reduce the level of procarcinogenic enzymes such as beta-glucuronidase, nitroreductase, and azoreductase [ 161 ]. L. casei was used in two trials of patients with superficial bladder cancer. In the first trial, the probiotic group had a 50% disease free time of 350 days, compared to 195 days for the control group [ 162 ]. The second trial also showed that the probiotics worked better than the placebo, except for multiple recurring tumors [ 163 ]. Except for the two studies noted above, most of the research of probiotics and cancer has been done in animals. Studies have looked at markers of tumor growth or at animals with chemically induced tumors. Studies in rats have shown that probiotics can inhibit the formation of aberrant crypt foci, thought to be a pre-cancerous lesion in the colon. Some of the best results were obtained with a probiotic strain consumed with inulin, a type of fructooligosaccharide. Total aberrant crypt foci, chemically induced, were reduced 74% by the treatment of rats with inulin and B. longum , but only 29 and 21% by B. longum and inulin alone, respectively [ 164 ]. There was a synergistic effect in using both products together. Similar synergy was seen in rats with azoxymethane-induced colon cancer in another study. Rats fed Raftilose, a mixture of inulin and oligofructose, or Raftilose with Lactobacilli rhamnosus (LGG) and Bifidobacterium lactis (Bb12) had a significantly lower number of tumors compared to the control group [ 165 ]. A probiotic mixture, without any prebiotic, given to rats fed azoxymethane reduced colon tumors compared to the control (50% vs 90%), and also reduced the number of tumors per tumor-bearing rat [ 166 ]. In lab mice bred to be susceptible to colitis and colon cancer, a probiotic supplement, Lactobacillus salivarium ssp. Salivarius UCC118, reduced fecal coliform levels, the number of potentially pathogenic Clostridium perfringens , and reduced intestinal inflammation. In this small study two mice died of fulminant colitis and 5 mice developed adenocarcinoma in the control group of 10 mice, while there was no colitis and only 1 mouse with adenocarcinoma in the probiotic test group [ 167 ]. The research on probiotics and disease is still an emerging field. There is a high degree of variation of health benefits between different strains of bacteria. As new methods for selecting and screening probiotics become available, the field will be able to advance more rapidly. Oral Enzymes Many people diagnosed with cancer have digestion or intestinal tract disorders as well. Impaired digestion will greatly hinder a nutritional approach to treating cancer. If the nutrients cannot be released from the food and taken up by the body, then the excellent food provided by the Hallelujah Diet will go to waste. Digestive enzyme supplements are used to ensure proper and adequate digestion of food. Even raw foods, which contain many digestive enzymes to assist in their digestion, will be more thoroughly digested with less of the body's own resources with the use of digestive enzymes. So, the enzymes taken with meals do not have a direct effect upon a tumor, but assist the body in getting all of the nutrition out of the food for healing and restoring the body to normal function. Recently, an in vitro system was used to test the use of supplemental digestive enzymes. The digestive enzymes improved the digestibility and bioaccessibility of proteins and carbohydrates in the lumen of the small intestine, not only under impaired digestive conditions, but also in healthy human digestion [ 168 ]. There is evidence that indicates the presence of an enteropancreatic circulation of digestive enzymes [ 169 ]. Digestive enzymes appear to be preferentially absorbed into the bloodstream and then reaccumulated by the pancreas for use again. There appears to be a mechanism by which digestive enzymes can reach systemic circulation. Enzymes, especially proteases, if they reach systemic circulation, can have direct anti-tumor activity. Wald et al [ 170 ] reported on the anti-metastatic effect of enzyme supplements. Mice inoculated with the Lewis lung carcinoma were treated with a proteolytic enzyme supplement, given rectally (to avoid digestion). The primary tumor was cut out, so that the metastatic spread of the cancer could be measured. After surgical removal of the primary tumor (day 0), 90% of the control mice died by day 18 due to metastasized tumors. In the first group, which received the rectal enzyme supplement from the time of the tumor-removal surgery, 30% of the mice had died from metastasized cancer by day 25. In the second group, which received the enzymes from 6 days prior to removal of the primary tumor, only 10% of the animals showed the metastatic process by day 15. In the third group, which received the enzyme treatment since the initial inoculation of the Lewis lung carcinoma, no metastatic spread of the tumor was discernible. One hundred day-survival rates for the control, first, second, and third groups were 0, 60%, 90%, and 100%. In a similar experiment, an enzyme mixture of papain, trypsin, and chymotrypsin, as used in the preparation Wobe-Mugos E, was rectally given to mice that were inoculated with melanoma cells. Survival time was prolonged in the test group (38 days in the enzyme group compared to 24 days in the control mice) and 3 of the 10 enzyme-supplemented mice were cured. Again, a strong anti-metastatic effect of the proteolytic enzymes was seen [ 171 ]. Further evidence of the efficacy of oral enzyme supplementation is available from clinical trials in Europe. Two different studies have demonstrated that two different oral proteolytic enzyme supplements were able to reduce high levels of transforming growth factor-β, which may be a factor in some cancers [ 172 , 173 ]. In the Slovak Republic an oral enzyme supplement was tested in a placebo-controlled trial of multiple myeloma. For stage III multiple myeloma, control group survival was 47 months, compared to 83 months (a 3 year gain) for patients who took the oral enzymes for more than 6 months [ 174 ]. Enzyme supplements have also been shown to reduce side effects of cancer therapy. Enzyme supplementation resulted in fewer side effects for women undergoing radiation therapy for carcinomas of the uterine cervix [ 175 ], for patients undergoing radiation therapy for head and neck cancers [ 176 ], and for colorectal cancer patients undergoing conventional cancer treatments [ 177 ]. In a large multi-site study in Germany women undergoing conventional cancer therapy were put into a control group or a group that received an oral enzyme supplement. Disease and therapy related symptoms were all reduced, except tumor pain, by the enzyme supplement. Also, survival was longer with less recurrence and less metastases in the enzyme group [ 178 ]. In all of these studies the oral enzyme supplements were well tolerated, with only a small amount of mild to moderate gastrointestinal symptoms. Even though these few studies don't give a lot of evidence of the effectiveness of oral enzyme supplementation, it is clear that there are some circumstances that will be helped by enzyme supplementation, with very little danger of negative side effects. At the least, enzymes will improve digestion and lessen the digestive burden on the body, leaving more reserves for disease eradication. However, as the research indicates, the effect may be much greater than that, with the potential for direct anti-tumor activity. Whole Diet Studies A diet-based cancer therapy, the Gerson Therapy, was used to treat melanoma cancer. The five-year survival rates from their therapy compared very favorably to conventional therapy reported in the medical literature, especially for more advanced stages of melanoma [ 179 ] (see Table 7 ). Table 7 Gerson Therapy for Melanoma [179]. Stage of melanoma Gerson Historical controls I – II 100% (N = 14) 79% (N = 15,798) IIIA 82% (N = 17) 39% (N = 103) IIIA + IIIB 70% (N = 33) 41% (N = 130) IVA 39% (N = 18) 6% (N = 194) An Italian cohort of 8,984 women was followed for an average of 9.5 years, with 207 incident cases of breast cancer during that time. Their diets were analyzed by patterns – salad vegetables (raw vegetables and olive oil), western (potatoes, red meat, eggs and butter), canteen (pasta and tomato sauce), and prudent (cooked vegetables, pulses, and fish). Only the salad vegetable diet pattern was associated with a significantly lower risk of breast cancer, about 35% lower. For women of normal weight (BMI <25) the salad vegetable pattern was even more protective, about a 61% decreased risk of breast cancer [ 180 ]. The overall dietary pattern does make a very significant difference. In US-based studies the "prudent" diet has been shown to be protective for colon cancer, while the "western" diet has been shown to be detrimental. The "western" dietary pattern, with its higher intakes of red meat and processed meats, sweets and desserts, French fries, and refined grains, was associated with a 46% increase relative risk of colon cancer in the Nurses' Health Study [ 45 ]. Slattery et al [ 17 ] found a two-fold increase in relative risk of colon cancer associated with a "western" dietary pattern, and a 35–40% decrease in relative risk associated with the "prudent" pattern, especially among those diagnosed at an earlier age (<67 years old). The "salad vegetable" pattern is still more likely to be protective compared to the prudent dietary pattern, but this pattern did not exist in this study population. In an analysis of the colon cancer data from the Health Professionals Follow-up Study, Platz et al [ 56 ] found that there was a 71% decrease in colon cancer risk when men with none of six established risk factors were compared to men with at least one of these risk factors (obesity, physical inactivity, alcohol consumption, early adulthood cigarette smoking, red meat consumption, and low intake of folic acid from supplements). So, if all men had the same health profile as these healthier 3% of the study population, colon cancer rates would have been only 29% of what they measured. A plant-based dietary pattern in being currently tested in the Women's healthy Eating and Living (WHEL) Study. About 3,000 women who were treated for an early stage of breast cancer have been randomized into two groups. The dietary goals for the test group of the study are 5 servings of vegetables, 16 oz of vegetable juice, 3 servings of fruit, 30 g of fiber, and <20% of energy from fat. No guidelines were given for animal product intake, and initial results seem to confirm, since there were no changes in body weight, total cholesterol, or LDL cholesterol [ 181 ], which would be affected by animal protein intake. However, over the first year of follow-up vegetable intake did increase to seven servings/day, fruit intake increased to 3.9 servings/day, energy from fat decreased from 28% to 23%. Also, plasma carotenoid concentrations increased significantly in the intervention group, but not in the control group. α-Carotene increased 223%, β-carotene increased 87%, lutein increase 29%, and lycopene increased 17% [ 182 ], indicating that a substantial dietary change had been made by these women. It will be very interesting to follow the results of this study. Conclusions What is the result when all of these things are put together? What if all of these factors reviewed here were taken into account and put into practice? This anticancer diet would have: • adequate, but not excessive calories, • 10 or more servings of vegetables a day, including cruciferous and allium vegetables; vegetable juice could meet part of this goal, • 4 or more servings of fruits a day, • high in fiber, • no refined sugar, • no refined flour, • low in total fat, but containing necessary essential fatty acids, • no red meat, • a balanced ratio of omega 3 and omega 6 fats and would include DHA, • flax seed as a source of phytoestrogens, • supplemented with ~200 μg/day selenium, • supplemented with 1,000 μg/day methylcobalamin (B-12), • very rich in folic acid (from dark green vegetables), • adequate sunshine to get vitamin D, or use 1,000 IU/day supplement, • very rich in antioxidants and phytochemicals from fruits and vegetables, including α-carotene, β-carotene, β-cryptoxanthin, vitamin C (from foods), vitamin E (from foods), • very rich in chlorophyll, • supplemented with beneficial probiotics, • supplemented with oral enzymes As reviewed above, reductions of 60 percent in breast cancer rates have already been seen in human diet studies, and a 71 percent reduction in colon cancer for men without the known modifiable risk factors. These reductions are without taking into account many of the other factors considered in this review, such as markedly increased fruit and vegetable intake, balanced omega 3 and 6 fats, vitamin D, reduced sugar intake, probiotics, and enzymes – factors which all are likely to have an impact on cancer. Certainly cancer prevention would be possible, and cancer reversal in some cases is quite likely. Competing Interests Michael Donaldson is a research scientist at the Hallelujah Acres Foundation, a foundation for investigations pertaining to the Hallelujah Diet. Funding for this review was provided by the Hallelujah Acres Foundation.

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517833

Keeping Proteins on Target

To keep a cell healthy, proteins must go to the right places within a cell. To direct proteins to specific areas of the cell, they're marked with tags akin to zip codes on mail. These tags can direct one protein to the cell's nucleus, where it regulates gene expression, say, while another is sent to the hinterlands of the cell membrane, where it receives environmental signals. Protein targeting is crucial even as cells are building proteins—otherwise, for example, proteins won't fold into the proper shape. There's a pair of proteins that, across all organisms, plays a key role in protein targeting. Called Ffh and FtsY in bacteria, these proteins each have an active and inactive state; only when they're in their active state can they bind each other and deliver other proteins to their proper location. Both these proteins are GTPases, a class of proteins that work as molecular switches. However, among GTPases, Ffh and FtsY are unique. Other GTPases switch between active and inactive states by binding different forms of a small, energy-carrying molecule—either GTP or GDP. Such GTPases often require the help of other proteins to switch states. Ffh and FtsY, on the other hand, almost always have GTP bound. And when they interact, they change each other's state—allowing each other to convert GTP to GDP—without the help of other proteins. But researchers didn't know exactly how these proteins interacted or how they switched between their active and inactive forms. Now, as reported in this issue of PLoS Biology, Shu-ou Shan and colleagues have found that when Ffh and FtsY bind and then activate each other, they likely go through an unusual, multi-step process in which the proteins change shapes, flexing so that different parts of the proteins become active at each step. The researchers mutated 45 different sites in the gene that encodes the FtsY protein to produce a bevy of mutant proteins with different properties. The researchers chose mutations that produced amino acid substitutions at sites in the FtsY protein that have been preserved through evolution, and so are presumably crucial for the protein's function. These sites, it turns out, are all on the protein's surface where it interacts with Ffh. The mutant versions of FtsY varied in how well they bound to the normal version of Ffh and how quickly the two proteins activated each other. The researchers were able to sort the different mutations into four different classes based on the type of problem the proteins had: some bound Ffh very loosely, some bound Ffh well but only weakly turned on its GTPase activity, and so on. All of these mutations would presumably foul up the protein targeting system, so this explains why certain amino acids have been preserved through evolution. Both Ffh and FtsY change shapes as they bind, activate each other's GTPase activity, then cleave GTP and release each other, the researchers infer. They don't have direct evidence for these shape changes, but the postulated bends and twists during interaction are consistent with the build of the proteins. These shape changes, they speculate, could switch different parts of each protein between active and inactive states. By showing how this unique type of GTPase switch likely works, Shan and colleagues have helped explain how cells target proteins to specific areas—and perhaps have paved the way for others to find similar switches elsewhere within cells.

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387276

Neural Basis of Solving Problems with Insight

If you're one of those insufferable people who can finish the Saturday New York Times crossword puzzle, you probably have a gift for insight. The puzzles always have an underlying hint to solving them, but on Saturdays that clue is insanely obtuse. If you had all day, you could try a zillion different combinations and eventually figure it out. But with insight, you'd experience the usual clueless confusion, until—voilà—the fog clears and you get the clue, which suddenly seems obvious. The sudden flash of insight that precedes such “Aha!” moments is characteristic of many types of cognitive processes besides problem-solving, including memory retrieval, language comprehension, and various forms of creativity. Although different problem-solving strategies share many common attributes, insight-derived solutions appear to be unique in several ways. In this issue, researchers from Northwestern and Drexel Universities report on studies revealing a unique neural signature of such insight solutions. Insight lights up the brain Mark Jung-Beeman, John Kounios, Ed Bowden, and their colleagues recount the storied origin of the term Eureka! , which Archimedes reportedly shouted upon realizing that water displacement could be used to compute density. Illustrating the strong emotional response elicited by such a sudden insight, Archimedes is said to have run home from the baths in euphoric glee—without his clothes. Among other characteristics that typically distinguish insight from “noninsight” solutions, people feel stuck before insight strikes; they can't explain how they solved the problem and might say they were not even thinking about it; the solution appears suddenly and is immediately seen as correct. But are the neural processes involved in arriving at a solution through insight actually distinct from those related to more mundane problem-solving? Recent findings suggest that people think about solutions, at an unconscious level, prior to solving insight problems, and that the right cerebral hemisphere (RH) appears to be preferentially involved. Jung-Beeman et al. predicted that a particular region of the RH, called the anterior superior temporal gyrus (aSTG), is likely involved in insight because it seems critical for tasks that require recognizing broad associative semantic relationships—exactly the type of process that could facilitate reinterpretation of problems and lead to insight. To test this hypothesis, Jung-Beeman et al. mapped both the location and electrical signature of neural activity using functional magnetic resonance imaging (fMRI) and the electroencephalogram (EEG). In the first experiment, thirteen people were given three words ( pine , crab , sauce ) and asked to think of one word that would form a compound word or phrase for each of the words (can you figure it out?). Neural activity was mapped with fMRI while the participants were given 124 similar word problems—which can be solved quickly with or without insight, and evoke a distinct Aha! moment about half the time they're solved. Subjects pressed a button to indicate whether they had solved the problem using insight, which they had been told leads to an Aha! experience characterized by suddenness and obviousness. While several cortical regions showed about the same heightened activity for both insight and noninsight-derived solutions, only the aSTG in the RH showed a robust insight effect. Given that neural activity in this area also increased when subjects first encountered the problem (perhaps reflecting unconscious processing), the authors conclude that the increase does not simply reflect the emotional jolt associated with insight. In a second experiment, 19 new participants engaged in the same type of problem-solving tasks as the first group while their brain waves were measured with an EEG. The researchers then analyzed the EEG recordings to look for differences between insight and noninsight solutions in brain wave activity. The researchers found that 0.3 seconds before the subjects indicated solutions achieved through insight, there was a burst of neural activity of one particular type: high-frequency (gamma band) activity that is often thought to reflect complex cognitive processing. This activity was also mapped to the aSTG of the RH, providing compelling convergence across experiments and methods. Problem-solving involves a complex cortical network to encode, retrieve, and evaluate information, but these results show that solving verbal problems with insight requires at least one additional component. Further, the fact that the effect occurred in RH aSTG suggests what that process may be: integration of distantly related information. Distinct neural processes, the authors conclude, underlie the sudden flash of insight that allows people to “see connections that previously eluded them.”

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514889

Verifying Sequences that Enhance Splicing

Identifying the causative mutation for a disease can be the first step to a potential cure. This task is not always trivial. Often the initial strategy is to look for the variations within a mutated gene that alter its protein coding sequence, as these mutations often alter the gene's function. However, in a growing number of cases, the causative mutation is a “synonymous” mutation—a change in the coding sequence of a gene that doesn't change the sequence of the protein coded by the gene. This type of mutation may be responsible for Seckel syndrome, a human disease characterized by dwarfism. In Seckel syndrome, the mutation doesn't alter the protein sequence itself but instead results in the skipping over of a portion of the protein coding sequence (an exon), a process called altered splicing. The disease-causing potential of this type of splicing mutation has only recently gathered attention. Splicing assembles the exons of a transcribed gene (the RNA copy) into the right order while removing the non-coding sequences of the RNA (introns). This highly regulated process is coordinated by a number of sequences within a gene, including splice sites that precede and follow the exon, as well as by exonic splicing enhancers (ESEs), which help recruit the factors (proteins) necessary to insure proper splicing. Although splice sites have optimal (consensus) sequences, there is some variability amongst individual splice site sequences that allows splicing to take place to a greater or lesser extent. ESEs facilitate splicing, especially when a gene's splice sites vary from the consensus sequence. Candidate ESEs have previously been identified based on their more frequent occurrence in exons that are adjacent to non-consensus splice sites. In this issue of PLoS Biology , William Fairbrother et al. investigated the functionality of these putative ESEs. If they are functional, the authors reasoned, then mutations that disrupt them would be selected against—that is, these mutations would tend to be discarded—in the human genome. To this end Fairbrother et al. developed a computational method, which they call VERIFY (for “variant elimination reinforces functionality”), to evaluate the selective pressure on ESEs. They took advantage of a public database of all single nucleotide polymorphisms (DNA changes at a single point) within the human genome and compared them to the chimpanzee genome; this allowed the authors to infer the identity of the ancestral gene (or allele). By determining which allele is ancestral and which is the variant, the researchers could then distinguish the mutations that created ESEs from mutations that disrupted ESEs. Mutations that altered or disrupted ESEs were under-represented, leading the authors to conclude that predicted ESE sequences evolve under a more stringent level of selection than exonic sequences with no predicted ESEs. This selective pressure was greater for predicted ESEs located near the splice signals than for ESEs that were located within the exon. This result was consistent with experimental findings that ESE strength diminishes with distance from the splice site. As more vertebrate genomes are sequenced and the public database of single nucleotide polymorphisms continues to grow, this type of computational method will become increasingly valuable. It can help confirm the functionality or role of candidate regulatory elements thought to control various aspects of gene expression, and in so doing, offer insights into the complex machinations required to maintain the healthy operation of the human genome.

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549556

Treatment outcome of new culture positive pulmonary tuberculosis in Norway

Background The key elements in tuberculosis (TB) control are to cure the individual patient, interrupt transmission of TB to others and prevent the tubercle bacilli from becoming drug resistant. Incomplete treatment may result in excretion of bacteria that may also acquire drug resistance and cause increased morbidity and mortality. Treatment outcome results serves as a tool to control the quality of TB treatment provided by the health care system. The aims of this study were to evaluate the treatment outcome for new cases of culture positive pulmonary TB registered in Norway during the period 1996–2002 and to identify factors associated with non-successful treatment. Methods This was a register-based cohort study. Treatment outcome was assessed according to sex, birthplace, age group, isoniazid (INH) susceptibility, mode of detection and treatment periods (1996–1997, 1998–1999 and 2000–2002). Logistic regression was also used to estimate the odds ratio for treatment success vs. non-success with 95% confidence interval (CI), taking the above variables into account. Results Among the 655 patients included, the total treatment success rate was 83% (95% CI 80%–86%). The success rates for those born in Norway and abroad were 79% (95% CI 74%–84%) and 86% (95% CI 83%–89%) respectively. There was no difference in success rates by sex and treatment periods. Twenty-two patients (3%) defaulted treatment, 58 (9%) died and 26 (4%) transferred out. The default rate was higher among foreign-born and male patients, whereas almost all who died were born in Norway. The majority of the transferred out group left the country, but seven were expelled from the country. In the multivariate analysis, only high age and initial INH resistance remained as significant risk factors for non-successful treatment. Conclusion Although the TB treatment success rate in Norway has increased compared to previous studies and although it has reached a reasonable target for treatment outcome in low-incidence countries, the total success rate for 1996–2002 was still slightly below the WHO target of success rate of 85%. Early diagnosis of TB in elderly patients to reduce the death rate, abstaining from expulsion of patients on treatment and further measures to prevent default could improve the success rate further.

Background The key elements in tuberculosis (TB) control are to detect the disease as early as possible and to ensure that those diagnosed complete their treatment and get cured. The World Health Organization (WHO) target for treatment success is 85 percent of all detected smear-positive cases [ 1 ]. Even where free medication is available, many patients are not successfully treated [ 2 , 3 ]. Main reasons for non-success are death (while on treatment or before start of treatment) and loss to follow-up. Incomplete treatment may result in prolonged excretion of bacteria that may also acquire drug resistance, cause transmission of disease and lead to increased morbidity and mortality [ 4 ]. Norway has about 4.5 million inhabitants, with foreign-born residents comprising 6.9% of the total population in 2002. The proportion of TB cases from foreign-born residents has increased from 19 % in 1986 to 76 % in 2002 [ 5 ]. However, the total number of cases has remained relatively stable. The reporting of treatment outcome for all TB cases has been obligatory since 1996. A study on treatment outcome for culture positive pulmonary TB in Norway in 1995 showed a high death rate and a high rate of loss to follow-up. Only 76% of patients who were included in that study completed treatment [ 6 ]. While the incidence rate of TB is low in Norway compared to most countries in the world, challenges still remain in achieving the WHO target for treatment success. However, there are some problems with the WHO definition of success rate and other measures have been proposed. A working group from the WHO, the International Union Against Tuberculosis and Lung Disease (IUATLD) and the Royal Netherlands Tuberculosis Association (KNCV) have defined a reasonable target for treatment outcome in low-incidence countries as to reduce the proportion of patients with a potentially bacteriologically unsuccessful outcome (failure, default, transfer) to less than 10% [ 7 ]. The aims of this study were to evaluate the treatment outcome for new culture positive pulmonary TB cases registered in Norway during the period 1996–2002, and to identify factors associated with non-successful treatment. Methods Setting and study population This was a register-based cohort study. In Norway, there is compulsory nominative notification of all TB cases directly to the National TB Registry. Both suspected and confirmed cases have to be reported by clinicians. Laboratories of clinical microbiology are also required to report all isolates of Mycobacteria . A total of 25 microbiological laboratories isolate M. tuberculosis from patient samples. The laboratory at the Norwegian Institute of Public Health functions as a national reference laboratory for TB. The Pharmacy at the National Hospital is the only pharmacy distributing drugs for TB treatment, and TB prescriptions are compared with the National TB Registry in order to identify cases that may not have been notified. The notification is therefore considered to be quite complete [ 8 ]. There is also compulsory notification of all treatment outcomes to the Registry. Nine months after treatment start, the Register sends a special form to the clinician in charge to be filled out with details of the treatment outcome. In this study, we included all new cases with culture positive pulmonary TB notified during 1996–2002. The sputum smear results were not recorded or reported for all cases, especially in the first years of the study period. We therefore based our analysis of treatment outcome on new cases with culture positive pulmonary TB. The mode of detection was determined by information on the notification form. Data on susceptibility to TB drugs was obtained from the notification forms and laboratory reports. In addition to the information in the TB Registry, we used data from the Cause of Death Registry at Statistics Norway to determine the cause of death for those who died under treatment or before start of treatment. Recommended regime for TB treatment in the study period consisted of isoniazid (INH), rifampicin and pyrazinamide in the intensive phase (two months). Ethambutol was added when resistance was suspected such as in foreign-born patients and in previously treated patients. The continuation phase consisted of four months with INH and rifampicin [ 9 ]. Directly Observed Therapy (DOT) was used before 2003 on an individual basis. However, we have no data to show how many patients in this study received DOT. Definition The treatment outcome was divided into six categories according to WHO guidelines, with some modifications [ 10 ]. These categories were: cured (finished treatment with negative bacteriology result at the end of treatment), completed treatment (finished treatment, but without bacteriology result at the end of treatment), failure (remaining smear/ culture positive at five months despite correct intake of medication), defaulted treatment (patients who interrupted their treatment for two consecutive months or more after registration), died (patients who died due to TB or other cause before or during treatment), transferred out (patients whose treatment results are unknown due to emigration before or during treatment). Patients who changed treatment due to multi-drug resistant TB (MDR-TB), i.e. resistance to both INH and rifampicin, were also defined as failures [ 11 ]. Treatment success was defined as the sum of the cases that were cured and that completed treatment. The proportion of patients with a potentially bacteriologically unsuccessful outcome (failure, default, transfer) was also calculated [ 7 ]. Statistical analysis We used the statistical package SPSS, version 11.0 for data analysis. To estimate the odds ratio for treatment outcome (success vs. non-success), logistic regression analysis was used. Confidence interval (CI) for the odds ratio has also been given. Variables such as sex, birthplace, age group, INH susceptibility, mode of detection and treatment periods (1996–1997, 1998–1999 and 2000–2002) were entered into both univariate and multivariate logistic regression model. P values of less than 0.05 were considered statistically significant. Results Six hundred and fifty-five new culture positive pulmonary TB patients were included in the study (table 1 ). Of these, 397 patients (61%) were foreign-born. Three hundred and twenty three patients (49%) were cured and 221 patients (34%) completed treatment. This gives a treatment success rate of 83% (95% CI 80%–86%). The treatment success rates for women and men were 86% (95% CI 82%–90%) and 81% (95% CI 77%–85%) respectively. For those born in Norway and abroad, the rates were 79% (95% CI 74%–84%) and 86% (95% CI 83%–89%) respectively. The rates for the treatment periods 1996–1997, 1998–1999 and 2000–2002 were 83% (95% CI 77%–89%), 82% (95% CI 76%–88%) and 84% (95% CI 80%–88%) respectively. Table 1 Number of new culture positive pulmonary tuberculosis patients by patient characteristics and treatment outcome, Norway, 1996–2002 Cured Completed treatment Failure* Defaulted treatment Died Transferred Out † Total Sex Women 144 90 2 7 17 11 271 Men 179 131 3 15 41 15 384 Birthplace Norway 119 84 1 5 49 0 258 Abroad 204 137 4 17 9 26 397 Age group (yrs) 0–14 6 19 0 1 0 0 26 15–39 170 105 3 13 4 14 309 40–64 59 45 2 4 11 10 131 65+ 88 52 0 4 43 2 189 INH-resistance ‡ No 295 210 0 15 53 22 595 Yes 21 10 5 7 2 4 49 Mode of detection # (due to symptoms) Yes 221 154 3 16 47 12 453 No 100 67 2 6 11 13 199 Treatment periods 1996–1997 67 48 0 3 19 2 139 1998–1999 83 58 1 7 20 3 172 2000–2002 173 115 4 12 19 21 344 *All patients are MDR-TB patients † All patients have left the country ‡ Information for INH susceptibility was available for 98.3% of the patients # Information for mode of detection was available for 99.5% of the patients In our study, the proportion of patients with a potentially bacteriologically unsuccessful outcome (failure, default, transfer) was 8%. The average duration of residence in Norway for foreign-born patients at TB registration was 3.8 (range 1–31) years. There were no differences in the treatment success rates for those who had lived in Norway for less than three years and for those who had stayed longer than three years. Among the 22 patients who defaulted treatment (3%) (table 1 ), four had MDR-TB and three additional patients had isolated INH resistant strains at the start of the treatment. The default rate was higher among foreign-born and male patients. There were 26 patients (4%) who were transferred out (table 1 ). Of these, 21 were reported in 2000–2002. All of them have left the country. Twenty-two were on treatment when they left the country and four had not started treatment. Among the 26, seven were expelled from the country. Six of these were on treatment and one had not started treatment at the time of expulsion. For those who left the country while on treatment, the average duration of treatment was 78 days. Twelve of them had treatment only for two months or less. Of the 26 patients who were transferred out, three had isolated INH resistant strains and one had MDR-TB when they left the country. Altogether 58 patients (9%) died, 19 (3%) before treatment start and 39 (6%) while on treatment. Of those who died before treatment start, four were diagnosed after death at autopsy. Eighty four percent of those who died were born in Norway. The median age for all who died was 80 years. For those who died before treatment start, the median age was 71 years. For those who died while on treatment, the median age was 81 years and the average duration of treatment was 67 days. Sixteen patients died within 14 days of treatment start. For 23 patients, TB was the primary cause of death. It was the only cause of death for nine of those 23 patients. For further 20 patients, TB was a contributing factor to their death. Fourteen patients died from other diseases than TB. The cause of death was unknown for one patient (table 2 ). Table 2 Primary causes of death among new culture positive pulmonary tuberculosis patients, Norway, 1996–2002* Tuberculosis mentioned on the death certificate Yes (N = 43) No (N = 14)) Tuberculosis (N = 23) † Heart failure (N = 1) Acute myocardial infarction (N = 3) Unspecified HIV disease (N = 1) Heart failure (N = 2) Unspecified non-Hodgkin's lymphoma (N = 1) Unspecified cardiac arrest (N = 1) Amyloidosis (N = 1) Stroke, not specified as haemorrhage or infarction (N = 2) Multiple myeloma (N = 1) Chronic ishaemic heart disease (N = 2) Mental and behavioural disorders due to use of opoids (N = 1) Paroxysmal tachycardia (N = 1) Mental and behavioural disorders due to multiple drug use and use of other psychoactive substances (N = 1) Other chronic obstructive pulmonary disease (N = 1) Malignant neoplasm of bronchus and lung (N = 5) Unspecified respiratory failure (N = 1) Liver cell carcinoma (N = 1) Unspecified HIV disease (N = 1) Other ill-defined and specified causes of mortality (N = 1) Other acute viral hepatitis (N = 1) Unspecified sepsis (N = 1) Unspecified non-Hodgkin's lymphoma (N = 1) Spinal muscular atrophy and related syndromes (N = 1) Malignant neoplasm of breast (N = 1) Malignant neoplasm of bronchus and lung (N = 1) *The cause of death was unknown for one patient † Including respiratory and miliary tuberculosis There was no systematic registration of TB/ human immunodeficiency virus (HIV) co-infection at the TB Registry. For those who died while on treatment or before treatment started, HIV disease was registered as the cause of death for two patients (table 2 ). Table 3 gives the odds ratio from logistic regression. The effect of birthplace in the treatment success changed from the univariate to the multivariate analysis. Patients who were born abroad had higher odds of success in the univariate analysis, but this changed to lower odds for success in the multivariate analysis. This is due to a strong confounding effect of age. The effect of age was distinct and graded, slightly stronger in the multivariate analysis. Both in the univariate and multivariate analysis, the odds for success were lower for those with INH resistant strains than for those with INH susceptible strains. Table 3 Odds ratio (OR) for treatment success vs. non-success among new culture positive pulmonary tuberculosis patients notified in Norway, 1996–2002 Treatment Success Univariate Multivariate* Yes No OR 95% CI OR 95% CI Sex Women 234 37 ref. ref. ref. ref. Men 310 74 0.7 04–1.0 0.7 0.4–1.1 Birthplace Norway 203 55 ref. ref. ref. ref. Abroad 341 56 1.7 1.1–2.5 0.7 0.3–1.3 Age group (yrs) 0–14 25 1 8.7 1.2–65.6 11.8 1.5–92.6 15–39 275 34 2.8 1.7–4.6 4.8 2.3–10.0 40–64 104 27 1.3 0.8–2.3 2.0 1.1–3.9 65+ 140 49 ref. ref. ref. ref. INH-resistance No 505 90 ref. ref. ref. ref. Yes 31 18 0.3 0.2–0.6 0.2 0.1–0.4 Mode of detection (due to symptoms) Yes 375 78 ref. ref. ref. ref. No 167 32 1.1 0.7–1.7 1.1 0.7–1.7 Treatment periods 1996–1997 115 24 0.9 0.6–1.6 1.2 0.7–2.2 1998–1999 141 31 0.9 0.5–1.4 0.9 0.5–1.5 2000–2002 288 56 ref. ref. ref. ref. *In the multivariate analysis, all variables in the univariate analysis were considered Susceptibility testing for the main TB drugs was done in most of the patients. However, susceptibility testing for streptomycin was done for fewer patients than other main TB drugs. Resistance to INH and streptomycin was most common, and resistance was frequent among foreign-born patients. Of those with MDR-TB, two were born in Norway and eight abroad. Of the 655 patients included in this study, 453 were detected through passive case finding (due to their symptoms). Another 101 patients were discovered through the immigration TB screening program. A total of 52 patients were detected through following ups of close contacts of identified infectious cases (22 cases) and of previous abnormal mass miniature radiology (MMR) (32 cases). The remainder was discovered through other screening programs. No information on mode of detection was available for three patients. Discussion In our study, the total treatment success rate for new culture positive pulmonary TB for the period 1996–2002 was 83%. This is close to the WHO target of success rate of 85% of all smear positive cases. However, subgroups of patients contributing to low success rate warrant special attention such as those who defaulted treatment, those who were transferred out and those who died. The first two subgroups mainly comprise patients who were born abroad and the last subgroup mainly comprises patients who were born in Norway. Despite these problems, Norway has reached the reasonable target for treatment outcome in low-incidence countries [ 7 ]. Our study shows a default rate of 3%. Higher default rates have been described in other studies such as Vaud County, Switzerland (16%) [ 2 ], Hamburg, Germany (10%) [ 12 ], and Sweden (7%) [ 3 ]. Although the default rate in Norway is lower than in these countries, some of the patients who defaulted treatment, including patients with MDR-TB, have been the cause of small on-going outbreaks [ 13 ]. Default can constitute a major public health problem. Although incomplete treatment can prevent patients from dying from TB, the patients may remain infectious and even develop MDR-TB. It is therefore worrying that several patients in our study who defaulted treatment had isolated INH resistant strains or MDR-TB prior to treatment. Language problems, lack of understanding of the patients' cultural background, lack of communication between primary health care and hospitals, frequent change of address and stigma related to TB might be some of the reasons for defaulting. DOT was used on an individual basis during the study period, especially when an increased risk of non-adherence was suspected, but it became mandatory in Norway from 2003 according to the new TB regulations [ 14 ]. Adoption of this strategy will hopefully improve treatment adherence further. In the transferred out group, the majority of patients left the country on their own initiative, but seven were expelled. Most of the patients who left the country were on treatment, but we do not have information about their treatment outcome. It is worrying that some of the patients who were expelled moved to countries with political unrest and poorly functioning TB programs. In this group three patients already had isolated INH resistant strains and one had MDR-TB. Expelling patients with TB before completion of treatment is unfortunate, unless it can be guaranteed that adequate treatment will be provided elsewhere. Efforts should be made to ensure the continuity of treatment for patients who move out of the country and, if possible, to allow them to start and complete their treatment, even if they have to leave the country later. The Netherlands have adopted a system where patients are not expelled from the country as long as they are on treatment. According to the new Norwegian manual for TB control and prevention [ 11 ], health personnel should encourage patients who are at risk to be expelled from the country to inform the Norwegian Directorate of Immigration through their legal representatives about their disease, and they may then be allowed to stay until treatment is completed. However, we believe there is a need for more awareness among health personnel, immigration authorities, the police and the legal representatives of the patients about this possibility. The death rate in our study was 9%. Other studies from low TB incidence regions of the world showed death rates among TB patients of 24%, 14% and 6% in Baltimore City, USA [ 15 ], Vaud County, Switzerland [ 2 ] and Hamburg, Germany [ 12 ] respectively. Common for these studies and our study is that most patients who died were old, and many of them also had other illnesses. But if we only include only patients who started treatment, the actual death rate for our study was 6%. When considering the treatment outcome of TB, many studies including ours, include patients who never started treatment. This might seem contradictory, but it is an important issue that shows a deficiency in TB control. This is also in line with the recommendations of a working group of the WHO and the European Region of the IUATLD for uniform reporting by cohort analysis of treatment outcome in TB patients [ 16 ]. It has been suggested that acceptable treatment success rates need to be revised under such circumstances. It is difficult to know to what extent the death of the nine patients whose only cause of death was TB could have been prevented. Diagnosis of pulmonary TB can be especially difficult in older patients with co-existing illness. In one study, it was suggested that treatment of latent TB in such high risk elderly patients should be a high priority although advanced age is a relative contraindication [ 17 ]. Other studies recommend the start of anti-TB treatment on suspicion whilst awaiting results of diagnostic tests in elderly patients, provided there is no other obvious cause of their illness [ 18 , 19 ]. This makes sense as our study shows that 19 patients died before treatment start and four of them were diagnosed at autopsy. Two studies from Canada [ 20 ] and former Yugoslavia [ 21 ] have concluded that delay in diagnosis of TB was the main factor contributing to death from TB. Delay in diagnosis is outside the scope of this study. But autopsy rates in Norway are low [ 18 ] and therefore it is likely that there is under-diagnosis of deaths due to TB. As indicated in the results section, TB/HIV co-infection is not a common cause of death for TB patients in Norway as most of the patients born in Norway were elderly persons with low risk of HIV and most foreign-born patients were from countries with low levels of HIV infection [ 22 ]. Therefore, we believe that it had a minor impact on the treatment outcome. In the logistic regression model, increasing age and INH resistance were significant risk factors for non-successful treatment. We expected to find that age played a role, since old age in itself will contribute towards higher mortality, partly through co-existing illness. INH is a powerful bactericidal drug and resistance to the drug might reduce the effectiveness of standard short-course treatment [ 23 ]. Treatment was fairly standardized even in the previous TB manual from 1996: Four drugs to all foreign-born and to all previously treated TB patients and three drugs to patients born in Norway who were not likely to have been infected with drug resistant strains abroad. However, an analysis made by the National TB register in 1999 showed that only 75% of foreign-born patients received four drugs at the start of their treatment (Heldal E, personal communication, National TB Registry of Norway). Conclusion Although the TB treatment success rate in Norway has increased compared to previous studies and although it has reached a reasonable target for treatment outcome in low-incidence countries, the total success rate for 1996–2002 was still slightly below the WHO target of success rate of 85%. Early diagnosis of TB in elderly patients to reduce the death rate, abstaining from expulsion of patients on treatment and further measures to prevent default could improve the success rate further. List of abbreviations CI- Confidence interval HIV- Human immunodeficiency virus INH- Isoniazid IUATLD- International Union Against Tuberculosis and Lung Disease KCNV-Royal Netherlands Tuberculosis Association MDR-TB- Multi-drug resistant tuberculosis, i.e. resistance to both isoniazid and rifampicin TB- Tuberculosis WHO- World Health Organization Competing interests The author(s) declare that they have no competing interests. Authors' contributions MGF participated in all phases of preparation of the manuscript (collection of data, analysis and interpretation of results and writing of the manuscript) and is corresponding author. AT, TWS, EH, ABB and GB have participated the interpretation of results and writing of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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526185

Peroxynitrite decomposition catalyst ameliorates renal damage and protein nitration in cisplatin-induced nephrotoxicity in rats

Background Oxidative stress is involved in cisplatin-nephrotoxicity. However, it has not completely established if reactive nitrogen species and nitrosative stress are involved in this experimental model. The purpose of this work was to study the role of peroxynitrite, a reactive nitrogen specie, in cisplatin-nephrotoxicity using the compound 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) (FeTPPS), a soluble complex able to metabolize peroxynitrite. Results In rats treated with cisplatin (a single intraperitoneal dose of 7.5 mg/kg body weight), renal nitrosative stress was made evident by the increase in 3-nitrotyrosine on day 3. In addition, cisplatin-induced nephrotoxicity was evident by the histological damage of proximal tubular cells and by the increase in (a) serum creatinine, (b) blood urea nitrogen, and (c) urinary excretion of N-acetyl-β-D-glucosaminidase and total protein. Cisplatin-induced nitrosative stress and nephrotoxicity were attenuated by FeTPPS-treatment (15 mg/kg body weight, intraperitoneally, every 12 hours for 3 days). Conclusions Nitrosative stress is involved in cisplatin-induced nephrotoxicity in rats. Our data suggest that peroxynitrite is involved, at least in part, in cisplatin-induced nephrotoxicity and protein nitration.

Background Cisplatin (cis-dichlorodiammine-platinum II) is an effective antineoplastic agent in the treatment of various solid tumours [ 1 ] including cancers of the ovary, testis, bladder, head, neck, lung, cervix, and endometrium [ 2 ]. Nevertheless, its full clinical utility is limited due to some adverse side effects including acute renal failure. The major site of renal injury is the S3 segment of the proximal tubule, located in the outer stripe of the outer medulla of the kidney [ 1 ]. The production of reactive oxygen species (ROS) and oxidative stress in kidney have been implicated in the pathogenesis of cisplatin-induced renal injury [ 3 ]. It has been shown that superoxide anion (O 2 •- ) [ 4 ], hydrogen peroxide (H 2 O 2 ) [ 5 ], and hydroxyl radical ( • OH) [ 6 ] are involved in cisplatin-induced nephrotoxicity. In addition, it has been found that renal lipid peroxidation [ 5 , 7 ] is increased and glutathione is decreased [ 8 ] in this experimental model. The involvement of oxidative stress is further supported by the fact that the antioxidants melatonin [ 9 ] and vitamins C and E [ 5 , 10 ] prevent cisplatin-induced nephrotoxicity. Interestingly, overexpression of heme oxygenase-1 ameliorates [ 11 ] and heme oxygenase-1 deficiency [ 12 ] aggravates renal damage induced by cisplatin, supporting additionally the involvement of oxidant stress in this experimental model. On the other hand, the role of reactive nitrogen species (RNS) and nitrosative stress has been less explored in cisplatin-induced nephrotoxicity. In this context, it has been studied the role of nitric oxide ( • NO) and nitric oxide synthase (NOS) [ 13 - 19 ]. It has been found that the renal content of total nitrate/nitrite is increased in cisplatin-treated rats [ 18 , 19 ] suggesting that • NO production is enhanced in these animals. Furthermore, the inhibition of NOS by L-NAME [ 14 ] or by aminoguanidine [ 13 ] decreased renal damage induced by cisplatin, suggesting that • NO is playing a toxic role in this experimental model. However, it is unknown if peroxynitrite (ONOO - ), a RNS that is generated by the reaction of • NO and O 2 •- , is involved in the renal damage induced by cisplatin. It has been shown that ONOO - , which is not a free radical, is involved in the pathogenesis of many diseases [ 20 - 25 ]. ONOO - can react with different biomolecules including amino acids such as cysteine, methionine, tryptophan, and tyrosine leading to changes in protein structure and function [ 26 ]. ONOO - has been shown to cause lipid peroxidation, chemical cleavage of DNA, and reduction in cellular defenses by oxidation of thiol pools [ 27 ]. In this work, we studied if ONOO - is involved in the nephrotoxicity induced by cisplatin by using 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) (FeTPPS). This compound is a water-soluble Fe (III) porphyrin complex that catalyzes rapid isomerization of ONOO - to nitrate (NO 3 - ) under physiologically relevant conditions (pH 7.4, 37°C) [ 28 ]. The cytoprotective actions of FeTPPS have been characterized [ 29 ]. Results Body weight and urinary volume Body weight decreased 8.5% in cisplatin (Cis) group on day 3 and FeTPPS tended to prevent this decrease in Cis+FeTPPS group, however there was no significative difference between Cis and Cis+FeTPPS groups. Body weight was similar in control (Ct), FeTPPS, and Cis+FeTPPS groups. Urinary volume was not significative difference among the four groups along the study and on day of sacrifice (Table 1 ). Table 1 Body weight and urinary volume in the 4 groups of rats studied on day 3. Ct Cis FeTPPS Cis+ FeTPPS Body weight (g) 235 ± 5 a 215 ± 4 b 238 ± 3 a 231 ± 4 a Urinary volume (mL/24 h) 5.7 ± 1.4 a 7.4 ± 0.8 a 3.5 ± 1.1 a 7.4 ± 1.6 a Values are mean ± SEM. n = 6. Groups with different letter are significantly different (P < 0.05). Markers of glomerular and tubular damage Serum creatinine increased 4.9 times and blood urea nitrogen (BUN) increased 5.5 times in Cis group compared to control one (Fig 1 ). FeTPPS prevented partially the increase in serum creatinine and BUN levels in Cis+FeTPPS group. Cisplatin increased urinary excretion of total protein (4.6 times) and N-acetyl-β-D-glucosaminidase (NAG) (9.6 times) (Fig 2A and 2B ). The increase in both parameters was prevented by FeTPPS in Cis+FeTPPS group (Fig 2 ). Serum creatinine, BUN, and urinary excretion of total protein and NAG were similar in Ct and FeTPPS groups (Figs 1 and 2 ). Figure 1 (A) Serum creatinine and (B) BUN on day 3 in the four groups of rats studied. Ct: control group, Cis: cisplatin group; FeTPPS: 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato iron (III) group, and Cis+FeTPPS: cisplatin+5,10,15,20-tetrakis(4'-sulfonatophenyl) porphyrinato iron (III) group. Data are mean ± SEM. n = 6. a P < 0.001 vs. Ct; b P < 0.001 vs. Ct, c P < 0.05 vs Cis (Panel A); a P < 0.001 vs. Ct; b P < 0.001 vs. Ct, c P < 0.001 vs Cis (Panel B). Serum creatinine and BUN increased in cisplatin group and FeTPPS prevented these increases in the Cis+FeTPPS group. Figure 2 Urinary excretion of (A) total protein and (B) NAG on day 3 in the four groups of rats studied. Data are mean ± SEM. n = 5–6. a P < 0.001 vs. Ct, b P < 0.05 vs. Cis. Cisplatin-treated rats increased urinary excretion of total protein and NAG and these increases were prevented by FeTPPS administration in Cis+FeTPPS group. Histological analysis After three days of cisplatin-treatment, the epithelium from proximal convoluted tubules (tubules with small lumen area and taller epithelial cells) showed cytoplasmic vacuolization, intracellular edema and extensive damage which affected 87 ± 4% of their surface area (Fig 3B ). The cisplatin toxic activity was higher in the straight portion of proximal convoluted tubules located in the inner area of the kidney cortex, where more than 90% of the epithelial surface suffered damage (Fig 4B ). Interestingly, FeTPPS administration partially decreased the damaged area from 87 ± 4 to 44 ± 6% (p < 0.0001) in proximal convoluted tubules (Fig 3D ) and from 93 ± 2 to 68 ± 10 (p < 0.0001) in the straight portion (Fig 4D ). The administration of FeTPPS did not produce any histological alteration in the kidneys (Figs 3C and 4C ). At the light microscopy level, glomeruli structure remained unchanged in all groups. Figure 3 Representative histological abnormalities in the external cortical kidney area after three days of cisplatin administration and their partial prevention by FeTPPS. (A) Normal kidney histology from control rat. (B) After three days of cisplatin administration, many cortical convoluted tubules are revisted by necrotic epithelial cells (arrows) or vacuolated swell cells (arrow heads), glomeruli do not show apparent damage. (C) FeTPPS administration does not produce histological kidney abnormalities. (D) The administration of FeTPPS partially prevents the cytotoxic damage induced by cisplatin; arrows indicate middle cellular vacuolization of cortical convoluted tubules. Figure 4 Representative histological abnormalities in the inner part of the cortical kidney after three days of cisplatin administration and their partial prevention by FeTPPS. (A) Normal kidney histology from control rat. (B) After three days of cisplatin administration, the straight portion of many cortical tubules are revisted by necrotic cells (arrows). (C) FeTPPS administration does not produce histological abnormalities. (D) The administration of FeTPPS partially prevents the cytotoxic damage induced by cisplatin; arrows indicate tubules with focal necrotic cells. Immunohistochemical localization of 3-nitro-L-tyrosine (3-NT) A strong 3-NT immunostaining was observed in the straight portion of the proximal convoluted tubules located in the inner area of the kidney cortex of cisplatin-treated rats (Fig 5B ). Interestingly, in the Cis+FeTPPS group, FeTPPS administration partially prevented the cisplatin toxic damage in the epithelium from the proximal convoluted tubules and its straight portion respectively, in coexistence with an evident decrease of 3-NT immunostaining (Fig 5D ). Figure 5 Nitrotyrosine (3-NT) expression determined by immunohistochemistry in the inner part of the cortical kidney after three days of cisplatin administration and its partial prevention by FeTPPS. (A) There is no 3-NT immunostaining in the kidney of control rat. (B) In contrast, three days after cisplatin administration there is a strong 3-NT expression in the necrotic cells from the straight portion of the proximal convoluted tubules (arrows). (C) FeTPPS administration does not induce 3-NT expression. (D) The administration of FeTPPS strongly decreases 3-NT expression induced by cisplatin-treatment (Cis+FeTPPS group). Discussion Cisplatin in an effective chemotherapeutic agent for a wide variety of tumors, nevertheless, nephrotoxicity is the major complication of this antineoplasic treatment [ 1 ]. The mechanism by which cisplatin causes renal damage is unclear, however, it has been postulated that oxidative stress is involved in this process [ 2 , 3 , 13 , 30 ]. The protective effect of overexpression of Mn-SOD [ 4 ] or the in vivo administration of some antioxidants such as vitamins C and E [ 5 , 10 ], melatonin [ 9 ], or selenium [ 31 ] in cisplatin-induced nephrotoxicity as well as the protective effect of tiron (a cell permeable O 2 •- scavenger), pyruvate and catalase (H 2 O 2 scavengers), and dimethylthiourea and thiourea ( • OH scavengers) in renal proximal tubular epithelial cells (LLC-PK1 cells) treated with cisplatin also strongly support the role of ROS in cisplatin renal toxicity [ 30 ]. In contrast the role of • NO and RNS in cisplatin-induced nephrotoxicity has not been completely established. It has been shown that the renal content of nitrate/nitrite is increased in cisplatin-treated rats suggesting that • NO is increased in these animals [ 18 , 19 ]. In fact it has been shown that renal NOS activity is increased in cisplatin-treated rats [ 14 ]. In addition, the following two experiments suggest a toxic role of • NO on cisplatin-induced renal toxicity: (a) aminoguanidine, an inhibitor of inducible NOS, decreased nephrotoxicity and prevented kidney lipid peroxidation and reduction of antioxidant enzymes induced by cisplatin [ 13 ], and (b) the administration of N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, reduced renal and gastrointestinal toxicity along with a significant inhibition in lipid peroxidation induced by cisplatin [ 14 ]. In contrast, Mansour et al . [ 15 ] and Li et al . [ 16 ] have found that L-NAME administration resulted in no protection and Saad et al . [ 17 ] found that this NOS inhibitor aggravates cisplatin-induced renal dysfunction. These data may suggest that • NO is not playing a toxic role in cisplatin-induced nephrotoxicity. The above mentioned disagreement justify the performance of additional experiments to clarify the role of NOS and • NO in cisplatin-induced nephrotoxicity. On the other hand, • NO is able to react with O 2 •- to produce ONOO - , which is a powerful oxidant more, reactive than its precursors, and has been implicated in an increasing list of diseases: hyperlipidemia [ 32 ], Alzheimer [ 20 ], acute renal ischemia [ 25 ], neurotoxicity induced by methamphetamine [ 33 ], and diabetes [ 23 ]. The ONOO - decomposition catalyst FeTPPS is a water-soluble Fe (III) porphyrin complex able to block ONOO - toxicity [ 28 , 29 ] and to protect against toxic insults in several experimental models. In focal cerebral ischemia-reperfusion in rats, massive production of • NO and O 2 •- results in continuous formation of ONOO - even several hours after ischemia-reperfusion insult [ 34 ]. Significant reduction of 3-NT in brain sections and prominent neuroprotection was observed by FeTPPS (30 mg/kg) [ 34 ]. In a model of sepsis induced by injection of endotoxin (10 mg/kg) in rats, FeTPPS prevented the accumulation of ONOO - as measured by plasma rhodamine fluorescence and heart 3-NT staining [ 35 ]. Interestingly, FeTPPS improved endotoxin-induced myocardial contractile dysfunction, which was associated with reduced degradation of nuclear factor kappa B inhibitory protein I-kappa-B, plasma TNF-alpha levels, and microvascular endothelial cell-leukocyte activation [ 35 ]. In this work it was found that FeTPPS partially prevented the increase in BUN and serum creatinine (markers of glomerular damage) and urinary excretion of NAG and total protein (markers of tubular damage) induced by cisplatin-treatment. The increase in urinary NAG and total protein excretion could be associated with necrosis of the proximal convoluted tubules, the primary site of drug accumulation [ 1 ]. FeTPPS prevented these alterations induced by cisplatin. This may be secondary to the ability of FeTPPS to catalyze the decomposition of ONOO - which could be responsible, at least in part, of the alterations induced by cisplatin. This ameliorative effect of FeTPPS was associated with the decrease in 3-NT staining suggesting that ONOO - is involved in protein nitration in cisplatin-nephrotoxicity. It is known that another RNS such as N 2 O 4 , HONOO, • NO 2 [ 36 ], and nitryl chloride (NO 2 Cl) [ 37 ], are involved in protein nitration. Nitryl chloride is formed by the reaction of NO 2 - and HOCl-derived myeloperoxidase [ 37 ]. Studies in animals have established that tubular injury plays a central role in the reduction of glomerular filtration rate in acute tubular necrosis. Two major tubular abnormalities could be involved in the decrease in glomerular fucntion in cisplatin-treated rats: obstruction and backleak of glomerular filtrate. The alteration in glomerular function can not be attributed to structural damage since glomeruli structure is normal in cisplatin-treated rats. The alterations in glomerular function in cisplatin-treated rats may also be secondary to ROS [ 38 ] which induce mesangial cells contraction, altering the filtration surface area and modifying the ultrafiltration coefficient, factors that decrease the glomerular filtration rate. In addition our data suggest that ONOO - may also be involved in the glomerular alterations in cisplatin-treated rats. The increase in renal ONOO - induced by cisplatin may be secondary to the increase in • NO and O 2 •- production. In fact, there are evidences of the renal increase in • NO production in cisplatin nephrotoxicity [ 18 , 19 ] and O 2 •- generation in cisplatin-treated LLC-PK1 cells [ 30 ]. The O 2 •- increase in cisplatin-nephrotoxicity may be simply consequence of the mitochondrial dysfunction [ 39 ] and the decrease in superoxide dismutase activity [ 5 ]. Conclusions Nitrosative stress is involved in cisplatin-induced nephrotoxicity in rats. The ameliorative effect of FeTPPS on cisplatin-induced nephrotoxicity in rats was associated with the decrease in protein nitration suggesting that ONOO - is involved in both protein nitration and nephrotoxicity in these animals. Methods Reagents Cisplatin (catalogue # P-4394) was from Sigma-Aldrich (St. Louis MO, USA). FeTPPS (catalogue # 341492) was from (Calbiochem, San Diego, CA, USA). Rabbit anti-3-NT polyclonal antibodies (Catalogue # 06–284) were from Upstate (Lake Placid, NY, USA). Anti-rabbit Ig horseradish peroxidase antibodies (Catalogue # SAB-300) were purchased from Stressgen (Victoria BC, Canada). Commercial kits to measure creatinine and urea were from Spinreact (Girona, Spain). All other chemicals were reagent grade and commercially available. Experimental design Male Wistar rats (Harlan Teklad, Mexico City, Mexico) initially weighing 200–250 g were used. Experimental work was approved by DGAPA (IN227103) and followed the guidelines of Norma Oficial Mexicana (NOM-ECOL-087-1995). All animals had free access to water and commercial rodent diet (Harlan Teklad, catalogue 2018S), and were randomly divided in four groups (n = 6 rats/group) as follows: (1) CT, injected intraperitoneally (i.p.) with isotonic saline solution; (2) Cis, treated with a single dose of cisplatin (7.5 mg/Kg b.w./i.p.) [ 40 ]; (3) FeTPPS, treated with FeTPPS (15 mg/kg/i.p./12 h) [ 32 ] for 3 days; and (4) Cis+FeTPPS, treated with Cis and with FeTPPS. During the study rats were maintained with a 12-h light:dark cycle in stainless steel metabolic cages to collect urine. On day 3, the animals were sacrificed by decapitation and blood was collected to obtain serum and to measure creatinine and BUN. Total protein and NAG were measured in 24-h urine. The kidneys were removed to obtain cortex samples for histological and immunohistochemical studies. Markers of glomerular and tubular damage The markers of glomerular damage, creatinine and urea, were measured using commercial kits. BUN was obtained by correcting the urea value by a 2.14 factor [ 41 ]. As markers of tubular damage, we measured urinary excretion of NAG and total protein. NAG activity was measured using p-nitrophenyl-N-acetyl-β-D-glucosaminide as substrate and total protein was measured by a turbidimetric method [ 42 ]. Histological analysis Thin slices of kidney tissue with cortex and medulla were fixed by immersion in buffered formalin (pH 7.4), dehydrated and embedded in paraffin [ 43 ]. Sections of 3 μm were stained with hematoxilin and eosin. The histological profile of twenty proximal tubules randomly selected per rat (6 rats per experimental group) was recorded using a Leica Qwin Image Analyzer (Cambridge, England). The percentage of tubular area with histopathological alterations like swelling, cytoplasmic vacuolization, desquamation or necrosis was obtained. The percentage of damaged area of Cis and Cis+FeTPPS groups was compared. Immunohistochemical localization of 3-nitro-L-tyrosine (3-NT) For immunohistochemistry, 3 μm sections were deparaffined with xylol and rehydrated with ethanol. Endogenous peroxidase was quenched/inhibited with 4.5% H 2 O 2 in methanol by incubation for 1.5 h at room temperature. Nonspecific adsorption was minimized by leaving the sections in 3% bovine serum albumin in phosphate buffer saline for 30 min. Sections were incubated overnight with a 1:700 dilution of anti-3-NT antibody. After extensive washing with phosphate buffer saline, the sections were incubated with a 1:1000 dilution of a peroxidase conjugated anti-rabbit Ig antibody for 1 h, and finally incubated with hydrogen peroxide-diaminobenzidine for 10 s. Sections were counterstained with hematoxilin and observed under light microscopy. All the sections from the four studied groups were incubated under the same conditions with the same antibodies concentration, and in the same running, so the immunostaining was comparable among the different experimental groups [ 43 ]. Statistics Results are expressed as the mean ± SEM. Data were analyzed by one-way ANOVA followed by Bonferroni's multiple comparisons. Non-paired t-test was used to compare the quantitative histological damage data using the software Prism 3.02 (GraphPad, San Diego, CA, USA). P ≤ 0.05 was considered statistically significant. Authors' contributions YICH performed animal experimentation, biochemical determinations, statistical analyses, light microscopy and immunohistochemical studies. RHP supported the light microscopy and immunohistochemical studies. JPCH conceived, designed and coordinated the study. All authors read and approved the final manuscript.

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539258

Factors associated with herbal use among urban multiethnic primary care patients: a cross-sectional survey

Background The use of herbal supplements in the United States has become increasingly popular. The prevalence of herbal use among primary care patients varies in previous studies; the pattern of herbal use among urban racially/ethnically diverse primary care patients has not been widely studied. The primary objectives of this study were to describe the use of herbs by ethnically diverse primary care patients in a large metropolitan area and to examine factors associated with such use. The secondary objective was to investigate perceptions about and patterns of herbal use. Methods Data for a cross-sectional survey were collected at primary care practices affiliated with the Southern Primary-care Urban Research Network (SPUR-Net) in Houston, Texas, from September 2002 to March 2003. To participate in the study, patients had to be at least 18 years of age and visiting one of the SPUR-Net clinics for routine, nonacute care. Survey questions were available in both English and Spanish. Results A total of 322 patients who had complete information on race/ethnicity were included in the analysis. Overall, 36% of the surveyed patients (n = 322) indicated use of herbs, with wide variability among ethnic groups: 50% of Hispanics, 50% of Asians, 41% of Whites, and 22% of African-Americans. Significant factors associated with an individual's herbal use were ethnicity other than African-American, having an immigrant family history, and reporting herbal use by other family members. About 40% of survey respondents believed that taking prescription medications and herbal medicines together was more effective than taking either alone. One-third of herbal users reported using herbs on a daily basis. More Whites (67%) disclosed their herbal use to their health-care providers than did African-Americans (45%), Hispanics (31%), or Asians (31%). Conclusions Racial/ethnic differences in herbal use were apparent among this sample of urban multiethnic adult primary care patients. Associated factors of herbal use were non-African-American ethnicity, immigrant family history, and herbal use among family members. Whereas Hispanics and Asians reported the highest rates of herbal use, they were the least likely to disclose their use to health-care professionals. These findings are important for ensuring medication safety in primary care practices.

Background The use of complementary and alternative medicine (CAM) in the United States gained greater popularity in the 1990s. Two national telephone surveys of 1,539 and 2,005 adults, respectively, demonstrated an increasing trend in the use of CAM, including relaxation techniques, herbal medicine, massage, chiropractic, and acupuncture[ 1 , 2 ]. Specifically, the use of these unconventional treatments rose from 33.8% in 1990 to 42.1% in 1997. These surveys found that use of herbal medicine within the past year increased from 2.5% in 1990 to 12.1% in 1997[ 2 ]. CAM use was also found to be more frequent among females, persons 35 to 49 years of age, persons of ethnicities other than African-American, persons who were college educated, and persons whose annual income was greater than $50,000[ 2 ]. In a separate study also conducted in the 1990s, the American Botanical Council estimated that one-third of the nation's adults use herbal remedies[ 3 ]. Efficacy studies of herbal supplements are on the rise, but most data published to date are preliminary and do not provide strong evidence for the clinical effectiveness of herbs. Nevertheless, about 15 million American adults (18%) are thought to use prescription medications concurrently with herbal or vitamin products[ 4 ], and as many as 70% of persons who use herbal remedies do not discuss their use of such remedies with their physicians or pharmacists[ 1 , 5 - 7 ]. By not communicating about herbal use, they may put themselves at increased risk for adverse drug-herb interactions[ 8 ] and make it extremely difficult for health-care professionals to monitor them for such interactions[ 9 ]. Likewise, patients do not know what symptoms they should report to their health-care provider that indicate potential adverse effects of drug-herb interactions. Consequently, unintentional medication errors could occur. The prevalence of herbal use among racially/ethnically diverse primary care patients varies from study to study[ 2 , 3 , 5 - 7 , 10 - 12 ], ranging from 30%[ 5 , 6 ] to 77%[ 7 ]. Since patients must interact with their primary care providers and pharmacists for illnesses to be diagnosed and quality medical care to be provided, a better understanding of variations in herbal use patterns among primary care patients is needed. To this end, we conducted a study with two objectives: 1) to describe the herbal use of ethnically diverse patients in a large metropolitan area and to examine factors associated with herbal use; and 2) to investigate perceptions about and patterns of herbal use among those patients. Methods Setting and study population We implemented this cross-sectional study within the Southern Primary-care Urban Research Network (SPUR-Net) from September 2002 to March 2003. SPUR-Net is a practice-based research network in Houston, Texas, that consists of five constituent member organizations affiliated with a county health system, a managed care organization, or a private practice clinic. SPUR-Net clinicians provide care to patients from diverse ethnic and socioeconomic backgrounds, with approximately one million patient visits per year. A total of six primary care clinics were included in this study that varied according to socioeconomic status (SES) of their patients as measured by income level and insurance type. For the purposes of this study, we defined "clinic SES" according to the insurance status of the majority of patients; "high SES" means that most patients have insurance (i.e., private insurance and/or Medicare), and "low SES" means most patients are indigent (i.e., county health-care coverage and/or Medicaid). Human subject approvals were obtained from the Institutional Review Boards at all of the SPUR-Net constituent organizations. Permission to conduct the study was also obtained from the medical directors and applicable patient advisory groups at each of the six participating clinics. To be eligible for participation in the study, patients had to be at least 18 years of age and to be visiting one of the participating clinics for routine, nonacute care. A target of 50 surveys in each of the six clinics was collected from a convenience sample of patients. The decision regarding the number of patients to be surveyed was limited by our resources, including availability in funding and personnel. A research assistant approached potential subjects in the clinic setting to determine their willingness to complete a 23-item questionnaire about herbal use in either English or Spanish. Those patients who consented to participate were either given the survey to complete on their own or had the survey administered to them by the research assistant. Research assistants were available on-site to answer any questions the patients had, helping to improve patients' understanding of the terms used in the survey. Recruitment methods were the same in all of the participating clinics. The research assistants stopped recruiting patients when a minimum of 50 surveys was collected in each clinic. Survey instrument Survey questions were adopted and modified from previously developed and validated surveys on CAM use, including national telephone surveys conducted by Eisenberg et al.[ 1 , 2 , 13 ], a family practice survey by Elder et al.[ 5 ], a research clinic survey by Johnson et al.[ 3 ], and a national mail survey by Astin et al[ 14 ]. We modified these questions for use among our multiethnic patient population; we also translated the survey questions into Spanish. The survey instrument was pilot tested with 54 English-speaking subjects and 10 Spanish-speaking subjects before the study. The survey was reviewed by several groups of patient representatives in the community health centers to ensure consistency in responses. For example, some members of a patient advisory group representing a homeless clinic perceived herbal use to be marijuana use; for this reason, we decided not to include this patient population in our study. The final survey instrument had three components. First, all participating patients answered questions regarding sociodemographic characteristics (e.g., gender, age, race, ethnicity, education, immigrant family history, herbal use by other family members, spoken language other than English, and clinic location). Immigrant family history and spoken language were elicited with the following questions: "Are your family members immigrants to the United States (Y/N)?," and "Do you speak another language other than English?" After completing the demographic questions, respondents answered a series of questions regarding their belief in herbal use and their herbal information sources. The questions pertained to their personal use of herbs (Y/N); their belief in the benefit of herbal remedies (Y/N), the source of their herbal information (physician, pharmacist, family, friends, etc.); their preferred content of herbal information (e.g., effectiveness, side-effects, interactions with other medications), and their preferred methods for obtaining herbal information from physicians or pharmacists (e.g., handout, World Wide Web site, consultation). Patients who reported using herbal supplements answered additional questions related to their patterns of and reasons for herbal use. In open-ended questions, the participating patients were asked about the herbs they specifically used and the health conditions for which they took the herbal products. Related questions included frequency of herbal use (daily, frequently-few times/month, occasionally—few times/year); duration of use (< 1 year, 1–2 years, 3–5 years, > 5 years); expenditure on herbal products; reported concomitant use of prescription medications; disclosure of herbal use to physicians or pharmacists; and any experiences of adverse reactions from using herbs. For the purposes of this study, we used the definition of dietary supplements stipulated in the 1994 Dietary Supplement and Health Education Act (DSHEA) to differentiate herbs from vitamins and minerals. Herbal use was defined as having ever used herbal products or natural medicines for health maintenance or treatment of health conditions. To measure herbal use, we asked the following question: "Do you use any of the following?" Response options included: herbs/herbal products or natural medicine (e.g., echinacea, St. John's wort, ginseng, ginkgo biloba, soy supplements), folk medicine or home remedy, vitamins, minerals, or none. Herbal use did not include the use of folk medicine, home remedies (such as honey), vitamins, or minerals. Data analysis Data from the paper-based survey were entered into an ACCESS database and were imported into SAS 9.1 for Windows. The study variables were summarized by using one-way frequencies to examine the sociodemographic characteristics of the study sample, the belief in and information source for herbal use, and the patterns of and reasons for herbal use among urban multiethnic primary care patients. The frequencies of use of specific herbs were counted, and the health conditions for which herbs were used were further coded into three types—acute, chronic, and health maintenance. Based on findings from previous studies, we used the following independent variables as reference variables for both the univariate and multivariate logistic regression analyses: male gender, age less than 30 years, African-American ethnicity, less than a college education, no immigrant family history, no herbal use by other family members, and visiting a high SES clinic. A Chi-square test of proportions was used to determine the association between herbal use and each of the independent variables related to demographic characteristics; a p value ≤ 0.05 was considered to be statistically significant. In order to assess factors associated with herbal use, all hypothesized variables (age, gender, race and ethnicity, education, immigrant family history, herbal use by other family members, and clinic clientele stratified by SES) were included in both the univariate and the multivariate logistic regression analyses. These independent variables were entered as dichotomous variables in the model: gender (male vs. female), age (< 50 years, ≥ 50 years), ethnicity (African-American vs. other, including Whites and Hispanics), education (less than college vs. college and greater), immigrant family history (yes vs. no), herbal use by family members (yes vs. no), and clinic clientele (high SES vs. low SES). Significant variables identified by backward elimination of the main effects from the multivariate analysis were further evaluated in two-way interactions. Thus, the final model contained all of the significant main effects and the two-way interaction terms. Odds ratios and 95% confidence intervals were calculated to determine the effects of the significant variables on herbal use. Since the sample size for Asians was small, Asians were not included in the logistic regression analyses. Furthermore, the language variable was excluded from the regression analyses because the survey question was not clearly answered by many patients; for example, 10 Spanish-language forms had "no language other than English" indicated. In addition, some answers were possibly indicative of an exclusive language other than English instead of the bilingual capability of the respondent. Results Description of sample Of the 327 patients who agreed to participate in the survey, only 322 completed the race/ethnicity information and were included in the analysis. The characteristics of the study sample are summarized in Table 1 . Two-thirds of the patients were female, and approximately half of all the patients had less than a college education. More than a third (37%) of the patients reported having an immigrant family history, and 50 patients (15%) used the Spanish-language form to complete the survey. Table 1 Descriptive Characteristics of the Study Sample (n = 322) Variables White n (%) Hispanic n (%) African American n (%) Asian n (%) Totals 68(21.1) 98(30.4) 136(42.2) 20(6.2) Gender Male 20(29.4) 34(34.7) 37(27.4) 7(35.0) Female 48(70.6) 64(65.3) 98(72.6) 13(65.0) Age (yrs) < 30 13(19.1) 17(17.3) 27(19.8) 4(20.0) 30–49 34(50.0) 34(34.7) 47(34.6) 5(25.0) 50+ 21(30.9) 47(48.0) 62(45.6) 11(55.0) Education < High School 3(4.4) 52(53.0) 17(12.6) 0 High School 16(23.5) 23(23.5) 54(40.0) 5(25.0) ≥ College 49(72.1) 23(23.5) 64(47.4) 15(75.0) Immigrant Family History No 57(86.4) 41(41.8) 101(77.1) 0 Yes 9(13.6) 57(58.2) 30(22.9) 20(100.0) Herbal Use by Other Family Members No 43(63.2) 42(42.9) 95(69.9) 11(55) Yes 25(36.8) 56(57.1) 41(30.1) 9(45) Clinic Type High SES Clinic 40(58.8) 14(14.3) 52(38.2) 9(45) Low SES Clinic 28(41.2) 84(85.7) 84(61.8) 11(55) Herbal use Overall, 36% of our study sample reported ever using herbs. The proportions of herbal users varied across racial/ethnic groups, with use being reported by 50% of Hispanics, 50% of Asians, 41% of Whites, and 22% of African-Americans. Herbal use by other family members was reported to be 41% (57% among Hispanics, 45% among Asians, 37% among Whites, and 30% among African-Americans). Patients who reported using herbs indicated that they received information about those herbs mainly from family members and relatives. Nevertheless, most patients reported that they preferred receiving herbal information (e.g., on effectiveness, side-effects, and drug interactions) through handouts or brochures from their physicians or pharmacists, followed by having access to a consultation service or a Web site. About 40% of all of the survey respondents, but especially Asians (55%) and Whites (47%), believed that taking prescription medications and herbal medicines together was more effective than taking either alone. About 41% of Hispanic respondents believed that herbal medicines were superior to prescription medications, as compared to 12% of Whites. These differences in beliefs about herbal use among the ethnic groups were found to be statistically significant ( p < 0.05). Nearly half of the patients who reported using herbs (46%), particularly Hispanics (63%) and Asians (57%), also reported taking prescription medications concomitantly with the herbs (Table 2 ). Since our survey question was designed to measure self-reported concomitant herbal use and prescription drug use, we cannot confirm whether or not those who reported taking both were actually using both. Table 2 Patterns of and Reasons for Herbal Use Among Urban Multiethnic Primary Care Patients (n = 322) Variables White n (%) Hispanic n (%) African American n (%) Asian n (%) Herbal Use 28(41.2) 49(50.0) 30(22.1) 10(50.0) Daily Herbal Use* 14(48.3) 13(22.8) 13(33.3) 4(30.8) Herbal Use 3+ Years* 12(41.1) 45(78.9) 17(45.9) 7(53.8) Report Taking Herbs and Prescription Medications for the Same Health Problems* 10(33.3) 36(63.2) 17(32.7) 8(57.1) Told Physicians/Pharmacists About Herbal Use* 20(66.7) 17(30.9) 21(44.7) 4(30.8) Had a Bad Reaction* 2(7.4) 1(2.0) 3(11.1) 0 Believed Both Prescription Medications and Herbal Medicines Are Better Than Either Alone** Agree 32(47.1) 28(28.6) 54(40.6) 11(55.0) Disagree 14(20.6) 44(44.9) 48(36.1) 6(30.0) Neither 22(32.4) 26(26.5) 31(23.3) 3(15) Believed Herbal Medicines Are Superior to Prescription Medications*** Agree 8(12.3) 40(41.2) 31(23.1) 6(30.0) Disagree 36(55.4) 20(20.6) 62(46.3) 8(40.0) Neither 21(32.3) 37(38.1) 41(30.6) 6(30.0) Received Herbal Information (multiple) Family or relatives 20(29.4) 60(61.2) 43(31.6) 10(50.0) Magazines 24(35.3) 19(19.4) 38(27.9) 5(25.0) Television 13(19.1) 24(24.5) 45(33.1) 0 Internet 12(17.7) 7(7.1) 9(6.6) 4(20) Physician 10(14.7) 8(8.2) 12(8.8) 2(10.0) Pharmacist 2(2.9) 2(2.0) 6(4.4) 0 Preferred Herbal Information (multiple) Effectiveness 53(77.9) 72(73.5) 87(64.0) 9(45.0) Side-effects 42(61.8) 76(77.6) 82(60.3) 12(60.0) Interactions 46(67.7) 67(68.4) 75(55.2) 9(45.0) Preferred Method for Obtaining Herbal Information (multiple) Handout/Brochure 45(66.2) 80(81.6) 84(61.8) 11(55.0) Website 25(36.8) 11(11.2) 20(14.7) 6(30.0) Consultation 29(42.7) 20(20.4) 47(34.6) 5(25.0) *Indicates only those patients who reported herbal use ** p = 0.008;*** p < 0.0001 Factors associated with herbal use Variables demonstrating a significant univariate association ( p < 0.05) with herbal use were ethnicities other than African-American, immigrant family history, and herbal use by other family members (Table 3 ). In the multivariate logistic regression model, non-African-American race/ethnicity (OR = 2.42, 95% CI, 1.33–4.40), immigrant family history (OR = 2.23, 95% CI, 1.20–4.14), and reported herbal use by other family members (OR = 7.98, 95% CI, 4.48–14.18) remained significant predictors of reported herbal use ( p < 0.05). In addition, interactions between immigrant family history and herbal use by other family members were found to be significant terms in the model (Table 3 ). With the race/ethnicity variable adjusted, having an immigrant family history was associated with a 19 times greater likelihood of herbal use among those whose family members also use herbs. When the analyses were run with the Asian group included, the results did not change. Table 3 Univariate Analysis of Factors Associated with Herbal Use Among Urban Multiethnic Primary Care Patients (n = 302) Variables Total Herbal Use n (%) X 2 p -value Gender Male 91 32(35.2) 0.9 Female 210 75(35.7) Age (yrs) < 30 106 34(32.1) 0.4 ≥ 30 196 73(37.2) Race/Ethnicity African-American 136 30(22.1) <0.0001 White & Hispanic 166 77(46.4) Education < College 165 57(34.6) 0.8 ≥ College 136 49(36.0) Immigrant Family History No 199 56(28.1) 0.0001 Yes 96 49(51.0) Herbal Use by Other Family Members No 180 31(17.2) <0.0001 Yes 122 76(62.3) Clinic Type High SES Clinic 106 35(33.0) 0.5 Low SES Clinic 196 72(36.7) Perceptions about and patterns of herbal use The reasons given by the study subjects for herbal use included faster resolution of symptoms (47%), the desire to try alternative therapies (33%), and preference for having their own methods to care for their health (20%). Among the herbal users, 32% reported taking herbs on a daily basis, and 60% reported using herbs for longer than three years. Usage varied by race/ethnicity; for example, 48% of Whites reported taking herbs on a daily basis, and 79% of Hispanics reported using herbs for longer than three years. Even though Hispanics and Asians used herbs more frequently, they were the least likely to disclose their herbal use to their physicians or pharmacists. More Whites (67%) told their health-care professionals about their herbal use than did the African-Americans (45%), Hispanics (31%), or Asians (31%). The reasons given for nondisclosure generally fell into two main categories: 1) "They (the provider) never asked," and 2) "It wasn't important for them to know." While few respondents (5.3%) reported having experienced an adverse reaction to herbs, many of them (43%) did not inform their physicians of it. The specific herbs used by the patients covered a wide spectrum and varied by ethnicity. The herbs used most commonly by White patients were echinacea (32.1%), St. John's wort (21.4%), ginkgo biloba (14.3%), and chamomile (14.3%). Hispanic patients most often reported using chamomile (61.2%), aloe vera (44.9%), and garlic (20.4%). African-American patients reported primarily using garlic (40%), ginseng (30%), and ginkgo biloba (10%). The herbs used by Asian patients were garlic (50%), ginkgo biloba (30%), and ginger (30%). Other herbs that were reported by patients—albeit infrequently—included Yun Zhi, black cohosh, dong quai, guggle phosphate, bee pollen, cat claws, and "a shot of whiskey." The patients who reported using herbs used them for a wide range of health problems, such as boosting the immune system, improving memory, and treating insomnia, depression, or diabetes. For conditions considered to be chronic, 44% of the White patients reported herbal use versus 32% of African-American patients. For conditions considered to be acute, 71% of Hispanic patients used herbs versus 10% of Asians. For health maintenance, 50% of Asian patients used herbs versus 16% of Hispanic patients. Discussion Our data show that herbal use is common (36%) among urban multiethnic primary care patients, but has a wide variability among racial/ethnic groups. Hispanics and Asians reported the highest rates of herbal use (50%), and African Americans reported the lowest (22%). Previous research conducted in the western United States found that the prevalence of herbal use among racially/ethnically diverse primary care patients varies[ 2 , 3 , 5 - 7 , 10 - 12 ], ranging from 30% among primary care patients residing in urban settings on the west coast of the United States[ 5 , 6 ] to 77% among primary care patients residing in the largest United States—Mexico border city[ 7 ]. As expected, factors associated with herbal use included race/ethnicity, having an immigrant family history, and herbal use by other family members. In addition, we found interactions between having an immigrant family history and herbal use by other family members. Previous studies did not examine such interactions and found age to be predictive of herbal use[ 2 , 6 , 7 ]. Unlike other investigators, we did not find a significant relationship between age and herbal use. Other investigators, however, did not account for interactions such as those addressed in our analysis. One study (n = 113) found no significant differences in the use of CAM therapies that could be attributable to gender, educational level, age, race, or clinic attended[ 5 ]. Another study (n = 542) found an association between the use of CAM therapies, high education level, and female gender[ 6 ]. In addition, a recent study conducted in a large United States—Mexico border city revealed that 77% of the residents surveyed (n = 547) use all modalities of CAM therapies and that such use was associated with a high education level[ 7 ]. When the residents reported specifically using herbal and home remedies (59%), however, herbal use was found to be associated with a low education level[ 7 ]. We found that nearly half of herbal users (46%) reported taking herbal medicines and prescription medications concomitantly. More importantly, 43% of herbal users reported not disclosing their herbal use to their physicians or pharmacists. Interestingly, Hispanics and Asians used herbs the most frequently but disclosed their herbal use to their physicians or pharmacists less often than did Whites and African Americans. This lack of communication about herbal use is an area of concern because of the potential for medication errors and untoward reactions to herb-drug interactions. Adverse drug-herb interactions pose a great danger for patients. For example, ginkgo biloba, garlic, and ginseng all may interact with Coumadin ® (warfarin sodium) and cause an increase in bleeding time[ 15 , 16 ]. Echinacea, an immunostimulant, can counteract the action of the immunosuppressants (e.g., the corticosteroids prednisone, methotrexate, and cyclosporine) used to treat immune disorders[ 17 , 18 ]. The interaction between St. John's wort and cyclosporine—which is used to treat rheumatoid arthritis and psoriasis and to prevent the rejection of a transplanted organ—could result in decreased availability of cyclosporine and, consequently, to the worsening of arthritis or psoriasis or the rejection of a transplanted organ [ 19 - 23 ]. St. John's wort may also interact with antidepressants, such as monoamine oxidase inhibitors (e.g., Nardil ® , Parnate ® ) and potentiate the effects of selective serotonin reuptake inhibitors (e.g., Paxil ® , Prozac ® , Zoloft ® )[ 24 ]. Moreover, drug-herb interactions might adversely affect the monitoring of certain drug therapies and might even cause life-threatening complications. For example, ginseng, hawthorn, licorice, kyushin, plantain, and uzara root have the potential to interfere with the monitoring of Lanoxin ® (digoxin)[ 25 ]. In addition, kava has been associated with hepatitis[ 26 ] and has resulted in coma when used with Xanax ® (alprazolam)[ 27 ]. As these detrimental effects have been realized, concern about the increased use of herbal supplements has grown[ 2 , 28 - 33 ]. Two-thirds of the patients we surveyed reported wanting to receive information on herbal medicines from their physicians or pharmacists, preferably in the form of a handout or a brochure. These findings suggest that future studies are warranted to develop and test educational materials to 1) deepen our understanding of racial/ethnic variation in herbal use among primary care patients; 2) educate health-care professionals about the variations in the use patterns and the rationales for use that may help to reduce medication errors and increase the quality and safety of medical care; and 3) educate patients regarding evidence-based herbal information and encourage patients to communicate their herbal use to their physicians/pharmacists. Our study results should be interpreted in the context of several limitations. First, our estimates of herbal use frequency are imprecise because we used a convenience sample instead of identifying patients by randomized sampling. Secondly, even though we adopted the DSHEA definition of herbs, some patients had difficulty understanding this definition. Specifically, a small group of patients thought that herbs were equivalent to prescription medications such as digoxin and aspirin; the patients' level of understanding of herbs was improved after the research assistants provided further explanation and clarification. Third, we discovered that asking questions, such as "What do you take when you run out of your medications?," was more effective in eliciting answers from the study subjects than when asking them, "Do you use herbs, herbal products or natural medicine?." For these reasons, we had research assistants on-site to help facilitate the survey process. Fourth, the patients surveyed reported their concomitant use of herbs with prescribed medications based on their perceptions and memories. Last, we did not include measures of quality of life or questions about patient satisfaction with herbal use, which would be helpful in future studies, especially when comparing multiethnic and socioeconomically diverse patient groups. Conclusions Despite these limitations, our findings confirm the increasing frequency of herbal use as reported in previous studies. Our study also gives a unique perspective by focusing on factors associated with reported herbal use among an urban multiethnic primary care patient population. In particular, we found that patients with immigrant family history—especially those with family members who use herbs—are most likely to report herbal use. Perhaps most disconcerting was our finding that while an increasing number of primary care patients report taking herbal medicines concomitantly with prescription medications, many of them do not disclose their herbal use to their physicians or pharmacists. These findings suggest that primary care clinicians need to understand the extent and patterns of herbal use by their multiethnic patients and efforts to elicit information from patients about herbal use may be warranted. Future studies are needed to develop effective interventions for primary care health-care professionals and patients to improve medication safety by eliminating potential adverse herb-drug interactions and medication errors. Competing interests The author(s) declare that they have no competing interests. Authors' contributions GMK conceived of the study, designed the survey questionnaires, coordinated and managed the data-collection process, directed data analysis, and drafted the manuscript. STH participated in drafting the manuscript and helped with data analysis. LTW performed the statistical analysis and participated in drafting the manuscript. RB helped with data analysis. RJV reviewed the questionnaires and data analysis, and participated in drafting the manuscript. All authors read and approved the final manuscript. Table 4 Multiple Logistic Regression Analysis of Factors Associated with Herbal Use Among Urban Multiethnic Primary Care Patients (n = 302) Variable OR 95% CI Main Effects Gender Male 1.00 Female 1.12 (0.60–2.11) Age (yrs) < 30 1.00 ≥ 30 1.37 (0.74–2.55) Race/Ethnicity African-American 1.00 White & Hispanic 2.42* (1.33–4.40) Education < College 1.00 ≥ College 1.11 (0.58–2.15) Immigrant Family History No 1.00 Yes 2.23* (1.20–4.14) Herbal Use by Other Family Member No 1.00 Yes 7.98* (4.48–14.18) Clinic Type High SES Clinic 1.00 Low SES Clinic 0.80 (0.40–1.60) Interactions Immigrant Family History * Herbal Use by Other Family Members 19.39 (8.11–46.38) * p < 0.05 Pre-publication history The pre-publication history for this paper can be accessed here:

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One year soy protein supplementation has positive effects on bone formation markers but not bone density in postmenopausal women

Background Although soy protein and its isoflavones have been reported to reduce the risk of osteoporosis in peri- and post-menopausal women, most of these studies are of short duration (i.e. six months). The objective of this study was to examine if one year consumption of soy-containing foods (providing 25 g protein and 60 mg isoflavones) exerts beneficial effects on bone in postmenopausal women. Methods Eighty-seven eligible postmenopausal women were randomly assigned to consume soy or control foods daily for one year. Bone mineral density (BMD) and bone mineral content (BMC) of the whole body, lumbar (L1-L4), and total hip were measured using dual energy x-ray absorptiometry at baseline and after one year. Blood and urine markers of bone metabolism were also assessed. Results and Discussion Sixty-two subjects completed the one-year long study. Whole body and lumbar BMD and BMC were significantly decreased in both the soy and control groups. However, there were no significant changes in total hip BMD and BMC irrespective of treatment. Both treatments positively affected markers of bone formation as indicated by increased serum bone-specific alkaline phosphatase (BSAP) activity, insulin-like growth factor-I (IGF-I), and osteocalcin (BSAP: 27.8 and 25.8%, IGF-I: 12.8 and 26.3%, osteocalcin: 95.2 and 103.4% for control and soy groups, respectively). Neither of the protein supplements had any effect on urinary deoxypyridinoline excretion, a marker of bone resorption. Conclusion Our findings suggest that although one year supplementation of 25 g protein per se positively modulated markers of bone formation, this amount of protein was unable to prevent lumbar and whole body bone loss in postmenopausal women.

Background It is estimated by the year 2010, 35 million women in the United States either will have osteoporosis or be at risk of developing the disease if appropriate preventive measures are not taken [ 1 ]. Aside from existing drug therapies, certain lifestyle and nutritional factors are known to reduce the risk of osteoporosis [ 2 - 5 ]. Additionally, there are a considerable number of women that would prefer dietary supplements as an alternative/adjunctive to conventional therapeutic options [ 5 ]. Examples of these alternative therapies include the use of natural or plant-based substances such as soy isoflavones [ 6 - 13 ]. Soy isoflavones have received considerable attention due to their estrogen-like properties on certain tissues such as bone, leading some investigators [ 14 , 15 ] to refer to them as naturally occurring selective estrogen receptor modulators (SERMs). Epidemiological data suggest that populations with high intakes of soy, i.e. Asians, have a lower incidence of osteoporotic fractures [ 16 , 17 ]. Asian women typically consume about 20 g of soy daily which provides approximately 40 mg isoflavones [ 18 , 19 ]. However, lower rates of fractures in these populations may not be fully attributed to soy consumption as there are a number of other confounding factors which can influence skeletal health. From the research point of view, there are a number of animal studies which have shown that soy protein and/or its isoflavones positively influence bone mineral density (BMD) [ 8 , 20 - 26 ]. In terms of human studies, there are limited numbers of trials that have examined the effects of soy and its isoflavones on bone. Some of these clinical trials [ 27 - 29 ] are of short duration varying between 3 to 6 months, making the findings questionable since periods less than one year may not be sufficient to detect clinically relevant changes in bone mass. Nonetheless, even the findings of the few clinical studies of one to two year duration that have been conducted [ 30 - 34 ] are inconclusive. For instance, Vitolins et al. [ 30 ] reported that daily consumption of 25 g soy protein with 5, 42 or 58 mg isoflavones had no bone preserving effects in peri- and post-menopausal women in a two-year study. Recent findings from two groups [ 31 , 32 ] have shown similar effects of soy protein and its isoflavones on bone. In contrast, other studies have suggested that isoflavone-rich soy milk delivering 80 to 90 mg isoflavones [ 33 ] or soy products delivering 40 to 60 mg isoflavones on a daily basis [ 34 ] have some bone protective effects. The purpose of the present study was to examine the effects of one-year supplementation of soy-based products containing 25 g protein and 60 mg isoflavones on BMD, bone mineral content (BMC), serum and urinary markers of bone turnover in postmenopausal women. The rationale for choosing this amount of soy protein was based on the average intake of Asians [ 18 , 19 ] and the recommended amount in the FDA approved health claim [ 35 ]. Methods Subjects Postmenopausal women younger than 65 years who were not on HRT or any prescription medications or herbal supplements, including soy isoflavones, known to positively influence bone were recruited. Women with cancer, liver disease, hypo- or hyperthyroidism, gastrointestinal disorders, insulin-dependent diabetes mellitus, pelvic inflammatory disease, and endometrial polyps were excluded from the study. The study protocol was approved by the Institutional Review Board at Oklahoma State University. Subjects signed a consent form after being provided with oral and written descriptions of the study. A complete medical history was obtained from all subjects before initiating the treatments. Subjects were also given routine physical and gynecological examinations. Subjects were independent living and were advised to maintain their usual physical activity. Study Design Eighty-seven eligible postmenopausal women were randomly assigned to one of two dietary treatments in a double-blind parallel study. The dietary treatments consisted of 25 g protein from soy products (donated by DrSoy Nutrition Irvine, CA) or comparative control. The test foods were in the form of a snack bar, drink mix or cereal and were consumed daily for a period of one year. The soy products were soy protein-based and delivered 60 mg isoflavones per day whereas the control regimen was devoid of soy protein and isoflavones. To ensure double-blinding, the study participants were randomly assigned to one of the two treatments and the study supplies were provided to the study participants in unlabeled packages. Additionally, the identity of each treatment was revealed to the investigators and research personnel involved in the collection and analyses of the data only after all analyses were completed. For this study compliance was measured in two forms. First, study participants were provided with customized calendars for subjects to record how much of each of the cereal, the snack bar, or the drink mix they consumed, if any. Second, study participants were asked to return any unconsumed foods to the study site so they could be tallied. The study participants were advised by a registered dietitian to make appropriate adjustments in their daily food consumption to account for the additional energy and nutrients supplied by the treatment regimen. Dietary assessment and anthropometric measurements For each subject, medical and nutrition histories were obtained at the beginning of the study. One-week food frequency questionnaires were completed via interview by a registered dietitian at the beginning and at the end of the study. Nutrient analysis was performed using food analysis software (Food Processor version 7.50, ESHA Research, Salem, OR). Anthropometric data were collected at the beginning, six months, and at the end of the study by a single trained staff member, as described elsewhere [ 36 ]. Height and weight were used to calculate body mass index (BMI). Abdominal and hip circumferences were used to calculate waist-to-hip ratio. Bone Density Assessments Bone density was assessed at the beginning and at the end of treatment using dual energy x-ray absorptiometry (DXA; Hologic QDR-4500C, Waltham, MA) equipped with appropriate software for whole body BMD and BMC. Additionally, select regional sites, i.e. total hip and lumbar spine (L1-L4) were analyzed using high resolution software. The intra- and inter- assay coefficients of variations were 3.4% and 5.1% and 2.5% and 4.7% for BMC and BMD, respectively. Gynecological exam and blood and urine collection Study participants were provided with routine physical and gynecological exams including a pap smear at baseline and at the end of the study. A venous blood sample was obtained after an overnight fast from each subject at the beginning, six months, and at the end of the study for various analyses. Blood samples were centrifuged at 2500 × g for 15 min at 4°C, serum samples were separated and stored at -20°C until analyses. Each study participant collected a 24-h urine specimen, excluding the first void, at the beginning, after six months, and at the end of the study. Urine volume was recorded and aliquots were stored at -20°C for later analyses. Analytical methods To assess whether soy isoflavones modulate sex steroids and their availability, serum levels of 17β-estradiol (E 2 ), estrone (E 1 ), estrone sulfate, follicle stimulating hormone (FSH), and sex hormone-binding globulin (SHBG) were assessed as we have previously described [ 37 ] using radioimmunoassay kits from Diagnostic Systems Laboratories Inc. (Webster, TX). Serum bone-specific alkaline phosphatase (BSAP) activity, a specific marker of bone formation [ 38 ], was quantified by immunoassay in a microtiter format (Metra Biosystems, Mountain View, CA). Alkaline phosphatase (ALP), a nonspecific marker of bone formation [ 38 ], was determined colorimetrically using a commercially available kit (Roche Diagnostics; Branchburg, NJ) and analyzed with a Cobas-Fara II Clinical Analyzer (Montclair, NJ). Additional serum biomarkers of bone formation i.e. osteocalcin, insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 (IGFBP-3), which usually increases parallel to IGF-I, were also measured using kits from Diagnostic Systems Laboratories Inc. Urinary creatinine was measured colorimetrically with commercially available kits (Roche Diagnostics) using a Cobas Fara II clinical analyzer. Urinary deoxypyridinoline (Dpd), a specific marker of bone resorption [ 39 ], was measured by competitive enzyme immunoassay in a microassay stripwell format (Quidel Corporation, Mountain View, CA). The intra- and inter-assay CVs were 4.3% and 4.6 %, and 6.5% and 8.6%, for creatinine and Dpd, respectively. Statistical Analyses Data were analyzed using analysis of variance methods with PROC MIXED in PC SAS (Version 8.2, SAS Institute, Cary, NC) analyzing the main and interaction effects of the two factors, treatment (soy protein or control) and time (baseline or after treatment), using the SLICE option. Since each subject was measured at baseline and after treatment, a split plot (repeated measures) model was utilized. The mean changes in endpoints for the soy protein and control treatment groups were compared by analyzing interaction effects of the two factors, treatment and time, using the SLICE option. Data are reported as least square mean ± standard error (SE); unless otherwise indicated, P < 0.05 was regarded as significant. Results Baseline characteristics, anthropometric measurements, and dietary intake Sixty-two of the 87 women completed the one year study resulting in an attrition rate of approximately 29%. Reasons for dropping from the study included medical conditions preventing continuation in the study (2 women in the soy group and 1 in the control group), starting HRT (1 woman in the soy group and 3 in the control group), noncompliance (2 women in the soy group), dislike of the volume or flavor of the food (3 women in the control group), gastrointestinal side effects (2 women in the control group), food was causing headaches (1 woman in the control group), and personal reasons (3 women in the soy group and 2 in the control group). Five additional women in the soy group decided to discontinue the study without citing a particular reason. Women in both treatment groups had similar baseline characteristics (Table 1 ). Body weight and BMI significantly increased in both treatment groups after one year of supplementation. On average, women in the soy group experienced a 1.6% increase in body weight, while a 3.3% increase was observed in women in the control group. Nonetheless, there was no significant change in waist to hip ratio in either treatment group. In terms of dietary intake, protein levels significantly increased in both treatment groups, as expected, due to the study supplements. Total caloric and carbohydrate intake increased in the control group, but decreased in the soy group after one year of supplementation (Table 2 ). No change in fat intake was found in either group. Bone mineral density and bone mineral content Subjects in both the soy and control groups lost whole body and lumbar BMD and BMC after one year, but no change was observed in the total hip (Table 3 ). Whole body BMD decreased by approximately 1.3% in both treatment groups, while lumbar BMD decreased by 1.0% in the soy group and 0.9% in controls (Figure 1 ). As shown in Figure 2 , whole body BMC decreased by 1.4% and 1.0%, and lumbar BMC was reduced by 1.5% and 1.1% in the control and soy groups, respectively. Although the change in whole body bone mineral area (BMA) of the soy-treated group was increased and the control group remained relatively unchanged, neither of these alterations reached the level of statistical significance (data not shown). The decrease in whole body BMD of subjects on soy may have resulted from the small increase in whole body area and a significant decrease in BMC, whereas the change in BMD in the control group was primarily a reflection of a decrease in BMC. Lumbar and total hip BMA was unaltered by either dietary treatment ( data not shown) . Serum and urinary parameters of relevance to bone and calcium metabolism Both dietary protein supplements significantly increased serum markers of bone formation, i.e. osteocalcin, BSAP, IGF-I and ALP (Table 4 ). However, IGFBP-3, which usually increases parallel to IGF-I, was only significantly increased in the women consuming the soy products. Neither proteins had any effect on bone resorption as indicated by urinary Dpd (Table 4 ). Since there are some reports [ 40 - 42 ] indicating that soy isoflavones may modulate sex steroids, we assessed serum levels of FSH, E 2 , E 1 , and estrone sulfate. None of the treatment significantly influenced these sex hormones (Table 5 ). However, soy but not control supplementation significantly decreased SHBG concentrations by 14.5%. Discussion The role of soy protein and its isoflavones in the maintenance of health such as the prevention of cardiovascular disease, certain types of cancer, and menopausal symptoms is now widely recognized [ 43 - 50 ]. In terms of bone, there are animal [ 20 - 26 ] and human [ 27 - 34 ] studies that have explored the role of soy in maintaining or increasing bone mass. In general, animal studies have shown that isoflavones in the context of soy protein have positive effects on BMD [ 20 - 26 ]. The findings of clinical trials have ranged from no significant changes [ 27 - 32 ] or a slight increase [ 28 , 33 , 34 ] in BMD. Nonetheless, the bone protective effects of soy and/or its isoflavones are at best inconclusive. In the present study, the daily consumption of 25 g protein for one year irrespective of the source resulted in no significant changes in hip BMD and BMC. Other investigators [ 51 , 52 ] have reported that diets high in protein were associated with higher BMD in femoral neck. We speculate that higher dietary protein may have a protective effect on hip BMD over the long term. This notion, however, seems somewhat paradoxical because high protein diets, especially proteins rich in sulfur-containing amino acids, are known to increase urinary calcium that may result in accelerated bone loss [ 53 ]. Nonetheless, a counter-argument has been made that protein-associated hypercalciuria is due to enhanced intestinal calcium absorption and not the breakdown of bone [ 54 , 55 ]. Our findings do not support a bone protective role for soy protein and its isoflavones at the level used in this study. Whether higher amount of soy protein and/or its isoflavones can reverse bone loss remains to be illustrated. Nonetheless, higher doses of soy protein with varying levels of isoflavones have not consistently shown to exert beneficial effects on bone. For instance, Gallagher et al. [ 32 ] supplemented the diets of postmenopausal women for nine months with 40 g soy protein delivering three levels of isoflavones (0, 52, and 96 mg) but all three groups experienced bone loss. On the other hand, six-month studies by Potter et al. [ 28 ] and Alekel and colleagues [ 29 ] reported positive effects of soy protein supplementation on BMD. Potter et al. [ 28 ] showed that 40 g of soy protein containing 90 mg isoflavones was able to attenuate lumbar spine (L1-L4) BMD, however, the same amount of protein with 56 mg isoflavones had no such an effect. Although Alekel et al. [ 29 ] suggested that 40 g soy protein supplementation with 80 mg isoflavones was able to attenuate bone loss from lumbar spine, women still lost 0.2% BMD in six months. Their data [ 29 ] imply that soy protein or its isoflavones are incapable of increasing bone mass in perimenopausal women. As for an effect of soy isoflavones alone, Chen et al. [ 56 ] recently reported that supplementing postmenopausal women with soy isoflavones (40 and 80 mg/d) for one year resulted in favorable increases in BMC of the hip in women who are at least four years postmenopausal and are of low body weight or have low levels of dietary calcium. Similar to that study [ 56 ], the majority of the women in our present study were four or more years postmenopausal; however, our study participants had adequate calcium intakes and did not have low body weights. It is possible that this difference in the nutrition status of the study participants between the two studies may be responsible for the discrepancy in the observed effects on bone. As far as which of the many isoflavones in soy is responsible for the effects on bone, to date, the most convincing data on the effect of a single isoflavone, genistein, on bone have been reported in a one-year study by Morabito and colleagues [ 57 ]. They demonstrated that both genistein at a dose of 54 mg/d and HRT increased BMD in early postmenopausal women. In that study [ 57 ], genistein significantly increased BMD of the femoral neck by 3.6% and lumbar spine by 3.0% while HRT increased femoral neck and lumbar spine BMD by 2.4 and 3.8%, respectively. These authors [ 57 ] suggested that genistein reduces bone resorption markers and enhances new bone formation parameters resulting in a net gain of bone mass. Isolated isoflavones derived from other sources such as red clover have also been found to positively affect bone. For example, Clifton-Bligh and colleagues [ 58 ] reported that clover-derived isoflavones at doses of 57 and 85 mg isoflavones/day were able to significantly increase BMD of the proximal radius and ulna by 4.1 and 3%, respectively after 6 months. Similarly, Atkinson et al. [ 59 ] showed that red-clover derived isoflavones (26 mg biochanin A, 16 mg formononetin, 1 mg genistein, and 0.5 mg daidzein) slowed the loss of lumbar spine BMC and BMD. These data suggest that isoflavones from sources other than soy, also have osteoprotective effects. In the present study, biomarkers of bone formation, i.e. osteocalcein, IGF-I, and BSAP were all significantly elevated in both groups. However, the specific marker of bone resorption, urinary Dpd, was not altered. A number of clinical studies have evaluated the effects of soy protein with its isoflavones on bone biomarkers. Overall, the effects of soy and/or its isoflavones have produced no consistent effects on biomarkers of bone turnover. For instance, biomarkers of bone formation have been reported to either increase [ 60 , 61 ] or not change [ 29 , 62 ] as result of soy supplementation. Similarly, biomarkers of bone resorption have been reported to decrease [ 62 - 64 ], not change [ 61 ] or even increase [ 60 ]. We have previously reported that 40 g of soy protein providing 90 mg of isoflavones/day reduced Dpd in postmenopausal women not on HRT [ 62 ]. In the present study, participants were asked to consume a lower amount of soy protein (only 25 g providing 60 mg of isoflavones/day). It is possible that the reduced dose of soy in this study may have contributed to the lack of effect on bone resorption (as assessed by Dpd excretion) and that a dose-response study may be justified in order to achieve both increases in markers of bone formation and reductions in markers of bone resorption. Nevertheless, the positive changes in biomarkers of bone formation in the present study have not translated to increases in BMD and BMC. Whether the positive effects of protein supplementation on bone biomarkers would translate to better bones needs to be assessed in a longer term study. Although in this study, soy supplementation for one year did not produce any estrogenic effects as assessed by circulating sex hormone levels, it did decrease SHBG concentrations Decreases in SHBG result in increases in the availability of circulating estrogens [ 65 ]. Thus, soy supplementation may have increased the availability of estrogens without affecting actual concentrations. However, this is speculative and measurement of bioavailable estradiol is necessary to confirm this statement. From the findings of our study and the collective review of existing literature, it is too early to state whether soy protein or its isoflavones can be substituted for estrogen in preventing the bone loss induced by ovarian hormone deficiency. Future studies are needed to address numerous questions including but not limited to whether: 1) isoflavones independent of soy protein can prevent ovarian hormone deficiency-associated bone loss; 2) consumption of soy containing food or intake of isoflavones on a daily basis is necessary to observe the expected beneficial effects on bone or simply intermittent use will produce the same results; 3) the effect of soy protein or its isoflavones on bone is transitory; and 4) the combination of soy isoflavones and lower doses of antiresorptive agents can prevent postmenopausal bone mineral loss. As these and other questions are answered, the efficacy of soy protein and its isoflavones as alternative and/or adjunctive treatments for postmenopausal osteoporosis can be determined. Competing interests The author(s) declare that they have no competing interests.

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Genome-wide microarray analysis of TGFβ signaling in the Drosophila brain

Background Members of TGFβ superfamily are found to play important roles in many cellular processes, such as proliferation, differentiation, development, apoptosis, and cancer. In Drosophila , there are seven ligands that function through combinations of three type I receptors and two type II receptors. These signals can be roughly grouped into two major TGFβ pathways, the dpp /BMP and activin pathways, which signal primarily through thick veins (tkv) and baboon (babo) . Few downstream targets are known for either pathway, especially targets expressed in the Drosophila brain. Results tkv and babo both affect the growth of tissues, but have varying effects on patterning. We have identified targets for the tkv and babo pathways by employing microarray techniques using activated forms of the receptors expressed in the brain. In these experiments, we compare the similarities of target genes of these two pathways in the brain. About 500 of 13,500 examined genes changed expression at 95% confidence level (P < 0.05). Twenty-seven genes are co-regulated 1.5 fold by both the tkv and babo pathways. These regulated genes cluster into various functional groups such as DNA/RNA binding, signal transducers, enzymes, transcription regulators, and neuronal regulators. RNAi knockdown experiments of homologs of several of these genes show abnormal growth regulation, suggesting these genes may execute the growth properties of TGFβ. Conclusions Our genomic-wide microarray analysis has revealed common targets for the tkv and babo pathways and provided new insights into downstream effectors of two distinct TGFβ like pathways. Many of these genes are novel and several genes are implicated in growth control. Among the genes regulated by both pathways is ultraspiracle , which further connects TGFβ with neuronal remodeling.

Background TGFβ pathways are conserved between primitive animals, such as sponges and sea anemone [ 1 , 2 ] and vertebrates, thus representing an ancient signal transduction pathway. In both vertebrates and invertebrates, TGFβ family members play fundamental roles in proliferation, pattern formation, apoptosis, and specification of cell fate. Mutations of various TGFβ signaling components are associated with human diseases including cancer [ 3 ]. In recent years, the core signaling components of the TGFβ pathways have been elucidated by a combination of genetics and biochemical approaches. Unique to these signaling pathways are transmembrane receptor serine-threonine kinases that are novel in animals. Signaling is initiated when dimeric ligands bind to the type I receptor or a complex of the type I and type II receptors. The type II receptor phosphorylates the type I receptor, which renders it active. R-Smads are phosphorylated by the type I receptor, the complex with a co-Smad, and translocate to the nucleus. Smads bind DNA promoter elements weakly and require co-factors for efficient regulation of target genes. In Drosophila , seven ligands have been identified from the genomic sequence [ 4 - 6 ]. These ligands act through a receptor complex comprised of heterodimeric combinations of type I and type II receptors. Three type I receptors, thick veins (tkv) , saxophone , baboon (babo) and two type II receptors, punt and wishful thinking (wit) , interact with either of two R-Smads, mothers against dpp (mad) or dSmad2 [ 7 - 12 ]. Although different heteromeric combinations of receptors exist, in general, tkv transmits a dpp /BMP signal through mad , and babo transmits an activin signal through dSmad2 . The dpp and activin pathways have known functions in the brain, although our understanding of it role is rudimentary. dpp is expressed in two areas adjacent to the outer proliferation center (OPC), where it modulates wingless expression [ 13 ]. To acquire the adult pattern of projections, extensive remodeling occurs in neurons of the larval neural circuits during metamorphosis [ 14 ]. Proper neuronal remodeling is important for transformation of the larval mushroom bodies (MBs) to the adult MBs [ 15 , 16 ]. babo and dSmad2 (activin pathway components) are involved in neuronal remodeling, which occurs in the larval-pupal transition [ 17 ]. One target of the activin pathway identified in these studies is a subunit of the ecdysone receptor, EcR-B. Neuronal remodeling is essential for brain development in most animals and this result raises the question of possible conservation of neuronal targets in vertebrates. In spite of intense study using classical genetic approaches and biochemical methods, very few targets of the pathway have been identified. A better understanding of the growth and patterning properties of the pathway require a more complete list of target genes. Using activated receptors ( tkv and babo ), we have used microarray technology to identify common targets of the BMP and activin pathways in the Drosophila brain. Results and discussion Identifying targets of babo and tkv in Drosophila brains Few targets of tkv signaling and even fewer targets of babo signaling are known in Drosophila . Though multiple ligands and type II receptors may interact with these type I receptors, the use of ligand/receptor combinations is not yet established with certainty. However, in a simplified view, tkv and babo send dpp (BMP) and activin signals. These pathways and receptors are conserved through evolution, but few downstream targets are known for these pathways in any organism. To learn more about the growth regulatory and pattering properties of these signals in the fly brain, we used microarray technology to identify downstream targets. In these experiments, Affymetrix™ chips containing the entire protein coding capacity of the Drosophila genome (about 13,500 genes) were screened. Genomic-wide microarray analysis allows us to examine similarities and differences between two signaling pathways in a tissue where both are known to function. Constitutively active forms of the receptors were made by single amino acid substitutions [ 18 ], rendering them active in the absence of ligand. Transformants were generated which could be transcriptionally expressed using the heat shock GAL4 driver ( hs -GAL4) [ 19 , 20 ]. To assay for the best induction protocol, animals were heat shocked and monitored for the presence of a UAS- gfp reporter. Since additional time is required for the induction of downstream signaling targets versus the time required for appearance of the GFP reporter, we collected RNA samples from third instar larvae in a broad time period roughly 30 minutes after the peak of GFP expression. Data resulting from induced ectopic expression of tkv , and babo were compared with each other and to the control (UAS- gfp; hs -GAL4). Three independent replicates for each treatment were generated. Hierarchical clustering (Fig 1A ) and Principal Component Analysis (Fig 1B ) indicated that the microarray data is highly reproducible. Only transcripts that show an expression level above 1.5 fold change at significance values of P < 0.05 (Anova) were considered to be differentially expressed. These experiments identified genes that are either regulated by both tkv and babo pathways or by one of the pathways only. Figure 1 Clustering of microarray data. A. Agglomerative Hierarchical Clustering of microarray data (P < 0.05, Group T, C, B represent individual samples with ectopic expression of tkv , control, and babo respectively); B. Principal Component Analysis (PCA) of microarray data (P < 0.05, Spheres in red, blue and yellow represent individual samples with ecotopic expression of babo , tkv , and control respectively). To verify the differential expression levels in response to ectopic expression of tkv and babo on microarrays, semi-quantitative real-time RT-PCR was performed on selected genes. Six among the 27 genes were picked for validation. Real time RT-PCR showed similar results similar to the microarray results for four of the six genes (Fig 2 ). These PCR results are consistent with those of other reported microarray experiments [ 21 , 22 ]. This data, with the reproducibility of the individual samples analyzed, establish the validity of our microarray data and provide a comparison of two signaling pathways in the Drosophila brain. Figure 2 Validation of microarray data by real time RT-PCR. The X-axis indicates fold changes (FC) of gene expression levels between tkv/babo ectopic expression and control (Positive values indicate that the relative expression level of a gene is increased and negative values indicate a decrease). Array /PCR ( tkv , babo ) represents the fold changes of transcripts with ectopic expression of tkv , babo in microarray /Real time PCR respectively. Overview of gene expression following ectopic expression of babo and tkv Upon ectopic expression of tkv , 91 transcripts are detected with differential expression values in brain tissues when compared with the control (Fig 3 ). This corresponds to about 0.7% of the transcripts on the array. More transcripts are down regulated (n = 60) than up regulated (n = 31) in abundance levels, indicating that ectopic expression of tkv causes both repression and activation of downstream genes. Induction of activated babo results in 216 genes with differential expression values in brain tissues. Interestingly, expression levels of more transcripts are decreased (n = 126) than increased (n = 90) (Fig 3 ). This corresponds to about 1.6% of the transcripts on the array. Most importantly, there are 27 genes co-regulated by induction of both babo and tkv – 17 of these transcripts are down regulated and 10 of them are up regulated (Fig. 3 ). Figure 3 Distribution of differential regulated genes by ecotopic expression of tkv and babo . Upon ectopic expression of tkv , 60 transcripts are downregulated and 31 are upregulated. Similarly, at ectopic expression of babo , we detected 216 genes with differential expression values in brain tissues and expression levels of 126 transcripts are decreased and 90 transcripts are increased. There are 27 genes coregulated at ectopic expression of both babo and tkv . 17 of these transcripts are downregulated and 10 of transcripts are upregulated. Role for TGFβ signaling in neuronal remodeling The fact that both DPP and activin signaling pathways share some common features in differentiation and growth control in various tissues suggests that both pathways might share some downstream target genes. Microarray experiments identified 27 genes (Table 1 ) co-regulated by the induced expression of both tkv and babo . Among these 27 co-regulated transcripts, there are transcription factors, enzymes, transporters, signal transducers, miscellaneous proteins and four unknown genes (Fig 4 ). The transcription factor ultraspiracle ( usp ) gene has the highest expression level increase (8.1-fold for babo , 27.3-fold for tkv ), which is a subunit of a nuclear receptor [ 15 ]. USP forms a heterodimer with the nuclear ecdysone receptor (EcR) and participates in neuronal remodeling [ 15 , 23 ]. Table 1 Changes in transcript levels of the coregulated genes by both tkv and babo pathways after ectopic expression of tkv and babo . (*FC represents the fold changes in gene expression levels between tkv/babo ectopic expression and control. Positive values indicate that the relative expression level of a gene is increased (upregulation) and negative values indicate a decrease (downregulation)). Gene/synonym Signal FC* Molecular function P babo tkv babo tkv Transcription factors usp 41 137 8.1 27.3 transcription factor, DNA binding, ligand-dependent nuclear receptor, ecdysteroid hormone receptor 0.0001 CG7839 36 34 1.7 1.6 transcription factor 0.0007 TfIIFβ 208 249 1.2 1.5 RNA polymerase II transcription factor 0.0000 CG14422 9 12 -3.8 -2.9 RNA binding /nucleic acid binding/transcription regulator 0.0115 Antp 24 25 -1.5 -1.4 specific RNA polymerase II transcription factor 0.0228 Enzymes and enzyme regulators ia2 351 276 7.3 5.8 protein tyrosine phosphatase 0.0000 CG1827 32 26 2.3 1.8 N4-(beta-N-acetylglucosaminyl)-L-asparaginase 0.0010 ninaC 22 22 1.8 1.9 myosin ATPase, protein serine /threonine kinase 0.0127 G-iα65A 322 259 1.8 1.4 heterotrimeric G-protein GTPase 0.0028 Sucb 148 142 1.6 1.5 succinate-CoA ligase 0.0096 CG7288 117 125 1.5 1.6 ubiquitin-specific protease 0.0040 CG8913 50 69 1.1 1.5 peroxidase 0.0000 CG9236 7 5 -2.6 -3.4 calcium-dependent protein serine/threonine phosphatase 0.0199 Transporters CG8533 16 12 -2.6 -3.5 glutamate-gated ion channel 0.0000 CG6293 28 49 -2.5 -1.4 L-ascorbate:sodium symporter 0.0004 Atpa 105 126 -1.5 -1.3 sodium/potassium-exchanging ATPase 0.0003 Fatp 240 219 -1.4 -1.5 long-chain fatty acid transporter 0.0003 Signal transducers usp 41 137 8.1 27.3 transcription factor, DNA binding, ligand-dependent nuclear receptor, ecdysteroid hormone receptor 0.0001 ninaC 22 22 1.8 1.9 myosin ATPase, serine/threonine kinase, calmodulin binding 0.0127 CG8533 16 12 -2.6 -3.5 glutamate-gated ion channel 0.0000 Structural protein CG14889 50 50 -2.8 -2.8 extracellular matrix /structural molecule 0.0023 Miscellaneous proteins CG2807 78 105 -2.3 -1.7 pre-mRNA splicing factor 0.0034 CG32423 431 428 -2.1 -2.1 RNA binding 0.0000 XRCC1 56 20 -1.4 -3.8 DNA repair protein 0.0000 Cyp9f2 121 104 -1.5 -1.8 cytochrome P450 0.0012 Cyp9f3 77 70 -1.5 -1.7 pseudogene 0.0038 Unknown CG3857 237 186 -2.4 -3.0 NA 0.0000 CG7986 130 117 -1.5 -1.7 NA 0.0009 CG31150 66 69 -1.5 -1.4 NA 0.0160 CG33187 148 125 -1.3 -1.6 NA 0.0035 Figure 4 Gene ontology of coregulated genes by both DPP and activin signaling pathways. The X-axis indicates the number of genes in each group. Previous studies have shown that the Drosophila activin signaling pathway partially mediates neuronal remodeling through regulating EcR-B1 expression [ 17 ]. Two independent mutations that block neuronal remodeling in the mushroom bodies (MBs) during pupation were found to reside in babo and dSmad2 [ 17 ], both of which have been shown to participate in the activin signaling pathway [ 7 , 9 ]. Further, mutations in these signaling components reduce the expression of EcR-B1, and restoration of EcR-B1 expression rescues neuronal remodeling defects. These observations led to the model that the Drosophila activin signaling results in induction of the EcR-B1 isoform. Upon binding of ecdysone to the EcR-B1/USP heterodimeric receptors, neuronal remodeling is initiated via transcriptional activation of downstream target genes [ 17 ]. Our microarray analysis shows that high level expression of usp is also induced by ectopic expression of tkv and babo . In addition, we find that EcR-A expression is repressed by the induction of babo . Using real-time PCR, we confirmed that EcR-B1 is induced by ectopic expression of babo (1.5 fold), a more modest change than the increases on usp by tkv and babo . These finding suggest that Drosophila activin signaling mediates neuronal remodeling by regulation of both EcR-B1 and usp expression, while inhibiting EcR-A induction. BMP-like pathways, as well as activin pathways, have been implicated in neuronal remodeling [ 13 , 17 ]. PUNT and WIT have been shown to have a redundant function in inducing EcR-B1 expression during brain development. In mutant clones, levels of EcR-B1 were unaffected, unless both receptors were mutant. These results are consistent with our findings that activated tkv and babo both induce EcR-B1, although it is not known which receptor combinations or ligands are responsible for these effects. dpp (and presumably tkv ) has other known roles in organizing the visual center of the brain [ 13 ]. It has been shown that wingless , acting through dpp , is an important participant in organizing the optical centers of the brain [ 13 ]. wingless is expressed at the tips of the crescent shaped OPC. Fourteen hours later, wingless induces dpp expression in adjacent cells, in two spots in each brain hemisphere. These dpp expressing cells also express fasciclin II. BrdU staining shows that wingless , dpp , and fasciclin II expressing cells proliferate throughout larval development. However, a reduction of wingless or dpp results in a reduction in the rate of proliferation in the OPC, resulting in smaller optic lobes of the brain. Loss of wingless also results in a severe reduction of the medulla, where the photoreceptor axons R7 and R8 migrate. Another defect noted in wingless mutant animals is that the OPC derived precursor cells had failed to assume their proper neuronal fate. Transcription factors regulated by both DPP and activin pathways Besides usp , two other transcription factor genes, CG7839 and TfIIFβ, are up regulated by tkv and babo . Both are implicated in growth processes. CG7839 has 30% homology over 1016 residues to C. elegans F23B12.7, which shows a slow growing phenotype in RNAi experiments [ 24 ]. TfIIFβ is part of the RNA transcriptional machinery, and 28% of glioblastomas and 80% of astrocytomas show amplification of this gene. Perhaps part of the growth potential of the tkv and babo TGFβ pathways operate through these transcription factors. Two transcription factors, CG14422 and Antennapedia ( antp ), are down regulated by both pathways during brain development. antp is a well-studied Hox gene in Drosophila , which controls many developmental decisions, most notably, the differentiation of the antennae and legs from homologous structures [ 25 ]. The enormous diversity of body plans in animals is partially due to the variations that Hox transcription factors regulate gene expression. Most animals have one or more clusters of Hox genes, and each Hox gene controls the development of a specific region of the body plan [ 26 ]. In Drosophila , differences between segments, such as the presence or absence of appendages, are often controlled by Hox transcription factors. The role of antp in brain development is not known, but it is tempting to speculate that both dpp and activin might regulate brain development, at least partially, through interaction with the Hox gene antp . Determining the mechanisms by which Hox proteins regulate gene expression will be important for understanding animal development and pattern formation. Other genes regulated by tkv and babo pathways Many of the other genes that are significantly regulated by tkv and babo are evolutionarily conserved throughout animal phyla. Quantitative analysis of transcript levels indicates that TGFβ controls some genes that encode kinases and phosphatases that might be involved in signaling pathways. For example, ia2 , a transmembrane receptor protein phosphatase [ 27 ], has the highest level of transcriptional change among these kinases and phosphatases. Antibodies to the human version of the gene are often indicative of diabetes [ 28 - 30 ]. NinaC is a protein serine/threonine kinase [ 31 ] with calmodulin binding activity [ 32 ]. CG9236 is a calcium-dependent protein serine-threonine phosphatase, which is down regulated. It is strongly related to C. elegans F30A10.1, which is involved in negative regulation of body size. If the function of the protein has also been conserved, then down-regulation by the TGFβ-like pathways would allow growth in the developing brain. Other kinases and phosphatases co-regulated by both TGFβ pathways are G-iα65A (G-ialpha65A), a G-protein coupled receptor protein involved in neuroblast cell division and cell size control [ 33 , 34 ], and CG 9236, a calcium-dependent protein serine/threonine phosphatase [ 27 ]. CG3857 and CG7986 are two novel proteins that have homologs in C. elegans and in vertebrates. While their molecular functions are not currently known, the C. elegans CG7986 homolog F41E6.13 is involved in positive regulation of growth. RNAi experiments with the C. elegans homolog of CG3857, Y54E2A.2, revealed no mutant phenotype. Four transporters (Atpa, Fatp, CG8533, CG6293) are transporters regulated by the tkv and babo pathways. Fatp is a long-chain fatty acid transporter. Atpa is a sodium/potassium-exchanging ATPase, while CG8533 is a glutamate-gated ion channel and CG6293 is a L-ascorbate:sodium symporter. Conclusions Microarray experiments revealed that 27 genes are co-regulated in both tkv and babo signaling pathways in the developing Drosophila brain. One of the most striking developmental events in the fly brain is neuronal remodeling. These results indicate usp is positively regulated by tkv and babo , and thus adds another important link to their roles in brain remodeling. Many of the 27 genes are strongly conserved in other species. If their biological functions are also conserved, then the RNAi experiments in their C. elegans counterparts show that several of them are involved in growth regulation. This is particularly useful since few downstream targets of BMP or activin signaling pathways are known, particularly the targets that execute their growth regulatory properties. Not surprisingly, mutational analysis of several of these genes has not been done, but the genetic tools in Drosophila make this relatively straightforward. Further characterization of these downstream genes may provide insights into the integration of tkv and babo signaling pathways in Drosophila brain development, and provide hints into their functions in other organisms. Methods Fly stocks For over-expression of constitutively activated tkv , virgin females from UAS-CA- tkv were crossed to hs -Gal4 males. For over-expression of constitutively activated babo and the control, UAS-CA- babo and UAS- gfp were crossed to hs -Gal4 flies. The larvae were raised in standard medium at 25°C. Heat shock treatment and RNA purification Wandering third-instar larvae were heat-shocked to induce ectopic expression of tkv, babo , and the gfp control (UAS- gfp; hs -GAL4). Animals were heat shocked at 37°C for 1 hour, followed by cooling to room temperature for 30 minutes, and then kept at 25°C for one hour to allow expression before dissection. Approximately 150-200 larvae were dissected and the brains were collected in a drop of PBT (PBS, 0.01% Tween-20, pH 7.4) on Sylgard (Dow Corning). Total RNA was extracted from the tissue using the Trizol™ reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Preparation of labeled cRNA Total RNA from each of nine independent samples (three tkv , three babo and three gfp ) was prepared for hybridization according to the Affymetrix GeneChip ® Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). The Superscript Choice System kit (Invitrogen, Gaithersburg, MD) was used to make complementary DNA (cDNA) from 5 μg. First strand synthesis was primed with a T7-(dT) 24 oligonucelotide primer containing a T7 RNA polymerase promoter sequence on the 5' end (Genset Oligos, La Jolla, CA). Second strand products were cleaned with the GeneChip ® Sample Cleanup Module (Affymetrix, Santa Clara, CA) and used as a template for in vitro transcription (IVT) with biotin-labeled nucleotides (Bioarray High Yield RNA Transcript Labeling Kit, Enzo Diagnostics, Farmindale, NY). The copy RNA (cRNA) product was cleaned with the GeneChip ® Sample Cleanup Module (Affymetrix, Santa Clara, CA) and a 20 μg aliquot was heated at 94°C for 35 min in fragmentation buffer provided with the Cleanup Module (Affymetrix, Santa Clara, CA). Microarray hybridization Fifteen μg of adjusted cRNA from each sample was hybridized for 16 hr at 45°C to an Affymetrix (Santa Clara, CA) Drosophila Genechip 1 array. After hybridization, each array was stained with a streptavidin-phycoerythrin conjugate (Molecular Probes, Eugene, Oregon), washed and visualized with a Genearray™ Scanner (Agilent Technologies, Palo Alto, CA). Images were inspected visually for hybridization artifacts. In addition, quality assessment metrics were generated for each scanned image and evaluated based on empirical data from pervious hybridizations and on the signal intensity of internal standards that were present in the hybridization cocktail. Samples that did not pass quality assessment were eliminated from further analyses. Generation of expression values Microarray Suite version 5 (Affymetrix, Santa Clara, CA) was used to generate *.cel files. Probe Profiler™ version 1.3.11 software (Corimbia Inc, Berkeley, CA) was used to convert cel file intensity data into quantitative estimates of gene expression for each probe set. For each probe set, a probability statistic is generated. Genes not significantly expressed above background in any of the samples (P > 0.05) were considered absent. Absent genes were removed from the data set and not included in further analyses. Data analysis Tests of Significance Gene expression levels were subjected to a 1-way analysis of variance (Anova) for 3 treatments (B, C, T) and 3 replications using AnalyzeIt Tools, a custom software program developed by the Interdisciplinary Center for Biotechnology Research (ICBR, University of Florida), for the analysis of microarray data. In this software, the statistical package, R, serves as the backend for Anova. Genes were considered to have a significant treatment effect if P-level was less than 0.05. The expression values of those genes that were considered to have a significant treatment effect were normalized by performing a Z- transformation [ 35 ], thereby generating a distribution with mean 0 and standard deviation of 1 for each gene. Hierarchical clustering, K-Means clustering and Principal Component Analysis were performed on normalized values using GeneLinker™ Gold 3.1 (Predictive Patterns, Kingston, Ontario). To eliminate noise from low-level expression, spots quantified less than 5 were replaced by value 5. The following criteria were used to filter the data. Only transcripts with the fold change difference over 1.5 ( tkv (or babo ) average/Control average or Control average/ tkv (or babo ) average) and statistically significant (P <= 0.05, analysis of variance (Anova)) were considered as differentially expressed. AnalyzeIt Tools and notations in Flybase were used for classification of genes by gene ontology in molecular function and biological process categories. Real time RT-PCR Two independent total RNA samples were generated for each of the three experimental conditions (two tkv , two babo and two gfp ). Each of the samples were analyzed three independent times, resulting in six repeats. These six repeats were averaged and the tkv and babo samples were compared with the gfp controls. Approximately 1 μg of the each total RNA was used for first strand cDNA reaction using Superscript First Strand Synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. For real-time PCR, the reaction consisted of cDNA first strand template, primer mix, Rox (Invitrogen, Carlsbad, CA) and SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA) in a total volume of 25 μl. Three reactions per template were performed in parallel. Actin 42F was used as an internal standard to generate a standard curve and to normalize the amount of cDNA samples. The fold change (as presented in Fig 2 ) was calculated from the average real time PCR data: ( tkv or babo ) average/Control average or Control average/( tkv or babo ) average. The experiments were performed using a Rotor Gene 3000 (Corbett Research, Sydney, Australia). To validate the specificity of PCR reaction, a melting curve was produced by denaturation of PCR end products from 60 to 99°C at 0.5°C/min steep and the end products were also assayed with 1.5% agarose gel electrophoresis after cycling. Authors' contributions MY and DN carried out experiments in the project and YF assisted in the experimental design. RWP implemented and supervised the project. RWP and MY prepared the manuscript.

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539066

Hirsute or Hairless? Two Proteins May Spell the Difference

If you're a cat fancier, you're well aware that hair follicles are expendable. The product of a spontaneous mutation that caught a cat breeder's eye, le chat nu , would quickly succumb in the wild—its winter coat consists of little more than a ridge of fur down the midback and tail—and needs special care to thrive as a pet. Hairless animals in the lab, on the other hand, can be very instructive. Understanding how hair develops sheds light on the fundamental processes that generate a wide range of tissues and organs, including the lungs, cornea, and mammary glands. How complex, three-dimensional structures emerge from single sheets of cells is a fundamental question in developmental biology. The dispensability of hair follicles makes them the perfect model system for studying this question—specifically, how structures and organs develop from buds. In a new study, Elaine Fuchs and colleagues use a three-pronged approach—involving gene expression analysis, transgenic mice, and cell cultures—to study how epithelial buds, the precursors of hair follicles, form. Their experiments point to two key actors in a signaling pathway that molds a targeted cluster of cells into a hair bud. During the budding process, overlapping signaling pathways from two adjacent embryonic cell layers—the epithelium and the mesenchyme—direct morphogenesis. The mesenchymal cells initiate the cell-to-cell “crosstalk” that controls bud formation by first directing a small cluster of epithelial cells to form a placode, the pouch that forms hair plugs. The placode in turn directs underlying mesenchymal cells to form the base of the hair follicle, called the dermal papilla, and both structures contribute to the mature hair follicle. During development, cells are constantly bombarded with external signals. The trick is figuring out which signals trigger the transcriptional and behavioral properties in cells that spur bud formation. In previous experiments, Fuchs and colleagues showed that reducing expression of E-cadherin—a membrane protein that forms the adhesive junctions between epidermal cells—is essential for allowing the cell remodeling required for bud formation. Here, the authors analyze the timing of external signals against the response of targeted cells to determine how targeted cells translate signals into changes in cell adhesion and remodeling, proliferation, and differentiation—the agents of most types of organogenesis. Transgenic epidermis expressing Snail (red) results in expanded keratin 1 expression (green) Since Snail, a protein that impedes the transcription of a subset of genes, functions in many developmental processes requiring epithelial remodeling, the authors reasoned it might do the same in hair bud formation. Working with developing mouse embryos, they saw a spike in Snail expression on embryonic day 17.5, coinciding with hair bud formation, enhanced cell proliferation, and the down-regulation of E-cadherin. Artificially sustaining Snail expression in the skin of transgenic mice caused abnormal levels of cell proliferation in the epidermis and reduced cell adhesion. Working with skin keratinocytes, precursors of hair fibers, Fuchs and colleagues explored several signaling proteins known to be involved in bud formation as possible activators of Snail expression. When the authors treated keratinocytes with small amounts of one stimulator, TGF-β2, they saw “rapid and transient induction of Snail.” Snail proteins were absent from 17.5-day-old knockout mice lacking TGF-β2 but not from their nonmutant littermates. Conversely, transgenic mice with elevated TGF-β2 signaling activity displayed ectopic expression of Snail. Knockout mice lacking TGF-β2 also showed higher levels of E-cadherin—normally down-regulated by Snail—than their nonmutant littermates. Altogether, these findings suggest that TGF-β2 signaling transiently induces Snail, which in turn down-regulates E-cadherin and activates a proliferation pathway in the developing bud. Reduced E-cadherin, the authors conclude, appears to contribute to Snail-mediated enhanced proliferation by allowing proteins normally sequestered at the membrane to operate in a proliferation pathway after the number of cellular junctions diminishes. By identifying which molecules are active in specific cell types at specific developmental stages, this study lays the foundation for dissecting the mechanisms that connect two key processes—intercellular remodeling and proliferation—in epithelial development. And since the consequences of TGF-β2 activity seen here in the hair bud more closely resemble certain types of skin cancer progression than skin development, a mechanistic understanding of hair follicle development promises to shed light on how skin cancer develops as well.

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526191

Effect of granulocyte colony-stimulating factor in experimental methicillin resistant Staphylococcus aureus sepsis

Background Methicillin resistant Staphylococcus aureus (MRSA) is the leading pathogenic cause of nosocomial infections, especially in bacteraemia and sepsis. The essential therapy for MRSA infection is glycopeptides. Therapeutic failure can be seen with this therapy and the mortality is still high. The aim of this study was to evaluate the additional effect of G-CSF on the traditional antibiotic treatment in an experimental MRSA sepsis. Methods Experimental sepsis was performed in mice by intraperitoneal injection of MRSA isolate. Inoculum dose was estimated as 6 × 10 9 /ml. Mice were randomised for the study into four group; control group (not receive any therapy), G-CSF group (1000 ng/daily, subcutaneously for 3 d), antibiotic group (vancomycin 25 or 50 mg/kg intraperitoneally every 12 hours for 7 d), and vancomycin+G-CSF group (at the same concentrations and duration). Autopsy was done within one hour after mice died. If mice was still alive at the end of seventh day, they were sacrificed, and autopsy was done. In all groups, the effect of G-CSF therapy on the survival, the number of the MRSA colonies in the lung, liver, heart, spleen, and peritoneal cultures, the histopathology of the lung, liver, heart and spleen was investigated. Results One hundred and six mice were used. There were no significant differences in survival rates and bacterial eradication in G-CSF group compared with control group, and also in antibiotic +G-CSF group compared with antibiotic alone group. These parameters were all significantly different in antibiotic alone group compared with control group. Histopathologically, inflammation of the lung and liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF and vancomycin (50 mg/kg)+G-CSF subgroups, respectively (p < 0.01). The histopathological inflammation of the other organs was not significantly different in antibiotic+G-CSF group compared with antibiotic group and, also G-CSF group compared with control group. Conclusion G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. G-CSF treatment is very expensive, likewise glycopeptides. The more interest in infection control measures, and prevent the spread of MRSA infections is more rational.

Background Staphylococcus aureus is an extremely virulent pathogen, and causes serious and deep-seated infections (e.g. endocarditis, osteomyelitis) [ 1 ]. In recent years, S. aureus is the leading pathogenic cause of both community-acquired and nosocomial infections, especially in bacteremia and sepsis [ 2 - 4 ]. Despite advances in antistaphylococcal drugs, S. aureus sepsis is one of the most important causes of death [ 5 ]. Furthermore, methicillin resistant strains are increasing in community and hospitals during the past decades, and many investigators proposed methicillin resistance as an independent predictor of adverse outcome [ 6 , 7 ]. The essential therapy for methicillin resistant S. aureus (MRSA) infections is glycopeptides (vancomycin or teicoplanin). However glycopeptides are intrinsically less effective against staphylococci than are antistaphyloccal β-lactams [ 1 ]. This may explain therapeutic failure and high mortality of MRSA infections. Advances in the pathophysiology of sepsis and septic shock suggest new therapeutic agents and approaches. Granulocyte-colony stimulating factor (G-CSF), a potent stimulator of neutrophil counts and functions, is one of these new strategies in sepsis. Despite a number of studies in sepsis, especially in neonatal sepsis, clinical efficacy of G-CSF is still controversial [ 8 ]. The aim of this experimental study was to evaluate the role of G-CSF in the treatment of MRSA sepsis. Methods Animal care and use Male BALB-C mice (8–10 weeks old, weighing 20–25 g) were obtained from Erciyes University Hakan Cetinsaya Experimental and Clinical Research Center. The Animal Care Committee of Erciyes University approved the experimental protocol used in this study. The animals were kept in a cage and allowed to feed and drink water. Bacterial strain The clinical MRSA isolate, obtained from a patient's blood and pleural effusion admitted in General Surgical Intensive Care Unit of Erciyes University with nosocomial sepsis, was used. The strain was subcultured on blood agar at 37°C over night. On the day of experiment, bacterial suspension was prepared by sodium chloride 0.9% solution and the concentration was adjusted by spectrophotometer. The bacterial account needed for experimental sepsis was determined by a fore study. The fore study was begun with the inoculum dose of 1 × 10 7 /ml bacterial suspension and gradually increased until experimental sepsis developed. Experimental sepsis was defined as the growth of MRSA in two or more organs. The inoculum dose of this study was estimated as 6 × 10 9 /mL. Treatment First dose of antibiotic was given at the sixth hour of bacterial inoculation. Vancomycin (DBL, 500 mg flacon), diluted in 5% dextrose, was given at 25 mg/kg or 50 mg/kg concentrations intraperitoneally every 12 hours for 7 days. rhG-CSF (Roche, Neupogen flk 30 mu/ 1 mL), diluted in 5% dextrose, was received 1000 ng/daily subcutaneously for 3 days [ 9 ]. Study design Animals were randomised to four groups; control group, G-CSF group, vancomycin group, and vancomycin +G-CSF group. The last two groups were divided into two subgroups for different dosage of vancomycin (25 mg/kg or 50 mg/kg). Each group had at least 15 mice and were kept in different cage. In control group, only bacteria suspension was given and no treatment was received. Bacterial suspension was given intraperitoneally to the mice, and when mice died, autopsy was done within one hour in aseptic conditions. If the mice was still alive at the end of seventh day, mice were sacrificed by servical dislocation and autopsy was done. The samples were taken from lung, liver, heart, spleen, and peritoneum for microbiological investigation, and from lung, liver, heart and spleen for histopathological examination. In each group, survival days were noted. Samples were taken from the organs with swab by one rotation on its axis, and were cultured on blood agar over night. The colonies on the agar were counted. The colonies more than 300 cfu in a plate noted as >300 cfu. The same pathologist examined tissue samples stained with hematoxylin and eosin. The degree of inflammation was graded by on a scale of 0 to ++++ (0, no inflammation; focal interstitial inflammation +; more diffuse interstitial inflammation ++; intense interstitial inflammation or microabscesses +++; more extensive abscess formation with tissue necrosis ++++) [ 10 ]. The pathologist was unaware about the groups. Survival days, semiquantitative bacterial count and histopathologic findings in the tissues of the treatment groups were compared with the control group. Statistics Survival was assessed using a log-rank test. Fisher's extract test and chi-square test were used to compare the groups. Mann Whitney U test was used to compare the histopathology. A p value less than 0.05 was considered significant. Results This study included 110 mice. Three mice that developed intraabdominal hemorrhage, and one mouse that developed Esherichia coli sepsis were excluded. 106 mice were evaluated. Six mice in control group, six mice in G-CSF group, 15 mice in vancomycin 25 mg, 15 mice in vancomycin 25 mg+G-CSF group, 18 mice in vancomycin 50 mg group and 14 mice in vancomycin 50 mg+G-CSF group were sacrificed at the end of seventh day. The death day and the survival rate of the mice in the groups are shown by the survival curve in figure 1 , 2 , 3 . The comparison of the groups between G-CSF and control, vancomycin (25 mg/kg) and vancomycin (25 mg/kg)+G-CSF, vancomycin (50 mg/kg) and vancomycin (50 mg/kg)+G-CSF showed no differences (p >0.05). Culture and histopathology results are shown in Table 1 . Antibiotic administration decreased bacterial count, and decreased inflammation rate in the organs. Culture and histopathological results in control and G-CSF group were not statistically different (p > 0.05). However, all these parameters were significantly different in antibiotic groups compared with control group (p < 0.01). Cultures of the organs in antibiotic groups were not statistically different from antibiotic+G-CSF group (p > 0.05). Only the inflammation degree in the lung and the liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF group and vancomycin (50 mg/kg)+G-CSF group, respectively (p < 0.01). The inflammatory changes were not significantly reduced in the other organs in two groups (p > 0.05). Discussion In recent years, MRSA has become widespread around the world, and become highly endemic in some hospitals. In the United States, the proportion of MRSA isolates increased from 2.4% in 1975 to up to 55% in recent years [ 11 , 12 ]. Similarly, in Europe the resistance rates increased from 12.8% to 26.3% [ 12 , 13 ]. Also, it is extremely high (>60%) in some regions of the world [ 12 ]. The recent studies, conducted in our hospital, showed 66% methicillin resistance in nosocomial S. aureus strains, isolated from the bloodstream infections [ 2 , 14 ]. Unfortunately, community-acquired MRSA also increases during the past decades [ 15 ]. G-CSF is a cytokine that stimulates myeloid progenitor cell proliferation and increases the bone marrow storage pool and the number of circulating mature neutrophils, which are important component of the host defense [ 16 ]. Also, it enhances neutrophil activities (chemotaxis, phagocytosis, antibody-mediated cytotoxicity, etc.) [ 17 ]. An inappropriate endogenous G-CSF response may be associated with an adverse outcome to sepsis. Low serum G-CSF concentrations (0 to 125 pg/mL) on admission are supposed to be associated with fatal outcome in patients with bacterial infections [ 18 ]. Investigators proposed to use G-CSF in infections in which neutrophil number and function are important to resolution and survival, also in patients which may have reduced neutrophil numbers or function because of underlying disease or physiologic state. The low toxicity and the beneficial effect on survival in animal studies have led to several clinical trials of rhG-CSF as an adjuvant therapy in treatment of infection in nonneutropenic patients [ 19 , 20 ]. Its beneficial effect was shown in clinical studies in diabetic foot infections, wound infections, extensive burns and fungal infections [ 21 - 23 ]. Also neutrophils in sepsis demonstrate a number of functional abnormalities (e.g. reduced bacterial killing, superoxide production, and migration) [ 24 ] and it can be hypothesized that these abnormalities can be corrected with G-CSF. The previous experimental therapeutic studies were mostly carried out in gram-negative sepsis, and in this experimental study, we investigated the effect of G-CSF in MRSA sepsis. The survival rates were not significantly different in G-CSF group compared with the control group, and also in antibiotic+G-CSF group compared with antibiotic alone group. Likewise, most of the other experimental studies in gram negative sepsis, showed that prophylactic rhG-CSF administration reduced endotoxemia and serum TNF-α levels and also improved cardiac function and survival, whereas therapeutic rhG-CSF (i.e. administered after the onset of infection) did not improve outcome and at very high dosages appeared harmful [ 25 , 26 ]. A recent multicenter, double-blind, randomised and placebo controlled study in patients hospitalised with pneumonia and severe sepsis, demonstrated that G-CSF had no beneficial effect in reducing mortality rates or complications from severe sepsis [ 27 ]. In this study, we did not measure the WBC count, so not have subgroups with neutropenia. In an experimental gram-negative sepsis in rabbits, Smith and colleguaes [ 28 ] showed the beneficial effect of therapeutic rhG-CSF only in early sepsis due to gram negative bacteria when complicated by leukopenia, no significant difference in nonneutropenic group. Bacteria are rapidly cleared from blood and tissues following intravenous antibiotic therapy [ 10 ]. In our study, antibiotic received group had significantly low bacterial count in the organ cultures compared with the other groups, however G-CSF had no beneficial effect on the bacterial clearance. The histopathologic findings of invasive S. aureus infections are leukocytic infiltration, focal pneumonitis, edema, microabscesses, etc. In an experimental study, significant pathologic changes during and after the elimination of bacteria from the blood and tissues were noted. Also, expression of cytokines (TNF, IL-1, IL-6) was observed in all of the infected tissues, and correlated tissue damage after clearance of bacteria from the blood and tissues[ 10 ]. However, in our study, G-CSF significantly reduced only the inflammation in the lung and liver in vancomycin +G-CSF subgroups (p < 0.01). This effect of G-CSF could not be explained. There was no significant effect of G-CSF on the other organs inflammation. In conclusion, G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. Furthermore, G-CSF treatment is very expensive, likewise glycopeptides. Because of high mortality and morbidity rates and excess costs, more interest in infection control measures, and prevent the spread of MRSA infections is more rational. Competing interests This study was supported by Erciyes University Research Fund. There was no non-financial competing interest. Authors' contributions EA and SG were the primary researchers. OC and BK are the pathologist and examined tissue samples. MD was the director of the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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524037

Editorial: Wishful thinking

As a supplement to our lead editorial, the editors of the new journal, Epidemiologic Perspectives & Innovations , provide a partial list of specific analyses and topic areas they would like to see submitted to the journal.

In our lead editorial [ 1 ], the editors of Epidemiologic Perspectives & Innovations ( EP&I ) present the underlying goals and philosophy of the journal, along with the types of articles we hope to publish. Here we clarify our goals with some specific ideas about articles we would like to see submitted to EP&I and with a "wish list" of methodological research and selected other innovations and perspectives we believe the field needs. This list is far from complete, and we want to emphasize that our intention is not to discourage submissions on topics not specifically mentioned here. EP&I welcomes submissions of any papers in and of epidemiology, with the exception of those that solely report research results. "In and of" epidemiology includes papers about epidemiology and those grounded in other disciplines that might be useful for epidemiology. Many of our articles will be highly quantitative, but non-quantitative analyses are also welcome. We welcome submissions whose premises about how the field should be advancing differ from those of this editorial. Bridging the gap EP&I hopes to help bridge the gap from best-practice methods to epidemiologic practice and teaching, and from there to epidemiology-based decision making. There is a gap between what methodologists know about doing epidemiology and standard epidemiologic practice. We believe that narrowing this gap is as important as the development of new methodological ideas. Papers that would narrow this gap are encouraged. These could take different forms, including: clear presentations of advanced methods that are underutilized (perhaps written as tutorials or applications), surveys of epidemiologic practice to assess where the gaps might be, papers that function as advanced textbook chapters, and teaching articles. We will give the highest priority to papers that achieve excellence in explanation. On a similar note, we would like to publish methods articles that fall into the category we call, "Don't Reinvent the Wheel." Other fields have different sets of methods that could be useful to epidemiologists, including observational methods from econometrics and related social sciences, uncertainty analysis methods from engineering and business, and historical perspectives from sciences with a longer history. Also on this theme, we plan to commission updates to classic methods papers from the original authors. Methodological knowledge is ever advancing, and it would be interesting and useful to see what changes an author would make to a must-read paper if that paper were written today. We welcome suggestions of candidates for this series. At the other end of the bridge, we invite methods and teaching papers that show how quantitative health researchers can present results that are more decision-relevant. We are especially interested in methods that go beyond statistical measures that emphasize merely whether an elevated risk has been found to ones that emphasize how much we ought to care about that elevated risk. New methodology The editors of EP&I have identified several specific methodological areas that we believe need attention. Without intending to restrict methodology submissions to these topics, we are particularly interested in submissions that deal with: (1) full and proper disclosure of uncertainty in study results, (2) decision-making in the face of this full disclosure, (3) identification and control of confounding, (4) quantifying the effect of random error on study results, and (5) specification error (i.e., artifacts of statistical methods). Papers that fall into category (1) have begun to appear in the epidemiologic literature [ 2 - 11 ] but much work remains to be done. For example, we especially encourage papers that deal with the specification of probability distributions for uncertainty-model parameters ("priors"), dependencies between priors, and uncertainty-analysis models for complicated (but realistic) situations. While some fields have many papers that fall into category (2), including clinical decision making, there are very few in the epidemiologic literature. As a fundamental challenge for causal inference and a potential source of error in all observational study results, confounding is, and perhaps will always be, a thorn in the side of observational epidemiology [ 12 ]. Papers that fall into category (3) continue to be published in the epidemiologic and statistical literature [ 13 , 14 ], but no completely satisfying method has emerged. Questions of how to think about, and quantify the effect of, random error in nonrandomized studies remain unresolved. A standard analysis pretends that researchers or nature randomized the study exposure to study subjects [ 15 , 10 ]. Is this the best we can do? Study results are a function of both the data being analyzed and assumptions made in the process of the analysis [ 16 - 18 ]. But surprisingly little attention has been paid in the epidemiologic literature to category (5), the impact of incorrect statistical assumptions on study results. Vandenbroucke's [ 19 ] question, "Should we abandon statistical modeling altogether?" and Greenland's [ 10 ] question, "Are conventional statistics anything other than misleading?" deserve serious attention. Philosophies of the science There is a substantial amount of epistemologic discussion in epidemiology, mostly relating to how we know if we are seeing a causal relationship and, to a lesser extent, what causation means. We hope that scientists in the field will become more actively engaged in practical epistemology (and learn to recognize such inquiry as valid scholarly analysis, rather than commentary), and we invite them to submit their work to EP&I . Epistemology in health science is often treated as a matter of statistical rules of thumb, with litigation serving as the ultimate arbiter. Neither statistical rules nor the results of lawsuits that go to trial are a particularly good source of knowledge. Also falling under the broad category of philosophy is analysis of ethics. In the health sciences, such discussion is dominated by the issue of protecting human research subjects and patients. What is largely missing, and what we would like to invite submissions about, is the ethics of the core of our science: study design, data analysis, and result reporting. Since it is impossible to report every result that might be gleaned from a dataset, what constitutes an ethical choice of what to present? This question is central to most everything that most epidemiologists do, and yet there is remarkably little discussion about it [ 20 ]. How many results need to be presented to justify the social expense of doing the research? Should we consider unethical the reporting of results in ways that make them seem larger or more important? Many epidemiologists consider the field per se to include policy advocacy based on health science findings. Whether or not one takes that position, discussions of and about epidemiology should address questions of what society ought to do with our findings. (If we do not bring careful analysis to such questions, who will?) One interesting and timely set of questions involves the relative merits of decision-analysis or risk-analysis based decisions, "precautionary-principle" based decisions, and actual current practices. Exemplars, reconsiderations, and debates We seek to acknowledge the best epidemiologic studies and papers with articles that discuss what sets these studies apart from the rest: a "Best of Epidemiology" series. Much of methods training involves finding the worst in a study. We believe it would be useful to give students and other epidemiologists examples of how to do epidemiology well. What studies should we most want to emulate, and why? In a similar vein, we hope to receive submissions that remind us about the historical foundations of epidemiology: a "History of Epidemiology" series. What historical studies, insights, or methodology are important for understanding the field, and why, and which are simply interesting enough to be worth highlighting? History-of-science type analysis of our field, almost completely absent from the history-and-philosophy-of-science literature, would be particularly welcome. A complement to the "best of" and recounting of historical high-points is reanalysis of particular study results or aspects of the field's literature more generally. Many substantive letters to the editor about published papers reflect a full article's worth of analysis by the letter authors. But those authors have little opportunity to publish that analysis, even when it has substantial practical or methodological importance; we encourage submission to EP&I of such papers, whether or not a letter to the editor has been published. We encourage authors to take advantage of the speed of online publishing to submit re-analyses of important findings for publication while the implications of the original study are still being debated. (Important re-analyses, based on either the original data or sometimes merely what was published, need not be immediate, however, and can still be interesting years later.) Readers of the health science literature may often find themselves asking a question about a study's results beyond the few specifically addressed in the published reports. A new analysis might be more relevant to a particular decision, or even out-and-out more informative than the original publication. Finally, we hope to publish "Point-Counterpoint" collections on single topics. Authors considering writing a counterpoint (or simply a related point) to an EP&I article are encouraged to contact us in advance to see if we might solicit other related submissions to create a larger collection. Topics of particular interest to us, where we would like to encourage the starting of point-counterpoint series, include the usefulness of causal inference in epidemiology and the scientific basis for a particular regulatory decision (e.g., the U.S. Food and Drug Administration decisions to ban phenylpropanolamine and ephedra). We especially encourage students to suggest potential topics. Wishful wish list? We will grant that this is an ambitious research agenda. Perhaps it will be years before journals (this one or others) see articles on all these topics. But it is also possible that the next 100 dissertations in the field will include chapters that cover most of this ground. And perhaps those of us well past our dissertations can contribute a few also. We hope that asking (and providing a venue for publication) will make this much more likely.

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524023

CytoJournal joins 'open access' philosophy

Welcome to CytoJournal ! We would like to introduce you to your journal, one that is run by and for the scientific cytopathology community with incontestable benefits of Open Access, and support from Cytopathology Foundation, Inc. CytoJournal is a peer-reviewed, PubMed indexed, online journal, publishing research in the field of cytopathology and related areas, with world wide free access. Authors submitting to CytoJournal retain the copyright to their hard earned work.

Welcome to CytoJournal ! We would like to introduce you to your journal, one that is run by and for the scientific cytopathology community with incontestable benefits of Open Access . CytoJournal is a peer-reviewed cytopathology journal is owned and supported by Cytopathology Fondation, Inc. It is a PubMed indexed, online journal, publishing research in the field of cytopathology and related areas with world wide free access. Authors submitting to CytoJournal retain the copyright to their hard earned work. Why CytoJournal is needed One could argue whether we need more journals in cytopathology, but without a shadow of doubt there is a global need for greater access to scientific information in this field. As an Open Access journal CytoJournal will meet this need, by removing subscription barriers. Communication in general has been revolutionized in the last decade. With the emergence of the internet, entire libraries of scientific information are potentially just a mouse click away. Open Access to quality controlled, scientific information to the general public and scientific community alike is extremely valuable for harvesting the fruits of hard work by academicians. However, to date little has been done to realize the potential of this technological revolution. It is now affordable to make our hard earned scientific information available to a much wider audience. Millions of students, teachers, physicians, scientists, general public, and other potential readers can have free access to the gold mine of this scientific information. The traditional model for journal publication has performed an admirable job of disseminating research and advancing science. Unfortunately, it neither can fully utilize recent technology nor extend its benefits to the scientific community and the general public. Principally, why should scientific information generated by academicians and published without charge, not be freely available? Giving away the copyrights to the original research reports and then paying for access of the same material is paradoxically anachronistic. With the advent of the internet and image digitization, the time has come for the development and implementation of a new model – a model beneficial to the scientific community and general public, alike. The recent adoption of an Open Access model by the Public Library of Science (PLoS) [ 1 ] and its recognition by other scientific organizations, including the National Institute of Health (NIH), has generated significant community interest. This has provided impetus for the creation of new journals such as CytoJournal with increasing support to open access philosophy by some of the existing leading journals. The benefits of an online journal can include rapid turnaround time, real time publication, significant cost savings, and a reduction in the environmental burden engendered in the production and disposal of a print publication. Presumably in the future, the majority of research publications will be of this type [ 2 ]. Free flow of scientific information is crucial for advancements in diagnosis and management of diseases in both the developed and developing world. Simply providing a conduit of information is not enough. There is one matter involving the human element that must be dealt with – peer review. We do not see a future where authors post their manuscripts on the web without peer-review. Science requires that publications be properly vetted (peer-reviewed) and experimental findings logically presented. In brief, standards must be maintained! This critical role is played by peer review. Acting on behalf of the scientific community, it is peer review that helps the scientific community distinguish between legitimate scientific work and quackery [ 3 - 7 ]. Thus, to maintain the quality and confidence, peer review will continue to be the keystone of scientific publishing. The peer-review process at CytoJournal combines the expertise of both professional editors, who are available to survey the broad landscape of cytopathology, and academic editors, who understand deeply the strengths and limitations of their specific area of research. Every article that is published in CytoJournal will be reviewed carefully by selected reviewers, in addition to evaluation by a managing/academic editor and professional editor, who work together throughout the editorial process. A summary of your peer review process is illustrated in Figure 1 . Figure 1 CytoJournal's online peer review process. Definition of Open Access CytoJournal's Open Access policy changes the way in which articles are published. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone, free of charge. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced [ 8 ]. Third, a copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal. CytoJournal's articles are archived in PubMed Central [ 9 ], the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam [ 10 ] in Germany, at INIST [ 11 ] in France and in e-Depot, the National Library of the Netherlands' digital archive of all electronic publications [ 12 ]. Benefits of Open Access Open Access has four broad benefits for science and the general public. First, authors are assured that their work is disseminated to the widest possible audience, given that there are no barriers to their work. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. It has been suggested that free online articles are more highly cited because of their easier availability [ 13 ]. Second, the information available to researchers will not be limited by their library's budget, and the widespread availability of articles will enhance literature searching [ 14 ]. Third, the results of publicly funded research will be accessible to all taxpayers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law [ 15 ]. Fourth, a country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones (although creating access to the internet is another matter) [ 16 ]. Open Access Publishing Model and Finance Open Access facilitates the transformation of scientific literature from rows of printed journals on library shelves to an instantly searchable archive of data. Liberating scientific literature from the vestiges of paper publication introduces the potential of various opportunities such as navigating, integrating, mining, annotating, and mapping connections in the high-dimensional space of scientific knowledge. To provide Open Access, CytoJournal will use a new business model. Our editorial expenses (managing peer review, providing editorial insight, and ensuring the highest production standards) will be supported by corporate sponsors through Cytopathology Foundation, Inc. and honorary pro bono services by the academicians (Figure 2 ). The publication cost for our publishers, BioMed Central, will be recovered by imposing a modest charge (currently US$ 525) to authors for each article accepted for publication after peer review. There is no charge for the submission of a manuscript and therefore, if the manuscript is submitted but not accepted for publication, the authors will not be charged. Figure 2 Core Principles of 'CytoJournal' and 'Cytopathology-Foundation' Our sponsors do not influence in any way the content of CytoJournal and it's editorial and publication decisions. However, they will be acknowledged and their links will be displayed on the home page of CytoJournal as a thank you note. Fee Waivers We understand that there are many scientists who might wish to publish in our journal but do not have access to grant funds or institutional support. For such authors, a decision can be made by the Editors-in-Chief to waive publication fees. Furthermore, the fee will automatically be waived for authors from institutes that are members of BioMed Central . We never want our publication charges to be a barrier to publication and are committed to publishing any manuscript that our editors and reviewers deem to be appropriate for the journal; we will treat the costs of handling these papers as a fundamental expense of running a high-quality journal through support from Cytopathology Foundation Inc. Joining Forces CytoJournal is your journal and you can lead the way. However, we have to face and overcome a few traditional obstacles. First, an unfamiliarity to the Open Access model. Second, lack of the benefits of an established "brand name" aura. Because of this, despite stringent standards and an extraordinary editorial team, CytoJournal may have an uphill task. Private foundations with a commitment to science and education have contributed generously to the cause. Like any new business, Cytopathology Foundation Inc. needed to raise funds to cover our startup costs. Initial support from Cytopathology Foundation Inc. and the publisher, BioMed Central, has enabled us to launch CytoJournal . Other individuals and organizations have also provided generous and welcome support. The start-up support made it possible to assemble an outstanding editorial board and staff, who have accomplished an extraordinary feat of launching this new premiere journal in cytopathology. We wish to thank and applaud the efforts and spirit of the pioneering authors who chose to send their articles to CytoJournal . In the end, it is the contributions by authors like you that will make CytoJournal a collective success. We encourage you to participate in the future of CytoJournal by reading, citing, submitting manuscript, sending any suggestions, and joining the panel of core reviewers. Bookmark the home page of Cytojournal for your quick reference! You may also link CytoJournal web site or recommend its linking through various other web sites.

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539270

Sick leave among home-care personnel: a longitudinal study of risk factors

Background Sick leave due to neck, shoulder and back disorders (NSBD) is higher among health-care workers, especially nursing aides/assistant nurses, compared with employees in other occupations. More information is needed about predictors of sick leave among health care workers. The aim of the study was to assess whether self-reported factors related to health, work and leisure time could predict: 1) future certified sick leave due to any cause, in nursing aides/assistant nurses ( Study group I ) and 2) future self-reported sick leave due to NSBD in nursing aides/assistant nurses ( Study group II ). Methods Study group I, comprised 443 female nursing aides/assistant nurses, not on sick leave at baseline when a questionnaire was completed. Data on certified sick leave were collected after 18 months. Study group II comprised 274 of the women, who at baseline reported no sick leave during the preceding year due to NSBD and who participated at the 18 month follow-up. Data on sick leave due to NSBD were collected from the questionnaire at 18 months. The associations between future sick leave and factors related to health, work and leisure time were tested by logistic regression analyses. Results Health-related factors such as previous low back disorders (OR: 1.89; 95% CI 1.20–2.97) and previous sick leave (OR 6.40; 95%CI 3.97–10.31), were associated with a higher risk of future sick leave due to any cause. Factors related to health, work and leisure time, i.e. previous low back disorders (OR: 4.45; 95% CI 1.27–15.77) previous sick leave, not due to NSBD (OR 3.30; 95%CI 1.33–8.17), high strain work (OR 2.34; 95%CI 1.05–5.23) and high perceived physical exertion in domestic work (OR 2.56; 95%CI 1.12–5.86) were associated with a higher risk of future sick leave due to NSBD. In the final analyses, previous low back disorders and previous sick leave remained significant in both study groups. Conclusion The results suggest a focus on previous low back disorders and previous sick leave for the design of early prevention programmes aiming at reducing future sick leave due to any cause, as well as due to NSBD, among nursing aides/assistant nurses. A multifactorial approach may be of importance in the early prevention of sick leave due to NSBD.

Background Over the past century, sick leave, mainly attributed to musculoskeletal disorders (MSD), has increased in Sweden, especially among women [ 1 ]. In 2003, the proportion of women on sick leave was higher than in any previous year [ 1 ], indicating more individual suffering and also increased cost for the community. The prevalence of neck, shoulder and back pain is higher among health care workers, especially among nursing aides, compared with employees in other occupations [ 2 - 4 ]. In 2002, women working within the health-care sector in Sweden had the highest proportion of sick leave, mainly attributed to MSD [ 1 ]. The need for effective early prevention strategies is evident. However, if customised prevention programmes are to be possible, more information is needed about predictors of future sick leave among women working in the nursing sector. Many studies have focused on the association between risk factors and the reporting of neck, shoulder and back pain [ 5 - 8 ]. However, back pain is a recurrent problem, which may or may not influence participation in daily activities and the ability to work [ 9 , 10 ]. It has been shown that predictors of perceived pain may not be the same as predictors of future sick leave [ 11 ]. Thus, when constructing intervention programmes, focusing on individuals at risk of future sick leave, predictors associated with perceived pain should be distinguished from those of future sick leave. Studies have pointed to different factors in different populations to relate with future sick leave. Demographic factors such as age may be associated with sick leave mainly in individuals with chronic neck-or-back disorders [ 12 ]. In some studies this association was, however, not verified, which may be due to a "healthy worker effect" [ 11 , 13 ]. Medical factors, such as, for example, previous experience of back disorders, perceived health or prior sick leave predicted future sick leave [ 11 , 12 , 14 - 16 ]. Self-reported high physical load increased the risk of prolonged sick leave in populations already on sick leave due to back pain [ 17 , 18 ]. Perceived high work load in combination with low decision latitude, hereafter called high-strain work, is assumed to have negative consequences on health [ 19 , 20 ]. Low social support may be a risk factor for the development of back pain and also for future sick leave [ 19 , 20 ]. The association between high work load, low decision latitude and low social support and future sick leave is, however, unclear [ 14 , 19 ]. Mental problems, such as, for example, anxiety, also increased the risk of future sick leave among nurses [ 19 ]. Moreover, economic problems exacerbated the risk of prolonged sick leave among patients with low back disorders [ 21 ]. A strained economic situation also tends to lead to a decreased probability that woman suffering low back pain will seek medical attention [ 22 ]. The effects of physical exercising on future sick leave are not clear. Sedentary activities in leisure time were, however, associated with a higher prevalence of back symptoms and sick leave [ 23 ]. Eriksen et al. [ 16 , 24 ] found that regular physical activity, such as brisk walks, aerobics or other forms of exercise for 20 minutes or more at least once a week predicted fewer sick leaves after 3 and 15 months among nursing aides. To summarise, a large number of factors, demographic, medical, physical, psychosocial, psychological as well as socioeconomic, have been documented as risk factors for future sick leave in different working populations [ 14 ]. It has been concluded that there is a lack of studies on neck-and-back disorders among women working within special groups as for example the health-care sector [ 14 ]. Because studies on home-care personnel, analysing risk factors associated with neck, shoulder and back disorders, have mostly been cross-sectional, it is not possible to determine causality. It is of interest to analyse whether the above-mentioned factors associated with future sick leave in different populations, can also predict future sick leave among nursing aides/assistant nurses working within the home-care service. Working as a nursing aide/assistant nurse within the home-care service is generally physically heavy work, requiring repeated transfers and lifts of patients. The physical load on the spine depends on several factors, for example, the weight of the patient, work place design, work organisation, work technique, work equipment, the cooperation of the patient etc [ 25 ]. Even transfers of a light and cooperative patient imply high spinal loads and a risk of causing low back disorders [ 26 ]. An appropriate work technique may decrease the biomechanical load on the spine. However, work technique differs among individuals and should thus be studied on an individual level [ 25 ]. In the present study, self-reported measures were analysed and physical load on the spine were not included. Self-reported working positions did not associate with prolonged sick leave in individuals with chronic back pain [ 14 ]. In longitudinal studies on nurses and nursing aides, frequent lifting or repositioning of patients did not predict future sick leave [ 11 , 16 ]. However, the perception of physical exertion at work was a risk indicator for low back symptoms among nursing aides working in geriatric care [ 6 ]. The perception of physical exertion correlated also with the onset of low-back pain among nursing aides [ 7 ]. The risk of seeking care was higher among the nursing personnel who perceived high physical exertion in domestic work [ 27 ]. The impact of perception of physical exertion on future sick leave among nursing aides/assistant nurses is, to our knowledge, not known. The aim of the present study was to assess whether a selection of self-reported measures of factors related to health, work and leisure time could predict 1. future, certified sick leave after 18 months, due to any cause, in a group of working nursing aides/assistant nurses ( Study group I ). 2. future, self-reported sick leave after 18 months, due to neck, shoulder and/or back disorders, in a group of working nursing aides/assistant nurses ( Study group II ). The aim was based on the hypothesis that factors related to the workplace as well as factors related to perceived health and to leisure time are associated with future sick leave in female nursing aides/assistant nurses. It was also hypothesised that predictors of future sick leave due to neck, shoulder and/or back disorders are different from predictors of sick leave due to any cause. Methods This study was one part of a larger project, aiming at preventing or reducing disorders of the neck, shoulder or back among female nursing aides/assistant nurses, working within the home-care services. The definition of the titles of home-care personnel is heterogeneous. In the present paper, nursing aides had little or no formal training, while the assistant nurses had undergone two – three years secondary education in nursing or had long experience of the work as a nursing aide and about one-year further education. The study was approved by the Ethical Committee of the Faculty of Medicine, University of Lund, Sweden (LU 286-95). All participants gave their written consent before participation. Subjects The municipal home-care services were organised in six units situated in different geographically defined areas of a medium-sized town in the south of Sweden. Initially, all of the 659 women working in five of the six units were invited to participate in the prevention project. (One unit was excluded due to its participation in another study). The inclusion criteria were: Swedish speaking, permanently employed, not pregnant, in work and working at least 50% of full time. Participation was accepted by 534 (81%) of the women. The main reasons not to participate were: the opinion that the project was important only for younger staff, dissatisfaction with the work situation, participation in compulsory, further education for nursing aides to qualify as assistant nurse, lack of time or family reasons. As we wanted to elucidate predictors of future sick leave at an 18 month follow-up, women who at that time had retired from work, had left work or were off duty, were excluded from the analyses. Two women were deceased (n = 91). Thus, the final sample consisted of 443 nursing aides/assistant nurses (Figure 1 ). At baseline, totally 282 of the originally 534 participants were randomized to one of two intervention groups, aiming at preventing neck, shoulder and back disorders (SM = Stress Management or IT = Individual Physical Training programmes) or to a Control group. Intervention groups and the Control group were evaluated and compared at the 18-month follow-up. No significant differences between the groups could be shown [ 39 ]. Thus, in the present study the groups on intervention programmes and the Control group were treated as a whole and called intervention. Study group I Study group I comprised 443 women who were not on sick leave when they completed the baseline questionnaire. Information on certified sick leave due to any cause during the six months preceding the 18-month follow-up was obtained from the National Social Insurance Board. The number of women who participated in intervention was 241 (54%). Demographic data, disorders, sick leave and participation in intervention for Study group I are presented in Table 1 . Study group II Study group II comprised 274 women. At baseline, 383 women had reported no sick-leave due to symptoms from the neck, shoulder and/or back during the preceding 12 months. At the 18-month follow-up, 109 of these women did not fill in the questionnaire (Figure 1 ). The main known causes for dropping out at the 18-month follow-up were, not being able to fill in the questionnaires in time, mainly due to vacations, illnesses and refusal to take part, mainly due to lack of time. The reason for not participating was unknown for 69 of the women (18%). However, when further contacts were established with these women at a later stage, we were told that the reason for not responding was frequent reorganisations at work. Compared with participants, non-participants were, at baseline, more dissatisfied with social support at work (p = 0.03). No other differences between the participants and the drop-outs were shown. The number of women who participated in some intervention was 173 (64%). Demographic data, disorders, sick leave and participation in intervention for Study group II are presented in Table 1 . Assessments All participants were asked to fill in a questionnaire at the start of the study and after 6, 12 and 18 months. At baseline, the questionnaires were administered by the project nurse and filled in at the work place ( Study group I and Study group II ). At the 18-month follow-up, questionnaires were sent out to a contact person at each work place, who was made responsible for the distribution to the participants in the study. Each respondent received a stamped envelope in which she returned her own questionnaire to the project nurse ( Study group II ). Dependent variables Sick leave due to any cause at the 18-month follow-up (Study group I) Data about sick leave during the six months preceding the 18-month follow-up were obtained from the register of the National Social Insurance Board. In Sweden, the municipal authorities systematically record and report each day an employee is on sick leave to the National Social Insurance Board. Diagnoses are not reported to the Board. All days were counted as whole days irrespective of whether they were whole days or part of days. Due to skewed data, sick leave was dichotomized in 0 days/≥1 day (third quartile). Sick leave due to disorders of the neck, shoulders and/or back at the 18-month follow-up (Study group II) At all follow-ups the nursing aides/assistant nurses were asked: "Have you been on sick leave any time during the previous six months due to neck, shoulder and/or back disorders?". Response options were yes/no and constituted the dependent variable for Study group II at the 18-month follow-up. The validity of the responses was checked for all follow-up questionnaires against the general Nordic Musculoskeletal Questionnaire (NMQ) [ 28 ] i.e. the question concerning whether the disorder had been incapacitating during the preceding six months. A pattern was seen for women who reported sick leave due to neck, shoulder and/or back disorders at follow-ups by the fact that they reported in the NMQ that the disorders also had been incapacitating some time during the 6 months preceding the 18 month follow-up. Independent variables Self-reported measures associated with health Mental health Anxiety and depression were assessed by the Hospital Anxiety and Depression Scale (HAD) [ 29 ] which consists of two subscales – one for anxiety and one for depression. Anxiety and depression were closely related (p = 0.000) and as depression levels are generally lower compared with anxiety levels in working populations [ 30 ], which was also the case in the present study, we selected anxiety as a measure of mental well-being. The subscale contains seven items ranging from 0 – 3, with higher scores reflecting greater anxiety. A sum of eight or more has been shown in comparisons with psychiatric interviews to reflect anxiety [ 29 ]. The Swedish version was tested and evaluated by Lundqvist et al. [ 31 ]. Musculoskeletal disorders The prevalence of musculoskeletal disorders from the neck, shoulders and back was assessed by the general Nordic Musculoskeletal Questionnaire (NMQ) [ 28 ]. Participants were asked about pain, aches or discomfort some time during the preceding 12 months. The response options were yes/no. Sick leave during the 12 months preceding baseline Information on sick leave was obtained from the National Social Insurance Board. Individuals in Study group II , who had been on sick leave, but who in the questionnaires had reported no sick leave due to symptoms from the neck, shoulder and/or back were assumed to be on sick leave due to other reasons. Work loss was recorded as whole working days. As all data on sick leave were extremely skewed, we chose to dichotomise the material. The third quartile was used as cut-off point, which for Study group I was 0 days/≥1 days and for Study group II <9 days/≥9 days. Self-reported measures associated with work Perceived physical exertion at work The participants were asked: "What degree of physical exertion do you usually perceive in your present job?" [ 32 ]. The question was assessed according to Borg [ 33 ] and ranged from 6 (no exertion at all) to 20 (maximal exertion). High physical exertion at work was defined as 15 on the scale (corresponding "hard") or more (third quartile) [ 6 ]. Perceived work-related psychosocial factors Psychosocial factors at work, such as social support, decision latitude and psychological load were assessed by a questionnaire developed by Rubenowitz [ 34 , 35 ]. The questionnaire considers five psychosocial factors: "Influence and control over work", "Supervisor climate", "Stimulus from the work itself", "Relation to fellow workers" and "Psychological load". Each factor comprises five items and each item has five fixed response alternatives from 1 to 5, where 1 means very unsatisfactory and 5 very satisfactory. A separate score, ranging from 1 to 5, is calculated on the mean of each factor. In the present analysis, "Supervisor climate" and "Relation to fellow workers" were defined as "Social support" and "Influence and control over work" and "Stimulus from the work itself" were defined as "Decision latitude" [ 35 ]. "Psychological load" together with "decision latitude" were defined as strain. High strain was equal to high psychological load in combination with low decision latitude [ 36 ]. The first quartile of the psychosocial factors were categorised as poor [ 34 ]. Self-reported measures associated with leisure time Exercise and physical activity The participants were asked: "To what extent have you performed physical activities or fitness training during the previous six months?" [ 32 ]. The question comprised eight options. A sedentary life style was assumed by the response "No exercise and very little physical activity" (score = 1). Perceived physical exertion in domestic work The participants were asked about physical exertion in domestic work in the same way as for physical exertion at work: "What degree of physical exertion do you usually perceive in your daily domestic work?" [ 32 ]. Response options ranged from 6 (no exertion at all) to 20 (maximal exertion) [ 33 ]. The cut-off point was 13 (third quartile) corresponding "somewhat hard". Perceived psychological stress outside work This question, originally developed for the Malmo Diet and Cancer study on 53000 Swedish men and women, was defined by "Have you lately felt mentally stressed or been under psychological pressure due to problems outside work?" Response options were yes/no [ 37 ]. Economic situation Perceived satisfaction with her own economic situation was assessed by a seven-point scale where the first point represented "very bad" and the seventh point "Excellent, could not be better" [ 38 ]. Statistical analysis All logistic regression analyses were adjusted for age and intervention. Three models were tested, namely, for factors related to health, to work and to leisure time. The associations between the independent variables and the outcome variables were first analysed for each of the three models by univariate logistic regression. Secondly, each model was analysed with all variables included in a backward stepwise multivariate regression analysis with a likelihood ratio test. Finally, a multivariate model including all the variables from the three models together was tested. The criteria for inclusion and exclusion were p = 0.05. The logistic regression analyses were tested for goodness of fit by means of the Hosmer and Lemeshow method. Factors were dichotomized by using cut-off points described in the literature. When no such literature was found, cut-off points were taken as the first or the third quartile for frequencies for Study group II . There were minimal differences between the quartiles of the two study groups. Thus, for the purpose of comparing the two groups, the same cut-off points were used. However, for sick-leave before baseline we chose two different cut-off points, as the third quartile differed between the two study groups, being 0 days for Study group I and 9 days for Study group II . The sample size may vary in the different analyses due to missing values. Comparisons between drop-outs and participants were analysed by t-test or the chi-square test. Correlations between the exposure variables were calculated with Pearson correlation coefficients (r). All statistical calculations were performed using the SPSS 11.5.1 Software for Windows (SPSS Inc., Chicago, IL, USA). Results In the univariate logistic regression analysis, there were minimal differences between odds ratios adjusted and not adjusted for age and intervention. Thus, in Tables 2 , 3 , 4 , 5 , only adjusted odds ratios are presented. Predictors of future sick leave due to any cause (Study group I) Age or participation in intervention (presented under section Methods-Subjects) did not have any effect on future sick leave due to any cause. Self-reported factors related to health Perceived disorders of the low back and sick leave during the 12 months preceding baseline predicted future sick leave due to any cause in the unadjusted and the adjusted univariate analyses as well as in the multivariate analysis (Table 2 and 5 ). Perceived disorders of the neck or shoulders did not predict future sick leave. Neck, shoulder and back disorders at baseline correlated with each other. Of the women who reported neck disorders, 88% also reported shoulder disorders (r = 0.57) and 75% reported low back disorders (r = 0.33). Of the women who reported shoulder disorders 71% reported low back disorders (r = 0.28). Self-reported factors related to work Factors related to work did not predict future sick leave due to any cause (Table 3 and 5 ). Self-reported factors related to leisure time Factors related to leisure time did not predict future sick leave in the univariate or in the multivariate analysis (Tables 4 and 5 ). Final model Study group I When all variables from the three models were entered into the final model, disorders of the low back during the preceding 12 months (OR: 1.95; CI: 1.23–3.10) and sick leave during the 12 months preceding baseline (OR: 6.61; CI: 4.08–10.73) were the only factors predicting sick leave due to any cause after 18 months. Predictors of future sick leave due to disorders of the neck, shoulders and/or back (Study group II) A tendency towards a reduced risk of future sick leave due to neck, shoulder and/or back disorders was found for the women who participated in some intervention (OR: 0.46; CI: 0.21–1.01). Age did not have an impact on sick leave (OR: 1.02; CI: 0.98–1.06). Self-reported factors related to health Anxiety, low back disorders during the 12 months preceding baseline as well as sick leave due to reasons other than disorders of the neck, shoulders and/or back were significant predictors of sick leave after 18 months due to disorders of the neck, shoulders and/or back (p < 0.05) before and after adjustment for age and intervention (Table 2 ). When all characteristics related to health were entered into the multiple logistic regression model, disorders of the low back and sick leave due to reasons other than disorders of the neck, shoulders and/or back remained significant (Table 5 ). Neck disorders at baseline, correlated with shoulder disorders (r = 0.57) and with back disorders (r = 0.36). Of the nursing aides/assistant nurses who reported neck disorders, 87% also reported disorders of the shoulders and 75% reported disorders of the low back. Of those who reported disorders of the shoulders at baseline, 70% also reported disorders of the low back (r = 0.29). Self-reported factors related to work Before adjustment for age and intervention, high psychological load and high-strain work predicted future sick leave due to neck, shoulder and/or back disorders (OR: 2.28; CI:1.04–5.03 and OR: 2.42; CI: 1.09–5.37). However, after adjustments, only high-strain work demonstrated significant association with future sick leave (Table 3 ). Two multivariate regression analyses were performed: one by entering psychological load and decision latitude separately into the model and one by analysing the same factors together (called strain). Only high-strain work predicted future sick leave due to disorders in the neck, shoulder and/or back (Table 5 ). Self-reported factors related to leisure time High perceived physical exertion in domestic work and perceived psychological stress outside work were associated with future sick leave due to neck, shoulder and/or back disorders both before and after adjustments for age and intervention (Table 4 ). In the multivariate model, high perceived physical exertion in domestic work remained significant predictor of future sick leave (Table 5 ). Final model, Study group II In the final model, where all variables from the three models were entered into the logistic regression analysis, perceived low back disorders some time during the 12 months preceding baseline and sick leave of > 9 days in the year before baseline remained significant predictors of sick leave due to neck, shoulder and/or back disorders at 18 months after baseline (OR: 7.36; CI: 1.67–32.43 and OR: 2.84; CI: 1.13–7.11 respectively). Discussion The results of the present study indicated that only factors related to health were associated with future sick leave due to any cause in a group of nursing aides/assistant nurses. On the other hand, factors related to health, the workplace as well as leisure time were associated with future sick leave due to neck, shoulder and/or back disorders, indicating a multifactorial background for these disorders. Low back disorders and sick leave during the 12 months preceding baseline, were significantly associated with future sick leave in both Study group I and Study group II . Thus, the hypothesis that risk factors for future sick leave due to disorders in the neck, shoulders and/or back are different from sick leave due to any cause was only partly demonstrated. This is in concordance with other studies, which have also documented earlier experience of back pain and sick leave preceding baseline as predictors of future sick leave due to low back pain among nursing personnel [ 11 ] as well as in general populations [ 12 , 40 ]. Natvig et al. [ 15 ], showed that low back pain as a part of widespread pain predicted long-term disability due to any cause, while local low back pain did not. In the present study perceived disorders of the neck, shoulder and back correlated with each other, indicating wide spread disorders. Further, only disorders of the back and not of the neck or of the shoulders predicted sick leave in Study group I as well as in Study group II . One explanation for this finding may be the content of the work performed by a nursing aide/assistant nurse, with repeated repositioning, transfers and lifts of patients, which result in high spinal loads mainly on the low back [ 25 , 26 ]. Thus, it may be more difficult to perform this work with a vulnerable low back in comparison with vulnerable neck/shoulders. In the final analyses of Study group I and Study group II , including all variables from the three models, only low back disorders and previous sick leave remained significant. However, in Study group II , due to the small number of women on sick leave due to neck, shoulder and/or back disorders at the 18-month follow-up, results of this final analysis should be interpreted with caution. Generally, most studies on predictors of future sick leave are made on men and few studies focusing on nursing personnel have been published. Thus, comparisons of the results from the current study with similar studies are limited. The present study was performed during a period with frequent reorganisations and reductions of staff within the home-care services in the city studied, as in general in Sweden. A greater amount of work was performed faster than before and the category of patients, for whom the community home-care organisation took responsibility for, were more handicapped than previously. Thus, the physical and psychological stresses were generally exacerbated during the period studied. In the present study, among the factors related to work, only high-strain work (high psychological load in combination with low decision latitude) predicted future sick leave due to neck, shoulder and/or back. Marras et al. [ 41 ] showed that lifting combined with psychosocial stress, increases the load on the spine indicating an increased risk of back pain when working in physically demanding jobs in combination with high strain. Bourbonnais and Mondor [ 19 ] found that high-strain work reinforced the risk of future, short-term sick leave due to any cause among nurses. The inclusion of strain-reducing strategies in stress-management programmes for nursing aides/assistant nurses may, thus, decrease the risk of future sick leave due to back disorders, but should be further studied. Women still do more unpaid work than men. In a 23-year perspective study, the physical work load decreased among men but not among women [ 42 ]. In the present study, we found that high physical exertion in domestic work predicted sick leave due to neck, shoulder and/or back disorders. Josephson et al. [ 27 ] found that the tendency to seek care was greater among nursing personnel who perceived that they did far too much domestic work. These findings point to the importance of also including factors outside work, even when customising early prevention programmes at the work place with the aim to reduce future sick leave due to neck, shoulder and back disorders among female nursing aides/assistant nurses. We could not verify inactivity to be a predictor of future sick leave. The cut-off point was based on the assumption that a sedentary life style would predict future sick leave as shown in the study by Hildebrandt et al. [ 23 ]. In the study by Eriksen et al. [ 24 ], brisk walks, aerobics or gymnastics and other physical leisure-time activities for 20 minutes or more at least once a week protected against sick leave of more than 14 days, independent of diagnosis, after a 15-month follow-up period among nursing aides. The work as a nursing aide/assistant nurse is physically active, involving a great deal of walking. Few women reported a sedentary life at baseline. Thus, inactivity may not have been an appropriate cut-off point. Moreover, in the present study, social support did not predict future sick leave. Since there was a significant initial difference in perceived social support between participants and non-participants in Study group II , the conclusions of this analysis are limited. However, neither in the Study group I , was social support related to sick leave. This finding is contradictory to the results of the study by Eriksen et al. on 5563 Norwegian nursing aides [ 16 ]. These authors found that social support was the most important work-related predictor of future sick leave due to any cause. Bourbonnais and Mondor [ 19 ] could also state a relationship between low social support and future sick leave among Canadian nurses. However, after adjustment for job strain, this relationship was no longer significant. Methodological considerations Data on the dependent variable sick leave, for Study group I were obtained from the National Social Insurance Board and for Study group II were taken from the questionnaires. Systematically recorded, certified sick leave is assumed to be more valid than data on sick leave based on questionnaires [ 43 ]. For Study group II , questionnaires were filled in at the 6, 12 and 18 month follow-ups. In order to strengthen the validity of the question "Have you been on sick leave any time during the preceding six months due to neck, shoulder and/or back disorders", answers at the follow-ups were checked against the NMQ [ 28 ]. Participants who at baseline reported no sick leave due to neck, shoulder and/or back disorders could nevertheless, in the NMQ, indicate perceived disorders from the same regions but that these disorders had not been incapacitating. However, a pattern was seen for women who reported sick leave due to neck, shoulder and/or back disorders at follow-ups by the fact that they also, in the NMQ, reported that these disorders had been incapacitating. It may also be assumed that the recall bias was reduced and the sensitivity of the question about sick leave enhanced, as we did not ask the respondent to specify the number either of sick-days or of sick-leave episodes [ 43 ]. Age was not related to any reason for sick leave, which is in contrast with the results of a review study by Turner et al. [ 12 ] on people working in various occupations. They found an increased risk of sick leave with older age. However, the results of the present study are in concordance with other studies on home-care personnel and future sick leave [ 11 , 13 ], indicating a healthy worker effect among nursing aides/assistant nurses, which in turn may result in an underestimation of risk factors for future sick leave. In Study group II , a large number of participants dropped out at the 18-month follow-up, which constitutes a risk of selection bias. The number of drop-outs is comparable with that found in other studies on nursing personnel [ 19 , 30 , 44 ]. In addition, except for social support at work (discussed earlier), participants did not differ from non-participants on any of the dependent variables. Thus, we regard the 274 participants in Study group II to be representative as a working group of nursing aides/assistant nurses. The feasibility to generalise the results from Study group I to other women working as nursing aides/nursing assistants should be satisfactory, as all women participating at baseline were analysed at18 months and the data of the dependent variable, certified sick leave, were provided by the National Social Insurance Board. In Study group I , 54% and in Study group II , 64% of the nursing aides/assistant nurses had participated in some intervention. The main purpose of this intervention project was not to reduce sick leave. The participants were informed that the aim of the project was to prevent or reduce pain and discomfort of the neck, shoulder and back. Sick leave was not mentioned. Thus, in our opinion, participation in intervention groups should not have biased the reporting of sick leave due to neck, shoulder and back disorders. Conclusions The present study indicated that previous disorders of the low back and previous sick leave were the strongest predictors of future sick leave due to any cause as well as of future sick leave due to neck, shoulder and/or back disorders in a group of nursing aides/assistant nurses, who at baseline were at work. Previous neck or shoulder disorders did not predict future sick leave. Moreover, factors related to health, work and leisure time were all related to sick leave due to neck, shoulder and/or back disorders, while factors related only to health predicted sick leave due to any cause. The results point to the importance of a primary focus on previous low back disorders and previous sick leave when designing early prevention programmes for future sick leave among this working population. The results might also point to the importance of a multifactorial approach when customising early prevention programmes with the purpose to decrease future sick leave due to neck, shoulders and/or back disorders in a group of women working as nursing aides/assistant nurses. Competing interests The author(s) declare that they have no competing interests. Authors' contributions EHj participated in the design of the study, participated in collecting the data, performed the statistical analyses, and drafted the manuscript. IJ and EHm participated in the design of the study and in the progress and revision of the manuscript. CE participated in the progress and revision of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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The Rab5 Effector Rabankyrin-5 Regulates and Coordinates Different Endocytic Mechanisms

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity . In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.

Introduction In mammalian cells multiple mechanisms of endocytosis operate within a single cell to perform nutrient uptake, cellular homeostasis, neurotransmission, signal transduction, antigen presentation, and defence against pathogens. Endocytosis comprises two major categories, phagocytosis and pinocytosis, depending on the uptake of particles or fluid, respectively (reviewed in Conner and Schmid 2003 ). Pinocytosis encompasses various membrane entry routes and mechanisms. Clathrin-mediated endocytosis, the best-studied route at the molecular level to date, primarily serves receptor-mediated uptake. Caveolae assemble on sphingolipid and cholesterol rafts and may internalise molecules partitioning in these lipid microdomains. To a certain extent, ligand-receptor complexes and raft components can follow both entry routes but can also be internalised via clathrin- and caveolae-independent endocytosis. Large volumes of fluid are engulfed by the closure of plasma membrane protrusions in a process termed macropinocytosis. Although it was the first mode of pinocytosis reported ( Lewis 1931 ), little is known on the molecular mechanisms underlying this physiologically very important endocytic process. Constitutively active in immature dendritic cells, macropinocytosis favors antigen sampling ( Steinman and Swanson 1995 ). However, it can be transiently induced in most cells by growth factors ( Haigler et al. 1979 ; Shao et al. 2002 ), tumor-promoting chemicals such as phorbol 12-myristate 13-acetate (PMA), or oncogenes like H-Ras or v-Src ( Bar-Sagi and Feramisco 1986 ; Veithen et al. 1996 ), and it has been proposed to downregulate signalling molecules from the cell surface. Since macropinosomes arise from membrane ruffles, regions of intense actin remodelling which are also a trait of motile cells, they have been implicated in directed cell locomotion ( Carpentier et al. 1991 ). Macropinocytosis is exploited by several invasive pathogens as entry route ( Francis et al. 1993 ; Sansonetti 1997 ) but differs from phagocytosis with respect to regulation (e.g., receptor mediated) and cargo (e.g., uptake and degradation of opsonised particles) ( Galan and Zhou 2000 ). Macropinosomes are distinct from early endosomes, morphologically and biochemically. In epidermal growth factor (EGF)-stimulated A431 cells, whereas macropinosomes can fuse homotypically, they seldom fuse with early endosomes ( Hewlett et al. 1994 ). However, this partition is not absolute, since in dendritic cells macropinosomes do fuse with endosomes ( Racoosin and Swanson 1993 ). The intracellular trafficking properties of macropinosomes may therefore be governed by cell type-specific mechanisms and fulfil specialised functions. Finally, macropinocytosis shares mechanistic features with apical fluid-phase endocytosis in polarised cells ( Gottlieb et al. 1993 ; Jackman et al. 1994 ; Holm et al. 1995 ; Amyere et al. 2002 ). In kidney, apical pinocytosis contributes an essential function to the physiology of the renal system by contributing to vectorial fluid-phase transport across the membranes ( Goligorsky and Hruska 1986 ). Given the dependence on actin remodelling, it is no surprise that Rho GTPases ( West et al. 2000 ), ARF6 ( Radhakrishna et al. 1996 ), and type 1 phosphatidylinositol-3 kinases (PI3-Ks) ( Hooshmand-Rad et al. 1997 ) are involved in macropinocytosis, presumably through their role in membrane ruffling. Much less clear is the function of two Rab GTPases, Rab5 and Rab34/Rah, both of which are implicated in the formation of macropinosomes ( Li et al. 1997 ; Sun et al. 2003 ). Rab34/Rah colocalises with actin to membrane ruffles and nascent macropinosomes and its overexpression promotes macropinocytosis ( Sun et al. 2003 ). Rab5 regulates fluid-phase and receptor-mediated uptake ( Bucci et al. 1992 ; McLauchlan et al. 1998 ), and its activity has been linked to Ras-stimulated fluid-phase endocytosis ( Li et al. 1997 ). However, Rab5 colocalises to some, but not all, macropinosomes harbouring Rab34/Rah ( Sun et al. 2003 ). Furthermore, Rab5 together with its effector PI3-K, hVPS34, regulates the recruitment of a set of cytosolic FYVE-finger effector proteins on early endosomes. These molecules cooperate in the tethering and fusion of clathrin-coated vesicles (CCVs) with early endosomes and homotypic endosome fusion ( Gorvel et al. 1991 ; Bucci et al. 1995 ; Rubino et al. 2000 ) as well as the motility of early endosomes along microtubules ( Nielsen et al. 1999 ). However, in view of the function of Rab5 on the early endosomes, its role in macropinocytosis is difficult to assess. Specifically, it is unclear whether the large vacuoles induced by Rab5 activation, either via the expression of activated mutants ( Stenmark et al. 1994 ) or via signalling molecules that stimulate the GTP loading on Rab5 ( Lanzetti et al. 2000 ; Tall et al. 2001 ), correspond to either macropinosomes or enlarged endosomes originated by increased homotypic early-endosome fusion, or both ( Hewlett et al. 1994 ). Here, the identification of a novel Rab5 FYVE-finger effector, Rabankyrin-5, led us to revisit the role of Rab5 in fluid-phase (macro)pinocytosis in both nonpolarised and polarised cells. Results Identification of a Novel 130-kDa Rab5 Effector We have previously purified a large number of Rab5 effectors by an affinity chromatography approach based on glutathione S-transferase (GST)-Rab5-GTPγS (Christoforidis et al.1999a). Among the numerous proteins purified, we focused on a prominent 130-kDa protein ( Figure 1 A). The protein was digested with trypsin and the amino acid sequences of the tryptic fragments were determined by nanoelectrospray tandem mass spectrometry ( Wilm et al. 1996 ). The identified peptides matched the mouse sequence of Ankhzn (unpublished data). The corresponding human cDNA was obtained by PCR, using primers derived from the mouse DNA sequence and a random primed HeLa cDNA library as template ( Zerial et al. 2001 ). The sequence of the human cDNA matched the recently revised hAnkhzn gene sequence (NP 057460.2) ( Kuriyama et al. 2000 ). The predicted human p130 primary sequence consists of an N-terminal BTB/POZ domain, a C-terminal FYVE-finger, and 21 successive ankyrin (ANK) repeats in between these two domains ( Figure 1 B). In light of the fact that p130 is a Rab5 effector (this study), we propose to amend its name to Rabankyrin-5 ( Rab5 binding and ankyrin repeats containing protein). Figure 1 A Protein of 130 kDa Is a New Rab5 Effector (A) GST-Rab5-GDP and GST-Rab-GTPγS were loaded on beads and incubated with bovine brain cytosol. Bound proteins were eluted and analysed by SDS-PAGE followed by Coommasie Blue staining. The positions of the already known Rab5 effectors (EEA1, Rabaptin-5, hVps34, p110β, and Rabenosyn-5) and of the new Rab5 effector are indicated. (B) Schematic representation of the domain organisation in Rabankyrin-5. ANK, ankyrin repeats. (C) Bovine brain cytosol or HeLa cell cytosol was incubated with GST-Rab5-GDP– or GST-Rab5-GTPγS–loaded beads. Subsequently the beads were washed, and bound proteins were eluted and analysed by Western blotting using anti–Rabankyrin-5 antibodies. (D) GST-Rab4, -5, -7, and -11 fusion proteins were preloaded with GDP or GTPγS and incubated with in vitro-translated 35 S-methionine–labelled Rabankyrin-5 full-length protein. As a control, bound and unbound material was analysed by SDS-PAGE followed by phosphoimager analysis. (E) Rabankyrin-5 binds most strongly to PI(3)P. Recombinant full-length Rabankyrin-5 was incubated with liposomes containing 2% of the indicated phosphoinositide. Bound Rabankyrin-5 was detected by Western blotting. To investigate the function of human Rabankyrin-5, we raised antibodies against the recombinant full-length protein. By Western blotting, these antibodies detected a predominant band of 130 kDa in both bovine brain and HeLa cytosol, in the corresponding GST-Rab5-GTPγS but not GST-Rab5-GDP affinity column eluates ( Figure 1 C). Evidence for a direct interaction with Rab5 was obtained by incubating in vitro-translated Rabankyrin-5 with beads displaying GST-Rab5, -Rab4, -Rab7, and -Rab11, preloaded with either GDP or GTPγS. Figure 1 D shows that human Rabankyrin-5 binds to GST-Rab5-GTPγS, but neither to beads containing GST-Rab5-GDP nor to other Rab proteins. We conclude that Rabankyrin-5 binds to Rab5 specifically, directly, and GTP dependently. Association of Rabankyrin-5 with Two Types of Rab5-Positive Vesicles After EEA1 ( Stenmark et al. 1996 ) and Rabenosyn-5 ( Nielsen et al. 2000 ), Rabankyrin-5 is the third Rab5 effector containing a FYVE-finger. Since the FYVE-finger is a key determinant for targeting these proteins to early endosomes via its binding to phosphatidylinositol-3-phosphate [PI(3)P] ( Kutateladze et al. 1999 ), we tested whether this holds true also for Rabankyrin-5. First, recombinant Rabankyrin-5 interacted significantly with PI(3)P in a liposome-binding assay ( Figure 1 E). Second, endogenous Rabankyrin-5 colocalised significantly with EEA1 and Rab5-positive early endosomes (approximately 80%) in A431 cells by confocal immunofluorescence microscopy analysis (see Figure 3 A and 3 B below). Figure 3 Rabankyrin-5 Associates with Two Types of Rab5-Containing Vesicles in A431 Cells, Early Endosomes, and Macropinosomes (A) A431 cells stably transfected for GFP-Rab5wt were immunostained for endogenous Rabankyrin-5 and EEA1. While there is perinuclear overlap between Rab5, Rabankyrin-5, and EEA1 in nontransfected cells (arrowheads), some smaller peripheral structures are devoid of EEA1 (arrows). (B) Overexpression of Rabankyrin-5 in A431 by using a recombinant adenovirus construct of Rabankyrin-5 causes an accumulation of peripheral, enlarged Rab5-positive structures, costained mainly by Rabankyrin-5 (arrows) but not detectable for EEA1. (C) Rabankyrin-5 localises on EGF-induced and -enriched macropinosomes. Serum-starved A431 cells (16 h) were incubated for 7 min with 100 ng/ml rhodamine-conjugated EGF to induce macropinocytosis and 1 μg/ml Cy5-labelled transferrin. Endogenous Rabankyrin-5 localises to enlarged EGF-containing macropinosomes, indicated by the lack of transferrin labelling (arrows), but also to EGF- and transferrin-containing endosomes (arrowheads). (D) Rabankyrin-5 structures contain tyrosine-phosphorylated proteins. A431 cells, stably transfected for GFP-Rab5, were stimulated with 50 ng/ml EGF for 7 min and immediately processed for immunofluorescence. Costaining of Rabankyrin-5 and tyrosine-phosphorylated proteins (α-4G10) reveal the localisation of Rabankyrin-5 to plasma membrane ruffles. Scale bars represent 10 μm. We next investigated whether Rabankyrin-5 plays a role in homotypic early endosome fusion as well as fusion of CCVs with early endosomes, similar to EEA1 ( Christoforidis et al. 1999a ) and Rabenosyn-5 ( Nielsen et al. 2000 ). Quantitative immunodepletion of the cytosolic pool of Rabankyrin-5 using anti–Rabankyrin-5 antibodies ( Figure 2 A) had only minor effects on early endosome fusion in vitro compared to preimmune serum ( Figure 2 B). Considering the possibility that the early-endosome–associated pool of Rabankyrin-5 may still be sufficient for fusion, we attempted to neutralise its function by the addition of anti–Rabankyrin-5 antibodies on top of Rabankyrin-5–depleted cytosol. This treatment strongly inhibited early-endosome fusion ( Figure 2 B). The fusion of CCVs with early endosomes was minimally affected under these conditions ( Figure 2 C), suggesting that Rabankyrin-5 is not essential in this process, at least in vitro. However, addition of increasing amounts of recombinant Rabankyrin-5 (at concentrations in the range of the endogenous protein; see Materials and Methods ) on either immunodepleted cytosol or nontreated cytosol significantly enhanced both homo- and heterotypic fusion activity. We conclude from these experiments that endogenous Rabankyrin-5 colocalises with EEA1 on Rab5 endosomes in vivo, through the interaction with Rab5 (see Figure 1 D) and PI(3)P (see Figure 1 E). It plays a modulatory role in early-endosome fusion since it can stimulate both homotypic and heterotypic fusion of CCVs with early endosomes. However, it does not appear to be strictly required in the latter reaction in vitro. Figure 2 Rabankyrin-5 Stimulates Homotypic Fusion between Early Endosomes and Heterotypic Fusion between Early Endosomes and CCVs In Vitro (A) Rabankyrin-5 was efficiently immunodepleted from HeLa cytosol without affecting the EEA1 concentration. Twenty micrograms of HeLa cell cytosol, either treated with control IgGs and immunodepleted for Rabankyrin-5 or nontreated, was applied to SDS-PAGE and probed for the indicated antigens by Western blotting. Anti-γ tubulin was used as a loading control. (B and C) Addition of recombinant Rabankyrin-5 stimulates homotypic and heterotypic fusion. Fusion (B) between early endosomes (EE-EE fusion) and (C) of early endosomes to CCVs (CCV-EE fusion) in vitro was performed in the presence of 3 mg/ml HeLa cytosol and an ATP-regenerating system (basal) and in the absence or presence of the indicated reagents. For some conditions, cytosol was immunodepleted for Rabankyrin-5 (columns 8–13) and in addition supplemented with anti–Rabankyrin-5 antibodies to neutralise the function of membrane-associated proteins (column 10). Background fusion activity is demonstrated by ATP (−Energy) or cytosol (−Cytosol) omission. Surprisingly, we noted that some peripheral structures harbouring Rabankyrin-5 overlapped with Rab5 but either poorly or not at all with EEA1 in A431 cells ( Figure 3 A, arrows). Upon overexpression, Rabankyrin-5 induced the accumulation of several vesicles of variable size in the periphery of the cells, most of them lacking EEA1 ( Figure 3 B, arrows). The irregular size, intracellular distribution ( Swanson 1989 ), and lack of endosomal markers (see “ Rabankyrin-5 localises to macropinosomes ” below) ( Hewlett et al. 1994 ) of these structures were suggestive of (macro)pinocytic vesicles. We therefore set out to investigate the putative role of Rabankyrin-5 in macropinocytosis. Rabankyrin-5 Localises to Macropinosomes Macropinosomes have been so far defined by a combination of criteria rather than specific molecular markers. To test whether the Rabankyrin-5 vesicles fulfil the previously described requirements of macropinosomes, we determined whether they (1) are stimulated by growth factors ( Haigler et al. 1979 ; West et al. 1989 ; Amyere et al. 2000 ), (2) do not promote receptor-mediated endocytosis of transferrin, (3) engulf large amounts of extracellular fluid ( Hewlett et al. 1994 ; Amyere et al. 2000 ), (4) depend on PI3-K activity ( Araki et al. 1996 ; Zhou et al. 1998 ), and (5) follow actin rearrangements at the plasma membrane. A431 and NIH3T3 cells are established experimental systems that exhibit different features in the regulation of macropinocytosis. In A431 cells, macropinocytosis can be transiently induced upon growth factor stimulation ( Hewlett et al. 1994 ), whereas NIH3T3 cells exhibit constitutive macropinocytic activity ( Dharmawardhane et al. 2000 ). We first tested whether EGF can induce the formation of Rabankyrin-5–positive macropinocytic structures. Upon treatment of A431 cells with rhodamine-conjugated EGF, endogenous Rabankyrin-5 colocalised to enlarged structures which, importantly, were largely devoid of simultaneously internalised transferrin ( Figure 3 C). Interestingly, EGF was not excluded but rather enriched in these structures. In addition, we observed that such vesicles were also enriched in tyrosine-phosphorylated proteins, presumably EGF-receptor and downstream signalling molecules ( Figure 3 D). In NIH3T3 fibroblasts, the staining pattern of endogenous Rabankyrin-5 was similar to that of EGF-stimulated A431 cells. Rabankyrin-5 was detected on enlarged structures, predominantly at the outermost periphery and in membrane protrusions of the cell ( Figure 4 A, arrows). Consistent with these observations, immunoelectron microscopy revealed that antibodies to Rabankyrin-5 labelled electron-lucent vesicular structures underlying the plasma membrane in the cell periphery and in cell protrusions ( Figure 5 ). The labelled structures were sparse in untransfected cells, but in Rabankyrin-5– overexpressing cells fortuitous sections revealed concentrated areas of Rabankyrin-5–labelled structures. Unfortunately, we failed to distinguish these structures from conventional early endosomes by double immunolabelling experiments due to technical limitations. By light microscopy, the appearance of vacuole-shaped structures devoid of the early endosomal marker EEA1 was dramatically increased upon Rabankyrin-5 overexpression. In addition, after a 3-min pulse, these structures were intensively labelled by a fluid-phase marker (see Figure 4 B, arrows) but not by transferrin (unpublished data). Given the potent induction of macropinosomes by Rabankyrin-5 in NIH3T3 cells, we used this experimental system to explore further the regulatory parameters of macropinocytosis. Figure 4 Rabankyrin-5 Localises to Macropinosomes in NIH3T3 Cells (A) NIH3T3 cells directly fixed and immunostained for endogenous Rabankyrin-5 and EEA1 reveal segregation of Rabankyrin-5 from EEA1-containing structures (arrows) in the cell periphery, while there is colocalisation in the cell centre (arrowheads). (B) Overexpression of Rabankyrin-5 increases the number of peripheral enlarged structures devoid of EEA1. NIH3T3 cells were infected with recombinant adenovirus for Rabankyrin-5 for 18 h. Dextran (2,5 mg/ml) uptake was performed for 3 min at 37 °C, fixed, and immunostained for the indicated antigens. (C and D) Formation of enlarged Rabankyrin-5 structures requires PI3-K activity. NIH3T3 cells either (C) DMSO treated or (D) pretreated for 20 min with wortmannin (WM; 100 nM) were incubated for 8 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 10.000), fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy. (E) NIH3T3 cells transiently transfected for YFP-Rabankyrin-5 and CFP-actin were imaged using time-lapse video microscopy to visualise the formation of macropinosomes by actin-driven membrane ruffles. Images were taken for the indicated time points. The arrowhead points towards Rabankyrin-5 association to an enlarged vesicle driven by actin dynamics over time. Scale bars represent 10 μm. Figure 5 Immunolocalisation of Endogenous and Overexpressed Rabankyrin-5 in NIH3T3 Cells Untransfected NIH3T3 cells or cells transfected with Rabankyrin-5 were labelled with antibodies to Rabankyrin-5 followed by 10 nm protein A gold. (A) Transfected cell showing labelling of a group of vesicular structures underlying the plasma membrane (pm). (B and C) In control (untransfected) cells, low but specific labelling for Rabankyrin-5 (arrowheads) is associated with compartments close to the pm. Scale bars represent 200 nm. In NIH3T3 cells, Rabankyrin-5–enlarged structures were labelled by an 8-min uptake of fluorescein isothiocyanate (FITC)-dextran (MW, 10.000). Consistent with their macropinocytic nature, these structures were largely depleted of transferrin (see Figure 4 C, arrows) and susceptible to the PI3-K inhibitors wortmannin (see Figure 4 D) and LY 294002 (unpublished data). These drugs both reduced the formation of peripheral enlarged vesicles and almost completely blocked the internalisation of high-molecular-weight dextran (MW, 70.000) ( Figure S1 A) that enters the cell almost exclusively via macropinocytosis ( Dharmawardhane et al. 2000 ). Interestingly, we noted that, unlike EEA1, Rabankyrin-5 did not readily dissociate from membranes (see Figure 4 D) upon inhibition of PI3-K activity, suggesting that endosome-targeting determinants other than PI(3)P contribute to its localisation. Neither markers of late endosomes (Rab7) ( Chavrier et al. 1990 ), nor of caveosomes (caveolin) ( Pelkmans et al. 2001 ), nor of the clathrin-independent pathway which is used by glycosyl-phosphatidylinositol (GPI)-anchored proteins (GFP-GPI) ( Sabharanjak et al. 2002 ) overlapped significantly with the peripheral enlarged Rabankyrin-5 structures (unpublished data). To confirm that the Rabankyrin-5 structures originated from the plasma membrane through an actin-driven process, we employed time-lapse video fluorescence microscopy on NIH3T3 cells transiently coexpressing yellow fluorescent protein (YFP)-Rabankyrin-5 and cyan fluorescent protein (CFP)-β-actin. In the sequence depicted in Figure 4 E (see also Video S1 ), membrane ruffling driven by actin resulted in the formation of a large, plasma membrane-derived vesicle acquiring Rabankyrin-5. Some newly formed vesicles shed off Rabankyrin-5 over time and seemed to regurgitate back to the plasma membrane via an actin comet-tail. Altogether, the data support the hypothesis that the enlarged structures containing Rabankyrin-5 are macropinosomes and can be molecularly distinguished from early endosomes by being mostly, albeit not exclusively, devoid of EEA1. Moreover, and contrary to earlier findings ( West et al. 1989 ), we found that in A431 cells, EGF but not transferrin could be enriched in macropinosomes. Both Rab5 and Rabankyrin-5 Are Required for Macropinocytosis The GTP-dependent interaction with Rab5 suggests that Rabankyrin-5 may function as a downstream effector of Rab5 in the macropinocytic pathway. Accordingly, one would expect that Rab5 localises to macropinosomes and, together with Rabankyrin-5, is required for macropinocytosis. We found that Rab5 indeed colocalises with Rabankyrin-5 on macropinosomes (see Figure 3 B, 3 D, and 6 A; unpublished data), which are operationally defined as vesicles (1) enriched in fluid-phase marker but depleted of transferrin receptor ( Racoosin and Swanson 1992 ), (2) of large size (0,2–2 μm) ( Swanson 1989 ; Hewlett et al. 1994 ), and (3) negative for EEA1 but positive for Rabankyrin-5. Consistent with a requirement for Rab5 in macropinocytosis, overexpression of RN-tre, a GTPase-activating protein (GAP) for Rab5 ( Lanzetti et al. 2000 ), significantly reduced (by 60% ± 10%) the uptake of large fluorescent dextran (MW, 70.000) into transferrin- and EEA1-negative structures in NIH3T3 cells ( Figure 6 B and 6 C). Similar results were also obtained when NIH3T3 cells were cotransfected for Rab5S34N and Rabankyrin-5 (unpublished data), implying that Rabankyrin-5 induces macropinocytosis Rab5-GTP dependently. Figure 6 Inhibition of Rab5 Activity Decreases Fluid-Phase Uptake NIH3T3 cells transiently transfected for either (A) GFP-Rab5wt or (B) RN-tre were subjected to a 30-min uptake of rhodamine-conjugated transferrin (1 μg/ml) or dextran (MW, 70.000; 3 mg/ml) at 37 °C and further processed for confocal imaging with indicated antibodies. (A) Rab5wt transfected cells show colocalisation of Rabankyrin-5–labelled macropinocytic structures, indicated by the lack of transferrin accumulation, with Rab5. (B) Cells transiently transfected for RN-tre (asterisk) show a significant reduction of fluid-phase uptake compared to nontransfected cells. (C) Fluid-phase dextran quantification of single cells transfected for RN-tre by measuring internalised fluorescence intensity ( p > 0,001). Values shown are means ± standard deviation of at least 15 cells. Scale bars represent 10 μm. Whereas the aforementioned results demonstrate a general requirement for Rab5 and Rabankyrin-5 in pinocytosis, they do not discriminate between clathrin-dependent and-independent endocytosis. To distinguish between the two pathways, we compared the effects of Rabankyrin-5 overexpression on transferrin and horseradish peroxidase (HRP) uptake in NIH3T3 cells, biochemically. A recombinant adenovirus was generated to overexpress Rabankyrin-5 in these cells. In comparison with mock-infected cells, Rabankyrin-5 overexpression increased the uptake of HRP, particularly after longer (10 min) incubation times ( Figure 7 A). In contrast, the rate of transferrin uptake was unaffected, suggesting that the increase of fluid phase was not due to increased clathrin-mediated endocytosis ( Figure 7 C). The increased intracellular accumulation of HRP induced by Rabankyrin-5 seems to be due to increased uptake rather than inhibited recycling, as the latter process was only slightly decreased ( Figure 7 B). Figure 7 Rabankyrin-5 Overexpression Increases, whereas Knock-Down Decreases Fluid-Phase Uptake, Specifically (A–C) NIH3T3 cells were either mock-infected or infected with recombinant adenovirus encoding Rabankyrin-5. Simultaneous uptake of HRP (5 mg/ml) and biotinylated transferrin (2 μg/ml) was performed at 37 °C for the indicated time points. (C) NIH3T3 cells were pulsed with HRP (10 mg/ml) for 10 min. Recycled HRP into the medium was determined after the indicated time points. (D–F) A431 cells were treated with esiRNA against Rabankyrin-5 or control treated for 4 d. (D) Western blot analysis revealed a 50% reduction of Rabankyrin-5 in whole-cell lysate. (E and F) Serum-starved cells (1 h) were stimulated with 50 ng/ml EGF in complete medium for the indicated time points in the presence of 5 mg/ml HRP and 2 μg/ml biotinylated transferrin. Values shown are means ± standard deviation and were performed in duplicates. The results are representatives of at least two independent experiments. To determine whether Rabankyrin-5 is also required for macropinocytosis, we attempted to ablate the protein by RNA interference using endoribonuclease-prepared small interfering RNA (esiRNA) ( Yang et al. 2002 ). Since we optimised this technique in human cells, we conducted these experiments in EGF-stimulated A431 cells where an approximately 55% knock-down of Rabankyrin-5 was achieved ( Figure 7 D). EGF stimulation of control-treated cells enhanced HRP uptake 2-fold versus control-treated, non-EGF-stimulated cells, as reported previously. Reduction of Rabankyrin-5 expression in esiRNA-transfected cells caused an inhibition of HRP of up to approximately 50% by selectively affecting the burst of pinocytosis induced by EGF (within 5 min) ( Figure 7 E). Under the same conditions, transferrin uptake was marginally inhibited ( Figure 7 F). We conclude from these experiments that inhibition of fluid-phase uptake occurs due to an inhibition of (macro)pinocytosis rather than of clathrin-mediated endocytosis. To confirm the requirement of Rabankyrin-5 for fluid-phase endocytosis we used an independent method: quantification of the uptake of fluorescent dextran in NIH3T3 cells microinjected with affinity-purified anti–Rabankyrin-5 antibodies. Following microinjection, FITC-dextran uptake was internalised for 30 min to label macropinosomes. Microinjection of anti–Rabankyrin-5 antibodies inhibited FITC-dextran uptake by 53% ± 5% when compared with cells injected with preimmune serum ( Figure S2 ), whereas the uptake of rhodamine-transferrin was not significantly changed. Altogether, these results suggest that Rabankyrin-5 is a Rab5 effector required for the formation of macropinosomes. Rabankyrin-5 Regulates Apical Fluid-Phase Endocytosis in Polarised Epithelial Cells Apical pinocytosis in polarised epithelial cells and growth factor-induced macropinocytosis share mechanistic properties, like dependence on actin ( Gottlieb et al. 1993 ) and PI3-K activity ( Tuma et al. 1999 ; Sandvig et al. 2000 ). In view of the role of Rab5 in endocytic trafficking of polarised cells such as epithelial cells and neurons ( Bucci et al. 1994 ; de Hoop et al. 1994 ), we explored the role of Rabankyrin-5 in apical endocytosis in polarised epithelial cells. First, we determined the intracellular localisation of Rabankyrin-5 on cryosections of adult mouse kidney, since the epithelial cells in this organ, especially proximal tubule cells ( Christensen et al. 1998 ), exhibit high levels of apical pinocytosis under physiological conditions. Apical endocytic vesicles were pulse-labelled by injecting mice with HRP followed by fixation of the kidney through perfusion after 5 min. Prominin-1 was used as marker for the apical brush border of proximal tubules ( Weigmann et al. 1997 ) in combination with affinity-purified anti–Rabankyrin-5 antibodies. Basement membrane staining was demonstrated by immunolabelling of endogenous mouse immunoglobulin G (IgG). As illustrated in Figure 8 , Rabankyrin-5 immunoreactivity was mainly detected on vesicle-like structures underneath the apical, prominin-1–labelled brush border. Most of the vacuole-like Rabankyrin-5 structures contained HRP, suggesting that they correspond to apical endocytic structures ( Figure 8 D, arrows). By further investigating the nature of these Rabankyrin-5– and HRP-labelled structures, we observed that some vesicles were immunoreactive for LAMP-1 ( Figure 8 D, arrowheads), suggesting that Rabankyrin-5 (macro)pinocytic vesicles can acquire characteristics of late endocytic compartments. Figure 8 Rabankyrin-5 Localises on Vesicular Structures Underlying the Apical Brush Border in Mouse Kidney Proximal Tubules Mice were perfused either (A–C) directly with 4% PFA or (D) 5 min after injection of HRP into the femoral vein. Immunofluorescence was performed on semi-thin cryosections (500 nm). (C) Higher magnification of the inset in (B). Scale bars represent 10 μm. This was confirmed and extended by immunogold electron microscopy on ultrathin frozen sections of mouse proximal tubule. Rabankyrin-5 showed weak labelling of the apical microvilli-covered surface of proximal tubule cells but labelled large electron-lucent structures underlying the apical surface ( Figure 9 A and 9 B). Consistent with the immunofluorescence analysis, double labelling with LAMP-1 antibodies confirmed that many of these structures were LAMP-1 negative or weakly labelled. However, a significant number of Rabankyrin-5–positive structures were also LAMP-1 positive. Rabankyrin-5 immunoreactivity was also observed in distal tubule cells and other segments of the nephron. Figure 9 Immunolocalisation of Rabankyrin-5 in the Mouse Kidney Mouse kidney cortex was processed for frozen section immunoelectron microscopy. Sections were (A and B) single labelled for Rabankyrin-5 (arrowheads, 10 nm) or (C and D) double labelled (arrows, 5 nm) for Rabankyrin-5 and LAMP-1. (A) Low-magnification view of the apical region of two proximal tubule cells demonstrates low labelling for Rabankyrin-5 on apical microvilli (M) but stronger labelling (arrowheads) of large subapical electron-lucent vesicular structures (asterisks). One of these structures is shown at higher magnification in (B). L, lateral membrane. (C) Rabankyrin-5 labels LAMP-1–negative subapical structures as well as compartments showing low LAMP-1 labelling (arrows and asterisk). (D) Rabankyrin-5 (arrowheads) associates with compartments, which show no or weak labelling for LAMP-1 (asterisks). In addition, low Rabankyrin-5 labelling is associated with more strongly labelled LAMP-1–positive compartments. Note that there is some nonspecific labelling of mitochondria (m). Scale bars represent 500 nm. To functionally address the role of Rabankyrin-5 in pinocytosis, we used filter-grown Madin-Darby canine kidney (MDCK) cells as an established system to study polarised trafficking. Alexa 488 dextran was internalised for 13 min from either the apical or the basolateral chamber. Although endogenous Rabankyrin-5 exhibited partial colocalisation with fluid-phase marker internalised from the basolateral side (unpublished data), a striking degree of overlap between the internalised tracer and Rabankyrin-5 on often ring-shaped structures underlying the apical region was observed ( Figure 10 A). These large vesicles were neither detectable in the nuclear area ( Figure 10 B) nor close to the basolateral domain ( Figure 10 C). We next tested whether Rabankyrin-5 overexpression could stimulate fluid-phase uptake from the apical plasma membrane domain. Rabankyrin-5 was overexpressed using the recombinant adenovirus system in MDCK cells grown on coverslips and the cells stained for Rabankyrin-5 and EEA1. As in A431 and NIH3T3 cells, and similar to endogenous Rabankyrin-5 in MDCK cells ( Figure 11 A), the overexpressed protein exhibited significant colocalisation with endogenous EEA1, but in addition, it induced the formation of enlarged structures devoid of EEA1 in the periphery of the cell ( Figure 11 B). The recombinant adenovirus was next applied to infect filter-grown MDCK cells. In these cells, it induced enlarged Rabankyrin-5 structures predominantly underlying the apical domain that contained dextran internalised from the apical side. Some of these structures were EEA1 positive, whereas others lacked this marker of early endosomes (see Figure 10 D, arrows). These structures were also detectable to some extent in the nuclear region (see Figure 10 E) and basolateral membrane domain (see Figure 10 F). In addition, there were some enlarged Rabankyrin-5 vesicles that were labelled for neither dextran nor EEA1. These may correspond to macropinosomes that were formed prior to the labelling with the fluorescent fluid-phase marker. Figure 10 Rabankyrin-5 Colocalises to Apically Labelled Structures in Filter-Grown MDCK Cells MDCK cells were seeded on polycarbonate filters for 4 d and either (A–C) mock-infected or (D–F) infected with an adenovirus construct expressing full-length Rabankyrin-5 for another 18 h. Cells were then incubated for 13 min with 5 mg/ml Alexa 488 dextran, added to the apical medium, fixed, and stained for the indicated antigens. Images represent confocal scans just beneath (A and D) the apical plasma membrane, (B and E) the nuclear area, and (C and F) the basal plasma membrane. Endogenous Rabankyrin-5 and EEA1 localise to several dextran-labelled structures, often with ring-shaped appearance in the apical region. Overexpression of Rabankyrin-5 seems to increase the number of enlarged and dextran-labelled structures, some of which are depleted of EEA1 (arrows) (B). Scale bars represent 10 μm. Figure 11 Overexpression of Rabankyrin-5 Stimulates Fluid-Phase Uptake from the Apical Plasma Membrane (A and B) MDCK cells plated on coverslips were (A) mock-infected or (B) infected with Rabankyrin-5 and processed for immunofluorescence. Under both conditions EEA1 colocalised almost completely with Rabankyrin-5, while there were some Rabankyrin-5 structures devoid of EEA1. Upon Rabankyrin-5 overexpression these EEA1-lacking structures were enlarged macropinocytic-like vesicles localised predominantly in the periphery of the cell, while other enlarged compartments containing EEA1 were centripetally located. (C–F) Filter-grown MDCK cells were either mock-infected or infected with recombinant adenovirus for full-length Rabankyrin-5. HRP (5 mg/ml) or biotinylated Fab fragments (50 μg/ml) were internalised from the indicated chambers for various time points. Whereas Rabankyrin-5 overexpression (C) specifically increased HRP uptake from the apical domain, (D) basolateral HRP uptake and (E and F) Fab uptake at both domains were unaffected ( p < 0,01). Values shown are means ± standard deviation of at least three independent experiments. Scale bars represent 10 μm. We next measured fluid-phase uptake biochemically by quantifying the internalisation of HRP either from the apical or the basolateral side for various periods of time. Filter-grown MDCK cells were either infected with adenovirus encoding Rabankyrin-5 or control virus prior to HRP internalisation. While internalisation of HRP from the basolateral plasma membrane did not significantly vary ( Figure 11 D), uptake from the apical membrane was increased up to 65% in cells overexpressing Rabankyrin-5 when compared with control cells ( Figure 11 C). Given that Rabankyrin-5 was overexpressed in only 50–60% of the cells, this value may be an underestimate. Since the kinetic profiles resembled the ones obtained in NIH3T3 cells, we also tested whether the increase in HRP uptake may have been due to an inhibition of recycling or transcytosis. Neither of these trafficking pathways was significantly affected (unpublished data). To verify that the increase in fluid-phase uptake was not due to stimulation of clathrin-mediated endocytosis, we measured the internalisation of FcLR 5–27, a chimeric receptor between the IgG Fc receptor and the LDL receptor which, when expressed in MDCK cells, is targeted to, and internalised from, both apical and basolateral plasma membrane domains ( Matter et al. 1992 ). Rabankyrin-5 overexpression did not affect significantly the internalisation of FcLR 5–27, as revealed by the uptake of a biotinylated Fab-fragment of the 2.4G2 monoclonal anti-FcRII antibody ( Figure 11 E and 11 F). Taken together, these findings indicate that Rabankyrin-5 is predominantly localised to apical endocytic structures and specifically stimulates apical, non-clathrin-mediated fluid-phase endocytosis upon overexpression in polarised epithelial cells. Discussion Rabankyrin-5 in Macropinocytosis Since the pioneering work of Bar-Sagi and Feramisco (1986) almost two decades ago, it has been established that certain oncogenes and signalling molecules induce actin-dependent membrane ruffling and macropinocytosis. Subsequent work has advanced our understanding of the signalling pathway that led to growth factor-dependent actin remodelling and membrane ruffling ( Ridley 1994 ). Despite macropinocytosis being the most ancient form of pinocytosis and an endocytic process of high physiological importance ( Amyere et al. 2002 ), progress in determining the molecular mechanisms that regulate the generation and trafficking of macropinosomes has advanced to a lesser extent. It has been demonstrated that macropinocytosis differs from clathrin-mediated endocytosis ( West et al. 1989 ) and that the two pathways lead to distinct endocytic structures ( Hewlett et al. 1994 ). Rab5, an established regulator of clathrin-mediated endocytosis and endosome dynamics ( Gorvel et al. 1991 ; Bucci et al. 1992 ; McLauchlan et al. 1998 ), has also been implicated in macropinocytosis ( Li et al. 1997 ). This hypothesis was based on the expression of mutants that induced or inhibited fluid-phase endocytosis and formation of giant endocytic vesicles. In those studies, however, an accurate assessment of whether the enlarged compartments corresponded to macropinosomes or were produced by the coalescence of multiple endosomes by homotypic fusion was not determined. Here, we provide evidence that Rabankyrin-5 is a novel Rab5 effector which, in addition to being localised to early endosomes, is associated with macropinosomes and promotes their formation according to previously established criteria. First, Rabankyrin-5 localised to enlarged vesicles that were stimulated by growth factors (e.g., EGF). Second, these vesicles differed from early endosomes by the lack of constitutively endocytosed molecules, such as transferrin, and components of the transport machinery, such as EEA1. Third, the vacuoles originated from the plasma membrane and engulfed extracellular fluid. Fourth, their formation depended on PI3-K activity, and fifth, they followed actin rearrangements at the plasma membrane. Possible Functions of Rabankyrin-5 Macropinosomes are formed by the closure of membrane protrusions generated upon actin-mediated membrane ruffling. Although membrane ruffling is required for macropinocytosis, it seems not to be sufficient for macropinosome formation ( Araki et al. 1996 ; West et al. 2000 ). Our data suggest that Rabankyrin-5 is a novel regulator of this endocytic mechanism. One possibility is that it could play an active role in the generation of macropinosomes (see Figure 7 ). The deduced primary structure of Rabankyrin-5 predicts the existence of several protein-protein interaction motifs, suggesting a role as a multifunctional adaptor protein. The N terminus of Rabankyrin-5 contains a BTB/POZ motif, which is present in proteins involved in signalling, development, and tumorigenesis and mediates homo- and heterodimerisation ( Bardwell and Treisman 1994 ; Kobayashi et al. 2000 ). It contains 21 ANK repeats which, by analogy with the function of ANKs ( Bennett and Chen 2001 ; Denker and Barber 2002a ), could interact with different proteins on the membrane, including Rab5, and mediate the assembly of a multiprotein complex. Such a scaffolding function has been postulated to bridge the membrane to the actin cytoskeleton but also to link proteins involved in endocytosis and signal transduction ( Pryciak and Hartwell 1996 ; Lubman et al. 2004 ). Rabankyrin-5 could assemble a membrane-cytoskeleton scaffold committing the ruffling membrane to generate a pinocytic vesicle or directly participate in the closure of the plasma membrane sealing the vesicle. Alternatively, Rabankyrin-5 could be recruited onto macropinosomes concomitantly or following their formation to prevent their back-fusion with the plasma membrane or regulate their onward trafficking. This possibility receives support from our time-lapse video microscopy analysis, where the dissociation of Rabankyrin-5 from the membrane precedes what appears to be the regurgitation of macropinosomes back to the plasma membrane (as shown in Video S1 ). Interestingly, it has recently been proposed that ANK repeats may not be simply anchoring domains but may be part of a mechanical sensory system that transmits tension from the cytoskeleton to ion channels in the membrane ( Corey and Sotomayor 2004 ; Howard and Bechstedt 2004 ). In the case of Rabankyrin-5, the ANK repeats may confer a sensory function to detect and adapt to rearrangements of the actin cytoskeleton during membrane ruffling and membrane closure. Interactions of the ANK repeats with ion channels may sense changes in water-salt homeostasis and pH, and respond to them via modulation of macropinocytosis and macropinosome trafficking ( de Baey and Lanzavecchia 2000 ; Morris and Homann 2001 ; Denker and Barber 2002b ). Functional Relation between Macropinosomes and Endosomes How does the function of Rab5 and Rabankyrin-5 in macropinocytosis relate to early endosome trafficking? We have previously proposed a model whereby Rab5, via the recruitment of its effectors, generates and maintains a spatially restricted and functionally specialised membrane domain on the early endosomes ( Zerial and McBride 2001 ). The Rab5 effector hVps34 ( Christoforidis et al. 1999b ) is a PI3-K that generates PI(3)P. Together with Rab5, this phosphoinositide serves as a binding determinant for the FYVE-finger Rab5 effectors EEA1 ( Simonsen et al. 1998 ) and Rabenosyn-5 on the early-endosome membrane ( Nielsen et al. 2000 ). The Rab5 effectors form oligomeric complexes ( McBride et al. 1999 ) and play distinct but cooperative roles in membrane tethering, fusion, and motility of early endosomes along microtubules ( Nielsen et al. 1999 ). Our data extend the list of PI(3)P-binding Rab5 effectors to Rabankyrin-5. Rabankyrin-5 colocalises with Rab5 and EEA1 to early endosomes and plays a role in homotypic early-endosome fusion, further underscoring the activity of Rab5 as organiser of an endosomal Rab5 domain enriched in PI(3)P-binding effector proteins. Rabankyrin-5 played a minor modulatory role in the fusion of CCVs with early endosomes, suggesting mechanistic differences between homotypic early-endosome fusion and the heterotypic fusion of CCVs with early endosomes. Clearly, Rabankyrin-5 exerted the most striking effects on fluid-phase rather than clathrin-mediated endocytosis. Clathrin-dependent endocytosis and macropinocytosis are independent but interconnected pathways. Whereas macropinosomes and early endosomes remain segregated in EGF-stimulated A431 cells ( Hewlett et al. 1994 ), they can fuse in other cells, such as dendritic cells and macrophages ( Racoosin and Swanson 1993 ). Rabankyrin-5 alone may not be sufficient for macropinosomes to fuse with endosomes, and this activity may require the additional recruitment of EEA1 and Rabenosyn-5. The extent to which macropinosomes can recruit Rabankyrin-5, release or retain it, and further acquire late endocytic components or other Rab5 effectors acting in endosome tethering and fusion may depend on the cellular context. In kidney cells, for example, several Rabankyrin-5–positive structures appeared also to contain EEA1 and late endocytic markers (LAMP-1), suggesting that macropinosomes can communicate with other endocytic organelles and undergo maturation as it has been described for phagocytosis ( Allen and Aderem 1996 ). Also intriguing is the weak interaction of Rabankyrin-5 with PI(5)P (see Figure 1 E) and recent findings that BTB domain-containing proteins interact with ubiquitin ligases ( Furukawa et al. 2003 ). Since both components have been implicated in the biogenesis of multivesicular bodies ( Katzmann et al. 2001 ), Rabankyrin-5 may function in the trafficking to late endocytic components, as it was described previously for PIKfyve, another FYVE-finger–containing protein ( Ikonomov et al. 2003 ). Stimulation of epithelial cells with either EGF or phorbol esters increases fluid-phase uptake while reducing internalisation via clathrin-mediated endocytosis ( Sandvig and van Deurs 1990 ). Our results provide a possible explanation for how the two endocytic routes can be quantitatively balanced. Since Rab5 appears to be rate limiting for both receptor-mediated and fluid-phase endocytosis, its activity could be shifted between these two endocytic pathways, depending on the stimuli and the cell type. The shared activity of Rab5 and Rabankyrin-5 would ensure coordination between endosome trafficking and macropinocytosis, regulate the kinetics, and limit the extent of both endocytic processes, thus preserving plasma membrane homeostasis. Rabankyrin-5 in Apical, Actin-Dependent Endocytosis in Polarised Epithelial Cells In addition to macropinocytosis in nonpolarised cells, we have found that Rabankyrin-5 regulates apical, non-clathrin-mediated pinocytosis in polarised epithelial cells. The role of apically stimulated pinocytosis is crucial for the physiology of various organs. For example, it plays an important role in the reabsorption of proteins from the glomerular filtrate in the renal proximal tubule ( Christensen et al. 1998 ). Rabankyrin-5 may be a regulator of this process and this possibility is supported by our findings, which show that it (1) localises to subapical compartments in kidney proximal tubules and (2) induces fluid-phase, clathrin-independent uptake from the apical but not basolateral side in polarised MDCK cells. As for macropinocytosis, apical endocytosis depends on actin remodelling mediated by Rho family GTPases ( Gottlieb et al. 1993 ; West et al. 2000 ) and ARF6 ( Altschuler et al. 1999 ). Since macropinocytosis appears to occur primarily from the apical side, the involvement of epidermal growth factor receptor as demonstrated for nonpolarised cells ( Haigler et al. 1979 ; West et al. 1989 ) is unlikely since it is primarily localised to the basolateral domain of polarised epithelial cells ( Gesualdo et al. 1996 ). However, apical pinocytosis depends on other components of receptor tyrosine kinase signalling pathways such as PI3-K ( Tuma et al. 1999 ) and protein kinase C and can be induced by oncogenes (e.g., v-Src) that stimulate macropinocytosis in nonpolarised cells ( Holm et al. 1995 ; Amyere et al. 2002 ). It is not known which PI3-K subtype functions in apical pinocytosis, but an attractive candidate is PI3-Kβ, as it has been identified as a Rab5 effector ( Christoforidis et al. 1999b ). Its activity could be regulated by Rab5 constitutively and/or be subjected to stimulation by apical or axonal signalling molecules ( Pillion et al. 1989 ; Kryl et al. 1999 ). In neurons, Rac- and actin-dependent endocytosis of Eph receptor-ephrin complexes is required to control repulsive versus attractive cell movement during tissue patterning in embryonic development ( Marston et al. 2003 ; Zimmer et al. 2003 ) and may play a role in the structural plasticity of synapses ( Holt et al. 2003 ). It will be interesting to determine whether Rabankyrin-5 and the macropinocytic machinery play a role in this event. In conclusion, the identification of Rabankyrin-5 opens new opportunities for investigating the molecular principles underlying macropinocytosis and its regulation by signalling molecules. The dual role of Rab5 and Rabankyrin-5 in endosome and macropinosome function argues for a role in coordinating these two different endocytic mechanisms. Future work is required to identify the molecular partners of Rabankyrin-5 and to establish the mechanisms responsible for its membrane targeting and its role in endocytic membrane dynamics. Materials and Methods Reagents and cell lines. Phospholipids were purchased from Sigma (St. Louis, Missouri, United States) and Calbiochem (San Diego, California, United States). FITC- or Texas Red-conjugated dextran (lysine fixable; MW, 10.000 and 70.000), rhodamine-conjugated EGF, and rhodamine-conjugated transferrin were purchased from Molecular Probes (Eugene, Oregon, United States). Wortmannin and LY 294002 were from Calbiochem. A431 and NIH3T3 cells were grown in DMEM (high glucose) supplemented with 10% (v/v) heat-inactivated foetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. MDCK II cells were grown in MEM plus 10% FCS. MDCK II cells stably transfected for FcLR 5–27 ( Matter et al. 1992 ) were a gift of Professor Ira Mellman. Amino acid sequence determination and Rabankyrin-5 cloning The 130-kDa protein band was excised from gels and enzymatically digested. The tandem mass spectroscopy protein sequencing procedure was performed as described previously ( Wilm et al. 1996 ). Bovine peptides determined from Rabankyrin-5 were from bovine brain and were used to identify corresponding expressed sequence tags using BLAST similarity searches at NCBI. A random primed HeLa cDNA library was used in a PCR reaction with primers based on the mouse cDNA to obtain the full-length clone of Rabankyrin-5. Preparation of recombinant Rabankyrin-5 Recombinant proteins of Rabankyrin-5 were expressed in SF plus insect cells according to the manufacturer's instructions using Rabankyrin-5 cDNA subcloned into pfastBAC vector or subcloned into pGEX-6P series and expressed in BL21 cells. Antibodies, plasmids, and recombinant adenovirus Human anti-EEA1 serum (1:10.000) was a gift from Ban Hok Toh (Monash Medical School, Adelaide, Australia), whereas a mouse monoclonal EEA1 antibody, a sheep polyclonal anti-HRP antibody and an antibody against phosphotyrosine (α-4G10) were purchased from BD Bioscience (Heidelberg, Germany), Abam (Cambridge, United Kingdom), and Upstate Biotechnology (Lake Placid, New York, United States), respectively. Fluorescently labelled secondary antibodies were purchased from Molecular Probes. Recombinant full-length Rabankyrin-5 was used to raise a polyclonal antibody in rabbit. Human full-length Rabankyrin-5 was cloned into pEYFP vectors containing an N-terminal tag (pEYFP-C1; Clontech, Palo Alto, California, United States). Recombinant adenovirus encoding full-length Rabankyrin-5 was generated according to the manufacturer's protocol (AdEasy). cDNAs encoding Rab5wt were fused to the amino termini of ECFP or EYFP, as previously described ( Sonnichsen et al. 2000 ). The antibody for the FcLR 5-27 chimeric receptor was purified from 2.4G2 hybridoma supernatant ( Unkeless 1979 ) by ammonium sulfate precipitation. For the preparation and biotinylation of Fab fragments of 2.4G2, 6 mg of purified 2.4G2 IgG was digested with insoluble papain enzyme as described by the manufacturer (Sigma). Fab fragments were biotinylated with NHS-LC-biotin or NHS-SS-biotin (Pierce, Rockford, Illinois, United States). In vitro binding assays The GST-Rab5 affinity chromatography was performed as in Christoforidis and Zerial (2000) . 35 S-Methionine–labelled proteins were transcribed and translated in vitro using a TnT-coupled transcription-translation kit (Promega, Madison, Wisconsin, United States). For Rab5 effector recruitment assays, in vitro-translated proteins were incubated with glutathione-sepharose beads complexed with GST-Rab5-GTPγS or GST-Rab5-GDP and eluted as described previously ( Christoforidis et al. 1999a ). Confocal immunofluorescence microscopy and transfection Cells were transfected with plasmids using FuGENE6 according to the manufacturer's instructions (Roche, Basel, Switzerland). After 16–24 h, transfected cells were washed twice with PBS and fixed with 3% paraformaldehyde and immunofluorescence labelling was performed according to standard procedures. Cells were mounted in moviol and examined on a confocal microscope (Leica TCS SP2; Leica, Wetzlar, Germany) with a 100×/1.40 plan-Apochromat objective. Fluorescent images were collected using the Leica IM500 Image Manager and processed using Adobe Photoshop v5.5 (Adobe Systems, San Jose, California, United States). Endocytosis of FITC-labelled dextran and rhodamine-labelled transferrin, and quantification NIH3T3 cells grown on glass coverslips and transfected with plasmids encoding RN-tre were incubated with 3 mg/ml FITC-labelled dextran (MW, 70.000) in DMEM supplemented with 1% FCS and 20 mM HEPES (pH 7,4), fixed, and stained with rhodamine-conjugated antirabbit secondary antibody. Dextran uptake was determined by quantifying grey values of thresholded, fluorescent images of at least 15 cells using MetaVue 6.1r3 and ImageJ (NIH). In vitro endosome fusion and early endosome/liposome recruitment assays In vitro fusion assays were performed using early endosomes labelled with biotinylated transferrin or antitransferrin antibody as well as CCVs labelled with biotinylated transferrin prepared from HeLa cells as described previously ( Horiuchi et al. 1997 ). The concentration of Rabankyrin-5 in HeLa cell cytosol in the in vitro early-endosome fusion assay was estimated to be approximately 50 nM, as determined by comparison with a serial dilution of recombinant Rabankyrin-5 analysed by SDS-PAGE, followed by Western blotting analysis. Recruitment of cytosolic proteins to early endosomes was performed as described in Christoforidis et al. (1999b) . Recombinant Rabankyrin-5 was recruited to phosphoinositides as described previously ( Nielsen et al. 2000 ) using liposomes (98% phosphatidylcholine, 2% phosphoinositides; 2 mg/ml final concentration in 30 mM HEPES-NaOH [pH 7,2], 120 mM NaCl, 0,5 mM EGTA) prepared as reported previously ( Otter-Nilsson et al. 1999 ). Time-lapse video microscopy and data processing Time-lapse epifluorescence video microscopy was performed using an Olympus IX70 (Olympus, Hamburg, Germany) inverted microscope equipped with a polychrome II monochromator (TILL Photonics, Martinsried, Germany), a custom filter block for simultaneous visualization of YFP and CFP (AHF Analysentechnik, Tübingen, Germany), a 100× oil-immersion objective (NA 1.35, Olympus) attached to a PIFOC z-SCAN (Physik Instrumente, Waldbronn, Germany), an incubation chamber (37 °C), and a 12-bit CCD digital camera IMAGO (0,134 μm pixel-1; 2 times 2 binning) (TILL Photonics), controlled by TILLvisION v3.3 software (TILL Photonics). Time-lapse video sequences of YFP and CFP images were merged as RGBs using TILLvisION v3.3. They were exported as single TIFF files and either further processed using Adobe Photoshop 5.5 and Illustrator 8.0 or converted into QuickTime movies using ImageJ (NIH). HRP staining of the apical endocytic machinery in mouse proximal tubules Anaesthetized mice were perfusion fixed with 4% PFA, 100 mM HEPES (pH 7,25), and 0,2% sucrose either directly or 5 min after injection with 5 mg/g body weight HRP (Sigma) into the femoral vein. Kidneys were excised and postfixed 6 h at 4 °C. Some tissue pieces were cryoprotected in 2,3 M sucrose overnight at 4 °C, frozen in liquid nitrogen, and cryosectioned on a Leica UCT ( Tokuyasu et al. 1984 ). Adenovirus infection and continuous uptake measurement with HRP, biotinylated transferrin, and biotinylated Fab (2.4G2). MDCK cells were grown on 12-mm, 0,4-μm pore Transwell polycarbonate filters (Corning Costar, Cambridge, Massachusetts, United States). Ninety-six hours after seeding, cells were infected from the apical side for 4 h at 37 °C with adenoviruses in 250 μl of complete medium. After changing medium, cells were incubated for 18 h at 37 °C and then used for biochemical assays or microscopy. NIH3T3 and MDCK cells grown on coverslips in 24-well plates were infected in 500 μl of medium. HRP uptake was performed as described previously ( Bomsel et al. 1989 ). In brief, cells were incubated in incubation medium (IM; DMEM, 1% FCS, 24 mM HEPES [pH 7,4]) at 37 °C for 1 h. For internalisation experiments coverslips or Transwell filters (Costar) were incubated with IM containing 5–10 mg/ml HRP, 2 μg/ml biotinylated transferrin, or 5 μg/ml biotinylated Fab (2.4G2). In the case of MDCK cells, HRP and Fab were applied from either the apical or the basolateral side. After internalisation, cells were briefly washed with warm IM followed by three washes with ice-cold PBS supplemented with 1 mM CaCl 2 and 1 mM MgCl 2 (PBS ++ ) containing 2 mg/ml BSA. Cell surface-bound transferrin was efficiently removed (95%) by three incubations over 10 min in IM (pH 3,4). Extracellular HRP was quenched in the cold by incubating the cells for 20 min with 20 mM MESNA in 100 mM NaCl, 50 mM Tris (pH 8,7), followed by two washes with ice-cold PBS and a further incubation for 10 min with ice-cold 50 mM iodoacetamide in PBS. For recycling experiments, cells were transferred again to 37 °C in fresh medium that was collected at the indicated time points. Thereafter, cells were washed once more with PBS and extracted for 15 min at 4 °C with 300 μl of lysis buffer (1% w/v Triton X-100, 0,1% w/v SDS, 20 mM HEPES [pH 7,4], and 100 U/ml DNase). Total HRP activity present in the cells was determined in duplicates out of at least three independent experiments using the previously described enzymatic method ( Steinman et al. 1976 ) and standardised to the protein content of each well. Fab and transferrin uptake assay was performed as described previously ( Zacchi et al. 1998 ). Knock-down of Rabankyrin-5 in A431 cells by RNA interference A431 cells were seeded in 24-well plates the day prior to transfection. Cells were transfected using oligofectamine (Invitrogen, Carlsbad, California, United States) with either 300 ng/ml double- or single-stranded (control) esiRNA derived from the I.M.A.G.E. clone (IMAGE:258664) according to the protocol of Yang et al. (2002) . After 4 d, cells were starved in MEM containing 2 mg/ml BSA and 24 mM HEPES (pH 7,3) for 1 h and stimulated with 50 ng/ml EGF in complete medium containing 24 mM HEPES (pH 7,3), 5–10 mg/ml HRP, and 2 μg/ml biotinylated transferrin for the indicated time points (see “ Adenovirus infection and continuous uptake measurement with HRP, biotinylated transferrin, and biotinylated Fab (2.4G2) ” above). Electron microscopy Cells or tissues were fixed with 8% PFA in PHEM buffer and then processed for frozen sectioning according to published methods ( Liou et al. 1996 ). Thawed sections were either single or double labelled. For single labelling a polyclonal anti–Rabankyrin-5 antibody followed by protein A 10 nm gold (University of Utrecht) was used. For double labelling the polyclonal anti–Rabankyrin-5 antibody was incubated together with rat anti–LAMP-1 (1D4B; courtesy of Professor M. Desjardins, University of Montreal, Montreal, Canada). The sections were then incubated with a mixture of anti-rabbit 10 nm gold and anti-rat 5 nm gold (BBI International, Cardiff, United Kingdom). Supporting Information Figure S1 Wortmannin Treatment Inhibits Fluid-Phase Uptake into Enlarged Rabankyrin-5 Structures NIH3T3 cells were pretreated for 20 min with wortmannin (100 nM) and incubated for 25 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 70.000), fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy. Scale bar represents 10 μm. (8.4 MB EPS). Click here for additional data file. Figure S2 Microinjection of a Function-Blocking Antibody against Rabankyrin-5 Decreases Fluid-Phase Uptake NIH3T3 cells were either microinjected with (A and D) preimmune or (B and C) affinity-purified polyclonal α–Rabankyrin-5 antibodies and subjected to a 30-min incubation at 37 °C with (A and B) 3 mg/ml FITC-conjugated dextran (MW, 70.000) or (C and D) 1 μg/ml rhodamine-conjugated transferrin. Cells injected with immune but not preimmune antibodies showed a significant reduction of the fluid-phase marker, while the uptake of transferrin was not significantly perturbed. (D) Fluid-phase dextran quantification of single cells microinjected for indicated antibodies by measuring internalised fluorescence intensity ( p > 0.001). Values shown are means ± standard deviation of at least 15 cells. Scale bars represent 10 μm. (44.0 MB EPS). Click here for additional data file. Video S1 Rabankyrin-5 Localizes to Macropinosomes Generated by Actin Dynamics Images were taken every 3 s over a time period of 15 min. RGB stacks were converted to QuickTime format using ImageJ (NIH). The movie is played at 15 frames per second. (11.1 MB MOV). Click here for additional data file. Accession Numbers The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink ) accession numbers of proteins discussed in this paper are H-Ras (ID 3265), v-Src (ID 6714), Arf6 (ID 382), hVps34 (ID 5289), p110β (ID 5291), Rab34 (ID 83871), Rab5 (ID 5868), Ankhzn (ID 51479), Rab4 (ID 5867), Rab7 (ID 7879), Rab11 (ID 8766), Rabenosyn-5 (ID 64145), EEA1 (ID 8411), Caveolin-1 (ID 857), RN-tre (ID 9712), Transferrin (ID 7018), EGF (ID 1950), LAMP-1 (ID 16783), Prominin-1(ID 8842), PIKfyve (ID 200576), RhoA (ID 387), and PKC-α (ID 5578).

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549595

Unregistered Trials Are Unethical

Current journal requirements forcing clinical trials to be registered [ 1 ] are insufficient and are unlikely to solve the problem of negative trials never even making it to a journal. Most of the patients consenting to clinical trials do so out of altruism. It is a great betrayal of their trust to suppress clinical trial data. I suggest that institutional review boards refuse to allow human experimentation unless the protocol is filed in a central (online) repository. The primary data should also be required to be in the public domain (say, within 1–2 years after completion). Data obtained by appealing to altrusitic instincts, similar to money in public charities, are not proprietary information, nor can physicians cash out the trust of their patients. In reality, it is the pharmaceutical industry that stands to gain the most if data are made public as such data inform future research and help smaller, innovative companies avoid redundancy. Voluntarily sticking to higher standards of ethics will raise societal respect for the industry (currently being battered for greed) and attract a more talented workforce, and may even help the current efforts to reform the tort law.

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548674

Human breast cancer cell-mediated bone collagen degradation requires plasminogen activation and matrix metalloproteinase activity

Background Breast cancer cells frequently metastasize to the skeleton and induce extensive bone destruction. Cancer cells produce proteinases, including matrix metalloproteinases (MMPs) and the plasminogen activator system (PAS) which promote invasion of extracellular matrices, but whether these proteinases degrade bone matrix is unclear. To characterize the role that breast cancer cell proteinases play in bone degradation we compared the effects of three human breast cancer cell lines, MDA-MB-231, ZR-75-1 and MCF-7 with those of a normal breast epithelial cell line, HME. The cell lines were cultured atop radiolabelled matrices of either mineralized or non-mineralized bone or type I collagen, the principal organic constituent of bone. Results The 3 breast cancer cell lines all produced significant degradation of the 3 collagenous extracellular matrices (ECMs) whilst the normal breast cell line was without effect. Breast cancer cells displayed an absolute requirement for serum to dissolve collagen. Degradation of collagen was abolished in plasminogen-depleted serum and could be restored by the addition of exogenous plasminogen. Localization of plasmin activity to the cell surface was critical for the degradation process as aprotinin, but not α 2 antiplasmin, prevented collagen dissolution. During ECM degradation breast cancer cell lines expressed urokinase-type plasminogen activator (u-PA) and uPA receptor, and MMPs-1, -3, -9,-13, and -14. The normal breast epithelial cell line expressed low levels of MMPs-1, and -3, uPA and uPA receptor. Inhibitors of both the PAS (aprotinin and PA inhibitor-1) and MMPs (CT1166 and tisue inhibitor of metalloproteinase) blocked collagen degradation, demonstrating the requirement of both plasminogen activation and MMP activity for degradation. The activation of MMP-13 in human breast cancer cells was prevented by plasminogen activator inhibitor-1 but not by tissue inhibitor of metalloproteinase-1, suggesting that plasmin activates MMP-13 directly. Conclusions These data demonstrate that breast cancer cells dissolve type I collagen and that there is an absolute requirement for plasminogen activation and MMP activity in the degradation process.

Background Breast cancer is the most frequent cancer in the female population of industrialized countries. Metastasis of breast cancer cells to the skeleton occur in >70% of patients with progressive disease, resulting in debilitating symptoms such as severe bone pain, fractures, hypercalcaemia and spinal cord or nerve compressions due to extensive bone loss and tumour cell growth and expansion. Such bone loss occurs as a result of increased bone matrix resorption but the mechanisms by which cancer cells mediate this increased degradation have not been fully elucidated. Obviously, tumour expansion in bone requires the removal of the extracellular matrix (ECM) that is particularly abundant in bone. Cancer cells express matrix metalloproteinases (MMPs) and the plasminogen activator system (PAS) [ 1 - 3 ] and their levels of expression increase with progression of the tumour. The matrix metalloproteinases (MMPs) constitute a large family of structurally related matrix degrading proteases that have pivotal roles in development, tissue remodelling, and cancer [ 4 - 6 ]. The gene family of MMPs includes the interstitial collagenases (MMPs-1 and -13), gelatinase A (MMP-2), gelatinase B (MMP-9), the stromelysins (MMPs-3, 10 and 11) and the membrane type-matrix metalloproteinases (MT-MMPs 14,15,16,17, 24 and 25) [ 6 ]. The MMPs have the combined ability to degrade the major components of the ECM including type I collagen, the principal organic constituent of bone [ 4 ]. The PAS comprises: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), their inhibitors, and receptors. T-PA is thought to be more important in fibrinolysis, due to its fibrin binding capacity, whilst u-PA, especially when it is bound to its specific cell surface receptor (u-PAR), is thought to be involved in tissue remodelling and cell migration processes [ 7 ]. Whereas uPA alone recognizes a narrow range of substrates, the enzyme can catalyze the conversion of the circulating zymogen, plasminogen to plasmin. Plasmin, in turn, is a broad- spectrum proteinase that can directly degrade multiple ECM targets and can also cooperate with other ECM-degrading enzymes including members of the MMP gene family. Regulation of the PA/plasmin system is achieved mainly via plasminogen activator inhibitor (PAI) type-1 and type-2 and by agents that stimulate bone resorption, e.g. parathyroid hormone (PTH) and interleukin (IL)-1 [ 8 ]. To date, emphasis has focused on the ability of breast cancer cells to stimulate the formation and activity of osteoclasts, the cell primarily responsible for bone resorption under physiological conditions. The ability of osteoclasts to degrade bone lies in their ability to secrete protons and specialized collagenolytic proteinases, the cysteine proteinases in the acidic microenvironment that underlies osteoclasts during bone resorption [ 9 ]. Experimental studies showing that increased expression of MMPs and the PAS is associated with increased cellular invasion in vivo support the idea that they play an important role in metastasis of tumour cells [ 10 , 11 ]. Obviously tumour expansion in bone necessitates the removal of the ECM that is particularly abundant and resistant to degradation. Synthetic inhibitors of MMPs have been developed and two recent reports on their use on in vivo breast cancer metastasis to bone show promise when given as a preventive treatment to mice [ 12 , 13 ]. However, the role of MMPs and the PAS in mediating breast tumour bone collagen dissolution has not been addressed. We have therefore assessed the ability of three human breast cancer cell lines, MDA-MB-231 (MDA-231), ZR-75-1 and MCF-7 to degrade bone collagen in vitro using matrix degradation assays and compared their effects with those of a normal breast epithelial cell line, HME. We correlated the degradation activity of the breast cancer cells with their expression of MMPs and the PAS and we assessed the ability of group-selective proteinase inhibitors to prevent degradation of the organic aspect of bone by breast cancer cells. Results Type I collagen degradation Bone degradation involves an initial phase of removal of the unmineralized type I collagenous layer followed by degradation of the mineralized matrix which also comprises type I collagen. The fibrillar integrity of the collagen layer was confirmed by incubation with collagenase that degraded the film whilst the collagen fibres were resistant to degradation by both trypsin and plasmin (data not shown). When the breast cancer cells (MDA-231, ZR-75-1 or MCF-7) were stimulated with TGFβ(10 -10 M) and cultured in the presence of 10% FCS the cancer cells induced significant degradation of type I collagen (range 70–80%), whereas minimal degradation was observed in the absence of serum (Fig. 1 ). To investigate the possible role of the PAS in collagen breakdown by breast cancer cells, plasminogen was depleted from FCS by lysine-Sepharose chromatography [ 14 ]. Interestingly, the depletion of plasminogen from serum also completely blocked breast cancer cell mediated collagen dissolution, implicating the PAS in breast cancer-mediated collagen degradation (Fig. 1 ). In accordance with this finding, the breast cancer cells degraded collagen under serum-free conditions only when supplemented by exogenous plasminogen (Fig. 1 ). The TGFβ-stimulated normal breast cell line, HME cultured in the presence of 10 % FCS demonstrated low type I collagenase activity (Fig. 1 ). Figure 1 Degradation of 14 C-labelled type I collagen films by breast cancer cells. Breast cancer cells (10 5 cells/well) stimulated with TGFβ (10 -10 M) were cultured for 24 h on 14 C-labelled type I collagen under the following conditions: serum-free conditions; presence of 10% serum; 10% plasminogen (Plg)-depleted serum; serum-free medium supplemented with 2 μg/ml of human Plg. After 24-h incubation, collagen degradation was measured as described in Materials and methods. 8–800 mU (Ploug units) of pure human uPA in 1 ml of serum-free medium with or without 2 ug/ml of plasminogen, incubated as a control in parallel wells in the absence of cells, released 2–4% of the total radioactivity. This experiment was repeated twice. The results are expressed as percentage release of 14 C. Each bar is the mean ± S.E.M of six wells. The stimulatory effects of plasminogen and TGFβ on breast cancer cell mediated 14 C release were statistically significant ***P < 0.001 compared with the unstimulated controls and the HME cells. The ability of the cancer cells to degrade the type I collagen is consistent with the expression of proteolytic activity. However, the midpoint melting temperature of reconstituted firbrillar type I collagen (47°C) is lower than that of authentic type I collagen in tissues (55°C to 60°C) [ 4 ]. Because the proteinase resistance of type I collagen can be compromised at temperatures within 10°C of that at which the helix reversibly unfolds [ 4 ], reconstituted fibrillar collagen may provide a less resistant substrate to proteolytic activity. Hence to determine whether breast cancer cell mediated degradation of type I collagen could be extended to a more physiological system, cancer cells were cultured on bone matrix as described below. Bone matrix degradation by breast cancer cells The described sequential gene expression of differentiating osteoblasts [ 15 ] was verified in MC3T3-E1 cell cultures so that non-mineralized matrix production was prepared after collagen production had commenced but before mineralization started. Mineralization of the matrices was confirmed by von Kossa staining (not shown). When TGFβ-stimulated breast cancer cell lines were cultured as a monolayer on either non-mineralized (Fig. 2A ) or mineralized (Fig. 2B ) bone matrix over a 24 h culture period there was significant degradation of the non-mineralized matrix (range 65–75%; Fig. 2A ) and to a lesser extent the mineralized matrix (range 40–45%; Fig. 2B ) only in the presence of plasminogen. In contrast, the TGFβ-stimulated breast cancer cell lines achieved a minimal amount of degradation of either matrix in the absence of plasminogen (Fig. 2A and 2B ). The normal breast cell line, HME achieved a minimal amount of degradation of both matrices (5–15%) in the presence of plasminogen. Figure 2 Degradation of 3 H-non-mineralized (A) and 3 H-mineralized (B) bone matrix by breast cancer cells . Breast cancer cells (10 5 cells/well) stimulated with TGFβ (10 -10 M) were cultured for 24 h on 3 H-labelled extracellular matrices in the presence (+) and absence (-) of 2 μg/ml of human plasminogen. After 24-h incubation, bone matrix degradation was measured as described in Materials and methods. 8–800 mU (Ploug units) of pure human uPA in 1 ml of serum-free medium with or without 2 μg/ml of plasminogen, incubated as a control in parallel wells in the absence of cells, released 3–4% of the total radioactivity. This experiment was repeated twice. The results are expressed as percentage release of 3 H labelled bone matrix. Each bar is the mean ± S.E.M. of six wells. The stimulatory effects of TGFβ on breast cancer cell mediated 3 H release were statistically significant ***P < 0.001 compared with the unstimulated controls and the HME cells. Expression of mRNA for MMPs and the PAS by breast tumour cells To characterize the profile of MMPs expressed constitutively and upon stimulation with TGFβ, breast cancer cells were cultured on type I collagen. Total RNA was isolated from 24-h cultures and screened by reverse transcription-PCR for MMP-1 through MMP-17. Under these conditions, four secreted MMPs were identified: MMP-1, MMP-3, MMP-9, and MMP-13 and the membrane-anchored MMP, MT1-MMP in all 3 breast cancer cell lines (Fig 3A ; MDA-231 cells shown, results similar in all 3 breast cancer cell lines). TGFβ upregulated MMP expression in all 3 cancer cells lines (Fig. 3B ; MDA-231 cells shown, results similar in all 3 breast cancer cell lines). The normal epithelial cell line, HME was found to express low levels of MMP-1 and MMP-3 upon stimulation with TGFβ (Fig. 3C ) Figure 3 RT-PCR of MMPs in breast cancer cells. Breast cancer cells were cultured as described in the Material and methods in the absence (A) and presence (B) of TGFβ (10 -10 M). Total RNA was isolated and RT-PCR performed with specific primers for MMPs-1,2,3,7,8,9,10,11,12,13,14,15,16,17. The housekeeping gene GAPDH was used as a positive control. Representative results for MDA-231 cells are shown (A) and (B): Lane 1 MMP-1; lane 2 MMP-13; lane 3 MMP-3; lane 4 MMP-9; lane 5 MMP-14; lane 6 GAPDH. The normal breast cell line HME stimulated with TGFβ is shown in (C). Band intensities were quantified by scanning densitometry and data expressed as a ratio (MMP/G3PDH) of the average optical density (OD) × area. The ratio of the intensity of the MMP mRNA band over the intensity of the G3PDH mRNA was arbitrarily designated as 1.0. All 3 breast cancer cell lines expressed u-PA and u-PAR (Fig. 4A ; ZR-75-1 cells shown, results similar with all 3 breast cancer cell lines) and upon stimulation with TGFβ there was increased expression of both u-PA and u-PAR by the breast cancer cells (Fig. 4B ; ZR-75-1 cells shown, results similar with all 3 breast cancer cell lines.). The intensity of the signal was greater for the breast cancer cells than the normal breast cell line, HME (Fig. 4C ). Figure 4 RT-PCR of uPA and uPAR in breast cancer cells. ZR-75-1 breast cancer cells were cultured as described in the material and methods in the absence (A) and presence (B) of TGFβ (10 -10 M). Total RNA was isolated and RT-PCR performed with specific primers for uPA, tPA and uPAR. The housekeeping gene GAPDH was used as a positive control. Lane 1 uPA; lane 2 uPAR; lane 3 GAPDH. The normal breast cell line HME stimulated with TGFβ is shown in (C). Band intensities were quantified by scanning densitometry and data expressed as a ratio (uPA or uPAR/G3PDH) of the average optical density (OD) × area. The ratio of the intensity of the uPA or uPAR mRNA band over the intensity of the G3PDH mRNA was arbitrarily designated as 1.0. MMP and uPA production by breast cancer cells To analyze the functional activities of MMPs and PAs expressed in breast cancer cells, collagenase activity in BCCM was measured by the degradation of FITC labelled type I collagen in the presence and absence of plasminogen. TGFβ markedly stimulated collagenase activity only in the presence of plasminogen (Fig. 5 ). uPA production was high in all cancer cells which degraded bone matrix but not in HME cells (Table 1 ) that did not degrade bone at all, suggesting that uPA may be necessary to accomplish this task. Figure 5 Collagenase activity in breast cancer conditioned media Breast cancer cells were cultured for 24 h in serum-free medium in the presence and absence of 2 μg/ml of human plasminogen and TGFβ (10 -10 M). Conditioned media were collected and incubated for 4 h with FITC-labelled type I collagen to detect collagenase activity, as described in Material and methods. The data are expressed as means SEM of 4–6 independent experiments, significantly different from control (**, P < 0.01; *** P < 0.001). Table 1 Production of uPA by Breast Cancer Cells Cells uPA (U/1 × 10 5 cells/24 h) MDA-231 2.3 ZR-75-1 1.9 MCF-7 1.6 HME Undetected uPA was measured by a chromogenic assay (see Materials and methods) in serum-free conditioned medium collected over a 24 h period. Breast cells were cultured in the presence of plasminogen (2 ug/ml) and TGFβ (10 -10 M). uPA was not detected in unstimulated breast cancer cells. Roles of MMPs and PAS in breast cancer cell mediated bone degradation We examined the effects of inhibitors of MMPs and the PAS on TGFβ-stimulated MDA-231 cell-mediated degradation of non-mineralized bone matrix under serum free conditions supplemented with plasminogen. The MMP inhibitors, CT1166 and TIMP-1 completely prevented TGFβ-stimulated breast cancer cell mediated bone collagen degradation (Fig. 6 ). When aprotinin, which inhibits both plasmin bound to the cell surface and plasmin in solution, was added, collagen degradation was also completely blocked (Fig. 6 ). Similar inhibitory effects were seen with function blocking antibodies to uPA or PAI-1 (Fig. 6 ). In contrast, the serpin α 2 -antiplasmin, which is a poor inhibitor of cell surface bound plasmin but an excellent inhibitor of plasmin in solution, did not prevent collagen degradation (Fig. 6 ). Since in the absence of cells, plasmin had no collagenolytic activity (see legend to Figures 1 and 2 ) and none of these inhibitors was cytotoxic, (data not shown), these results showed that bone collagen degradation by human breast cancer cells is dependent upon plasminogen activation and MMP activity. Western blot analysis demonstrated that neither CT1166 nor aprotinin influenced the production of MMPs (Fig. 7A ) or uPA (Fig. 7B ). Figure 6 Effects of MMP and PAS inhibitors on MDA-231 cell mediated degradation of non-mineralized matrix. MDA-231 breast cancer cells (10 5 cells/well) were cultured for 24 h on 3 H-labelled extracellular matrices in the presence of 2 ug/ml of human plasminogen, TGFβ (10 -10 M) with and without CT1166 (10 -5 M), TIMP-1 (50 ug/ml), aprotinin (10 -5 M), antibodies to human uPA (50 μg/ml) or human uPAR (50 μg/ml). After 24-h incubation, bone matrix degradation was measured as described in Materials and methods. This experiment was repeated twice. The results are expressed as percentage release of degradation of 3 H labelled bone matrix. Each bar is the mean ± S.E.M. of six wells. The effects of the inhibitors were statistically significant *P < 0.05;***P < 0.001. Figure 7 Immunological characterization of MMPs and uPA in Breast Cancer Cells. Breast cancer cells (10 5 cells/well) stimulated with TGFβ (10 -10 M) were cultured for 24 h in serum-free medium in the presence of 2 μg/ml of human plasminogen and CT1166 and aprotinin. Western blot analysis was undertaken as described in the Materials and methods section. Lane 1, MDA-231 cells; lane 2, ZR-75-1cells; lane 3 MCF-7 cells; lane 4, HME cells. Pro- and active forms of collagenase-1 gelatinase-B, and stromelysin-1 and proform of collagenase-3 were detected (A). Pro and active forms of uPA are shown (B). Discussion The data presented in this paper clearly demonstrate that human breast carcinoma cell lines have the capacity to degrade the organic aspect of bone matrix in vitro , and there is a dependency on the PA system for the cell-mediated collagen degradation. Furthermore, we have shown that plasmin associated with the cell surface is responsible for activating the fibrillar collagenase, MMP-13. The ECM in our experiments was produced by MC3T3-E1 mouse calvarial-derived cells. These cells display osteoblast-like characteristics, providing a suitable model of osteogenesis analogous to in vivo bone formation [ 17 ]. The bone nature is evident from the appearance of mineralization, resulting in the production of a solid sheet of mineralized matrix. Our evidence showing that breast cancer cells degrade bone matrix is in agreement with that of Eilon and Mundy [ 18 ] who reported that MCF-7 cells were capable of degrading the organic aspect of devitalized murine bone explants in vitro . More recently it has been demonstrated that prostate cancer cells and melanoma cells directly degrade mineralized bone matrix and that the degradation was reduced by generalized inhibition of MMP activity [ 19 , 20 ]. Whereas the induction of MMPs in TGFβ stimulated breast cancer cells that are actively engaged in tumour osteolysis has not been examined previously, this growth factor has been reported to increase the expression of MMPs-1, -3 and -9 [ 21 ]. In our study TGFβ induced a complex MMP expression profile that included MMPs-1, -3, -9 -13, and -14 as the principle products. Furthermore, MMPs are able to release and activate TGFβ, a very abundant bone matrix-bound factor [ 22 ]. The fact that breast cancer cells degraded the non-mineralized bone matrix to a greater extent than the mineralized substrate, suggests that access of the matrix degrading proteinases to the organic phase of the matrix may have been restricted by the mineralized phase. This investigation has demonstrated a significant difference in the ability of breast cancer cells and normal breast epithelial cells to degrade the organic aspect of bone matrix in vitro . Variations in the profile and amounts of proteinases expressed by the different cell lines may be responsible. Since the MMP inhibitors used in this study (CT1166 and TIMP-1) inhibit the activities of all MMPs, except the membrane-type MMPs in the case of TIMP-1, we cannot identify the contribution of individual MMP members to bone matrix degradation. However, our demonstration of a direct requirement of plasmin in the activation of proMMP-13 suggests that this fibrillar collagenase may play a prominent role in the degradative activity of breast cancer cells. However, the possibility of other known MMPs also being involved in plasminogen-dependent collagenolysis by breast cancer cells cannot be excluded. The participation of the membrane-type MMPs such as MMP-14, -15 and -16 is improbable due to the ability of TIMP-1 to effectively block collagen dissolution by breast cancer cells. MMP-2 and – 8 were not expressed in detectable amounts by breast cancer cells, as assessed by RT-PCR. The existence of very small, but functionally important, amounts of MMP-2 or – 8 however cannot be excluded unequivocally by the expression studies. MMP-1 is expressed by the human breast cancer cells in this study and may contribute to plasmin-dependent fibrillar collagen dissolution. Currently, little is known with regard to the physiological mechanisms by which MMPs undergo activation in intact cell systems [ 23 ]. However, a primary function of the uPA-uPAR couple is to focus the processing of the plasminogen zymogen to active plasmin on the cell surface. It has recently been demonstrated that binding of u-PA to its receptor increases pro-MMP-2,-9 [ 23 ] and -13 [ 16 ] activation, and that in the absence of cells, plasmin not only fails to activate pro-MMPs, but rapidly degrades them [ 23 ]. This would explain why interfering with only one element, plasmin or MMP activity, has such powerful biological effects. It should be stressed, however, that few of the cascades or activities have been unequivocally documented in intact cell systems. Nonetheless, because MMP-specific inhibitors are not yet available, the individual role of each of the cell-derived MMPs awaits the use of interventions based on RNA inhibition. The increased bone resorption that accompanies breast cancer cell infiltration of bone may arise as a result of (1) breast cancer cells stimulating osteoblast and osteoclast activity and (2) the production by breast cancer cells of proteinases that participate in degrading the organic aspect of bone matrix which would also facilitate the access of osteoclasts to the underlying mineralized matrix. Even if tumour-derived MMPs do not directly digest mineralized matrix, they may participate in osteolysis via a mechanism that is analogous to a known osteoblast function. It is believed that all endosteal surfaces are covered with a layer of nonmineralized matrix. In areas of bone formation, this layer is called osteoid and it is easily observed in stained sections [ 24 ]. Evidence is accumulating that degradation of this layer must occur before the attachment of osteoclasts to the underlying mineralized matrix [ 25 ]. In normal remodelling, digestion of this layer is apparently accomplished by MMPs produced by osteoblasts [ 25 - 27 ]. It is conceivable that breast cancer cells in bone adopt this osteoblast function; thus, the possibility that MMPs produced by cancer cells enhance osteoclastic degradation by prior removal of the overlying unmineralized layer. Furthermore, removal of this layer may be partially responsible for the recruitment and activity of osteoclasts due to the release of osteoclast attractants/stimulants. Bone resorption by osteoclasts is augmented experimentally by coating mineralized matrix with collagenase-cleaved collagen fragments [ 28 ]. As nonmineralized matrix is degraded, osteoclasts may be exposed to extracellular proteins, such as fibronectin, vitronectin, osteopontin or other cryptic epitopes. Osteoclasts bind to these proteins via integrins a process that may enhance bone resorption [ 29 ]. Conclusion In conclusion, we have shown that breast cancer cells can degrade the organic aspect of bone matrix in contrast to non-tumourogenic breast epithelial cells. There is an absolute requirement for both the PA system and MMPs in the degradation process. Materials and methods Reagents The MMP inhibitor CT-1166 was a generous gift from Dr A Docherty, Celltech (Slough, UK). The PAS inhibitor aprotinin, plasminogen and TGFβ were purchased from Sigma. The human mammary epithelial cell (HMEC) line, Mammary Epithelial Basal Medium (MEBM), insulin, hydrocortisone, gentamicin/amphotericin-B and Bovine Pituitary Extract (BPE) were purchased from Clonetics (Walkersville, MD, USA). Human breast tumour cell lines MDA-231, ZR-75-1 and MCF-7 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Gelatinase-A, TIMP-1 and polyclonal sheep antibodies to human MMP-1, -2,- 3, -9 and -13 were gifts from Dr J Reynolds, Strangeways Research Laboratory, Cambridge, UK. Neutralizing mAbs against human uPA or human uPAR were from American Diagnostica, Greenwich, CT, USA. Cell cultures Cell lines were cultured in 75-cm 2 plastic tissue culture flasks (Costar Corning, Cambridge, MA, USA) as follows: ZR-75-1, MDA-231, MCF-7 and MC-3T3-E1 in α-MEM and HME cells in MEBM medium. All media were supplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin (Sigma Chemical Co.). Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide and subcultured every third day. Type I collagen degradation assay 14 C-labelled collagen films were prepared as described previously [ 30 ]. Breast cells were seeded into collagen coated 16 mm tissue culture wells at a density of 1 × 10 5 /well. After 4 h incubation at 37°C in MEM supplemented with 10% FCS, the cells were washed twice with phosphate buffered saline (PBS) to remove residual FCS, and incubated for 24 h with 1 ml/well of serum-free MEM with or without the indicated concentrations of plasminogen, TGFβ and the proteinase inhibitors to be tested. At the end of the culture period the media were centrifuged (15 min, 1200 × g) to remove any collagen fibrils, and radioactivity released during collagen degradation quantified by liquid scintillation counting. Residual collagen was digested with bacterial collagenase (50 μg/ml) and assayed for radioactivity. Collagenolysis was expressed as radioactivity released from the films as a percentage of the total ± SEM. Formation of 3 H-labelled, non-mineralized and mineralized bone matrix The murine calvarial-derived MC3T3-E1 is a well characterized osteoblast culture system providing a suitable model of osteogenesis analogous to in vivo bone formation [ 15 ]. The cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide. Culture medium was changed on the first day after seeding and then every 72 h. The cells were split after 7–9 days in culture, before they reached confluence, and plated at a density of 1 × 10 4 cells/well on collagen-coated 24-well plates (Becton Dickinson, MA, USA). After 4–5 days, when the cultures had reached confluence and the formation of an ECM had started, fresh medium was added containing 1 μCi/ml 3 H amino acid mixture for 7 days (Amersham International, Aylesbury, UK) to create a non-mineralized radiolabelled ECM. In order to create a mineralized bone matrix, the medium was supplemented with 10 mM β-glycerol phosphate and the cells cultured for 14 days and the radiolabelled medium changed every 3 days. Unincorporated 3 H-radiolabelled amino acids were washed from the remaining ECM using 2 water washes and 75% (v/v) ethanol. The matrices were dried and stored at -20°C until use. Von Kossa staining for mineralization Mineralization of matrices was determined by von Kossa staining. The matrices were rinsed with cold PBS and fixed in 10% neutral buffered formalin for 15 min, rinsed with distilled water and left in distilled water for 15 min. Matrices were stained with 2.5 % silver nitrate solution for 30 min at room temperature. The silver nitrate was removed and matrices were rinsed with distilled water before the addition of sodium carbonate formaldehyde for 5 min. After rinsing, the matrices were counterstained with toluidine blue, rinsed with tap water and air dried. Bone matrix degradation assay Breast cancer cells (10 5 /16 mm culture well) were seeded onto the bone matrix as for the type I collagen degradation assay. After 4 h incubation at 37°C in MEM supplemented with 10% FCS, the cells were washed twice with phosphate buffered saline (PBS) to remove residual FCS, and incubated for 24 h with 1 ml/well of serum-free MEM with or without the indicated concentrations of plasminogen, TGFβ and the proteinase inhibitors that were tested. After 24 h incubation, the media were removed and the extent of degraded 3 H-radiolabelled matrix released into the medium was determined by liquid scintillation counting. The remaining matrix was degraded as for the type I collagen assay and matrix degradation expressed as above. PCR and RT-PCR Confluent breast tumour cells were stimulated with TGFβ (10 -10 M) in serum-free medium for 24 h. For PCR analysis, oligonucleotide primers were synthesized based on the published sequences for 14 MMPs (i.e., MMP-1, -2, -3, -7,-8,-9,-10, -11, -12, -13,-14,-15, -16, and -17) [ 4 ]. t-PA, u-PA and, u-PAR oligonucleotides were designed using Designer PCR (Research Genetics, AL, USA) and primers purchased from Life Technologies Ltd. The housekeeping gene GAPDH was used as a positive control for the RT-PCR methodology. Enzymes and buffers for the reverse transcriptase and PCR reactions were obtained from Perkin Elmer (CA, USA). Reverse transcriptase reactions were done according to the manufacturer's protocol using 1 μg of total RNA collected from untreated or TGF-β treated breast cells. PCR reactions were performed in an automated DNA thermal cycler (Perkin Elmer) for 25 cycles of denaturation (95°C, 1 min), annealing (variable time and temperature) and polymerisation (60°C, variable time). Amplification of the house-keeping gene, G3PDH mRNA (35 cycles), provided an internal control for the efficiency of the RT-PCR process. RT-PCR products were analyzed against molecular weight standards (pBR322 HaeIII digest) on a 2.5% agarose gel stained with ethidium bromide, electrophoresed in 0.5× TBE buffer at 100 V for 90 minutes. Gels were examined under ultraviolet light and photographed. The authenticity of the PCR products was verified by sequencing [ 31 ]. Western Blot analysis To identify MMP proteins and uPA present in breast cell conditioned medium, Western blot analysis was performed using their specific antibodies as previously described [ 32 ]. Briefly, samples were separated by 8.5% SDS-PAGE, transblotted on to PVDF membranes (Millipore Corp., MA, USA) and immunoblotted with polyclonal sheep antiserum to MMPs or polyclonal goat antiserum to uPA and secondary peroxidase-conjugated anti-sheep / anti-goat antibodies. Labelled proteins were detected with ECL detection solution and exposed to autoradiographic film (Hyperfilm ECL, Amersham International, UK). Assay of collagenase activity To measure collagenase activity, TGFβ (10 -10 M)-stimulated breast cells were cultured in the presence/absence of 2 ug/ml of human plasminogen for 24 h. Collagenase activity was measured by the degradation of fluorescein isothiocyanate (FITC)-labelled type I collagen using type I activity assay kits. One unit of these activities degrades 1 μg of collagen per min at 37°C. uPA Assay Cells were plated in 6-well trays and grown to near confluence, the medium was removed, and the cells were washed and incubated with 2 ml of serum-free medium for 24 hr. The medium was collected, centrifuged, and frozen until assayed. The cells were lysed in 300 ul of 0.1 M Tris (pH 8.1) and 0.1% Triton X-100. The u-PA (10 ul of conditioned medium and 10 μg of cell protein) was assayed as previously described [41] using plasminogen and S2251, the chromogenic substrate for plasmin. Statistical Analysis Differences between control and treatment groups were determined by the Mann Whitney U-test.

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499549

Stem cells and repair of lung injuries

Fueled by the promise of regenerative medicine, currently there is unprecedented interest in stem cells. Furthermore, there have been revolutionary, but somewhat controversial, advances in our understanding of stem cell biology. Stem cells likely play key roles in the repair of diverse lung injuries. However, due to very low rates of cellular proliferation in vivo in the normal steady state, cellular and architectural complexity of the respiratory tract, and the lack of an intensive research effort, lung stem cells remain poorly understood compared to those in other major organ systems. In the present review, we concisely explore the conceptual framework of stem cell biology and recent advances pertinent to the lungs. We illustrate lung diseases in which manipulation of stem cells may be physiologically significant and highlight the challenges facing stem cell-related therapy in the lung.

Introduction According to Greek mythology, the immortal Prometheus stole fire from the Gods as a gift for humankind. As punishment, he was shackled to a rock, whereupon each day for 30,000 years an eagle consumed as much of his liver as would regenerate. There is some debate whether the eagle ate his liver or heart, but what if the bird had a taste for lung? And what if Prometheus was a mere mortal? Analogous to Prometheus and the eagle, the ambient air-exposed lung is subject to an array of potentially damaging agents, including chemical oxidants and proteolytic enzymes. Presumably, daily oxidant and protease wear and tear on structural components such as elastin and collagen contributes to inevitable age-related declines in pulmonary function in normal individuals [ 1 , 2 ]. Acute and chronic lung disease, or its treatment with oxygen and positive pressure ventilation, may further damage lung tissue in excess of the capacity for orderly repair, resulting in characteristic pathologic changes including tissue destruction or fibrotic scarring [ 3 - 5 ]. But what determines the lungs' capacity for repair? Certainly, one factor must be the ability of stem cells to proliferate and differentiate to replace damaged cells and tissues. As discussed later in this review, the traditional view is that, during development, self-renewing tissues are imbued with resident, tissue-specific stem cells, so-called adult somatic stem cells. However, recent but highly controversial evidence suggests that stem cells from one type of tissue may generate cells typical of other organs. In this fashion, circulating cells derived from bone marrow may augment resident stem cells, and we comprehensively review such data from lung. Finally, there is great hope that embryonic stem cells, embryonic germ cells, or even adult somatic stem cells can be engineered as an unlimited source of cells to enhance organ-specific repair or replace lost tissues. Below, we concisely review stem cell biology, focusing on recent findings relevant to the lungs. Diseases in which alterations in stem cells contribute to lung dysfunction are discussed, as are the challenges facing the nascent field of pulmonary regenerative medicine. Embryonic and adult (somatic) stem cells For links to more in-depth information on general principles in stem cell biology, a comprehensive glossary, and the latest updates in this quick moving field, the reader is referred to the International Society for Stem Cell Biology . During embryonic development, the inner cell mass of the blastocyst forms three primary germ layers, which generate all fetal tissue lineages (reviewed in [ 6 ], illustrated in Figure 1 , path 1). Embryonic stem cells (derived from the blastocyst inner cell mass), or embryonic germ cells (derived from the gonadal ridge), when cultured on embryonic mouse fibroblast feeder cell layers in the presence of a differentiation-suppressing cytokine (leukemia inhibitory factor), proliferate indefinitely and remain pluripotent. Manipulation of culture conditions can coax the cells to undergo differentiation characteristic of many tissue types (Figure 1 , paths 2 and 3). Theoretically, pluripotent embryonic cells can serve as an unlimited resource for therapeutic applications [ 7 , 8 ]. Figure 1 Cell lineage determination during embryogenesis and generation of pluripotent embryonic cells . The three primary germ layers form during normal development (path 1). Embryonic stem cells from the inner cell mass (path 2) or embryonic germ cells from the gonadal ridge (path 3) can be cultured and manipulated to generate cells of all three lineages. General principles of tissue renewal by adult stem cells have been reviewed recently [ 9 ] and can be summarized as follows. The traditional view of cell lineages is that adult somatic stem cells maintain cell populations in adult tissues. The adult lung falls into the category in which cell proliferation is very low in the normal steady-state but can be induced dramatically by injury (see [ 10 , 11 ] for recent reviews of lung stem cells). The conditional nature of lung cell proliferation complicates the search for lung stem cells. Cell lineages are much better understood in continuously proliferating tissues such as the gut, skin and hematopoietic system (reviewed in [ 12 - 14 ], respectively). The long-standing view, developed from these other organs, is that stem cells reside in well-protected, innervated, and vascularized niches that provide cues regulating cell fate decisions such as proliferation, migration, and differentiation [ 15 ]. Adult stem cells are capable of abundant self-renewal and can also generate the specific cell lineages within the tissue compartment (Figure 2 ). Proportional to tissue needs, stem cells may undergo asymmetric cell division, in which they generate one stem cell and a committed progenitor. The capacity for self-renewal decreases progressively as committed progenitors differentiate. The wisdom of the body is to conserve stem cells. They cycle infrequently and the majority of cell replacement is accomplished by committed progenitors within the so-called transiently amplifying compartment. Eventually, individual cells become incapable of further cell division. In tissues, there are specific temporal and spatial hierarchic relationships between stem cells in their niches and their differentiated progeny. Within this axis, cell proliferation, migration, differentiation, function, death, and removal are tightly regulated to maintain tissue homeostasis. Figure 2 Traditional view of cell lineage in adult renewing tissues . Organ-specific (somatic) stem cells generate characteristic cell types through a linear set of commitment and differentiation steps. Arrow thickness represents self-renewal potential. Cell compartments in the lung and functional integration In the architecturally complex lung, cells of multiple germinal lineages interact both during morphogenesis and to maintain adult lung structure. Even within derivatives of a single germ layer, cells become subdivided into separate cell lineage "zones". For example, the endoderm generates least four distinct epithelial regions, each with a different cellular composition (Figure 3 ). Additional cell types, including airway smooth muscle, fibroblasts, and the vasculature, are derived from mesoderm. Airway and alveolar architecture, and in turn, function, result from interaction among epithelium, smooth muscle, fibroblasts, and vascular cells, all within an elaborate structural matrix of connective tissue. The complexity of even this oversimplified view, which omits pulmonary neuroepithelial cells and bodies, innervation, and classical hematopoietically-derived cells such as dendritic cells, mast cells, and macrophages, has hindered identification of lung stem cells and patterns of cell migration during tissue renewal. Nevertheless, the prevailing view is that airway basal and Clara cells and alveolar type II cells serve as epithelial progenitors [ 11 , 16 - 19 ]. Cell lineages in the mesodermal compartments remain less well understood. Figure 3 Stem cell compartments in the lungs . The endoderm-derived epithelium can be subdivided into at least 4 types whereas smooth muscle, fibroblasts, and vascular cells are derived from mesoderm. The coordinated interaction of multiple cell types, including alveolar epithelium, interstitial fibroblasts, myofibroblasts and pulmonary endothelium, is necessary to form alveolar septa. Stem cell plasticity and the lung Recent studies challenge the view that tissues are maintained solely by organ-specific stem cells. There is evidence that adult stem cells from a variety of sources can generate not only their own lineages, but those of other tissues, sometimes crossing barriers of embryonic derivation previously thought impenetrable [ 20 , 21 , 8 ]. There are a few controversial reports that adult stem cells from outside the bone marrow may reconstitute the hematopoietic system, but most of the evidence flows in the other direction- namely, that cells from the bone marrow can generate diverse non-hematopoietic cell types. Both experimental studies in animals and human clinical studies, summarized in Table 1 , provide evidence for, and against, circulatory delivery of lung progenitor cells. While bone marrow-derived cells, such as alveolar macrophages, dendritic cells, mast cells, and lymphocytes, normally migrate to the lung, the surprise in the recent literature is that under certain circumstances circulating cells can apparently generate lung resident cells, including epithelial, endothelial, and myofibroblast cells. The technical approach towards identification of these cells is often technically challenging and involves co-localization of a donor cell marker, for example, the Y chromosome, in sex-mismatched transplantation, or a genetically engineered marker in mouse experiments, and proteins characteristic of the differentiated cell type in the lung, for example, keratin in epithelial cells or collagen in fibroblasts. As discussed below, the results are highly variable and often contradictory, depending on factors including the starting cell population, the methods for marker detection, and the amount of injury to the lung. Table 1 Evidence for, and against, circulating progenitor cell generation of non-hematopoietic lung cell types. Study Type Disease or Model Tissue of Origin Lung Cell Type Formed / Frequency Method of Detection Ref. Animal, in-vivo BMT MSC Undefined mesenchymal cells / occasional PCR for collagen gene marker [30] Animal, in-vivo Bleomycin fibrosis MSC Type I pneumocytes / rare β galactosidase protein [23] Animal, in-vivo BMT HSC enrichment Type II pneumocytes / up to 20%, bronchial epithelium / 4% Y chromosome FISH, surfactant B mRNA [31] Animal, in-vivo Radiation pneumonitis Whole bone marrow Type II pneumocytes, bronchial epithelium / up to 20% of type II cells Y chromosome FISH, surfactant B mRNA [25] Animal, in-vivo BMT Whole bone marrow/EGFP retrovirus Type II pneumocytes / 1–7% EGFP, keratin immunostain, surfactant protein B FISH [33] Animal, in-vivo BMT and parabiotic animals HSC Hematopoietic chimerism but exceedingly rare lung cell types EGFP [32] Animal, in-vivo Bleomycin fibrosis MSC Type II pneumocytes / ~1% Y chromosome FISH [22] Animal, in-vivo Radiation fibrosis MSC or whole bone marrow Fibroblasts / common EGFP, Y chromosome FISH, vimentin immunostain [26] Animal, in-vivo BMT Bone marrow, EGFP labeled Fibroblasts, Type I pneumocyte / occasional to rare Flow cytometry [34] Animal, in-vitro and in-vivo Hypoxia-induced pulmonary hypertension Circulating BM-derived c-kit positive c-kit positive cells in pulmonary artery vessel wall; In hypoxia, circulating cells generate endothelial and smooth muscle cells in-vitro Flow cytometry and immunohistochemistry [27] Animal, in-vivo Ablative radiation and elastase induced emphysema GFP + fetal liver Alveolar epithelium and endothelium; frequency not reported but increased by G-CSF and retinoic acid Immunohistochemistry for CD45 - , GFP + cells [28] Animal, in-vivo Bleomycin fibrosis Whole marrow GFP + GFP + type I collagen expressing Flow cytometry and immunohistochemistry, RT-PCR [24] Human, in-vitro Heat shock in cell culture MSC and SAEC Cell fusion / common Immunostaining, microarray [39] Animal, in-vivo Human, in-vivo OVA-sensitized mouse model Allergen – sensitized asthmatics CD34 positive, collagen I expressing fibrocytes CD34 positive, collagen I expressing fibrocytes Myofibroblasts / ? Myofibroblasts / ? CD34-positive, collagen I, α-smooth muscle actin CD34-positive, collagen I, α-smooth muscle actin [29] Human, in-vivo Human heart and lung transplant Sex-mismatched donor lung or heart No lung cell types of recipient origin X and Y chromosome FISH, antibody stain for hematopoeitic cells [36] Human, in-vivo Human lung transplant Human BMT Sex-mismatched donor lung Sex-mismatched donor bone marrow Bronchial epithelium, type II pneumocytes, glands of recipient origin / 9 – 24% No lung cell types of donor origin Y chromosome FISH, short tandem repeat PCR Y chromosome FISH, short tandem repeat PCR [35] Human, in-vivo Human BMT Sex-mismatched donor bone marrow Lung epithelium and endothelium of donor origin / up to 43% X and Y chromosome FISH, keratin and PECAM immunostain [38] Human, in-vivo Human BMT Sex-mismatched donor bone marrow No nasal epithelium of donor origin Y chromosome FISH, cytokeratin immunostain [37] BMT = bone marrow transplant (with prior ablation), MSC = mesenchymal stem cells (bone marrow stromal cells, adherent bone marrow cells), EGFP = enhanced green fluorescent protein, HSC = hematopoietic stem cells, FISH = fluoresence in situ hybridization, SAEC = small airway epithelial cells Transplantation studies in mice can be performed using whole donor bone marrow, the fraction that adheres in culture, termed marrow stromal cells (MSC), or preparations enriched for hematopoietic stem cells (HSC). Whole body irradiation, which may injure lung tissue, is typically used to deplete the host bone marrow. Importantly, lung injury apparently enhances engraftment into lung [ 22 - 29 ]. Whole bone marrow, MSC, or HSC have all been reported to reconstitute lung parenchymal cells. MSC transplantation resulted in collagen I expressing donor cells in the lung [ 30 ], and in the presence of bleomycin injury, MSC reportedly generated type I [ 23 ] or type II pneumocytes [ 22 ]. Transplantation with HSCs yielded up to 20% donor-derived pneumocytes and 4% bronchial epithelial cells [ 31 ]. However, other investigators have identified only hematopoeitic chimerism by HSCs [ 32 ]. Whole bone marrow infusion generated type II pneumocytes [ 33 ], or fibroblasts and type I pneumocytes [ 34 ]. Radiation pneumonitis augmented whole bone marrow generation of type II pneumocytes and bronchial epithelial cells [ 25 ] or fibroblasts [ 26 ]. Bleomycin lung injury enhanced formation of type I collagen-producing cells [ 24 ] from whole bone marrow, whereas elastase-induced emphysema stimulated formation of alveolar epithelium and endothelium [ 28 ]. Lung injury alone, without bone marrow transplantation, may promote stem cell migration. For example, in the ovalbumin model of asthma, circulating fibrocytes were recruited into bronchial tissue [ 29 ], and in a bovine model of hypoxic pulmonary hypertension, cells capable of generating endothelial and smooth muscle cells in vitro were found in the circulation [ 27 ]. Sex-mismatched lung and bone marrow transplantation in humans provides a natural model for analysis of donor and recipient cell behavior. Bronchial epithelial and gland cells and type II pneumocytes of host origin were reported in one study of lung allografts [ 35 ], but not another [ 36 ]. After bone marrow transplantation, epithelial cells of donor origin were not detected in the nasal passages [ 37 ]. Similar to lung allografts, following bone marrow transplantation, epithelium and endothelium of donor origin were found in one study [ 38 ], but not another [ 35 ]. Many questions remain unanswered. The mechanism whereby cells assume lung cell phenotypes remains uncertain. Several studies have demonstrated that cell fusion occurs both in vitro and in vivo , which likely explains why some of the cells contain both donor and lung cell markers [see [ 39 ] for a study of fusion of MSCs and lung epithelium and [ 40 , 41 ] for recent reviews]. Alternatively, cells may reprogram in the lung environment- a concept termed "transdifferentiation", which is defined as the ability of a particular cell from one tissue type to differentiate into a cell type characteristic of another tissue. It has been suggested that many of the events previously attributed to transdifferentiation may actually represent cell fusions, particularly due to the influx of fusion-prone myeloid cells into damaged tissues from the repopulated bone marrow [ 40 ]. New, more stringent, criteria have been put forth for demonstration of transdifferentiation [ 41 ]. Bone marrow harbors a generalized pluripotent stem cell [ 42 ] and the bone marrow cell responsible for lung engraftment has not been identified with certainty. It is possible that rare transdifferentiation events represent migration of a pluripotent bone marrow cell type resembling an embryonic stem or embryonic germ cell still harbored in the adult bone marrow. It remains unknown whether bone marrow cells must transit through an intermediate compartment prior to lung colonization (Figure 4 ) or whether circulating stem cells can be mobilized from sources other than bone marrow. It is important to note that bone marrow derived cells of typical hematopoietic lineage, chimeric cells created by fusion, or lung cells generated by transdifferentiation may all play a role in lung repair by promoting the local production of stem cells or reparative function of lung-specific cell types. A compelling study suggests that mesenchymal stem cells from bleomycin-resistant mice can mitigate the pro-fibrotic effects of bleomycin in sensitive mice [ 22 ], while another study suggests that bone marrow cells actively contribute to the formation of fibrotic tissue [ 24 ]. Mitigating or exacerbating roles for bone marrow derived cells in lung repair or fibrosis are not mutually exclusive. The important concepts of whether the lungs' capacity for repair is dependent on circulating cells, and whether exogenously delivered cells can enhance resistance to injury or promote healing, remain unanswered and controversial. Figure 4 Evolving view of cell lineages in the lungs . The functional significance of circulating cells towards lung cell maintenance or tissue repair remains unknown, as does the precise mechanism whereby circulating cells generate lung cell types. Lung "stem cell" diseases Major lung diseases likely involving stem cells and the cellular targets for stem cell therapy are summarized in Table 2 . These may be broadly categorized whether they involve stem cell deficiency, hyper-proliferation or possibly, a combination of both. For example, impaired pulmonary endothelial and/or epithelial barrier function may contribute to the pathophysiology of adult respiratory distress syndrome. Mobilization of endogenous endothelial or epithelial stem/progenitor cells or delivery of adult somatic stem cells, embryonic stem cells, or embryonic germ cells may theoretically improve barrier function, supporting the notion of treating a "stem cell deficiency". Similarly, toxic, viral or alloimmune destruction of the bronchiolar epithelium suggests stem cell deficiency in bronchiolitis obliterans. However, fibrotic reactions and scarring in response to epithelial injury can be viewed as fibroblast "stem cell hyper-proliferation". The general concept is that augmentation of stem cells may minimize lung injury, augment repair, or possibly regenerate lost tissue. However, one must also consider that inhibiting excessive growth of stem cells may be a valid therapeutic goal when hyper-proliferation contributes to disease pathophysiology, as in fibrosis, smooth muscle hyperplasia or lung cancer. Table 2 Major lung diseases potentially treatable by stem cell manipulation. Disease Category Injured, Depleted, or Deranged Cellular Compartment* Therapeutic Goals Congenital lung hypoplasia Chronic lung disease of prematurity Pulmonary emphysema Alveolar epithelium, Interstitial fibroblast, Capillary endothelium, Generate alveolar septa Restore complex three dimensional structure Neonatal RDS Adult RDS Alveolar epithelium, Capillary endothelium Enhance surfactant production Reinforce endothelial and epithelial barriers Pulmonary fibrosis Alveolar epithelium, Interstitial fibroblast Prevent alveolar epithelial loss Inhibit fibroblast proliferation Asthma Airway epithelium, Myofibroblasts, Airway smooth muscle Create an anti-inflammatory environment Inhibit airway wall remodeling Inhibit smooth muscle hypertrophy and hyperplasia Cystic fibrosis Airway epithelium Deliver functional CFTR Bronchiolitis obliterans Airway epithelium Reinforce the epithelium against toxic, viral or immunologic injury Lung cancer Epithelium Detection, monitoring or treatment based on molecular regulation of stem cell proliferation and differentiation RDS = respiratory distress syndrome, CFTR= cystic fibrosis transmembrane conductance regulator *Each cell type listed in this column is affected in all of the specific conditions listed in the left hand column Challenges for lung regenerative medicine What are the realistic prospects for beneficial stem cell therapy of the lung? First, we must conclusively identify lung diseases/cases/timing in which cell and tissue damage occurs in excess of the capacity for timely endogenous repair. Second, we must establish standardized sources of relevant stem/progenitor cells and methods for their delivery to the appropriate lung sub-compartment. Once delivered, therapeutic cells must home to microscopic sites of need and integrate to serve a beneficial function. There is clearly potential for adverse effects, as exemplified by the propensity of embryonic stem cells to form teratomas when implanted in vivo [ 43 ]. Major lung diseases potentially addressable by stem cell therapy may pose unique challenges. Reversal of lung developmental anomalies resulting in hypoplasia, or repair of chronic lung disease of prematurity and advanced pulmonary emphysema in adults, will require neogenesis of alveolar septa in which the endogenous "tissue blueprint" never developed, or was completely destroyed. Until we gain a much better understanding of lung tissue morphogenesis, we must rely on stem cells intrinsically "knowing" where to go and "how" to recreate alveolar septal architecture to ultimately restore higher order complex three dimensional relationships amongst alveoli, airways, and vessels. Stem cell therapy to cure cystic fibrosis will require heterologous, or gene corrected autologous, stem cells to colonize the airway, proliferate, and differentiate into columnar cells covering a significant portion of the airway lumen. However, most evidence thus far suggests that cells from the circulation may generate isolated, single airway basal cells. Stem cell therapy to mitigate respiratory distress syndrome (RDS) will require cells capable of restoring alveolar endothelial and epithelial function in the face of evolving injury. Whereas injury is thought to promote stem cell recruitment, the relevant question is whether it can occur quickly enough to meaningfully reverse acute, widespread cellular dysfunction typical of RDS. Conclusion Provocative, but controversial, recent evidence suggests that circulating stem cells may home to the lung. There is great excitement and hope that exogenous and/or mobilized endogenous stem cells may be harnessed to prevent or treat acute and chronic lung diseases and even regenerate abnormally developed or lost tissue. Our understanding of lung stem cells and the regulation of lung morphogenesis is still rudimentary, and the complex, integrated function of multiple cell types underlying normal lung structure and function poses unique challenges. Thus, the therapeutic prospects for stem cell therapy in lungs appear more distant than in some other organs. This realization should stimulate meaningful new studies from the lung research community. Unlike the mythical hero Prometheus, patients with lung disease cannot wait 30,000 years! Competing interests None declared.

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176545

The Transcriptome of the Intraerythrocytic Developmental Cycle of Plasmodium falciparum

Plasmodium falciparum is the causative agent of the most burdensome form of human malaria, affecting 200–300 million individuals per year worldwide. The recently sequenced genome of P. falciparum revealed over 5,400 genes, of which 60% encode proteins of unknown function. Insights into the biochemical function and regulation of these genes will provide the foundation for future drug and vaccine development efforts toward eradication of this disease. By analyzing the complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of the HB3 strain of P. falciparum , we demonstrate that at least 60% of the genome is transcriptionally active during this stage. Our data demonstrate that this parasite has evolved an extremely specialized mode of transcriptional regulation that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium -specific functionalities, such as genes involved in erythrocyte invasion. The data reveal that genes contiguous along the chromosomes are rarely coregulated, while transcription from the plastid genome is highly coregulated and likely polycistronic. Comparative genomic hybridization between HB3 and the reference genome strain (3D7) was used to distinguish between genes not expressed during the IDC and genes not detected because of possible sequence variations. Genomic differences between these strains were found almost exclusively in the highly antigenic subtelomeric regions of chromosomes. The simple cascade of gene regulation that directs the asexual development of P. falciparum is unprecedented in eukaryotic biology. The transcriptome of the IDC resembles a “just-in-time” manufacturing process whereby induction of any given gene occurs once per cycle and only at a time when it is required. These data provide to our knowledge the first comprehensive view of the timing of transcription throughout the intraerythrocytic development of P. falciparum and provide a resource for the identification of new chemotherapeutic and vaccine candidates.

Introduction Human malaria is caused by four species of the parasitic protozoan genus Plasmodium . Of these four species, Plasmodium falciparum is responsible for the vast majority of the 300–500 million episodes of malaria worldwide and accounts for 0.7–2.7 million annual deaths. In many endemic countries, malaria is responsible for economic stagnation, lowering the annual economic growth in some regions by up to 1.5% ( Sachs and Malaney 2002 ). While isolated efforts to curb malaria with combinations of vector control, education, and drugs have proven successful, a global solution has not been reached. Currently, there are few antimalarial chemotherapeutics available that serve as both prophylaxis and treatment. Compounding this paucity of drugs is a worldwide increase in P. falciparum strains resistant to the mainstays of antimalarial treatment ( Ridley 2002 ). In addition, the search for a malaria vaccine has thus far been unsuccessful. Given the genetic flexibility and the immunogenic complexity of P. falciparum , a comprehensive understanding of Plasmodium molecular biology will be essential for the development of new chemotherapeutic and vaccine strategies. The 22.8 Mb genome of P. falciparum is comprised of 14 linear chromosomes, a circular plastid-like genome, and a linear mitochondrial genome. The malaria genome sequencing consortium estimates that more than 60% of the 5,409 predicted open reading frames (ORFs) lack sequence similarity to genes from any other known organism ( Gardner et al. 2002 ). Although ascribing putative roles for these ORFs in the absence of sequence similarity remains challenging, their unique nature may be key to identifying Plasmodium -specific pathways as candidates for antimalarial strategies. The complete P. falciparum lifecycle encompasses three major developmental stages: the mosquito, liver, and blood stages. It has long been a goal to understand the regulation of gene expression throughout each developmental stage. Previous attempts to apply functional genomics methods to address these questions used various approaches, including DNA microarrays ( Hayward et al. 2000 ; Ben Mamoun et al. 2001 ; Le Roch et al. 2002 ), serial analysis of gene expression ( Patankar et al. 2001 ), and mass spectrometry ( Florens et al. 2002 ; Lasonder et al. 2002 ) on a limited number of samples from different developmental stages. While all of these approaches have provided insight into the biology of this organism, there have been no comprehensive analyses of any single developmental stage. Here we present an examination of the full transcriptome of one of these stages, the asexual intraerythrocytic developmental cycle (IDC), at a 1-h timescale resolution. The 48-h P. falciparum IDC ( Figure 1 A) initiates with merozoite invasion of red blood cells (RBCs) and is followed by the formation of the parasitophorous vacuole (PV) during the ring stage. The parasite then enters a highly metabolic maturation phase, the trophozoite stage, prior to parasite replication. In the schizont stage, the cell prepares for reinvasion of new RBCs by replicating and dividing to form up to 32 new merozoites. The IDC represents all of the stages in the development of P. falciparum responsible for the symptoms of malaria and is also the target for the vast majority of antimalarial drugs and vaccine strategies. Figure 1 Parasite Culturing and Data Characteristics of the P. falciparum IDC Transcriptome Analysis (A) Giemsa stains of the major morphological stages throughout the IDC are shown with the percent representation of ring-, trophozoite-, or schizont-stage parasites at every timepoint. The 2-h invasion window during the initiation of the bioreactor culture is indicated (gray area). (B–D) Example expression profiles for three genes, encoding EBA175, DHFR-TS, and ASL, are shown with a loess fit of the data (red line). (E) MAL6P1.147, the largest predicted ORF in the Plasmodium genome, is represented by 14 unique DNA oligonucleotide elements. The location of each of the oligonucleotide elements within the predicted ORF and the corresponding individual expression profiles are indicated (oligo 1–14). A red/green colorimetric representation of the gene expression ratios for each oligonucleotide is shown below the graph. The pairwise Pearson correlation for these expression profiles is 0.98 ± 0.02. (F) The percentage of the power in the maximum frequency of the FFT power spectrum was used as an indicator of periodicity. A histogram of these values reveals a strong bias toward single-frequency expression profiles, indicating that the majority of P. falciparum genes are regulated in a simple periodic manner. This bias is eliminated when the percent power was recalculated using random permutations of the same dataset (inset). For reference, the locations of EBA175 (peak B), DHFR-TS (peak C), and ASL (peak D) are shown. Our laboratory has developed a P. falciparum– specific DNA microarray using long (70 nt) oligonucleotides as representative elements for predicted ORFs in the sequenced genome (strain 3D7) ( Bozdech et al. 2003 ). Using this DNA microarray, we have examined expression profiles across 48 individual 1-h timepoints from the IDC of P. falciparum . Our data suggest that not only does P. falciparum express the vast majority of its genes during this lifecycle stage, but also that greater than 75% of these genes are activated only once during the IDC. For genes of known function, we note that this activation correlates well with the timing for the respective protein's biological function, thus illustrating an intimate relationship between transcriptional regulation and the developmental progression of this highly specialized parasitic organism. We also demonstrate the potential of this analysis to elucidate the function of the many unknown gene products as well as for further understanding the general biology of this parasitic organism. Results Expression Profiling of the IDC The genome-wide transcriptome of the P. falciparum IDC was generated by measuring relative mRNA abundance levels in samples collected from a highly synchronized in vitro culture of parasites. The strain used was the well-characterized Honduran chloroquine-sensitive HB3 strain, which was used in the only two experimental crosses carried out thus far with P. falciparum ( Walliker et al. 1987 ; Wellems et al. 1990 ). To obtain sufficient quantities of parasitized RBCs and to ensure the homogeneity of the samples, a large-scale culturing technique was developed using a 4.5 l bioreactor (see Materials and Methods ). Samples were collected for a 48-h period beginning 1 h postinvasion (hpi). Culture synchronization was monitored every hour by Giemsa staining. We observed only the asexual form of the parasite in these stains. The culture was synchronous, with greater than 80% of the parasites invading fresh RBCs within 2 h prior to the harvesting of the first timepoint. Maintenance of synchrony throughout the IDC was demonstrated by sharp transitions between the ring-to-trophozoite and trophozoite-to-schizont stages at the 17- and 29-h timepoints, respectively ( Figure 1 A). The DNA microarray used in this study consists of 7,462 individual 70mer oligonucleotides representing 4,488 of the 5,409 ORFs manually annotated by the malaria genome sequencing consortium ( Bozdech et al. 2003 ). Of the 4,488 ORFs, 990 are represented by more than one oligonucleotide. Since our oligonucleotide design was based on partially assembled sequences periodically released by the sequencing consortium over the past several years, our set includes additional features representing 1,315 putative ORFs not part of the manually annotated collection. In this group, 394 oligonucleotides are no longer represented in the current assembled sequence. These latter ORFs likely fall into the gaps present in the published assembly available through the Plasmodium genome resource PlasmoDB.org ( Gardner et al. 2002 ; Kissinger et al. 2002 ; Bahl et al. 2003 ). To measure the relative abundance of mRNAs throughout the IDC, total RNA from each timepoint was compared to an arbitrary reference pool of total RNA from all timepoints in a standard two-color competitive hybridization ( Eisen and Brown 1999 ). The transcriptional profile of each ORF is represented by the mean-centered series of ratio measurements for the corresponding oligonucleotide(s) ( Figure 1 B–1E). Inspection of the entire dataset revealed a striking nonstochastic periodicity in the majority of expression profiles. The relative abundance of these mRNAs continuously varies throughout the IDC and is marked by a single maximum and a single minimum, as observed for the representative schizont-specific gene, erythrocyte-binding antigen 175 ( eba175 ), and the trophozoite-specific gene, dihydrofolate reductase–thymidylate synthetase ( dhfr-ts ) ( Figure 1 B and 1C). However, there is diversity in both the absolute magnitude of relative expression and in the timing of maximal expression (phase). In addition, a minority of genes, such as adenylosuccinate lyase ( asl ) ( Figure 1 D), displayed a relatively constant expression profile. The accuracy of measurements from individual oligonucleotides was further verified by the ORFs that are represented by more than one oligonucleotide feature on the microarray. The calculated average pairwise Pearson correlation ( r ) is greater than 0.90 for 68% (0.75 for 86%) of the transcripts represented by multiple oligonucleotides with detectable expression during the IDC ( Table S1 ). Cases in which data from multiple oligonucleotides representing a single putative ORF disagree may represent incorrect annotation. The internal consistency of expression profile measurements for ORFs represented by more than one oligonucleotide sequence is graphically shown in Figure 1 E for the hypothetical protein MAL6P1.147, the largest predicted ORF in the genome (31 kb), which is represented by 14 oligonucleotide elements spanning the entire length of the coding sequence. The average pairwise correlation ( r ) for these features is 0.98±0.02. Periodicity in genome-wide gene expression datasets has been used to identify cell-cycle-regulated genes in both yeast and human cells ( Spellman et al. 1998 ; Whitfield et al. 2002 ). Owing to the cyclical nature of the P. falciparum IDC dataset, a similar computational approach was taken. We performed simple Fourier analysis, which allowed us to calculate both the apparent phase and frequency of expression for each gene during the IDC (see Materials and Methods ). The fast Fourier transform (FFT) maps a function in a time domain (the expression profile) into a frequency domain such that when the mapped function is plotted (the power spectra), sharp peaks appear at frequencies where there is intrinsic periodicity. The calculated power spectra for each expression profile confirmed the observation that the data are highly periodic. The majority of profiles exhibited an overall expression period of 0.75–1.5 cycles per 48 h. We have used the FFT data for the purpose of filtering the expression profiles that are inherently noisy (i.e., that have low signal) or that lack differential expression throughout the IDC. Since the majority of the profiles display a single low-frequency peak in the power spectrum, we have taken advantage of this feature to classify profiles, similar to the application of a low-pass filter in signal processing. By measuring the power present in the peak frequency window (the main component plus two adjacent peaks) relative to the power present at all frequencies of the power spectrum, we were able to define a score (percent power) that we have used to stratify the dataset. The resulting distribution of expression profiles, scored in this way, is shown in Figure 1 F for all oligonucleotides. For reference, the positions of profiles corresponding to eba175 (peak B), dhfr-ts (peak C), and asl (peak D) are indicated. It is striking that 79.5% of the expression profiles have a very high score (greater than 70%). For comparison, we applied our FFT analysis to the Saccharomyces cerevisiae cell cycle data, yielding only 194 profiles (3.8%) above a 70% score ( Figure S1 ). In addition, we randomly permuted the columns of the complete dataset 1,000 times, each time recalculating the FFT, for a total of 5 million profiles (see inset in Figure 1 F). The randomized set exhibits essentially no periodicity: the probability of any random profile scoring above 70% is 1.3 × 10 −5 . P. falciparum Transcriptome Overview To provide an overview of the IDC transcriptome, we selected all 3,719 microarray elements whose profiles exhibited greater than 70% of the power in the maximum frequency window and that were also in the top 75% of the maximum frequency magnitudes. Although hierarchical clustering is extremely useful for comparing any set of expression data, regardless of the experimental variables, we sought to specifically address temporal order within the dataset. To accomplish this, the FFT phase was used to order the expression profiles to create a phaseogram of the IDC transcriptome of P. falciparum ( Figure 2 A). The overview set represents 2,714 unique ORFs (3,395 oligonucleotides). An additional 324 oligonucleotides represent ORFs that are not currently part of the manually annotated collection. Figure 2 Overview of the P. falciparum IDC Transcriptome (A) A phaseogram of the IDC transcriptome was created by ordering the transcriptional profiles for 2,712 genes by phase of expression along the y-axis. The characteristic stages of intraerythrocytic parasite morphology are shown on the left, aligned with the corresponding phase of peak gene expression. (B–M) The temporal ordering of biochemical processes and functions is shown on the right. Each graph corresponds to the average expression profile for the genes in each set and the mean peak-to-trough amplitude is shown in parentheses. The IDC phaseogram depicts a cascade of continuous expression lacking clear boundaries or sharp transitions. During the first half of the IDC, a large number of genes involved in general eukaryotic cellular functions are induced with broad expression profiles. This gradual continuum includes the transition from the ring to the early trophozoite stage and the trophozoite to the early schizont stage, encompassing approximately 950 and 1,050 genes, respectively. Next, the mid- and late-schizont stages are marked by a rapid, large amplitude induction of approximately 550 genes, many of which appear to be continually expressed into the early-ring stage. However, owing to the level of synchrony in the culture, the ring-stage signal may be partially attributed to cross-contamination from residual schizonts. In the final hours of the IDC, approximately 300 genes corresponding to the early-ring stage are induced, indicating that reinvasion occurs without obvious interruptions to initiate the next cycle. The expression profiles for developmentally regulated genes in the P. falciparum IDC transcriptome reveal an orderly timing of key cellular functions. As indicated in Figure 2 B–2M, groups of functionally related genes share common expression profiles and demonstrate a programmed cascade of cellular processes that ensure the completion of the P. falciparum IDC. Ring and Early-Trophozoite Stage In the following text, we have grouped the genes according to temporal expression phases based on their association with the common P. falciparum cytological stages. Following invasion, approximately 950 ORFs are induced during the ring and early trophozoite stage, including genes associated with the cytoplasmic transcriptional and translational machinery, glycolysis and ribonucleotide biosynthesis ( Figure 2 B–2E). Represented in this group are 23 ORFs involved in transcription, including the four subunits of RNA polymerase I, nine subunits of RNA polymerase II, three subunits of RNA polymerase III, and four transcription factors. The average expression profile for this group is shown in Figure 2 B. (See Table S2 for all functional group details.) Also in this set are three previously identified P. falciparum RNA polymerase genes: the large subunits of P. falciparum RNA polymerase I ( Fox et al. 1993 ) and RNA polymerase II ( Li et al. 1989 ) and RNA polymerase III ( Li et al. 1991 ). The cytoplasmic translation gene group ( Figure 2 C) consists of 135 ORFs including homologues for 34 small and 40 large ribosomal subunits, 15 translation initiation factors, five translation elongation factors, 18 aminoacyl-tRNA synthetases, and 23 RNA helicases. In addition to the manually annotated ORFs, the translation gene group contains three ORFs predicted only by automated annotation including two ribosomal proteins (chr5.glm_215, chr5.glm_185) and a homologue of eIF-1A (chr11.glm_489) ( PlasmoDB.org ). In one case, chr5.glm_185 overlaps with the manually annotated ORF PFE0850w, which is found on the opposite strand. Oligonucleotide elements for both of these ORFs are present on the array. The oligonucleotide corresponding to the automated prediction yielded a robust FFT score and a phase consistent with the translation machinery, yet no PFE0850w expression was detected. These results suggest that the automated prediction for chr5.glm_185 most likely represents the correct gene model for this genomic locus and illustrates the use of the IDC expression data for further verification of the P. falciparum genome annotations. Another set of 33 ORFs with homology to components of the translational machinery displayed an entirely distinct expression pattern, being induced during the late-trophozoite and early-schizont stage. This group includes 11 homologues of chloroplast ribosomal proteins, four mitochondrial/chloroplast elongation factors, and six amino acid tRNA synthetases ( Table S2 ). These ORFs also share a common pattern of expression, suggesting that these factors are components of the mitochondrial and/or the plastid translation machinery. This observation is supported by the presence of predicted apicoplast-targeting signals in 18 of these proteins ( PlasmoDB.org ). In addition, one of these factors, ribosomal protein S9, has been experimentally immunolocalized within the plastid ( Waller et al. 1998 ). These data suggest that the peak of expression for the cytoplasmic translation machinery occurs in the first half of the IDC, whereas plastid and mitochondrial protein synthesis is synchronized with the maturation of these organelles during the second half of the IDC. In addition to transcription and translation, genes involved in several basic metabolic pathways were also induced during the ring and early-trophozoite stage, including glycolysis and ribonucleotide biosynthesis ( Figure 2 D and 2E). Unlike the majority of P. falciparum biochemical processes, most of the enzymes involved in nucleotide metabolism and glycolysis have been identified ( Reyes et al. 1982 ; Sherman 1998 ). The glycolysis group ( Figure 2 D) is tightly coregulated throughout the IDC and contains all of the 12 known enzymes. Expression initiates after reinvasion and continues to increase toward maximal expression during the trophozoite stage, when metabolism is at its peak. The glycolytic pathway is very well preserved in P. falciparum and exemplifies how data from this study can complement the homology-based interpretation of the genome. First, the genome contains two putative copies of pyruvate kinase on chromosomes 6 and 10, MAL6P1.160 and PF10_0363, respectively ( Gardner et al. 2002 ). However, only one of these genes, MAL6P1.160, has a similar expression profile to the other known glycolytic enzymes, suggesting that this enzyme is the main factor of this step in the glycolytic pathway. Interestingly, PF10_0363 contains a putative apicoplast-targeting signal ( PlasmoDB.org ). In another case, the malaria genome sequencing consortium has predicted two homologues of triose phosphate isomerase, PF14_0378 and PFC0381w. The latter is not detected by our analysis, suggesting that this gene is utilized in another developmental stage or may be a nonfunctional, redundant homologue. P. falciparum parasites generate pyrimidines through a de novo synthesis pathway while purines must be acquired by the organism through a salvage pathway ( Gero and O'Sullivan 1990 ). The mRNA levels of 16 enzymes corresponding to members of the pyrimidine ribonucleotide synthesis pathway, beginning with carbamoyl phosphate synthetase and ending with CTP synthetase, were uniformly induced immediately after invasion ( Figure 2 E). The relative abundance of these transcripts peaked at approximately 18–22 hpi and then rapidly declined. Similar expression characteristics were detected for the enzymes of the purine salvage pathway, including the nucleoside conversion enzymes, hypoxanthine–guanine–xanthine phosphoribosyltransferase, and both guanylate and adenylate kinases ( Figure 2 E; Table S2 ). Trophozoite and Early-Schizont Stage The mRNA expression data indicate that ribonucleotide and deoxyribonucleotide production is clearly bifurcated into two distinct temporal classes. While ribonucleotide synthesis is required in the early stages of the IDC, deoxyribonucleotide metabolism is a trophozoite/early-schizont function. mRNA transcripts for enzymes that convert ribonucleotides into deoxyribonucleotides, including DHFR-TS and both subunits of ribonucleotide reductase, were induced approximately at 10 hpi, peaking at approximately 32 hpi ( Figure 2 F). This represents a temporal shift from the induction of ribonucleotide synthesis of approximately 8–10 h. The expression of the deoxyribonucleotide biosynthesis is concomitant with the induction of DNA replication machinery transcripts, reflecting a tight relationship between DNA synthesis and production of precursors for this process. Thirty-two ORFs with homologies to various eukaryotic DNA replication machinery components are transcribed during the late-trophozoite and early-schizont stage. The timing of their transcription presages cell division. This functional gene group ( Figure 2 G), with peak expression around 32 hpi, contains the previously characterized P. falciparum DNA Polα, DNA Polδ, and proliferating cell nuclear antigen, as well as the vast majority of the DNA replication components predicted by the malaria genome sequencing consortium ( Gardner et al. 2002 ). These additional components include eight predicted DNA polymerase subunits, two putative origin recognition complex subunits, six minichromosome maintenance proteins, seven endo- and exonucleases, seven replication factor subunits, and two topoiosomerases. Interestingly, a number of proteins typically required for eukaryotic DNA replication, including the majority of the subunits of the origin recognition complex, have not yet been identified by conventional sequence similarity searches of the P. falciparum genome. All genes necessary for the completion of the tricarboxylic acid (TCA) cycle were detected in the Plasmodium genome ( Gardner et al. 2002 ), although earlier studies indicate an unconventional function for this metabolic cycle. These studies suggest that the TCA cycle does not play a major role in the oxidation of glycolytic products. Instead, it is essential for the production of several metabolic intermediates, such as succinyl-CoA, a precursor of porphyrin biosynthesis ( Sherman 1998 ). The peak of expression for all TCA factors was detected during the late-trophozoite and early-schizont stage ( Figure 2 H). Consistent with the model suggesting a disconnection of the TCA cycle from glycolysis during the IDC, no expression was detected for the subunits of the pyruvate dehydrogenase complex, including the α and β chains of pyruvate dehydrogenase and dihydrolipoamide S-acetyl transferase, the typical links between glycolysis and the TCA cycle. On the other hand, expression of TCA cycle genes is well synchronized with the expression of a large number of mitochondrial genes, including the three ORFs of the mitochondrial genome ( Feagin et al. 1991 ), and several factors of electron transport ( Table S2 ). Although some of the TCA cycle proteins have been localized to the cytoplasm ( Lang-Unnasch 1992 ), the expression data suggest an association of this biochemical process with mitochondrial development and possibly with the abbreviated electron transport pathway detected in this organelle. Schizont Stage A transition from early to mid-schizont is marked by the maximal induction of 29 ORFs predicted to encode various subunits of the proteasome ( Figure 2 I). Seven α and six β subunits of the 20S particle and 16 ORFs of the 19S regulatory particle were identified in this gene group. The common expression profile for the subunits of both of the 26S particle complexes suggests the involvement of ubiquitin-dependent protein degradation in the developmental progression of the parasite. The peak of proteasome expression coincides with a transition in the IDC transcriptome from metabolic and generic cellular machinery to specialized parasitic functions in the mid-schizont stage. This suggests an association between transcriptional regulation and protein turnover during this and possibly other transitions during the progression of the P. falciparum IDC. In the schizont stage, one of the first specialized processes induced was expression from the plastid genome ( Figure 2 J). The essential extrachromosomal plastid (or apicoplast) genome contains 60 potentially expressed sequences, including ribosomal proteins, RNA polymerase subunits, ribosomal RNAs, tRNAs, and nine putative ORFs, including a ClpC homologue ( Wilson et al. 1996 ). Very little is known about the regulation of gene expression in the plastid, but it is thought to be polycistronic ( Wilson et al. 1996 ). In support of this observation, we find that 27 of the 41 plastid-specific elements present on our microarray displayed an identical expression pattern ( Figure 3 C). The remaining elements correspond mainly to tRNAs and failed to detect appreciable signal. The highly coordinated expression of the plastid genome, whose gene products are maximally expressed in the late-schizont stage, is concomitant with the replicative stage of the plastid ( Williamson et al. 2002 ). Note that not all plastid ORFs are represented on the microarray used in this study, and thus it is a formal possibility that the expression of the missing genes may differ from those shown in Figure 3 C. Figure 3 Coregulation of Gene Expression along the Chromosomes of P. falciparum Is Rare, While Plastid Gene Expression Is Highly Coordinated Expression profiles for oligonucleotides are shown as a function of location for Chromosome 2 ([A], Oligo Map). With the exception of the SERA locus (B), coregulated clusters of adjacent ORFs are seldom observed, indicating that expression phase is largely independent of chromosomal position. (C) In contrast to the nuclear chromosomes, the polycistronic expression of the circular plastid genome is reflected in the tight coregulation of gene expression. This is an expanded view of the plastid-encoded genes from Figure 2 J. Genomic differences between strain 3D7, from which the complete genome was sequenced, and strain HB3 were measured by CGH. The relative hybridization between the gDNA derived from these two strains is shown as a percent reduction of the signal intensity for 3D7 ([A], CGH Data). Differences between the two strains are predominately located in the subtelomeric regions that contain the highly polymorphic var, rifin, and stevor gene families. Intrachromosomal variations, as observed for the msp2 gene, were rare. Offset from the plastid by approximately 6 h, a set of approximately 500 ORFs exhibited peak expression during the late-schizont stage. Merozoite invasion of a new host cell is a complex process during which the parasite must recognize and dock onto the surface of the target erythrocyte, reorient with its apical tip toward the host cell, and internalize itself through invagination of the erythrocytic plasma membrane. The entire sequence of invasion events is facilitated by multiple receptor–ligand interactions with highly specialized plasmodial antigens ( Cowman et al. 2000 ). The merozoite invasion group contains 58 ORFs, including 26 ORFs encoding antigens previously demonstrated to be important for the invasion process (see Figure 2 K). These include integral membrane proteins delivered to the merozoite surface from the micronemes (AMA1 and EBA175), GPI-anchored proteins of the merozoite membrane (MSP1, MSP4, and MSP5), proteins extrinsically associated with the merozoite surface during their maturation in the PV (MSP3 and MSP6), and soluble proteins secreted to the parasite–host cell interface (RAP1, RAP2, and RAP3). In addition, late-schizont-specific expression was observed for several antigens whose functions are not completely understood, but which have been associated with the invasion process. These ORFs include the merozoite-capping protein (MCP1), erythrocyte-binding-like protein 1 (EBL1), reticulocyte-binding proteins (RBP1 and RBP2), acid basic repeat antigen (ABRA), MSP7, and a homologue of the Plasmodium yoelii merozoite antigen 1. As expected, peak expression of these antigens coincides with the maturation of merozoites and development of several apical organelles, including rhoptries, micronemes, and dense granules. Many of these proteins have been considered as vaccine candidates since antibodies against these antigens were readily detected in the immune sera of both convalescent patients as well as individuals with naturally acquired immunity ( Preiser et al. 2000 ). The sensitivity of invasion to protease and kinase inhibitors indicates an essential role for these activities in merozoite release as well as in the reinvasion process ( Dluzewski and Garcia 1996 ; Blackman 2000 ; Greenbaum et al. 2002 ). The merozoite invasion gene group contains three serine proteases, including PfSUB1, PfSUB2, and an additional homologue to plasmodial subtilases (PFE0355c), and two aspartyl proteases, plasmepsin (PM) IX and X. Peak expression during the mid-schizont stage was also observed for seven members of the serine repeat antigen (SERA) family, all of which contain putative cysteine protease domains. In addition to the proteases, expression of 12 serine/threonine protein kinases and three phophorylases was tightly synchronized with the genes of the invasion pathway, including six homologues of protein kinase C, three Ca + -dependent and two cAMP-dependent kinases, phosphatases 2A and 2B, and protein phosphatase J. Another functionally related gene group whose expression is sharply induced during the late-schizont stage includes components of actin–myosin motors (see Figure 2 L) ( Pinder et al. 2000 ). As in other apicomplexa, actin and myosin have been implicated in host cell invasion ( Opitz and Soldati 2002 ). Schizont-specific expression was observed for three previously described class XIV myosin genes, one associated light chain, two actin homologues, and three additional actin cytoskeletal proteins, including actin-depolymerizing factor/cofilin (two isoforms) and coronin (one isoform). Although the molecular details of plasmodial actin–myosin invasion are not completely understood, the tight transcriptional coregulation of the identified factors indicates that the examination of schizont-specific expression may help to identify additional, possibly unique elements of this pathway. Early-Ring Stage The expression data are continuous throughout the invasion process, with no observable abrupt change in the expression program upon successful reinvasion. However, a set of approximately 300 ORFs whose expression is initiated in the late-schizont stage persists throughout the invasion process and peaks during the early-ring stages (see Figure 2 M). It was previously determined that immediately after invasion, a second round of exocytosis is triggered, ensuring successful establishment of the parasite within the host cell ( Foley et al. 1991 ). One of the main P. falciparum virulence factors associated with this process is ring-infected surface antigen 1 (RESA1). RESA1 is secreted into the host cell cytoplasm at the final stages of the invasion process, where it binds to erythrocytic spectrin, possibly via its DnaJ-like chaperone domain ( Foley et al. 1991 ). The early stages of the IDC contain a variety of putative molecular chaperones in addition to RESA1, including RESA2 and RESAH3, plus five additional proteins carrying DnaJ-like domains. However, the functional roles of these chaperones remain unclear. Despite the cytoplasmic role of RESA1, abundant antibodies specific for RESA1 are present in individuals infected with P. falciparum , indicating that RESA1 is also presented to the host immune system ( Troye-Blomberg et al. 1989 ). Several genes encoding additional antigenic factors are found among the early ring gene group, including frequently interspersed repeat antigen (FIRA), octapeptide antigen, MSP8, and sporozoite threonine- and asparagine-rich protein (STARP). Like RESA1, antibodies against these antigens are also found in the sera of infected individuals, suggesting that the final stages of invasion might be a target of the immune response. Overall, the genes expressed during the mid- to late-schizont and early-ring stage encode proteins predominantly involved in highly parasite-specific functions facilitating various steps of host cell invasion. The expression profiles of these genes are unique in the IDC because of the large amplitudes and narrow peak widths observed. The sharp induction of a number of parasite-specific functions implies that they are crucial for parasite survival in the mammalian host and hence should serve as excellent targets for both chemotherapeutic and vaccine-based antimalarial strategies. IDC Transcriptional Regulation and Chromosomal Structure Transcriptional regulation of chromosomal gene expression in P. falciparum is thought to be monocistronic, with transcriptional control of gene expression occurring through regulatory sequence elements upstream and downstream of the coding sequence ( Horrocks et al. 1998 ). This is in contrast to several other parasites, such as Leishmania sp. , in which polycistronic mRNA is synthesized from large arrays of coding sequences positioned unidirectionally along the arms of relatively short chromosomes ( Myler et al. 2001 ). Recent proteomic analyses failed to detect any continuous chromosomal regions with common stage-specific gene expression in several stages of the P. falciparum lifecycle ( Florens et al. 2002 ). However, transcriptional domains have previously been suggested for Chromosome 2 ( Le Roch et al. 2002 ). The availability of the complete P. falciparum genome coupled with the IDC transcriptome allows us to investigate the possibility of chromosomal clustering of gene expression (see Figure 3 A). To systematically explore the possibility of coregulated expression as a function of chromosomal location, we applied a Pearson correlation to identify similarities in expression profiles among adjacent ORFs. The pairwise Pearson correlation was calculated for every ORF pair within each chromosome ( Figure S2 ). Gene groups in which the correlation of 70% of the possible pairs was greater than r = 0.75 were classified as putative transcriptionally coregulated groups. Using these criteria, we identified only 14 coregulation groups consisting of greater than three genes, with the total number of genes being 60 (1.4% of all represented genes) ( Table S3 ). In eight of the 14 groups, the coregulation of a pair of genes may be explained by the fact that they are divergently transcribed from the same promoter. A set of 1,000 randomized permutations of the dataset yielded 2.25 gene groups. Contrary to the nuclear chromosomes, there was a high correlation of gene expression along the plastid DNA element, consistent with polycistronic transcription (see Figure 3 C). The average pairwise Pearson correlation for a sliding window of seven ORFs along the plastid genome is 0.92±0.03. The largest group demonstrating coregulation on the nuclear chromosomes corresponds to seven genes of the SERA family found on Chromosome 2 (see Figure 3 B) ( Miller et al. 2002 ). Besides the SERA gene cluster and a group containing three ribosomal protein genes, no additional functional relationship was found among the other chromosomally adjacent, transcriptionally coregulated gene groups. The limited grouping of regional chromosomal expression was independent of strand specificity and, with the exception of the SERA group, did not overlap with the groups of “recently duplicated genes” proposed by the malaria genome sequencing consortium ( Gardner et al. 2002 ). Three major surface antigens, the var , rifin , and stevor families, have a high degree of genomic variability and are highly polymorphic between strains and even within a single strain ( Cheng et al. 1998 ; Afonso Nogueira et al. 2002 ; Gardner et al. 2002 ). Expression profiles for only a small subset of these genes were detected in the IDC transcriptome and were typically characterized by low-amplitude profiles. This could be due to two nonmutually exclusive possibilities: first, the HB3 DNA sequence for these genes may be substantially rearranged or completely deleted relative to the reference strain, 3D7; second, only a few of these genes may be selectively expressed, as has been proposed ( Deitsch et al. 2001 ). To identify regions of genomic variability between 3D7 and HB3, we performed microarray-based comparative genomic hybridization (CGH) analysis. Array-based CGH has been performed with human cDNA and bacterial artificial chromosome-based microarrays to characterize DNA copy-number changes associated with tumorigenesis ( Gray and Collins 2000 ; Pollack et al. 2002 ). Using a similar protocol, CGH analysis revealed that the majority of genetic variation between HB3 and 3D7 is confined to the subtelomeric chromosomal regions containing the aforementioned gene families ( Figure 3 A; Figure S3 ). Only 28.3% of rifin , 47.1% of var , and 51.0% of stevor genes predicted for the 3D7 strain were detected for the HB3 genomic DNA (gDNA) when hybridized to the 3D7-based microarray. Thus, the underrepresentation of these gene families in the HB3 IDC transcriptome is likely due to the high degree of sequence variation present in these genes. Excluding the three surface antigen families in the subtelomeric regions, 97% of the remaining oligonucleotide microarray elements exhibit an equivalent signal in the CGH analysis. However, 144 of the differences detected by CGH reside in internal chromosomal regions and include several previously identified plasmodial antigens: MSP1, MSP2 ( Figure 3 A), S antigen, EBL1, cytoadherence-linked asexual gene 3.1 (CLAG3.1), glutamine-rich protein (GLURP), erythrocyte membrane protein 3 (PfEMP3), knob-associated histidine-rich protein (KAHRP), and gametocyte-specific antigen Pfg377 ( Table S4 ). These results demonstrate a high degree of genetic variation within the genes considered to be crucial for antigenic variation between these two commonly used laboratory strains of P. falciparum . Implications for Drug Discovery The majority of the nuclear-encoded proteins targeted to the plastid are of prokaryotic origin, making them excellent drug targets ( McFadden and Roos 1999 ). Moreover, inhibitors of plastid-associated isoprenoid biosynthesis, DNA replication, and translation have been shown to kill the P. falciparum parasite, demonstrating that the plastid is an essential organelle ( Fichera and Roos 1997 ; Jomaa et al. 1999 ). The plastid has been implicated in various metabolic functions, including fatty acid metabolism, heme biosynthesis, isoprenoid biosynthesis, and iron–sulfur cluster formation ( Wilson 2002 ). It is clear that, within the plastid, functional ribosomes are assembled to express the ORFs encoded by the plastid genome ( Roy et al. 1999 ). However, nuclear-encoded components are required to complete the translational machinery as well as for all other plastid metabolic functions. A bipartite signal sequence is required for efficient transport of these nuclear proteins from the cytoplasm to the plastid via the endoplasmic reticulum ( Waller et al. 2000 ). Computational predictions suggest that the P. falciparum genome may contain over 550 nuclear-encoded proteins with putative transit peptides ( Zuegge et al. 2001 ; Foth et al. 2003 ). Given that over 10% of the ORFs in the P. falciparum genome are predicted to contain an apicoplast-targeting sequence, we sought to use the IDC transcriptome as a means to narrow the search space for candidate apicoplast-targeted genes. As mentioned above, the expression profiles for genes encoded on the plastid genome are tightly coordinated (see Figure 3 C). We reasoned that genes targeted to the plastid would be expressed slightly before or coincidentally with the plastid genome. Therefore, we utilized the FFT phase information to identify ORFs in phase with expression of the plastid genome (see Materials and Methods ) ( Table S5 ). Because the genes of the plastid genome are maximally expressed between 33 and 36 hpi, we searched for all genes in the dataset with an FFT phase in this time window and then cross-referenced the list of predicted apicoplast-targeted sequences ( PlasmoDB.org ), resulting in a list of 124 in-phase apicoplast genes ( Figure 4 A). Within this list are two ORFs that have been directly visualized in the apicoplast, acyl carrier protein and the ribosomal subunit S9 ( Waller et al. 1998 ), as well as many ORFs associated with the putative plastid ribosomal machinery, enzymes involved in the nonmevalonate pathway, additional caseineolytic proteases (Clps), the reductant ferredoxin, and replication/transcriptional machinery components. However, this list contains only 14 of the 43 proteins categorized in the Gene Ontology (GO) assignments at PlasmoDB.org as apicoplast proteins by inference from direct assay (IDA). In addition, 30% of the nuclear-encoded translational genes that are not coexpressed with the known cytoplasmic machinery are found within this small group of genes. More importantly, 76 ORFs (62%) are of unknown function, with little or no homology to other genes. This limited subgroup of putative plastid-targeted ORFs are likely excellent candidates for further studies in the ongoing search for malaria-specific functions as putative drug targets. Figure 4 Temporal Distribution of the Apicoplast-Targeted Proteins and P. falciparum Proteases, Potential Antimalarial Drug Candidates (A) The expression profiles of all putative plastid-targeted genes represented on our microarray are shown. The yellow box encompasses a highly synchronized group of genes, which are in-phase with plastid genome expression. The average expression profile for this in-phase group of genes is shown and includes most of the known apicoplast-targeted genes as well as many hypothetical genes. For reference, the average expression profile for the plastid genome is shown (dashed gray line). (B) Proteases represent an attractive target for chemotherapeutic development. The broad range of temporal expression for various classes of proteases and their putative functions are displayed. Abbreviations: HAP, histo-aspartyl protease (PM III); Clp, caseineolytic protease; sub1, 2, subtilisin-like protease 1 and 2. Similarly, P. falciparum proteases have received much attention, since they are candidates as drug targets and have been shown to play important roles in regulation as well as metabolism throughout the IDC ( Rosenthal 2002 ). A temporal ordering of expression profiles for several well-characterized P. falciparum proteases is shown in Figure 4 B, demonstrating the broad significance of these enzymes throughout the IDC. One of the principal proteolytic functions is considered to be the degradation of host cell hemoglobin in the food vacuole (FV) to produce amino acids essential for protein synthesis. This elaborate process is carried out by a series of aspartyl proteases, cysteine proteases, metalloproteases, and aminopeptidases ( Francis et al. 1997 ). A family of ten aspartyl proteases, the plasmepsins (PMs), has been identified in the P. falciparum genome, four of which have been characterized as bona fide hemoglobinases: PM I, II, III (a histo-aspartic protease [HAP]), and IV ( Coombs et al. 2001 ). Our data reveal that the PMs are expressed at different times throughout the lifecycle, suggesting that they are involved in different processes throughout the IDC. PM I, II, HAP, and PM IV are adjacent to one another on Chromosome 14 and have been localized to the FV. While HAP and PM II are expressed in the mid-trophozoite stage, during peak hemoglobin catabolism, PMI and IV are maximally expressed in the ring stage along with the cysteine protease falcipain-1 (FP-1). FP-1 has recently been implicated in merozoite invasion and has been localized to the interior of the PV ( Greenbaum et al. 2002 ). The coincident expression of these proteases implies that the development of the PV and the FV occurs during the very early-ring stage. This observation is corroborated by similar expression profiles for the PV-associated protein RESA1 and the FV protein PGH1. Subsequently, a second group of hemoglobinases, including the m1-family aminopeptidase, FP-2, and falcilysin, is expressed simultaneously with HAP and PM II during the trophozoite stage of the IDC. The expression of PM V and the newly identified FP- 2 homologue during this stage suggests they are also important in the trophozoite stage. The other known falcipain, FP-3, does not show a marked induction in expression throughout the IDC. We fail to detect any transcripts for PM VI, VII, and VIII during the IDC. These genes may have roles in any of the other sexual, liver, or mosquito stages of development. In addition to the hemoglobinases, P. falciparum contains a variety of proteases involved in cellular processing, including a group of Clps and signal peptidases that are all expressed maximally at the late-trophozoite stage ( Figure 4 B). The timing of these genes may play a key role in protein maturation during trafficking to various compartments, including the plastid. The three Clps contain putative leader peptides and may actually function within the plastid. Finally, a group of proteases are expressed in the schizont stage and include the P. falciparum subtilisin-like proteases PfSUB1 and PfSUB2 as well as PMs IX and X. PfSUB1 and PfSUB2 are believed to be involved in merozoite invasion and have been localized apically in the dense granules. Interestingly, there are two PfSUB1 protease homologues (PFE0355c and PFE0370c); PM X parallels the expression of PfSUB1 (PFE0370c), suggesting that aspartyl proteases may also be involved in merozoite invasion. In addition, the phase of the PfSUB1 homologue suggests a concomitant role, with PM IX slightly preceding merozoite invasion. In total, we have detected gene expression for over 80 putative proteases throughout the entire IDC ( Table S6 ). This set includes over 65 proteases from a group of recently predicted proteases ( Wu et al. 2003 ). The differing temporal expression of these proteases may allow for a multifaceted approach toward identifying protease inhibitors with efficacy at all stages of the IDC. Implications for New Vaccine Therapies Merozoite invasion is one of the most promising target areas for antimalarial vaccine development ( Good 2001 ). Many vaccine efforts thus far have focused primarily on a set of plasmodial antigens that facilitate receptor–ligand interaction between the parasite and the host cell during the invasion process ( Preiser et al. 2000 ) (see Figure 2 K and 2M). Merozoite invasion antigens are contributing factors to naturally acquired immunity, triggering both humoral and antibody-independent cell-mediated responses ( Good and Doolan 1999 ). Antibodies against these antigens have been demonstrated to effectively block the merozoite invasion process in vitro and in animal models ( Ramasamy et al. 2001 ). Owing to the highly unique character of merozoite surface antigens, homology-based searches have yielded only a limited set of additional invasion factors. We utilized the IDC transcriptome to predict a set of likely invasion proteins by identifying expression profiles with characteristics similar to previously studied merozoite invasion proteins. The expression profiles for all known invasion factors undergo a sharp induction during the mid- to late-schizont stage and are characterized by large expression amplitudes (see Figure 2 A). Among these proteins are seven of the best-known malaria vaccine candidates, including AMA1, MSP1, MSP3, MSP5, EBA175, RAP1, and RESA1. To identify ORFs with a possible involvement in the merozoite invasion process, we have calculated the similarity, by Euclidian distance, between the expression profiles of these seven vaccine candidates and the rest of the IDC transcriptome. A histogram of the distance values reveals a bimodal distribution with 262 ORFs in the first peak of the distribution ( Figure S4 ). This represents the top 5% of expression profiles when ranked by increasing Euclidian distance ( Table S7 ). In addition to the seven vaccine candidate genes used for the search, essentially all predicted P. falciparum merozoite-associated antigens were identified in this gene set ( Figure 5 ). These include the GPI-anchored MSP4; several integral merozoite membrane proteins, such as EBA140 and EBL1; three RBPs (RBP1, RBP2a, RBP2b); and a previously unknown RBP homologue. In addition, components of two proteins secreted from the rhoptries to the host cell membranes, RhopH1 and RhopH3, or to the PVs RAP1, RAP2, and RAP3 were found in the selected set. Surprisingly, CLAG2 and CLAG9 were also classified into the merozoite invasion group. Although the biological function of these genes is believed to be associated with cytoadherence of the infected erythrocyte to the vascular endothelium, a highly related homologue, CLAG3.1 (RhopH1), was recently detected in the rhoptries, suggesting a possible secondary role for these genes in merozoites ( Kaneko et al. 2001 ). Figure 5 Phaseogram of Putative Vaccine Targets The similarity of all expression profiles to seven known vaccine candidates (boxed) was calculated. The top 5% of similar profiles correspond to 262 ORFs, 28 of which have been previously associated with plasmodial antigenicity and the process of merozoite invasion. A number of antigens are presently in various stages of clinical trials and are yielding encouraging results ( Good et al. 1998 ). However, many single-antigen vaccine studies indicate that the most promising approach will require a combination of antigenic determinants from multiple stages of the complex plasmodial lifecycle ( Kumar et al. 2002 ). Searches for new target antigens in the P. falciparum genome are thus vital to the development of future vaccines, since no fully protective vaccine has been assembled thus far. Of the 262 ORFs whose expression profiles were closest to the profiles of the seven major vaccine candidates, 189 are of unknown function. These ORFs represent a candidate list for new vaccine targets. Discussion The transcriptome of the IDC of P. falciparum constitutes an essential tool and baseline foundation for the analysis of all future gene expression studies in this organism, including response to drugs, growth conditions, environmental perturbations, and genetic alterations. Essentially all experiments involving asexual intraerythrocytic-stage parasites must be interpreted within the context of the ongoing cascade of IDC-regulated genes. In our global analysis of the P. falciparum transcriptome, over 80% of the ORFs revealed changes in transcript abundance during the maturation of the parasite within RBCs. The P. falciparum IDC significantly differs from the cell cycles of the yeast S. cerevisiae ( Spellman et al. 1998 ) and human HeLa ( Whitfield et al. 2002 ) cells, during which only 15% of the total genome is periodically regulated. Instead, the P. falciparum IDC resembles the transcriptome of the early stages of Drosophila melanogaster development, which incorporates the expression of over 80% of its genome as well ( Arbeitman et al. 2002 ). Unlike the development of multicellular eukaryotes, there is no terminal differentiation and, with the exception of gametocytogenesis, the parasite is locked into a repeating cycle. In this respect, the P. falciparum IDC mirrors a viral-like lifecycle, in which a relatively rigid program of transcriptional regulation governs the progress of the course of infection. The lack of continuous chromosomal domains with common expression characteristics suggests that the genes are regulated individually, presumably via distinct sets of cis - and trans -acting elements. However, the extent and the simple mechanical character of transcriptional control observed in the IDC suggest a fundamentally different mode of regulation than what has been observed in other eukaryotes. It is plausible that a comparatively small number of transcription factors with overlapping binding site specificities could account for the entire cascade. While further experiments are ongoing, it may be the case that P. falciparum gene regulation is streamlined to the extent that it has lost the degree of dynamic flexibility observed in other unicellular organisms, from Escherichia coli to yeast. This observation also implies that disruption of a key transcriptional regulator, as opposed to a metabolic process, may have profound inhibitory properties. While a few putative transcription factors have been identified in the P. falciparum genome, no specific regulatory elements have been defined in basepair-level detail. A further analysis of the upstream regions of genes with similar phases should facilitate the elucidation of regulatory regions and their corresponding regulatory proteins. In general, the timing of mRNA expression for a given gene during the IDC correlates well with the function of the resultant protein. For example, replication of the genome occurs in the early-schizont stage and correlates well with the peak expression of all factors of DNA replication and DNA synthesis. Also, organellar biogenesis of several intracellular compartments such as mitochondria, the plastid, or the apical invasion organelles is concomitant with the maximal induction of mRNAs encoding proteins specific to these organelles. In addition, our data are generally in good agreement with proteomic analyses that have detected intraerythrocytic-stage proteins from the merozoite, trophozoite, and schizont stages. More than 85% of the 1,588 proteins detected in these studies were also expressed in our analysis ( Florens et al. 2002 ; Lasonder et al. 2002 ). However, a more detailed proteomic analysis at different stages of the IDC will be needed to ascertain the temporal changes of these proteins. We initially expected that a high percentage of the genome would be specialized for each lifecycle stage (mosquito, liver, blood), yet this was not observed; the mRNA transcripts for 75% of proteins determined to be gamete-, gametocyte-, or sporozoite-specific by mass spectrometry are also transcribed in the plasmodial IDC. These findings confirm previous studies demonstrating that not only genes used for generic cellular processes are present in multiple developmental stages, but also factors of highly specialized Plasmodium functions ( Gruner et al. 2001 ). This may indicate that only a small portion of the genome may actually be truly specific to a particular developmental stage and that the majority of the genome is utilized throughout the full lifecycle of this parasite. It is also feasible to speculate that a multilayer regulatory network is employed in the progression of the entire P. falciparum lifecycle. In this model, the same cis - and trans -acting regulatory elements driving the actual mRNA production in IDC are utilized in other developmental stages. These elements are then controlled by an alternate subset of factors determining the status of the lifecycle progression. These findings also outline two contrasting properties of the P. falciparum genome. The Plasmodium parasite devotes 3.9% of its genome to a complex system of antigenic determinants essential for host immune evasion during a single developmental stage ( Gardner et al. 2002 ). On the other hand, large portions of the genome encode proteins used in multiple stages of the entire lifecycle. Such broad-scope proteins might be excellent targets for both vaccine and chemotherapeutic antimalarial strategies, since they would target several developmental stages simultaneously. While there are certainly proteins specific to these nonerythrocytic stages, a complementary analysis of both proteomic and genomic datasets will facilitate the search. With malaria continuing to be a major worldwide disease, advances toward understanding the basic biology of P. falciparum remain essential. Our analysis of the IDC transcriptome provides a first step toward a comprehensive functional analysis of the genome of P. falciparum . The genome-wide transcriptome will be useful not only for the further annotation of many uncharacterized genes, but also for defining the biological processes utilized by this highly specialized parasitic organism. Importantly, candidate groups of genes can be identified that are both functionally and transcriptionally related and thus provide focused starting points for the further elucidation of genetic and mechanistic aspects of P. falciparum . Such biological characterizations are presently a major objective in the search for novel antimalarial strategies. The public availability of the dataset presented in this study is intended to provide a resource for the entire research community to extend the exploration of P. falciparum beyond the scope of this publication. All data will be freely accessible at two sites: http://plasmodb.org and http://malaria.ucsf.edu . Materials and Methods Cell culture. A large-scale culture of P. falciparum (HB3 strain) was grown in a standard 4.5 l microbial bioreactor (Aplikon, Brauwweg, Netherlands) equipped with a Bio Controller unit ADI 1030 (Aplikon, Brauwweg, Netherlands). Cells were initially grown in a 2% suspension of purified human RBCs and RPMI 1640 media supplemented with 0.25% Albumax II (GIBCO, Life Technologies, San Diego, California, United States), 2 g/l sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES (pH 7.4), and 50 μg/l gentamycin, at 37°C, 5% O 2 , and 6% CO 2 . Cells were synchronized by two consecutive sorbitol treatments for three generations, for a total of six treatments. Large-scale cultures contained 32.5 mM HEPES (pH 7.4). The bioreactor culture was initiated by mixing 25.0 ml of parasitized RBCs (20% late schizonts, approximately 45 hpi) with an additional 115.0 ml of purified RBC in a total of 1.0 l of media (14% hematocrit). Invasion of fresh RBCs occurred during the next 2 h, raising the total parasitemia from an initial 5% to 16%. After this period, the volume of the culture was adjusted to 4.5 l, bringing the final RBC concentration to approximately 3.3% to reduce the invasion of remaining cells. Immediately after the invasion period, greater than 80% of the parasites were in the ring stage. Temperature and gas conditions were managed by the Bio Controller unit. Over the course of 48 h, 3–4 ml of parasitized RBCs was collected every hour, washed with prewarmed PBS, and flash-frozen in liquid nitrogen. RNA preparation and reference pool. P. falciparum RNA sample isolation, cDNA synthesis, labeling, and DNA microarray hybridizations were performed as described by Bozdech et al. (2003 ). Samples for individual timepoints (coupled to Cy5) were hybridized against a reference pool (coupled to Cy3). The reference pool was comprised of RNA samples representing all developmental stages of the parasite. From this pool, sufficient cDNA synthesis reactions, using 12 μg of pooled reference RNA, were performed for all hybridizations. After completing cDNA synthesis, all reference pool cDNAs were combined into one large pool and then split into individual aliquots for subsequent labeling and hybridization. Microarray hybridizations were incubated for 14–18 h. DNA microarray hybridizations and quality control. In total, 55 DNA microarray hybridizations covering 46 timepoints were performed. Timepoints 1, 7, 11, 14, 18, 20, 27, and 31 were represented by more than one array hybridization. Data were acquired and analyzed by GenePix Pro 3 (Axon Instruments, Union City, California, United States). Array data were stored and normalized using the NOMAD microarray database system ( http://ucsf-nomad.sourceforge.net/ ). In brief, a scalar normalization factor was calculated for each array using unflagged features with median intensities greater than zero for each channel and a pixel regression correlation coefficient greater than or equal to 0.75. Quality spots were retained based on the following criteria. The log 2 (Cy5/Cy3) ratio for array features that were unflagged and had a sum of median intensities greater than the local background plus two times the standard deviation of the background were extracted from the database for further analysis. Subsequently, expression profiles consisting of 43 of 46 timepoints (approximately 95%) were selected. For those timepoints that were represented by multiple arrays, the ratio values were averaged. FFT analysis of the expression profiles. Fourier analysis was performed on each profile in the quality-controlled set (5,081 oligonucleotides). Profiles were smoothed with missing values imputed using a locally weighted regression algorithm with local weighting restricted to 12% using R ( http://www.R-project.org ). Fourier analysis was performed on each profile using the fft() function of R, padded with zeros to 64 measurements. The power spectrum was calculated using the spectrum() function of R. The power at each frequency ( Power() ), the total power (P tot ), and the frequency of maximum power (F max ) were determined. The periodicity score was defined as Power [(F max−1 ) + (F max ) + (F max+1 )]/P tot . The most frequent value of F max across all profiles was deemed the major frequency (m) and used in determining phase information. The phase of each profile was calculated as atan2\[−(I (m)],R (m)\, where atan2 is R's arctangent function and I and R are the imaginary and real parts of the FFT. Profiles were then ordered in increasing phase from −π to π. The loess smooth profiles were drawn through the raw expression data using the loess() function found in the modern regression library of R (version 1.5.1). The default parameters were used, with the exception that local weighting was reduced to 30%. For the averaged profiles of the functional groups (see Figure 2 B–2M), the loess smooth profiles were calculated for each expression profile individually and subsequently averaged to create the representative profile. These same methods were applied to both the randomized set (see the inset to Figure 1 F) and the yeast cell cycle dataset (see Figure S1 ). The raw results files ( Dataset S1 ), the fully assembled raw dataset ( Dataset S2 , the overview dataset ( Dataset S3 , and the quality control dataset ( Dataset S4 ) are available as downloads. Evaluation of coexpression along chromosomes. The evaluation of coexpression of genes along chromosomes was carried out as follows. The Pearson correlation coefficient was calculated for each pair of profiles. For ORFs with multiple oligonucleotides, the average profile was calculated. The neighborhood of each ORF profile was defined as a window of between one and ten adjacent ORF profiles. If any window in an ORF profile's neighborhood displayed more than 70% pairwise correlation of greater than 0.75, it was flagged as enriched. The length of the window was then recorded as a region of coexpression. This process was repeated without strand separation of ORFs and with randomly permuted datasets. Comparative genomic hybridization. P. falciparum strains 3D7 and HB3 were cultured as previously described at a concentration of 10% parasitaemia. Genomic DNA (gDNA) was isolated from a minimum of 500 ml of total culture for each P. falciparum strain, as previously described ( Wang et al. 2002 ). Isolated gDNA from each strain was sheared by sonication to an average fragment size of approximately 1–1.5 kb and then was purified and concentrated using a DNA Clean and Concentrator kit (Zymo Research, Orange, California, United States). Amino-allyl-dUTP first was incorporated into the gDNA fragments with a Klenow reaction at 37°C for 6–8 h with random nonamer primers and 3 μg of sheared gDNA. After purification and concentration of the DNA from the Klenow reaction, CyScribe Cy3 and Cy5 dyes (Amersham Biosciences, Buckinghamshire, United Kingdom) were coupled to HB3 DNA and 3D7 DNA, respectively, as previously described ( Pollack et al. 1999 ). Uncoupled fluorescent dye was removed using a DNA Clean and Concentrator kit. Labeled DNA fragments were hybridized to the oligonucleotide-based DNA microarrays. Fluorescence was detected and analyzed using an Axon Instruments scanner and GenePix Pro 3.0 software. Only features that had median intensities greater than the local background plus two times the standard deviation of the background in each channel were considered for further analysis. For each feature, the percent of the total intensity was determined using the signal in the 3D7 channel as the total amount of intensity for each oligonucleotide; intensity differences less than 50% were considered to be significant for subsequence analysis. Calculation for in-phase plastid-targeted genes. The range of FFT-based phases for the expression profiles of the plastid genome is between 0.32 and 1.05 (or roughly π/9 −π/3). Using the list of 551 apicoplast-targeted genes available at PlasmoDB.org , we first ordered these genes by phase and then grouped all genes with a phase range between 0.00 and 1.40 (0–4π/9), resulting in 124 genes represented by 128 oligonucleotides on the microarray. This select group represents the in-phase plastid targeted genes (see Table S6 ). Calculation for vaccine targets. To select the expression profiles most related to the AMA1, MSP1, MSP3, MSP5, EBA175, RAP1, and RESA1 vaccine candidates, we calculated the similarity of all expression profiles in the dataset to those of these antigens by Euclidian distance. The minimum Euclidian distance calculated for every profile was then binned into 60 bins and plotted as a histogram. A natural break in the histogram was seen that included the set of 262 ORFs (see Figure S2 ). Supporting Information Dataset S1 Raw GenePix Results (29.5 MB ZIP). Click here for additional data file. Dataset S2 Complete Dataset (3.7 MB TXT). Click here for additional data file. Dataset S3 Overview Dataset (2.4 MB TXT). Click here for additional data file. Dataset S4 Quality Control Set (3.1 MB TXT). Click here for additional data file. Figure S1 Histogram of the Percent Power at Peak Frequencies for the Yeast Cell Cycle Data The percent of power in the maximum frequency of the FFT power spectrum was used to determine periodicity of the yeast cell cycle data from Spellman et al. (1998 ). The histogram reveals periodic regulation of gene expression for only a small subset of genes (% power >70%). (223 KB EPS). Click here for additional data file. Figure S2 Pearson Correlation Maps for the P. falciparum Chromosomes A matrix of the pairwise Pearson correlations was calculated for every expression profile along the chromosomes. The analysis included all annotated ORFs. The gray areas correspond to a Pearson correlation d(x, y) = 0 and indicate ORFs with no detectable IDC expression or ORFs not represented on the microarray. The starting point (left) and the end point (right) of the chromosomes and the ORF order along the chromosomes are identical to the order in PlasmoDB.org . (30.9 MB EPS). Click here for additional data file. Figure S3 CGH of 3D7 versus HB3 for All Chromosomes Genomic differences between strain 3D7 and strain HB3 were measured by CGH. The relative hybridization between the gDNA derived from these two strains is shown as a percent reduction of the signal intensity for 3D7 along individual chromosomes. (1.7 MB ZIP). Click here for additional data file. (A) (216 KB EPS) Click here for additional data file. (B) (232 KB EPS) Click here for additional data file. (C) (237 KB EPS) Click here for additional data file. (D) (240 KB EPS) Click here for additional data file. (E) (252 KB EPS) Click here for additional data file. (F) (232 KB EPS) Click here for additional data file. (G) (235 KB EPS) Click here for additional data file. (H) (235 KB EPS) Click here for additional data file. (I) (249 KB EPS) Click here for additional data file. (J) (265 KB EPS) Click here for additional data file. (K) (283 KB EPS) Click here for additional data file. (L) (270 KB EPS) Click here for additional data file. (M) (305 KB EPS) Click here for additional data file. (N) (332 KB EPS) Click here for additional data file. Figure S4 Distribution of Euclidian Distances between Expression Profiles of the IDC Genes and Seven Vaccine Candidates The similarity between each IDC expression profile and the profiles of the seven selected vaccine candidate genes was evaluated by Euclidian distance calculations, d(x,y) = Σ(x i − y i ) 2 . The Euclidian distance value to the closest vaccine homologue was selected for each IDC profile and used to generate this plot. Genes with d(x,y) < 20 were selected for the phaseogram of putative vaccine targets (see Figure 5 ). (494.02 KB EPS). Click here for additional data file. Table S1 Pearson Correlation for ORFs Represented by Multiple Oligonucleotides This table contains all of the ORFs in the analyzed dataset that are represented by multiple oligonucleotides on the DNA microarray. The average Pearson correlation value has been calculated for the expression profiles of all oligonucleotides for each given ORF. (44 KB TXT). Click here for additional data file. Table S2 P. falciparum Functional Gene Groups This table contains all of the P. falciparum groups discussed. The groups include the following: transcription machinery, cytoplasmic translation machinery, the glycolytic pathway, ribonucleotide synthesis, deoxyribonucleotide synthesis, DNA replication machinery, the TCA cycle, the proteaseome, the plastid genome, merozoite invasion, actin–myosin motility, early-ring transcripts, mitochondrial genes, and the organellar translational group. (291 KB TXT). Click here for additional data file. Table S3 Coregulation along the Chromosomes of P. falciparum This table contains the regions of coregulation found in the chromosomes of P. falciparum determined by calculating the Pearson correlation between expression profiles for contiguous ORFs. The cutoff was 70% pairwise correlation of greater than 0.75 for each group. Only groups of two ORFs or more are listed. (6 KB TXT). Click here for additional data file. Table S4 3D7 versus HB3 CGH Data This table contains all of the intensity data from CGH of gDNA derived from the 3D7 and HB3 strains of P. falciparum . The averaged intensities from three microarray hybridization experiments are listed. (414 KB TXT). Click here for additional data file. Table S5 Putative Apicoplast-Targeted Genes and Expression Profiles This table contains all of the predicted apicoplast-targeted ORFs from PlasmoDB.org . The presence of each ORF on the DNA microarray is tabulated, as well as whether each ORF is present in the overview set. Finally, the plastid ORFs in-phase with plastid genome expression are listed, as well as the corresponding oligonucleotide identifiers. (147 KB TXT). Click here for additional data file. Table S6 Putative P. falciparum Proteases and Their Expression Data The table was constructed by searching the database for any putative protease annotations and contains all of the 92 proteases identified by Wu et al. (2003 ). (59 KB TXT). Click here for additional data file. Table S7 Vaccine Candidate Correlation Table The similarity of all expression profiles to seven known vaccine candidates was evaluated by a Euclidian distance calculation to all expression profiles measured. These 262 ORFs constitute the top 5% of genes in the IDC with minimum distance to these seven ORFs. The seven candidates used are AMA1, MSP1, MSP3, MSP5, EBA175, RAP1, and RESA1. (204 KB TXT). Click here for additional data file.

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544933

Introducing "Frontiers in Zoology"

As a biological discipline, zoology has one of the longest histories. Today it occasionally appears as though, due to the rapid expansion of life sciences, zoology has been replaced by more or less independent sub-disciplines amongst which exchange is often sparse. However, the recent advance of molecular methodology into "classical" fields of biology, and the development of theories that can explain phenomena on different levels of organisation, has led to a re-integration of zoological disciplines promoting a broader than usual approach to zoological questions. Zoology has re-emerged as an integrative discipline encompassing the most diverse aspects of animal life, from the level of the gene to the level of the ecosystem. The new journal Frontiers in Zoology is the first Open Access journal focussing on zoology as a whole. It aims to represent and re-unite the various disciplines that look at animal life from different perspectives and at providing the basis for a comprehensive understanding of zoological phenomena on all levels of analysis. Frontiers in Zoology provides a unique opportunity to publish high quality research and reviews on zoological issues that will be internationally accessible to any reader at no cost.

The revival of integrative zoology Zoology has a long history of more than 2000 years and is one of the natural sciences attracting most public attention. Nevertheless, as a scientific field, zoology occasionally is considered to be old-fashioned and in danger of being replaced by narrower sub-disciplines that focus on a few important and in vogue aspects of animal biology. The remarkable success of such specialized approaches has in the last decades promoted the breakdown of zoology into a number of autonomous research areas, some focussing on molecules and cells and others on whole organisms or ecosystems, each with its own specialized journals and with little exchange of concepts and empirical information between them. The advance of molecular methodology into "classical" fields of biology and the development of theories that have the power to explain phenomena on different levels of organisation urge for the re-integration of zoological disciplines and at the same time highlight the advantages of a broader approach in the study of proximate and ultimate aspects of animal life. Zoology thus fulfils any requirements as an integrative discipline encompassing all aspects of animal life, from the level of the gene to the level of the ecosystem. We feel that this renaissance of integrative zoology as a modern and vigorous field of research needs to be reflected in an up-to-date journal dedicated to zoology as a whole. We also believe that the best way to broadly disseminate the results of modern zoological science is provided by an Open Access, online journal. We therefore have launched Frontiers in Zoology , the only Open Access, online journal focussing on zoology. Research at the frontiers in zoology Frontiers in Zoology aims at re-uniting the various sub-disciplines that investigate animal life from different angles and providing the basis for a more complete understanding of animal life. Initiated and supported by the Deutsche Zoologische Gesellschaft, one of the largest national societies devoted to zoology as a whole, and based on an editorial board ( ) with renowned experts from different research areas, Frontiers in Zoology provides a novel opportunity to publish high quality articles and reviews on zoological phenomena with all the advantages provided by Open Access. The advantages of Open Access As Slade et al. [ 1 ] have summarized, Open Access policy generally changes the way in which articles are published. First, all articles become freely and universally accessible online, and so an author's work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced. Third, a copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal. Articles in Frontiers in Zoology are archived in PubMed Central, the US National Library of Medicine's full-text repository of life science literature, and also in repositories at the University of Potsdam in Germany, at INIST in France and in e-Depot, the National Library of the Netherlands' digital archive of all electronic publications. Open Access has four broad benefits for science and the general public. First, authors are assured that their work is disseminated to the widest possible audience, given that there are no barriers to access their work. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. It has been suggested that free online articles are more highly cited because of their easier availability [ 2 ]. Second, the information available for researchers will not be limited by their library's budget, and the widespread availability of articles will enhance literature searching [ 3 ]. Third, the results of publicly funded research will be accessible to all taxpayers and not just those with access to a library with a subscription. Note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law [ 4 ]. Fourth, a country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same materials as wealthier ones (although creating access to the internet is another matter [ 5 ]). The peer review policy of Frontiers in Zoology Manuscripts with all figures and tables shall be submitted online using any of the listed formats ( ). The Editors-in-Chief or one of the Specialist Editors will forward the manuscript to two or three referees for peer-review. Upon receiving the reports from the referees, the respective Editor may suggest rejection or acceptance without, with major or with minor changes to the Editors-in-Chief, who will finalise the decision based upon the reports and the editor's opinion. Articles will be published online immediately upon acceptance and soon after listed in PubMed. Decisions about a manuscript will be exclusively based on the quality of the work, not on whether the authors can pay the article-processing charge. Note that processing charges will be waived for all authors whose institution is a BioMed Central member. Furthermore, editors can grant discretionary waivers for 35% of articles submitted in any given period of 90 days. Conclusion Frontiers in Zoology provides an optimal way for the broad and fast dissemination of outstanding research from all areas of Zoology. We therefore invite all researchers in zoology to experience the manifold advantages provided by Open Access, online journals and to submit their high quality manuscripts to this new journal.

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529261

Active collaboration with primary care providers increases specialist referral in chronic renal disease

Background Late referral to specialist nephrological care is associated with increased morbidity, mortality, and cost. Consequently, nephrologists' associations recommend early referral. The recommendations' effectiveness remains questionable: 22–51% of referrals need renal replacement therapy (RRT) within 3–4 months. This may be due to these recommendations addressing the specialist, rather than the primary care providers (PCP). The potential of specialist intervention aiming at slowing progression of chronic renal failure was introduced individually to some 250 local PCPs, and referral strategies were discussed. To overcome the PCPs' most often expressed fears, every referred patient was asked to report back to his PCP immediately after the initial specialist examination, and new medications were prescribed directly, and thus allotted to the nephrologist's budget. Methods In retrospective analysis, the stage of renal disease in patients referred within three months before the introductory round (group A, n = 18), was compared to referrals two years later (group B, n = 50). Results Relative number of patients remained stable (28%) for mild/ moderate chronic kidney disease (MMCKD), while there was a noticeable shift from patients referred severe chronic kidney disease (SCKD) (group A: 44%, group B: 20%) to patients referred in moderate chronic kidney disease (MCKD) (group A: 28%, group B: 52%). Conclusion Individually addressing PCPs' ignorance and concerns noticeably decreased late referral. This stresses the importance of enhancing the PCPs' problem awareness and knowledge of available resources in order to ensure timely specialist referral.

Background Late referral to specialist care for renal failure is associated with increased morbidity, mortality, and cost (review in[ 1 , 2 ]). Consequently, nephrologists' associations recommend early referral[ 3 , 4 ]. The recently published ERA/ EDTA guideline states: "Referral to nephrology should be considered when the GFR is <60 ml/min and is mandatory when the GFR is <30 ml/min"[ 3 ]. The recommendations' effectiveness remains questionable: 22–51% of patients need renal replacement therapy (RRT) within 3–4 months [ 5 , 6 ] after first referral to specialist nephrological care. Chronic renal disease may be asymptomatic for a very long time. This stresses the importance of primary care providers in ensuring timely referral to a renal care center. Levin[ 1 ] reviewed aspects of the referral process and noted that the consequences of late referral were usually and most specifically described in specialist nephrological journals, thus not reaching the necessary target audience, the primary care providers (PCPs). The problem of late referral seems to be ubiquitous, if discussions with colleagues world- wide may be believed, and has led to numerous local, regional and national initiatives aiming at ensuring timely referral into specialist care. Some initiatives[ 7 , 8 ] successfully bypass the PCPs by introducing population screening programs, while others opt for the alternative of "managed care"[ 9 ]. This article summarizes findings from a local initiative started by one of the authors (K.H.) in 1997 in the city of Dortmund, Germany. In Germany, about 90% of the population are covered by the mandatory health insurance system ("Gesetzliche Krankenversicherung"). All physicians who wish to treat these patients are organized in associations, which negotiate a budget with the insurance companies. Depending on the specialty, a typical per capita budget is then assigned per patient for a period of three months. This budget covers consultation fees, and the cost of medication prescribed. If a physicians exceeds the total budget thus calculated for his practice, the insurance companies can demand restoration of funds. Typically, a GP's per patient budget would be much lower than a nephrologist's. During the time period described here, the nephrologist's budget per patient was about ten times as much than a general practitioner's. While this budget strategy is intended as a safeguard against excess prescriptions, it has been criticized by many physicians for inducing sub- optimal treatment as practice owners comply rather with budget demands than with best practice guidelines. Dortmund, a town of 589000, has a physician to patient ratio of 141/ 100000 (German average: 156/ 100000). There are five nephrological centers, including two hospitals. Currently, 172 general practioners ("Allgemeinärzte und Praktische Ärzte"), 123 internists ("Ärzte für Innere Medizin") and 21 urologists are listed in Dortmund. One of the internist practices supervises a dialysis center, but does not provide specialized nephrological consultancy. GPs, internists and urologists were considered primary care providers, as all referrals for treatment of chronic renal failures came from one of these specialties. There were no major fluctuations of physician numbers between 1997 and 1999, the period for which data was analyzed here. Joint initiatives by all five nephrology centres in Dortmund to give PCPs a series of lectures on treatment of chronic renal disease met with little response, as the presenters regularly outnumbered the audience. In the spirit of the Declaration of Helsinki, part II.1.: ("In the treatment of the sick persons, the doctor must be free to use a new therapeutic measure, if in the doctor's judgment it offers hope of saving life, reestablishing health, or alleviating suffering")[ 10 ], K.H. decided to take active measures to induce PCPs to refer patients at an earlier stage. In order to do so, education was brought to the primary care providers, as opposed to bringing the PCPs to education. Over a period of 18 months, K.H. introduced himself and the potential of timely nephrological care in delaying or even halting the progression of chronic renal disease to PCPs- General Practitioners, and specialists in Internal Medicine and Urology. This introductory round included participation in local PCPs' round tables and PCP organized continuous medical education events, but also individually meeting some 250 PCPs for discussion. During these sessions, the topic of renal disease was subtly introduced during discussions on the more common diseases and risk factors, such as cardiovascular disease, hypertension and diabetes mellitus. During these teaching sessions and discussions, three arguments were repeatedly brought forward by the PCPs: 1. Mild- moderate renal failure should be treated by the primary care providers, while the nephrologists' task was seen in providing renal replacement therapy. 2. Referral to specialist care meant risking the loss of the patient to the specialist's practice (In Germany, direct specialist access is possible, and patients tend to stay with one doctor if satisfied). 3. If patients did return from specialist consultation, they were usually carrying recommendations for costly permanent medication, such as ACE inhibitors, that put a heavy burden on the PCP's budget. The first of these arguments was addressed by the "teaching module" of the visits. PCPs were given an executive summary of the complexities of diagnostic and therapeutic procedures used in state- of- the- art nephrology, and the potential of intensified treatment by specialists aiming at slowing the progression of renal failure was explained in detail. Secondly, all patients referred were asked to report back to the referring PCP within one week of the consultation, by which time a summary of findings and appropriate counsel had been sent ahead. Thirdly, instead of drug recommendations , drug prescriptions were handed out directly and so allotted to the much higher nephrologist's budget, thus easing the financial pressure on the primary care providers. More importantly, also the follow- up prescriptions were done in the renal care center, so that at no time the PCPs budget became endangered. The present study investigates the question whether improving the PCP's knowledge about the potential of timely treatment of chronic renal failure, in combination with addressing their economic concerns, succeeded in encouraging timely referral to specialist care. Methods Retrospective analysis of patients' records. Analysis of patient records was carried out by a final year medical student (TDV). All patients had agreed to have their data used for quality control measures at the time of referral. Specifically informed consent was obtained from all patients still alive at the time of data collection. Two groups were selected by date of first contact with the nephrologist: Group A, third quarter 1997, immediately before the start of the initiative, and Group B, third quarter 1999, six months after the last visit to a primary care provider had taken place. All new patients whose records indicated referral for nephrological specialist treatment were included in the study. Criteria were subnormal creatinine clearance (ECC) or elevated serum creatinine, elevated blood pressure, proteinuria, or erythrocyturia. The patients in each of the two groups were divided into three subgroups according to their renal function: mild/moderate chronic kidney disease (MMCKD), ECC > 60 ml/min/1.73 m 2 , moderate chronic kidney disease (MCKD), 60 ml/min/1.73 m 2 > ECC > 20 ml/min/1.73 m 2 , and severe chronic renal disease (SCKD), ECC < 20 ml/min/1.73 m 2 . Due to small proband numbers in the subgroups, the null hypothesis ("no inter- group differences") was tested by the non- parametric, two sided Chi- square test. Survival was estimated by Kaplan- Meier analysis. Statistical analysis was carried out using the SPSS v11.5 package. Results Table 1 and 2 show the descriptive statistics of two groups, including gender and age distribution, diagnosis, prevalence of diabetes, and biopsy frequency. Individually addressing PCPs ignorance and concerns decreased late referral, from the high (SCKD: 44%) to the low (SCKD: 20%) end of the spectrum in published data[ 5 , 6 ] (Chi-square test: 2-sided p = 0.09), as detailed in Figure 1 . The relative share of patients seen at the stage of moderate CKD increased, while the relative percentage of patients seen in mild/moderate CKD remained stable. The health outcome (survival in MMCKD and MCKD groups) of patients was insignificantly improved: the mean survival time in group A is 1.71 yrs (1.30–2.12 yrs) compared to a mean survival of 1.90 yrs (1.78–2.02 yrs) in group B. Table 1 Age and gender distribution in the two cohorts. Group A: n= 18 MMCKD : n= 5 (28%) MCKD : n= 5 (28%) SCKD : n= 8 (44%) M F M F M F Gender (n/ %) 3/ (60%) 2/ (40%) 4/ (80%) 1/ (20%) 4/ (50%) 4/ (50%) Age (yrs ± SD) 59.7 ± 10.7 44.0 ± 11.3 59.5 ± 20.7 26.0 ± 0.0 55.3 ± 27.2 72.5 ± 8.3 Group B: n= 50 MMCKD : n= 14 (28%) MCKD : n= 26 (52%) SCKD : n= 10 (20%) M F M F M F Gender (n/ %) 11/ (78.6%) 3/ (21.4%) 17/ (65.4%) 9/ (34.6%) 1/ (10.0%) 9/ (90%) Age (yrs ± SD) 32.7 ± 16.1 25.0 ± 17.6 60.9 ± 12.6 54.8 ± 11.8 75.0 ± 0.0 65.4 ± 13.2 Table 2 Diagnosis of patients referred for specialist nephrological evaluation. Group A, n = 18 count (% within group) Group B, n = 50 count (% within group) Diagnosis No renal failure 0 (0%) 13 (26%) Glomerulonephritis 4 (22.2%) 5 (10%) Diabetes 3 (16.7%) 8 (16%) Nephrosklerosis 2 (11.1%) 4 (8%) Lupus 1 (5.6%) 0 (0%) Nephrectomy 2 (11.1%) 4 (8%) Renal cirrhosis 2 (11.1%) 4 (8%) Reflux 0 (.0%) 1 (2%) Polycystic disease 1 (5.6%) 1 (2%) Others 1 (5.6%) 3 (6%) Unknown cause 2 (11.1%) 7 (14%) Diabetes No 12 (66.7%) 30 (62.5%) Yes 6 (33.3%) 18 (37.5%) Biopsy No 15 (83.3%) 40 (83.3%) Yes 3 (16.7%) 8 (16.7%) Figure 1 Relative distribution (% of total) of patients into the three subgroups of MMCKD, MCKD and SCKD as defined under "methods". Distribution into MCKD and SCKD is inversed after intervention while MMCKD remains stable. Group A: patients referred in 1997; Group B: patients referred in 1999. Discussion The present study is a retrospective analysis of patient data from a single nephrological referral centre, and statistics are carried out on a data set of limited size (n = 18 only in the pre- intervention group A). As such, the statistical significance of the findings is doubtful, and the results might be interpreted as akin to a case report, or a medical anecdote. As such, however, it may have a value quite different from, but equal to that of a randomized, controlled study. Aronson recently discussed the value of medical anecdotes[ 11 ]; of the eight reasons listed there, the present study meets four: it generates a hypothesis (i.e., close communication with PCPs leads to earlier referral to specialist nephrological care), it suggests a method of management (i.e., individualized teaching sessions and adoption of financial incentives to increase early referral), it reminds and educates (i.e., of the benefit of early referral), and it hopes to stimulate a systematic review (i.e., the authors hope that the results will entice larger dialysis providers to design and carry out a prospective, randomized study). More often than not, observational studies give similar results to controlled randomized trials[ 12 , 13 ], and critical appraisal of this notion leads to the conclusion that, while good controlled randomized trials do provide the highest level of evidence, a flexible approach may be taken "in which randomised controlled trials and observational studies have complementary roles. High quality observational studies may extend evidence over a wider population and are likely to be dominant in the identification of harms and when randomised controlled trials would be unethical or impractical"[ 14 ]. The positive finding of the present study is that active recruiting strategies may improve the referral pattern. Following the discussions with individual PCPs, more patients were referred to specialist care at a stage where appropriate treatment may slow or even halt the progression of chronic renal failure. This should benefit first the patient, and then in the long run society as a whole. Recent studies[ 2 , 15 ] have shown that early referral causes a substantial decrease in hospitalization costs in the first year after referral. It is very probable that adequate treatment and therefore prolongation of the pre- dialysis moderate CKD stage will incur reduced overall spending; the increase in quality of life for the patients is immeasurable. Due to the relatively small number of patients available in this single centre study, combined with the short follow- up interval of just two years, only a slight trend towards prolonged "survival" as defined above could be demonstrated. An American multi- centre study[ 16 ] has recently shown, however, that late referral is clearly associated with higher morbidity. To what extend the timing of dialysis initiation influences survival remains as yet questionable. By contrast, Traynor[ 17 ] recently demonstrated that early initiation of dialysis may even be detrimental. There is currently one controlled, randomized study addressing this question[ 18 ], and the perspective may change once high level evidence is obtained. Late referral to specialist care in chronic renal disease remains a problem world- wide. As early as 1984, focusing on the question under which circumstances renal replacement therapy should be initiated or not, questionnaires showed significant differences in the attitudes of nephrologists and non- nephrologists towards referral[ 19 ] in the United Kingdom, and this difference in behavior has decreased, but not been eradicated over time[ 20 ]. Similar observations were made in Canada[ 1 ], and the United States [ 21 ]. It has been speculated that this may be because these recommendations address the specialist, rather than the primary care providers (PCP)[ 1 , 2 ]. At present, a medline search using the keywords "referral" and "chronic renal failure" listed a total of 81 references since 1974, with only 48 in specialist, not necessarily nephrological journals, while adding "guideline" reduced the reference list to a disappointing three references from the years 1998 and 1999, which included, however, a fully accessible online guideline in the Canadian Medical Association's journal. Neither the American, nor the European Renal Associations' guidelines[ 3 , 4 ] were found by these obvious search strategies. Only the former was found by the search terms "guideline" and "kidney disease", leading to the relevant article in the American Journal of Kidney Disease, accessible only to subscribers. Better results were obtained using the same search terms on a general search engine (Google), leading at first hit to the DOQI webpage, which includes the free access to full text guidelines. An extensive search using the same terms in German, however, showed no relevant results but an abundance of lay articles of dubious quality. This scarcity of qualified information might exclude those physicians not fluent in English from gathering relevant and up- to- date information. National renal care associations should therefore consider extending their educational efforts beyond their specialist members and selectively target PCPs and their respective professional associations. In the present study, two arguments brought forward by PCPs to explain their reluctance to refer early focused on the economic burden this presented to their own practice, rather than society as a whole. These two arguments concerned budget penalties if too much money was spent on the (costly) pre- dialytic patients with chronic kidney disease, and loss of patients after referral. This is an unusually frank statement. The majority of 22 medline hits using the search terms "economy" and "renal disease" address the question of best cost- efficiency of treatment strategies on a macro - economic level. Micro - economic considerations in the distribution of health care are only hinted at[ 2 ]. Health care planners increasingly stress economic factors. While their laudable aim is to provide affordable treatment options to the general population, the drawback of this approach becomes obvious in situations as these, where a conflict of interest arises between best practice and best revenue. Due to the retrospective design of this study, one cannot analyze to what extent the PCPs' economic considerations rather than their educational state were responsible for their referral pattern. The fact that financial concerns were the tenor of the individual discussions, however, indicates that health care planners may profit from taking this into account. The authors hope that this single- centre study will entice multi- centre RRT providers to conduct a prospective, large scale study to further investigate the relative contribution of the factors discussed here. Conclusions The initiative presented here shows that timely specialist referral in renal care can be achieved. National and regional differences in the organization of health care provision may lead to variant strategies to implement optimal health care, but close collaboration with the primary care providers is essential. List of abbreviations ECC: endogenous creatinine clearance MCKD: moderate chronic renal disease MMCKD: mild/moderate chronic renal disease PCP: primary care provider RRT: renal replacement therapy SCKD: severe chronic renal disease Competing interests The authors declare that they have no competing interests. Authors' contributions K.H. started the initiative, carried out all the teaching modules and gave access to the data. T.D.V. retrieved the data from archived files Z.A.L. did statistical analysis M.L. did statistical analysis A.M. did statistical analysis and wrote the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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535933

Bilateral Ramsay Hunt syndrome in a diabetic patient

Background Herpes zoster oticus accounts for about 10% cases of facial palsy, which is usually unilateral and complete and full recovery occurs in only about 20% of untreated patients. Bilateral herpes zoster oticus can sometime occur in immunocompromised patients, though incidence is very rare. Case presentation Diabetic male, 57 year old presented to us with bilateral facial palsy due to herpes zoster oticus. Patient was having bilateral mild to moderate sensorineural hearing loss. Patient was treated with appropriate metabolic control, anti-inflammatory drugs and intravenous acyclovir. Due to uncontrolled diabetes, glucocorticoids were not used in this patient. Significant improvement in hearing status and facial nerve functions were seen in this patient. Conclusions Herpes zoster causes severe infections in diabetic patients and can be a cause of bilateral facial palsy and bilateral Ramsay Hunt syndrome. Herpes zoster in diabetic patients should be treated with appropriate metabolic control, NSAIDS and intravenous acyclovir, which we feel should be started at the earliest. Glucocorticoids should be avoided in diabetic patients.

Background Varicella-zoster virus, member of Herpesviridae family has structural characteristics like a lipid envelope surrounding a nucleocapsid with icosahedral symmetry, a total diameter of 180–200 nm and centrally located double-stranded DNA about 125,000 bp in length. Varicella-zoster virus lies latent in sensory root ganglion for years in a patient who had chickenpox earlier. Some precipitating factor may reactivate it especially when immunity of patient wanes leading to Herpes zoster, a sporadic disease. Involvement of geniculate ganglion of sensory branch of facial nerve leads to Herpes zoster oticus also known as Ramsay Hunt syndrome. Involvement of facial nerve leads to otalgia, lower motor neuron homolateral facial paralysis and vesicular eruptions in auricle and external auditory canal. In severe cases of herpes zoster oticus, involvement of vestibulocochlear nerve leads to sensorineural hearing loss in 10% and vestibular symptoms in 40% patients. Herpes zoster oticus accounts for 10% cases of the facial palsy, paralysis is usually complete and full recovery occurs in only about 20% of untreated patients [ 1 ]. Herpes zoster rash is characterized by unilateral vesicular eruptions with in a single dermatome. In 16% of patients of zoster, vesicles develop beyond single dermatome [ 1 ]. Onset of disease is heralded by pain with in dermatome that may precede lesions by 48–72 hrs; total duration of disease is 7–10 days. In immunocompromised and elderly patients course of herpes zoster is more prolonged and severe. Rarely in such patients zoster may successively involve further dermatomes. Considering, rarity of bilateral herpes zoster, a diabetic patient presenting with bilateral Ramsay Hunt syndrome is being reported here. Case presentation Diabetic male, 57 year old presented to us with history of pain in left ear for the last 8 days. 48–72 hrs after the onset of otalgia, patient developed facial weakness on left side along with vesicular eruptions on left conchae and in left external auditory meatus. After another 24–48 hrs patient had similar episode on right side. On the day of reporting to us patient was having bilateral facial weakness, impaired taste sensation, dryness of eyes along with decreased hearing on both sides but there was no history of vertigo or any ear discharge. There was history of stressful life events in past 6 months before the onset of rash. On examination, there was bilateral lower motor neuron facial palsy which was complete. Bell's phenomenon was present on both sides Fig- 1 . There were adherent crusts and scabs in left conchae and external auditory meatus. While vesicular eruptions were present in right external auditory meatus. Tuning fork tests were showing bilateral sensorineural hearing loss. Figure 1 Patient at time of presentation, photograph showing bilateral lower motor neuron type of facial palsy and presence of Bell's phenomenon. The patient was admitted and investigated. His postprandial blood sugar was 339 mg%, HbAIc was 7%. Pure tone audiometery was showing mild to moderate bilateral sensorineural hearing loss, stapedial reflexes were absent on both sides on tympanometery. There was impaired taste sensation from anterior two third of tongue. ELISA and Western Blot tests for HIV infection were negative. Liver function tests, tumor markers, thyroid hormones; serum ACE levels were all with in normal limits. Lumbar puncture revealed normal pressure. Glucose, protein and white blood cell count were all with in normal limits in CSF. Plain X-ray views of mastoid, internal auditory meatus and chest were normal. Computerized tomography of brain stem, cerebellopontine angle, temporal bone and skull base were normal. A smear from floor of vesicle stained with Giemsa stain showed degenerating cells with multiple nuclei. This favoured the clinical diagnosis of herpes zoster oticus. This diagnosis was confirmed by detection of IgM antibodies to Varicella-Zoster virus by ELISA test. Diabetes of this patient was controlled with insulin. Intravenous acyclovir was given in dose of 10 mg/kg every 8 hr for 7 days. Glucocorticoids were avoided in this patient due to diabetes, but NSAIDS were given. After 2 weeks of treatment and diabetes control, pure tone audiometery showed improvement in hearing by 10 db in all frequencies. 8 weeks later in the follow up, patient was able to close his eyes completely Fig- 2 . and facial nerve functions on both sides recovered; recovery was more on right side. Figure 2 Patient after 8 wks of follow up, photograph showing complete closure of eyes. Discussion Severity and incidence of herpes zoster increases in elderly and in immunomodulated state like in AIDS, lymphoproliferative disorders, disseminated carcinomatosis, diabetes, during steroid therapy, during radio or chemotherapy [ 2 ]. Kubeyinje EP.(1995) found that varicella runs more aggressive course in diabetic patients as compared to otherwise healthy individuals [ 3 ]. Neu I et al (1977)in their study found basal metabolic disorders especially diabetes of particular significance inactivation and in primary and secondary manifestations of varicella zoster virus [ 4 ]. Muller C. et al (1989) concluded that metabolic derangement in diabetes leads to reversible disturbance in certain cellular immune functions which can be normalized by good metabolic control achieved by insulin treatment [ 5 ]. Postprandial blood sugar of our patient was 330 mg% at time of presentation and HbA 1c was 7.0%, so blood sugar control was not appropriate in this patient. Patient was also under severe psychological stress since last 6 months and psychological stress is identified as a potential risk factor for zoster that might operate by suppressing cell-mediated immunity [ 6 ]. So uncontrolled diabetes and psychological stress were two risk factors present in this patient, making him prone for severe and recurrent infection of herpes zoster. Hiroshige K et al (2002) conducted a study based on detection of varicella zoster virus DNA in tear fluid and saliva of patients with Ramsay Hunt syndrome and concluded that varicella zoster virus reactivation occurs in the unaffected side at the same level as in the affected side [ 7 ]. This explains the occurrence of bilateral Ramsay Hunt syndrome due to herpes zoster oticus in this patient. Shoji H et al (1980) also suggested that in Ramsay Hunt syndrome and its subgroups, bilateral involvement or wide spread of infection through nervous tissue can occur though its incidence is very rare[ 8 ]. In the management part, as metabolic control of diabetes improves leukocyte functions and overall immune status of patient [ 5 ] so insulin was started and dose was adjusted to achieve good metabolic control. As incidence of severe and disseminated infections of Herpes zoster is more in diabetes so intravenous acyclovir in dose of 10 mg/kg every 8 hr for 7 days was given. Recent reports suggest that treatment with i.v acyclovir decreases the incidence of permanent facial nerve palsy in Ramsay Hunt Syndrome. Results of relevant control trials have not yet been published [ 9 ]. Glucocorticoid therapy usually used in facial palsy due to herpes zoster oticus was avoided in this patient due to uncontrolled diabetes but NSAIDS and other analgesics were given in this patient. On follow up of this patient, facial nerve recovered on both sides, recovery was more on right side. This was documented by electroneurography which showed greater amplitude of muscle compound action potentials on right side as compared to left. This might have been due to lesser time gap between the onset of acyclovir therapy and attack of zoster oticus on right side. Time gap between the onset of acyclovir therapy and attack of zoster oticus is most relevant prognostic factor in recovery of these patients and this hypothesis is supported by findings in studies of Mcgrath N. (1997) [ 10 ], Raschilas F (2002) [ 11 ] and Strupp M. et al (2004) [ 12 ]. There was also a significant improvement in hearing status of patient. This result was in accordance with other studies which keep age 64 years or younger, mild initial hearing loss, a cochlear pattern of hearing loss and absence of vertigo as factors favorable for recovery of auditory function [ 13 ]. Conclusions Herpes zoster can cause severe infections in diabetic patient and can cause bilateral facial palsy and bilateral Ramsay Hunt syndrome. -Herpes zoster in diabetic patients should be treated with appropriate metabolic control, NSAIDS and intravenous acyclovir, which we feel should be started at the earliest. -Intravenous acyclovir therapy in cases of herpes zoster oticus is effective in control of disease and prevents the incidence of permanent facial palsy but treatment should be started early in the course of disease preferably with in 72 hrs from start of disease. -Glucocorticoids should be avoided in Herpes zoster patients having uncontrolled diabetes. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All three authors 1) Have made substantial contributions in management of this case and in conception, design, analysis and interpretation of results of this case report 2) Have been involved in drafting the article or revising it critically for important intellectual content. 3) Have given final approval to the version to be published. Pre-publication history The pre-publication history for this paper can be accessed here:

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535890

Autodissemination of the entomopathogenic fungus Metarhizium anisopliae amongst adults of the malaria vector Anopheles gambiae s.s.

Background The entomopathogenic fungus Metarhizium anisopliae is being considered as a biocontrol agent for adult African malaria vectors. In the laboratory, work was carried out to assess whether horizontal transmission of the pathogen can take place during copulation, as this would enhance the impact of the fungus on target populations when compared with insecticides. Methods Virgin female Anopheles gambiae sensu stricto were exposed to conidia whilst resting on fungus-impregnated paper. These females were then placed together for one hour with uncontaminated males in proportions of either 1:1 or 1:10 shortly before the onset of mating activity. Results Males that had acquired fungal infection after mating indicate that passive transfer of the pathogen from infected females does occur, with mean male infection rates between 10.7 ± 3.2% and 33.3 ± 3.8%. The infections caused by horizontal transmission did not result in overall differences in survival between males from test and control groups, but in one of the three experiments the infected males had significantly shorter life spans than uninfected males ( P < 0.05). Conclusion This study shows that autodissemination of fungal inoculum between An. gambiae s.s . mosquitoes during mating activity is possible under laboratory conditions. Field studies are required next, to assess the extent to which this phenomenon may augment the primary contamination pathway (i.e. direct contact with fungus-impregnated targets) of vector populations in the field.

Introduction Control of the main African malaria vector Anopheles gambiae (Diptera: Culicidae) continues to rely heavily on application of residual insecticides, either for indoor residual house spraying [ 1 ] or bednet impregnation [ 2 ]. These approaches have been highly effective in reducing malaria morbidity and mortality [ 2 ], but associated problems regarding environmental pollution [ 3 , 4 ], acceptability and cost [ 5 , 6 ] and the now widespread and continuing development of resistance [ 7 - 10 ] underscore the need for alternative strategies, such as vector control with biological agents [ 1 , 11 , 12 ]. Entomopathogenic fungi are among the biological control agents used against insect pests. Interest in using the hyphomycete Metarhizium anisopliae against adult African malaria vectors has recently increased [ 13 ]. The fungus has proven to be highly virulent for this vector, both in the laboratory [ 14 ] as well as in the field (Scholte et al ., in preparation). The principal method of contamination of the target insect population with the fungus is through application of conidia on indoor resting targets. However, in order to achieve the highest possible impact on the target population, it is desirable that contamination pathways other than the primary mode of contamination are utilised, for instance horizontal transmission. Horizontal transmission of pathogens within the same host/target species is called autodissemination, and this phenomenon has been suggested for biocontrol of several insect pests [ 15 , 16 ]. Successful transmission of M. anisopliae by honeybees for infection of the pollen beetle Meligethes aeneus [ 17 ], of Beauveria bassiana between adult flies of Delia radicum [ 18 ] and of M. anisopliae and B. bassiana between adult tsetse flies, Glossina morsitans morsitans [ 19 ] confirms the capability of insects to transmit fungi horizontally. Autodissemination of insecticidal biocontrol agents, such as insect-pathogenic fungi, provides an additional advantage over pesticides, as the impact on pest populations increases beyond direct contact. In several cases, autodissemination of entomopathogenic fungi within populations of insect pests, using attractant traps as the initial source of infection, has succeeded [ 18 , 20 - 22 ]. The strategy envisaged for the use of M. anisopliae against adult An. gambiae is that host-seeking females, and occasionally also males that rest indoors, will receive primary infections while resting indoors on fungus-impregnated resting targets. Under optimal circumstances, prior to death, this infection may be transmitted to conspecifics upon contact (e.g. during mating). These mosquitoes are, therefore, not infected through direct contact with fungus-impregnated materials, but indirectly, upon physical contact with infected counterparts. It is estimated that approximately half of newly hatched, virgin females take a blood meal before mating [ 23 , 24 ]. A female, with contaminated legs and mouthparts following the blood-feeding visit to a house containing fungus-impregnated resting targets, may contaminate male counterparts when she mates the following dusk period, thereby spreading the fungus through the population. The objective of this study was to investigate whether adult An. gambiae infected with M. anisopliae can transmit the fungus to uncontaminated mosquitoes of the opposite sex through physical contact during the mating process. Materials and Methods Bioassays Three bioassays were conducted to assess whether autodissemination of M. anisopliae can occur during the process of mating: 1) 30 fungus-contaminated virgin females and 30 uncontaminated males were placed in a standard (30 cm 3 ) netting cage for one hour during the normal mating period; 2) a single fungus-contaminated virgin female and a single uncontaminated male where placed together in a glass tube for one hour during the normal mating period; 3) a single fungus-contaminated virgin female and 10 uncontaminated males were placed in a standard (30 cm 3 ) netting cage for one hour during the normal mating period. A fourth bioassay assessed whether or not: 4) males that became infected with the fungus had acquired the infective propagules from contaminated females or rather from contact with the substrate where those females had rested previously. Mosquitoes The Anopheles gambiae sensu stricto strain used originates from Suakoko, Liberia (courtesy Prof. M. Coluzzi) and has been maintained in the Wageningen laboratories since 1989, under standardized conditions described by Mukabana et al . [ 25 ]. Experimental mosquitoes were four (females) or seven day old (males). Virginity of the females was assured by collecting them within 24 hrs after emergence from cages and keeping them separate from the males prior to the experiments. In all experiments, mosquito mortality was checked daily, the mosquito cadavers placed on moist filter paper and placed in Petri dishes that were sealed with parafilm. These were kept in an incubator at 27 ± 2°C and checked for fungal sporulation after three days. Fungus Metarhizium anisopliae var. anisopliae isolate ICIPE30 (courtesy Dr. N.K. Maniania) was originally isolated in 1989 from a stemborer, Busseola fusca Fuller, near Kendu Bay, Western Kenya. Conidia were inoculated on oatmeal agar (OA) and placed in an incubator to grow. Fungal virulence was maintained by passing it through An. gambiae mosquitoes every five cycles after growing on OA. Third instar larvae were infected by applying dry conidia on the water surface. After 48–72 hours, moribund larvae were removed and their thorax opened to remove tissue with blastospores. These were plated on OA and placed in a dark incubator at 27°C. One week after the onset of sporulation of the colonies, conidia were harvested using a 0.05% Triton-X solution and a glass rod. The solvent with conidia was concentrated by removing the supernatant after centrifuging for three minutes at 5000 rpm. Dilutions were made using 0.05% Tween80 to obtain a conidial concentration of 10 8 conidia ml -1 . Vegetable (sunflower) oil was added to obtain an 8% adjuvant oil formulation. Five ml of this suspension was pipetted evenly over a 240 cm 2 piece of filter paper resulting in conidial densities of 1.6 × 10 10 conidia m -2 . The impregnated paper was left to dry at 20°C and 75 ± 5% RH for 48 hours and was then placed on the inside of a plastic cylinder (height 11.3 cm, diameter 3.4 cm) in such manner that the paper neatly lined the upright wall of the tube. The top of the tube was covered with netting material. This setup was used only to infect female mosquitoes. Before any contamination, the viability of the impregnated conidia was checked by placing a 1 cm 2 piece of the impregnated paper on a Sabourad Dextrose Agar in an incubator at 27°C in the dark for 16–20 hours. After incubation, the piece of paper was carefully removed and placed under a microscope (X 400) to determine the proportion germinated. For direct contamination of the female mosquitoes with M. anisopliae , around 30 individuals were placed in the cylinder for 24 hours. Experimental procedures Bioassay 1 Thirty uninfected males were placed in a 30 cm 3 netting cage three hours before the onset of mating. Half an hour before (artificial) dusk, which for An. gambiae is the time when mating activity occurs [ 26 ], thirty contaminated females were added to this cage by releasing them from the cylinder (see above) where they had spent the previous 24 hours. By that time a large percentage of the males had the fibrillae on their antennae erect, which is considered a sign for impending mating activity [ 27 ]. One hour after introduction of the females, all males were gently removed from the cage using a 2 cm diameter glass vial and placed in a clean cage where they had access to 6% glucose ad libitum . The experiment was replicated three times, on different days. Control groups were treated similarly, with the difference that the paper lining the contamination tube was void of conidia. Mortality of males and females was checked daily to measure longevity. Dead mosquitoes (both sexes) were removed from the cages and placed on moist filter paper in a Petri dish, which was sealed and examined for fungal growth three days later. An additional similar experiment with only five females and five males was performed to determine whether conidia could be seen on the cuticle of the test males directly after the females were removed. Males were killed by a droplet of chloroform and placed under a microscope (X 400) and examined for attached conidia. Bioassay 2 A single, uncontaminated 7-day old male was placed in a clean glass vial (diameter 2 cm, height 10 cm, sealed off with netting), to which one M. anisopliae contaminated female was added 30 minutes before the onset of mating activity. After one hour the couple was separated by gently removing the male, which was placed and kept in a separate glass vial until it died. A wad of cotton wool moist with 6% glucose was placed on top of the vial. Females remained in the vial and were provided with glucose in the same way. Mortality of both sexes was recorded daily. The control group consisted of an equal number of pairs that were handled equally, with the difference that the females were not infected with the fungus. This was repeated with 35 male-female pairs on three different days. Bioassay 3 Ten uncontaminated 7-day old males were placed in a 30 cm 3 netting cage. Half an hour before the onset of mating activity, a single infected female mosquito was added to this cage with the males. After one hour each male was gently removed using a clean 2 cm diameter glass vial and kept alive individually as in bioassay 2. This was done 14 times for the test group and six times for the control group. Bioassay 4 To assess whether the males in the above bioassays had acquired fungal infection from the contaminated females during the process of mating, or from resting on the substrate where fungus-contaminated females had rested previously (glass and netting), two experiments were carried out. In one experiment a total of 46 contaminated females (in two separate trials) were gently transferred to a 500 ml glass beaker, sealed of with a glass Petri-dish. The females were killed after one hour by applying a droplet of chloroform in the beaker. They were then removed and placed into a Petri-dish on a piece of moist filter paper. The dish was sealed with parafilm, to be checked three days later for fungal infection. Directly after removal of the contaminated females, a total of 47 uncontaminated males (in two separate trials) were placed in the beaker for three days, after which they were killed, removed and checked for fungal infection as described above. During the three days inside the beaker they had continuous access to a cotton wool wad moist with a 6% glucose solution. The second experiment differed only in that a standard netting cage was used instead of a glass beaker, with 64 fungus-contaminated females and 67 uncontaminated males in two separate trials. Data analysis Mortality data were subjected to Kaplan-Meier pair-wise comparison survival analysis. Mosquito mortality data closely fitted the Gompertz distribution [ 28 ]. Comparisons of the average ages at the time of death were calculated and analyzed using GLM analysis. All analyses were carried out by using Genstat 7.0. Results In all four bioassays, female mosquitoes that had been exposed to conidia in the cylinder setup died significantly faster than the control females (F = 104.4; p < 0.001), with an average of 96.4 ± 2.0% of the cadavers having sporulating M. anisopliae . All three replicates of bioassay 1 indicated transfer of the fungus from contaminated females to uninfected males, with an average male infection rate of 26.1 ± 5.3% (Table 1 ). There was no difference in survival between the males of the control and test groups (F = 0.30; p = 0.5844), but when the group of test males was split into those that had been infected and those that had not, survival analysis showed a difference in survival approaching significance (F = 2.73; p = 0.098). Under a compound microscope, conidia were observed on four of the five males. Most were found on the lower parts (tibia, tarsi, uncinus (claws) and arolium) of the first and second pair of legs (Figure 1 ). A few conidia were found attached to the hairs of the tip of the wings. No conidia were found on the head, thorax, abdomen or the hind legs. Per mosquito 0–25 conidia were found. Female mosquitoes that had spent 24 hrs on fungus-impregnated paper had conidia on legs, tips of their wings and mouthparts, but not on the thorax or abdomen. Table 1 Autodissemation of M. anisopliae from females to males An. gambiae s.s .. The proportions of male mosquitoes infected are shown with the differences in survival of fungus-infected males compared to uninfected males within the test groups. Bioassay Ratio Average %(± SEM a ) of males infected Survival analysis (Kaplan Meier) Average age of males at time of death (days) infected uninfected 1 1:1 26.1 ± 5.3 F = 2.73; p = 0.098 10.3 ± 0.7 18.6 ± 0.8 b 2 1:1 34.0 ± 0.6 F = 3.39; p = 0.065 11.3 ± 1.9 15.6 ± 1.6 b 3 1:10 10.7 ± 3.2 F = 13.02; p = 0.001 13.1 ± 1.3 17.9 ± 0.6 b a : SEM = standard error of mean b : Significant difference (p < 0.05) between infected and uninfected males using GLM analysis (Genstat 7.0). Figure 1 Conidia of the entomopathogenic fungus Metarhizium anisopliae on the lower part of a male Anopheles gambiae s.s . leg, after having been horizontally transferred from an infected female during copulation. In the second bioassay, with individual pairs in glass vials, an average of 34.2 ± 0.6% of the males acquired and died of the fungal infection. Survival of these males as a group was not significantly different from the control group (F = 1.27; p = 0.259). When the males from the test group were split into those that became infected and those that did not, a decrease in survival of the infected test males compared to the uninfected males was observed, although this effect was not significant (F = 3.39; p = 0.065). However, the average age at death of 11.3 ± 1.9 days of the fungus-infected males was significantly lower than the 15.6 ± 1.6 days of the uninfected test males (p = 0.001). In the third bioassay, horizontal transmission occurred in nine of the 14 replicates (64.3%). In those nine trials an average of 16.7 ± 3.7% of the 10 males acquired an infection. Calculated over the 14 trials, the infection rate was 10.7 ± 3.2%. As in the other two bioassays, there was no significant difference in survival rates between the test and control groups (F = 2.19; p = 0.139). However, those males in the test group that were found with sporulating fungus died significantly faster than uninfected males (F = 13.02; p = 0.001) with the average age at the time of death of 8.4 ± 1.1 days for infected and 17.9 ± 0.6 days for uninfected males, respectively. None of the 114 males in bioassay 4 were found infected with the fungus. Discussion It is generally believed that fungal dissemination within a host population occurs due to activities and movements of the host. The fungus can exploit host behaviour like trophallaxis, tactile communication, grooming (in social insects) [ 29 , 30 ] and mating [ 31 ] to spread through a host population. Taking into account the physiological state of the females and the natural display of behaviour at the time of the bioassays, it is assumed that the observed autodissemination of M. anisopliae from female to male An. gambiae s.s . was the result of mating. This is strongly supported by the findings from experiment 4 where none of the males that had stayed on the surface area where fungus-contaminated females had rested previously acquired an infection. The average age at death of fungus-infected mosquitoes was quite high when compared to mosquito survival in Scholte et al . [ 14 ]. This is probably due to the relatively low level (a maximum of 25 conidia) of inoculum transferred. From those mosquitoes that were checked under the microscope for the presence of conidia, four out of five males contained conidia. It is thus likely that many males become contaminated, but that only a relatively low proportion of these males will actually succumb to the infection: in many cases the number of conidia was low, resulting in marginal infections that were successfully countered by the immune responses staged by the males. In order to achieve the highest possible impact of the fungus on the mosquito population, it is desirable that other pathways besides the primary mode of (direct) contamination are utilized. The results of this study show that under laboratory conditions horizontal transmission can occur, which suggests that it may occur in the field. When these experiments were carried out, it was presumed that predominantly females would be infected directly from the indoor resting targets in the field. From a recent field experiment (Scholte et al ., in preparation), however, it was found that a large proportion (44.9%) of the An. gambiae s.l . found indoors were males. This suggests that not only females can deliver fungal inoculum to uninfected males, but that also infected males may infect uninfected females. Further research is needed to determine to what extent this secondary pathway of fungal contamination may contribute to spreading the fungus within mosquito field populations. Conclusion This study has shown that horizontal transfer of fungal inoculum between mosquitoes is possible during copulation and may contribute to spreading of the fungus within target mosquito populations in the field. However, since conditions under which horizontal transmission is likely to occur are quite specific, field experimentation is required to measure the real impact that autodissemination may have. For now it is concluded that the relatively low infection levels recorded in this study suggest that the impact of biological control with M. anisopliae against African anophelines will predominantly depend on direct contamination of adult mosquitoes from conidia-impregnated resting targets such as walls, ceilings and sheets. Authors' contribution E-JS was directly involved in the experimental work, analysis of the data and drafting of the manuscript. BGJK conceived of the study, obtained funding for it in collaboration with WT and revised the manuscript prior to submission.

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523228

Whole-Genome Analysis of Temporal Gene Expression during Foregut Development

We have investigated the cis -regulatory network that mediates temporal gene expression during organogenesis. Previous studies demonstrated that the organ selector gene pha-4 /FoxA is critical to establish the onset of transcription of Caenorhabditis elegans foregut (pharynx) genes. Here, we discover additional cis -regulatory elements that function in combination with PHA-4. We use a computational approach to identify candidate cis -regulatory sites for genes activated either early or late during pharyngeal development. Analysis of natural or synthetic promoters reveals that six of these sites function in vivo. The newly discovered temporal elements, together with predicted PHA-4 sites, account for the onset of expression of roughly half of the pharyngeal genes examined. Moreover, combinations of temporal elements and PHA-4 sites can be used in genome-wide searches to predict pharyngeal genes, with more than 85% accuracy for their onset of expression. These findings suggest a regulatory code for temporal gene expression during foregut development and provide a means to predict gene expression patterns based solely on genomic sequence.

Introduction Formation of organs depends on successive programs of gene expression during development. Temporal regulation of transcription is critical to achieve spatial patterning, as seen during vertebrate somitogenesis ( Pourquie 2003 ). Temporal regulation is also essential to integrate the progression of events that accompany cell fate specification and differentiation ( Bruhn and Cepko 1996 ; Isshiki et al. 2001 ; Pearson and Doe 2003 ). The regulatory networks that guide these processes depend on a wide array of transcription factors, raising the question of how transcriptional circuitry dictates developmental timing. In some cases, tiers of transcription factors function hierarchically to establish sequential patterns of gene expression ( Kornberg and Tabata 1993 ; Maduro and Rothman 2002 ; Skeath and Thor 2003 ). These regulators are active for only a brief time during development and typically produce a uniform response in expressing cells. For example, three consecutive waves of GATA transcription factors establish the Caenorhabditis elegans midgut ( Maduro and Rothman 2002 ). Ectopic expression of at least some of these GATA factors can convert the entire embryo into midgut, suggesting a homogeneous and robust transcriptional response by these cells. In contrast, other transcriptional regulators function continuously during development ( Weatherbee et al. 1998 ; Bergstrom et al. 2002 ; Gaudet and Mango 2002 ). These proteins activate different target genes at different developmental stages, suggesting a more complex, heterogeneous transcriptional response. A critical question is how the second class of transcriptional regulators establishes consecutive programs of gene expression. The forkhead box (Fox) A family of transcription factors illustrates the second strategy of developmental control. These proteins are critical to form the digestive tract in Drosophila, mammals, and worms, and animals lacking FoxA have profound gut defects ( Weigel et al. 1989 ; Ang and Rossant 1994 ; Mango et al. 1994 ; Weinstein et al. 1994 ; Dufort et al. 1998 ). For example, inactivation of C. elegans pha-4 leads to a loss of foregut cells, which are transformed into ectodermal cell types such as glia and epidermis ( Mango et al. 1994 ; Horner et al. 1998 ). This dramatic phenotype reflects the global requirement for PHA-4 to transcribe genes selectively expressed in foregut cells throughout development. Direct PHA-4 targets include early-acting developmental regulators, such as ceh-22 /Nkx 2–5, and terminal differentiation genes that encode structural proteins and digestive enzymes ( Kalb et al. 1998 ; Gaudet and Mango 2002 ; Vilimas et al. 2004 ). FoxA members in other organisms have a similar range of early- and late-expressed targets, suggesting they, too, function during multiple stages of development ( Gualdi et al. 1996 ; Lehmann and Korge 1996 ; Duncan et al. 1998 ; Roth et al. 1999 ; Lee et al. 2002 ). The diversity of FoxA target genes raises the question of how these factors achieve appropriate temporal regulation of transcription during development. One answer is affinity for DNA. C. elegans PHA-4 recognizes sequences that conform to the consensus TRTTKRY (where R = A/G, K = T/G, and Y = T/C) ( Overdier et al. 1994 ; Gaudet and Mango 2002 ). Sequences that bind PHA-4 with high affinity in vitro are typically found in promoters of genes expressed early in development, whereas low-affinity sites are restricted to late promoters ( Gaudet and Mango 2002 ). Moreover, adjustment of a high-affinity binding site to a lower one shifts the onset of expression later, and, conversely, mutation to a higher-affinity site leads to earlier activation ( Gaudet and Mango 2002 ). These data demonstrate that binding-site affinity of PHA-4 for DNA is a critical determinant of gene expression. However, the affinity of PHA-4 for its recognition sequence is not an absolute predictor of gene activation. For example, the pharyngeal muscle myosin gene, myo-2, possesses high-affinity PHA-4 sites but is activated late in development ( Okkema et al. 1993 ; Gaudet and Mango 2002 ). These observations suggest additional factors function in combination with PHA-4 for temporal control of pharyngeal gene expression. In this study, we have combined bioinformatics and experimental approaches to investigate the cis -regulatory network for temporal gene expression within the pharynx. We identify sites that function in combination with PHA-4 elements to distinguish early from late expression. These elements can be used to build synthetic promoters with the expected expression profiles and to identify previously undiscovered pharyngeal genes within the genome. Results Our goal was to discover new regulators of pharyngeal transcription that would function in combination with PHA-4 ( Figure 1 A). To achieve this aim, we first identified candidate pharyngeal genes by microarray analysis and subdivided these genes into clusters based on their onset of expression (early versus late). Next, we searched for short sequences enriched within the predicted promoters of genes from the early or late clusters. These sequences were tested for enhancer or repressor activity in vivo using both natural and synthetic promoters. Figure 1 Strategy to Identify Temporal Regulatory Elements (A) Flowchart of the strategy used. (B) Northern blot of pha-4. pha-4 transcripts were approximately 25- to 100-fold enriched in par-1 compared to skn-1 embryos, but only approximately 5- to 10-fold enriched in wild-type compared to skn-1 embryos. Arrowheads indicate the three different pha-4 isoforms. (C) The same blot was probed with a fragment of the act-1 gene to demonstrate equal loading of RNA between lanes. Identification of Pharyngeal Genes We previously discovered pharyngeal genes using microarrays encompassing 62% of C. elegans genes ( Gaudet and Mango 2002 ). We extended this analysis by screening microarrays that covered 94% of C. elegans genes (17, 871 genes; Jiang et al. 2001 ). To maximize the sensitivity of detection, we compared gene expression profiles from embryos with excess pharyngeal cells ( par-1; Kemphues et al. 1988 ) to embryos with no pharyngeal cells ( skn-1; Bowerman et al. 1992 ). par-1 mutants affect the earliest embryonic cell divisions and produce cell fate transformations, such that par-1 mutant embryos lack gut cells, but have excess pharynx and body wall muscles. In contrast, skn-1 mutants lack both gut and pharynx, but have excess body wall muscle and epidermis. Thus, genes with a relatively high par-1/skn-1 ratio were likely to be selectively expressed in the pharynx. An advantage to using par-1 and skn-1 mutants was that they provided a broad range of expression differences. For example, pha-4 transcripts were approximately 25- to 100-fold enriched in par-1 versus skn-1 embryos, compared to only approximately 5- to 10-fold enriched in wild-type versus skn-1 embryos ( Figure 1 B and 1 C). We identified 339 genes with at least 2-fold greater expression in par-1 embryos compared to skn-1 mutants ( Materials and Methods ; Table S1 ). For genes whose expression was known, 81% (114/141) were selectively expressed in the pharynx ( Table S1 ). Importantly, the sensitivity of this approach enabled us to detect genes expressed at low levels (e.g., ceh-22; Okkema and Fire 1994 ; Vilimas et al. 2004 ) or in a small subset of pharyngeal cells (e.g., C49G7.4; Ao et al. 2004 ). Temporal Groups of Pharyngeal Genes We categorized the pharyngeal genes into early versus late temporal groups using previously defined expression patterns. The Nematode Expression Pattern Database ( Kohara 2001 ; http://nematode.lab.nig.ac.jp/db/index.html ) and green fluorescent protein (GFP) reporters enabled us to identify 37 early-onset and 34 late-onset pharyngeal genes ( Table S2 ). The set of early-expressed pharyngeal genes (Ph-E) contained genes whose expression initiated by mid-embryogenesis (the “bean” to “comma” stages; Sulston et al. 1983 ). At the end of this developmental stage, embryonic cell division is virtually complete, the pharynx primordium has formed, and cell fate patterning of the primordium has begun. The set of late-expressed pharyngeal genes (Ph-L) contained genes activated at the onset of terminal differentiation of the pharynx (the 3-fold stage; Sulston et al. 1983 ). Forty-three genes were associated with expression patterns but were not assigned to either category either because the onset of expression was ambiguous or because it fell between the early and late categories. The Ph-E cluster was enriched for genes predicted to encode transcription factors ( Figure 2 ; Table 1 ), consistent with Ph-E genes controlling early aspects of pharyngeal development such as cell fate specification. The Ph-L group was enriched for genes predicted to encode cytoskeletal or muscle proteins ( Figure 2 ; Table 1 ), consistent with Ph-L genes being involved in terminal differentiation and pharyngeal function. Intriguingly, Ph-L genes were more likely to be located on Chromosomes V and X ( p ≤ 0.05) at the expense of Chromosomes I and IV ( p ≤ 0.06; Table 2 ). Biases for gene placement on chromosomes have been observed previously. For example, genes expressed in the male germ line are excluded from the X chromosome ( Reinke et al. 2000 ), while muscle genes are often clustered along a chromosome ( Roy et al. 2002 ). Figure 2 Different Predicted Products of the Temporal Groups The Ph-E group is enriched for predicted transcription factors, while the Ph-L group is enriched for predicted structural or muscle proteins (see Table 1 ). Muscle proteins are proteins known or predicted to be involved in muscle function, including myosins, tropomyosins, and troponins. Table S2 provides a complete listing of the catergorization of the Ph-E and Ph-L genes. Table 1 Temporal Groups of Pharyngeal Genes Are Enriched for Different Kinds of Predicted Products Relative to both the genome and the total set of microarray positives, the Ph-E group is enriched for predicted transcription factors. In contrast, the Ph-L group is enriched for predicted structural or muscle proteins. Muscle proteins are proteins known or predicted to be involved in muscle function, including myosins, tropomyosins, and troponins ND, not determined Table 2 Ph-L Genes Are Enriched on Chromosomes V and X a Expected number if Ph-L genes are randomly distributed throughout the genome Identifying Regulatory Elements in Pharyngeal Promoters We examined predicted promoters of Ph-E and Ph-L members for candidate cis -regulatory elements that might contribute to temporal regulation. We first estimated the size of pharyngeal promoters by determining the sequence identity between pairs of C. elegans and C. briggsae orthologs. Conservation is a good indicator of functionally important regions for cis -regulation ( Kirouac and Sternberg 2003 ; Hwang and Sternberg 2004 ; Liu et al. 2004 ). We scored as “conserved” those regions of DNA with 75% or greater identity over 50 bp. Sixty-six percent of pharyngeal genes had conserved sequences within 500 bp upstream of the predicted start codon ( n = 64 genes; Table 3 ), whereas only 21% had conserved sequences from 500 to 1,000 bp ( n = 63 genes; Table 3 ). This landscape of sequence conservation agreed well with reporter studies in which 500 bp of upstream sequence was often sufficient to recapitulate the endogenous pattern of expression ( Gaudet et al. 1996 ; McGhee and Krause 1997 ; Gaudet and Mango 2002 ). Based on these observations, we chose to limit our motif searches to 500 bp upstream of predicted start codons. Table 3 Conservation of Non-Coding Sequence between C. elegans and C. briggsae Entries show the fraction of genes that have significant conservation of non-coding sequence (greater than 75% identity across 50 bp or more). “−1,000 to −501” is the region 501 to 1,000 bp upstream of the predicted ATG start codon; “−500 to −1” is the region within the first 500 bp upstream of the predicted ATG start codon. For a complete listing of conservation for each gene in the Ph-E and Ph-L groups, see Table S3 We used the Improbizer expectation maximization algorithm to search pharyngeal promoters for potential regulatory elements ( Ao et al. 2004 ). Improbizer detects short sequences that are over-represented within a cohort of genes. As a negative control, we examined groups of genes that were not specifically expressed in the pharynx (e.g., DNA synthesis genes or a set of 194 randomly selected genes) and removed motifs common to both the pharyngeal and negative control sets. This comparison identified nine candidate regulatory motifs for pharyngeal genes ( Figures 3 ; Dataset S1 ). Significantly, two motifs, Early-3 and P-3, conformed to the consensus sequences for the pharyngeal transcription factors CEH-22 and PHA-4, respectively ( Okkema and Fire 1994 ; Kalb et al. 1998 ; Gaudet and Mango 2002 ), indicating that our approach could successfully identify pharyngeal regulatory elements. Figure 3 Candidate Pharyngeal Motifs Identified by Improbizer Improbizer represents motifs as PWMs; the PWMs for the motifs are shown in Dataset S1 . We converted these matrices to the “sequence logos” shown here ( Schneider and Stephens 1990 ; Crooks et al. 2004 ). The threshold scores used by Cluster-Buster ( Frith et al. 2003 ) for each motif are shown below the sequence logos. “% Ph-E Genes” and “% Ph-L Genes” are the percentage of Ph-E and Ph-L genes that contain occurrences of a given motif within 500 bp upstream of the predicted ATG start codon, above the threshold shown. “% Neg Genes” is the percentage of DNA synthesis genes that contain occurrences of a given motif (above the threshold score) within 500 bp upstream of the predicted ATG start codon. Two other negative groups (carbohydrate synthesis genes [ Kim et al. 2001 ] and a set of 194 randomly selected genes) yielded similar results (data not shown). “Enhancer Activity” is the relative strength of expression generated by a motif present in three copies upstream of the Δ pes-10::GFP::HIS2B reporter. ND, not determined. Five of the remaining seven motifs were associated with either the Ph-E or Ph-L temporal expression groups ( Figure 3 ). The Early-1 and Early-2 motifs were found by screening promoters of the Ph-E gene group and occurred more frequently in Ph-E promoters (44% and 54% of promoters, respectively) than in Ph-L promoters (26% and 21% of promoters, respectively). Conversely, the Late-1, Late-2, and Late-3 elements were found in Ph-L promoter searches and were more likely to occur in Ph-L promoters (53%, 35%, and 15% of promoters) than in Ph-E promoters (27%, 16%, and 8% of promoters). Two other motifs named P-1 and P-2 (for pharyngeal gene motifs 1 and 2) were discovered by screening Ph-E and Ph-L promoters together. These elements occurred in Ph-E and Ph-L genes with comparable frequency and were more frequently represented in promoters of pharyngeal genes (P-1 in 34% and P-2 in 68% of promoters) than of non-pharyngeal control genes (P-1 in 21% and P-2 in 48% of promoters). These data suggest that P-1 and P-2 are pharyngeal regulatory elements that are not associated with temporal control. We reexamined Ph-E genes for additional elements present in genes that contained neither Early-1 nor Early-2 elements. Given that 48% of Ph-E genes contained one or more Early-1 or Early-2 elements ( Table S4 ), we examined the remaining 52% of Ph-E genes for the presence of additional motifs. This survey identified a single motif that appeared to be a variant of the Early-1 motif and was therefore named “E1var.” As with Early-1, E1var was enriched in the promoters of Ph-E pharyngeal genes compared to Ph-L pharyngeal and non-pharyngeal genes ( Figure 3 ). No other motifs were identified above background in these follow-up searches. We performed a similar analysis of Ph-L genes that did possessed neither Late-1 nor Late-2 elements, but found no additional motifs. These results suggest that if other temporal elements exist, they are represented by relatively degenerate sequences, are shared by small numbers of genes, occur in both pharyngeal and non-pharyngeal genes, or are outside the 500-bp regions examined. In Vivo Activity of Candidate Pharyngeal Regulatory Elements Two tests demonstrated that six of the candidate motifs had biological activity. First, we performed “enhancer assays” to determine whether a motif was sufficient to activate expression when introduced into a heterologous basal promoter. Second, we used site-directed mutagenesis to inactivate a motif within a native pharyngeal promoter and examine whether it was necessary for expression. We used the Δ pes-10 promoter for the heterologous enhancer assays ( Figure 4 ). This promoter does not activate GFP ( Figure 4 D– 4 F) but is competent to respond to enhancers in most or all tissues ( Seydoux and Fire 1994 ; Fire et al. 1998 ). Previous studies established that the PHA-4 binding site and the CEH-22 binding site could activate expression of Δ pes-10::GFP in pharyngeal and pharyngeal muscle cells, respectively ( Kuchenthal et al. 2001 ; Vilimas et al. 2004 ). We therefore used this reporter to determine whether our motifs could function as pharyngeal enhancers. Figure 4 Five Newly Identified Motifs Function as Pharyngeal Enhancers (A–C) Nomarski differential contrast interference images of embryos representing three different stages of embryonic development: (A) “early” development, when the pharynx primordium is formed, (B) “mid” development, when the pharynx has completed cell division and attached to the presumptive buccal cavity, and (C) “late” development, when pharynx development is almost complete and the embryo is about to hatch. Images on the left are of “early” embryos, images in the middle are of “mid” embryos, and images on the right are of “late” embryos. (D–U) Representative transgenic embryos showing expression from reporter constructs containing the Δ pes-10 promoter alone (D–F) or with insertion of three copies of Early-1 (G–I), Early-2 (J–L), Late-2 (M–O), P-1 (P–R), or P-2 (S–U). Dashed lines indicate the outline of the developing pharynx. Our heterologous promoter assay demonstrated that five of the seven pharyngeal motifs could function as enhancers in vivo. Early-1 and Early-2 activated pharyngeal expression early, after specification of pharyngeal precursors at the 200-cell stage but before formation of the pharynx primordium ( Figure 4 G– 4 L). In both cases, activity was confined to embryos. Early-1 was active in most or all pharyngeal cells, with occasional activity in non-pharyngeal cells. Early-2 was active in most or all pharyngeal cells and many non-pharyngeal cells in the head, suggesting that Early-2 could be a regulator of anterior or head-specific expression. The Late-2 element enhanced expression in the majority of pharyngeal cells, beginning at mid-embryogenesis, when the pharynx primordium initiates morphogenesis and differentiation ( Figure 4 M– 4 O; 1.5 fold stage; Sulston et al. 1983 ; Portereiko and Mango 2001 ). Late-2-dependent activity continued through embryogenesis but was relatively low or absent in larvae and adults. P-1 and P-2 functioned in pharyngeal cells after formation of the pharynx primordium, but both were relatively weak, variable enhancers compared to Early-1, Early-2, and Late-2 ( Figure 4 P– 4 U). Late-3 exhibited no enhancer activity. E1var, Early-3, and P-3 were not tested with this assay because of their similarity to other motifs (Early-1, CEH-22, and PHA-4, respectively). We conclude that five of the candidate elements can activate transcription broadly within the pharynx at distinct developmental stages. Tests of the Late-1 element revealed that this motif functions as a repressor rather than an enhancer ( Figure 5 ). We demonstrated repression using a modified version of the Δ pes-10 promoter in which three copies of the PHA-4 binding site were placed upstream of the Δ pes-10 basal promoter to activate expression within the digestive tract (3× pha-4 P::Δ pes-10 ::GFP). This construct expressed robustly in embryos and weakly in larvae and adults. To examine the Late-1 element for repressive activity, we inserted three copies of the Late-1 motif upstream of the PHA-4 sites. Remarkably, the presence of the Late-1 elements resulted in a marked decrease in embryonic GFP expression but had no effect on larval expression ( Figure 5 ). Embryos that expressed GFP from the 3×Late-1::3× pha-4 P::GFP reporter were late-stage embryos in which the pharynx was nearly or completely developed. Conversely, the Late-1 element was not active when tested for enhancer activity with the Δ pes-10 promoter (data not shown). Thus, Late-1 functioned as a repressor of early gut expression. Late-1 may also function in cells outside of the digestive tract, but this aspect of regulation was not tested. Figure 5 Late-1 Represses Early PHA-4-Dependent Expression Percent values indicate the percentage of transgenic animals exhibiting pharyngeal GFP expression. A reporter construct with three copies of a high-affinity PHA-4 site (TGTTTGC) upstream of the Δ pes-10 promoter (A) expresses GFP in pharyngeal cells of most transgenic embryos (B) and roughly one-third of transgenic larvae (C). The addition of three copies of the Late-1 element from R07B1.9 (CCTTGGCGGCGC) to this transgene (D) drastically reduces expression in transgenic embryos (E) but has no observable effect on transgenic larvae (F). Dashed lines indicate the outline of the pharynx. To characterize the temporal elements further, we examined their activity in endogenous pharyngeal promoters. We searched for examples of these motifs in predicted promoters that were conserved between C. elegans and C. briggsae to enrich for functionally relevant versions of motifs, rather than those that arose by chance alone. For Early-1 and Early-2, we tested the activity of these elements in the promoter of the early-onset pharyngeal gene K07C11.4. For Late-1, we tested the activity of this element in the promoter of the late-onset pharyngeal gene R07B1.9. We constructed a GFP reporter for K07C11.4 that faithfully recapitulated the expression observed by in situ hybridization experiments ( Figure 6 ; Kohara 2001 ). K07C11.4::GFP was expressed from the stage of pharynx primordium formation to adulthood in the pharynx, midgut, and hindgut. K07C11.4::GFP was also active in the proximal somatic gonad of late larvae and adults. Alignment of K07C11.4 promoter sequences (500 bp upstream of the predicted ATG) between C. elegans and C. briggsae revealed stretches of conserved sequences. These regions contain two predicted PHA-4 binding sites (at −151 to −157 and −141 to −147, relative to the ATG), an Early-1 motif (−114 to −123), and an Early-2 motif (−217 to −225). The distal predicted PHA-4 site had a relatively high affinity for PHA-4 in vitro, consistent with this gene being expressed early in pharyngeal development ( Gaudet and Mango 2002 ). Figure 6 Early-1 and Early-2 Elements Are Required for K07C11.4 Expression (A) A portion of the promoter sequence of K07C11.4 from C. elegans (bottom) aligned with its ortholog from C. briggsae (top). Boxed regions show conserved predicted PHA-4 binding sites and Early-1 and Early-2 elements. Site-directed mutations that disrupt Early-1 and Early-2 (“E2 + E1 Mut”) are shown below their respective wild-type (“E2 + E1 WT”) sequence from K07C11.4. (B–E) Confocal images of mid-stage embryos expressing GFP under the control of the wild-type K07C11.4 promoter (B) or promoters with a mutation in Early-1 (C), Early-2 (D), or both Early-1 and Early-2 (E). Percentages are the fraction of transgenic embryos expressing GFP; the remainder of embryos do not express GFP. (F) Expression of the wild-type K07C11.4 reporter in a subset of somatic gonad cells in an L4 animal (arrowheads). (G) Mutation of the Early-1 element eliminates gonadal expression but does not strongly affect expression in other tissues, such as intestinal cells (arrows). Dashed lines indicate the outline of the developing pharynx. Early-1 and Early-2 elements were both positive regulatory elements for K07C11.4. Mutation of either Early-1 or Early-2 elements resulted in a decrease in the strength of reporter expression, but did not affect timing or cell-type specificity ( Figure 6 C, 6 D, and 6 G). Interestingly, the two elements made distinct contributions to the activity of the K07C11.4 promoter: mutation of the Early-2 element had a relatively moderate effect on all aspects of K07C11.4 promoter activity, while mutation of the Early-1 element had only a mild effect on expression in the digestive tract but completely abolished expression in the somatic gonad. Simultaneous mutation of both elements virtually extinguished expression in all tissues, suggesting that the two elements functioned additively for K07C11.4 expression ( Figure 6 E). We conclude that Early-1 and Early-2 elements are bona fide cis -regulatory elements for early gene expression in the pharynx and some non-pharyngeal tissues. To assess Late-1 activity, we constructed a reporter for R07B1.9 that reproduced the endogenous pattern of expression (from in situ hybridizations; Kohara 2001 ; Figure 7 ). R07B1.9 was activated during the terminal stages of pharynx development, (the 3-fold stage; Sulston et al. 1983 ), and its expression was maintained throughout the life of the animal ( Figure 7 B and 7 C). The promoter of R07B1.9 contained two predicted PHA-4 binding sites (−243 to −249 and −221 to −227) and one Late-1 element (−170 to −180), all of which were conserved in C. briggsae ( Figure 7 A). Figure 7 The Late-1 Element Negatively Regulates R07B1.9 (A) A portion of the promoter sequence of R07B1.9 from C. elegans (bottom) aligned with its ortholog from C. briggsae (top). Boxed regions show conserved predicted PHA-4 binding sites and a Late-1 element. The site-directed mutation that disrupts Late-1 (“Mut”) is shown below the respective wild-type sequence from R07B1.9. (B and C) Confocal images of representative early and late embryos expressing GFP under the control of the wild-type R07B1.9 promoter. (D and E) Confocal images of representative early and late embryos expressing GFP under the control of the R07B1.9 promoter with a mutation in the Late-1 element. Note the early activation of R07B1.9 when Late-1 is inactivated. Dashed lines indicate the outline of the developing pharynx. The Late-1 element functioned as a repressor for R07B1.9, as predicted. Elimination of the Late-1 element led to precocious expression 2–3 h earlier than the wild-type ( Figure 7 D and 7 E). GFP was first visible when the pharynx primordium was formed and continued throughout the life of the animal. The mutant reporter was expressed in the same cells and at the same approximate strength as the wild-type, suggesting that disruption of the Late-1 element specifically affected the onset of gene expression. We conclude that six of the motifs (Early-1, Early-2, Late-1, Late-2, P-1, and P-2) display activity in vivo and that at least three of these contribute to temporal regulation of endogenous pharyngeal genes. Temporal Elements and PHA-4 Sites Determine Onset of Expression Our analyses suggested that the Early and Late elements function in combination with PHA-4 sites to modulate the onset of pharyngeal gene expression. We performed three tests to address the generality of this model. First, we surveyed the Ph-E and Ph-L gene clusters to determine the configurations of the five regulatory elements (Early-1, Early-2, Late-1, Late-2, and PHA-4 sites) within endogenous promoters. Second, we constructed synthetic promoters to examine the interplay between the temporal elements and PHA-4 binding sites. Third, we combined our observations from the synthetic and endogenous promoters to search the genome for genes containing similar configurations of the five cis -regulatory motifs. Temporal regulatory elements in Ph-E and Ph-L genes We examined Ph-E and Ph-L genes to see if their promoters contained distinct combinations of Early, Late, and PHA-4 sites ( Table 4 ; Table S4 ). For PHA-4 sites, we also examined the predicted affinity of these sites because the affinity of PHA-4 for its binding sites influences the onset of expression ( Gaudet and Mango 2002 ). For Ph-E and Ph-L, 22/33 and 19/33 genes, respectively, had temporal elements and predicted PHA-4 sites that were conserved in their C. briggsae orthologs. Interestingly, particular combinations of elements appear to be associated with Ph-E or Ph-L genes ( Table 4 ). For example, combinations of Early elements with PHA-4 sites of predicted high or medium affinity are far more frequent in Ph-E genes than in Ph-L genes (11/22 [50%] versus 2/19 [11%], respectively), suggesting that this configuration of elements promotes early pharyngeal expression. In contrast, combinations of Late elements with PHA-4 sites of varying affinity are frequent in Ph-L genes (9/19 [47%]) but do not occur at all in Ph-E genes. In addition, promoters with only low-affinity PHA-4 sites together with any temporal elements are more common in Ph-L than in Ph-E genes (4/19 [21%] versus 2/22 [9%], respectively). These trends suggest that late pharyngeal gene expression is promoted by the combination of either Late elements with any PHA-4 site, or low-affinity PHA-4 sites with any temporal elements. We conclude that expression of at least half of Ph-E and Ph-L genes can be accounted for by a combination of PHA-4 sites and temporal elements. Table 4 Combinations of Temporal Elements and PHA-4 Sites Predict Onset of Pharyngeal Gene Expression For temporal elements, “Early” indicates the presence of one or more of Early-1 and/or Early-2, and “Late” indicates the presence of one or more of Late-1 and/or Late-2. In all cases, motifs are present in both the C. elegans and C. briggsae orthologs of a gene. PHA-4 sites are as follows: “High” is TGTTTGC, TGTTGAC, or TGTTTAC; “Med” is TGTTTGT; “Low” is TATTTGT ( Gower et al. 2001 ; Gaudet and Mango 2002 ) (N. Gower and H. Baylis, personal communication); “Unk” is all other TRTTKRY except TATTTAT, which shows no appreciable binding of PHA-4 in vitro (unpublished data). We predict onset of expression for particular combinations of elements based on the prevalence in the promoters of Ph-E and Ph-L genes a Onset of expression of these genes will depend on the relative affinity of the PHA-4 sites. For a complete description of individual genes and their elements, see Table S4 Synthetic promoters recapitulate temporal expression The combination of elements observed in natural promoters suggests that a gene with a high-affinity PHA-4 site and an Early element will be expressed early in development, while a gene with a high-affinity PHA-4 site and a Late element will be expressed later in development. From the existing Ph-E and Ph-L promoters, however, the output of certain promoter configurations cannot be reliably predicted. For example, when a low-affinity PHA-4 site is paired with an Early element, is the gene activated early or late in development? A low-affinity PHA-4 site in the presence of an Early element might be expected to activate expression relatively late, as determined by the binding of PHA-4 to the low-affinity site, or early, if the Early element potentiates earlier expression. To test these ideas, we constructed artificial promoters within the context of the Δ pes-10 promoter and examined their expression patterns. We first investigated the behavior of the high- and low-affinity PHA-4 binding sites in synthetic promoters, because the configuration of Ph-E and Ph-L promoters together with previous work ( Gaudet and Mango 2002 ) suggested that onset of expression was influenced by the affinity of PHA-4 sites. To test this idea, we compared the activity of three copies of either a high- or low-affinity PHA-4 site placed in front of the Δ pes-10 promoter ( Figure 8 ). As observed previously ( Okkema and Fire 1994 ), three copies of a high-affinity PHA-4 site were sufficient to activate pharyngeal expression beginning early in development, prior to formation of the pharynx primordium ( n = 34/36). In contrast, three copies of a low-affinity PHA-4 site were sufficient to activate pharyngeal expression later in development. Early expression was observed significantly less frequently with the low-affinity constructs than with the high-affinity constructs ( n = 28/71). Notably, the strength of expression of both constructs was comparable in late embryos. We conclude that promoter activation depends on the affinity of PHA-4 for its binding sites and cannot be compensated for by multimers. This finding agrees with data that show FoxA factors bind DNA as monomers, not cooperatively ( Clark et al. 1993 ). Figure 8 High-Affinity PHA-4 Sites Activate Pharyngeal Expression Earlier Than Low-Affinity Sites Percent values indicate the percentage of transgenics exhibiting pharyngeal GFP expression. Dashed lines indicate the outline of the developing pharynx. (A–C) A reporter construct with three copies of a high-affinity PHA-4 site (TGTTTGC) upstream of the Δ pes-10 promoter reproducibly activates pharyngeal expression from the time of pharynx primordium formation (“early”) through embryogenesis. (D–F) A reporter construct with three copies of a low-affinity PHA-4 site (TATTTGT) upstream of the Δ pes-10 promoter activates pharyngeal expression from the time of attachment of the pharynx to the mouth (“mid”) through embryogenesis. We next tested synthetic promoters containing a single PHA-4 site (either high or low affinity) with a single temporal element (either Early-1 or Late-2) to determine how these elements behave in combination. The synthetic promoter constructs differed from our initial Δ pes-10 ::GFP reporter survey in that they resembled the configuration of endogenous promoters. While temporal elements and PHA-4 sites do not appear to exhibit conserved spacing or order within pharyngeal promoters, each element typically occurs in one or two copies per promoter. We therefore constructed reporters in which one copy of either an Early-1, Early-2, or Late-2 element was paired with one copy of either a high- or low-affinity PHA-4 site, separated by approximately 100 bp. The onset of expression for artificial promoters containing an Early-1 site depended on the relative affinity of the accompanying PHA-4 site ( Figure 9 ), as predicted. The promoter with a high-affinity PHA-4 site was reliably expressed at all embryonic stages examined, including early pharyngeal development ( n = estimated number of transgenic embryos scored = 17/23; see Materials and Methods for an explanation of the estimate). In contrast, the promoter with the low-affinity PHA-4 site was consistently expressed in late embryos, but only infrequently in earlier stages ( n = 4/58). Because both artificial promoter constructs activated expression comparably in late embryos, we conclude that the differences in early expression reflect a genuine difference in the onset of expression between constructs rather than a difference in the strength or penetrance of expression. These data support a model in which the Early-1 element is able to mediate early pharyngeal gene expression but where the onset of gene expression is ultimately limited by the relative affinity of the PHA-4 binding site. We similarly tested Early-2 together with a high-affinity PHA-4 site, but did not observe any expression above background, suggesting that this combination or configuration of sites was not sufficient to activate pharyngeal expression. Figure 9 Temporal Elements Combined with PHA-4 Sites Regulate the Onset of Pharyngeal Expression “Early,” “mid,” and “late” are as defined in Figure 3 . E1, Early-1; L2, Late-2; High, high-affinity PHA-4 site (TGTTTGC); Low, low-affinity PHA-4 site (TATTTGT). Percent values indicate the percentage of transgenics exhibiting pharyngeal GFP expression; the remainder of embryos do not express GFP. A reporter construct with one copy of the Early-1 element and one copy of a high-affinity PHA-4 site is expressed in “early” to “late” embryos (A–C). In contrast, a reporter with one copy of the Early-1 element and one copy of a low-affinity PHA-4 site is not consistently expressed until the “mid” to “late” stages (D–F). Reporters with one copy of Late-2 and one copy of either a high-affinity (G–I) or low-affinity (J–L) PHA-4 site are expressed in “mid” and “late” stage embryos. Dashed lines indicate the outline of the developing pharynx. Artificial promoters containing the Late-2 element were not expressed until mid-embryonic to late embryonic development, regardless of the relative affinity of the PHA-4 site ( Figure 9 ; n = 1/73 early embryos for constructs with a high-affinity PHA-4 site, n = 2/37 early embryos for constructs with a low-affinity site). Because the Late-2 element behaved as an enhancer, we hypothesize that the factor or factors that act through Late-2 are not available until late in development and that their absence in early development results in delayed expression of Late-2 dependent genes. Genome searches identify additional pharyngeal genes The temporal motifs offered an opportunity to discover new pharyngeal genes based solely on predicted cis -regulatory sites. We searched the C. elegans and C. briggsae genomes for conserved occurrences of the temporal motifs, together with PHA-4 sites, to determine whether combinations of these sites were predictive for pharyngeal genes. We required the presence of three elements within a 500-bp stretch because Ph-E and Ph-L averaged a total of 3.7 elements in their promoters and because artificial promoters with only two elements generated weaker expression than endogenous promoters ( Figure 9 ). We chose a stringent threshold score for each element that maximized the ability to distinguish elements in control runs of early versus late pharyngeal genes (see Materials and Methods ). Although these thresholds reduced the number of elements identified in the positive control set (e.g., the Early-1 element in Ph-E promoters) they also dramatically lowered the false positive rate (e.g., the Early-1 element in Ph-L promoters). The combination of Early-1, Early-2, and PHA-4 sites provided a powerful approach to predict early pharyngeal genes. Of 40 genes with conserved copies of all three elements, 20 had available expression data, and 70% of these were expressed in the pharynx (14 genes; Table S5 ). In contrast, of 194 randomly selected genes, 57 had expression data and only 12% (7/57) of these were expressed in the pharynx (data not shown), indicating that the results of the genomic searches are significantly enriched for pharyngeal genes ( p = 6 × 10 −7 ). Furthermore, of the 14 pharyngeal genes identified by the genomic search, 86% (12/14) were expressed early in pharyngeal development, as predicted. The 14 pharyngeal genes included ten genes not identified in our microarray experiments, reflecting the power of bioinformatics in predicting gene expression. Given the possibility that E1var is a functional temporal element, we performed genomic searches for genes that contain conserved E1var plus Early-2 plus PHA-4 elements together within 500 bp of their predicted start codon. This search identified 120 genes with some overlap with the Early-1 search output. Of these 120 genes, 53 had expression data ( Table S5 ), and 47% of these (25/53) were expressed in the pharynx, a significant enrichment compared to the random set ( p = 6 × 10 −5 ), suggesting that, like Early-1, E1var in combination with other sites is predictive for pharyngeal expression. Of these pharyngeal genes, 70% (16/23; onset of expression not determined for two genes) were expressed early, as predicted. Combining the results of the Early-1 and E1var searches, we identified a total of 35 pharyngeal genes, 73% of which (24/33) were expressed early. For late expression, 61 genes had conserved Late-1, Late-2, and PHA-4 sites, and these were also enriched for pharyngeal genes. Thirty of these genes had expression data, of which 33% (ten genes) were pharyngeally expressed ( Table S5 ), showing significant enrichment compared to the random gene set ( p = 0.02). Strikingly, the Late elements accurately predicted the timing of expression in the pharynx, as all ten pharyngeal genes were expressed late in pharyngeal development. Considering the Early and Late genomic searches together, we identified 45 pharyngeal genes, 36 of which were not present in our microarray positives. Furthermore, the onset of expression of these 45 pharyngeal genes was predicted with 88% overall accuracy. There are three different reasons for our microarray experiments not identifying the 36 new pharyngeal genes: (1) some genes are expressed in both pharyngeal and non-pharyngeal cells and would not be significantly enriched in the par-1 versus skn-1 samples, (2) some genes are expressed post-embryonically and would not have been present in the embryonic samples used for the microarray experiments, and (3) some genes scored just below the 2-fold enrichment threshold for inclusion in our positive set. Discussion The advent of microarray technology and bioinformatics has provided a powerful tool to dissect the transcriptional regulatory circuits that guide complex developmental processes. We have analyzed pharynx organogenesis and defined novel regulatory elements that contribute to temporal gene expression. Our screens differ from previous examples of bioinformatic analyses in that the regulatory motifs were identified in unbiased searches of promoter sequences and subsequently tested for biological activity. We used three assays to demonstrate a function in vivo. First, we tested whether the regulatory elements were necessary for expression of native pharyngeal genes. Second, we determined whether they were sufficient for pharyngeal expression from synthetic promoters. And third, we did genome-wide searches based on these elements, a process that identified 19 new pharyngeal genes, of which 88% displayed the expected onset of expression. The cis -regulatory motifs discovered here, combined with the PHA-4 binding site, establish a regulatory network that can account for the timing of activation of at least half of C. elegans pharyngeal genes. A Model for Temporal Control of Pharyngeal Gene Expression We previously proposed a model in which the relative affinity of PHA-4 for its binding sites controls the onset of pharyngeal gene expression. Because PHA-4 protein levels increase during development, we proposed that initially PHA-4 levels are low and only high-affinity sites are sufficiently occupied by PHA-4 to result in gene activation. As PHA-4 levels increase over time, lower-affinity sites also become occupied, leading to the expression of those genes. In addition, binding site affinity likely affects PHA-4 occupancy even at stable PHA-4 concentrations. The degree of PHA-4 occupancy would likely alter the probability of productive transcription of a target gene. However, the affinity model could not explain all temporal expression because some late-expressed pharyngeal genes had high-affinity PHA-4 binding sites (e.g., myo-2 and R07B1.9). In this study, we have identified additional regulatory elements that establish the onset of pharyngeal gene expression in combination with PHA-4. We suggest that the affinity of PHA-4 for its binding site determines the earliest possible time of pharyngeal gene expression but that other factors must be present for a promoter to be active. The Early elements likely represent binding sites for transcription factors that are available throughout embryonic pharyngeal development. In contrast, the Late-2 element is probably recognized by a transcription factor that is not available until midway through pharynx development, thereby delaying expression of these pharyngeal genes regardless of the quality of the PHA-4 binding sites. We note that our searches were restricted to genes with similar onset of expression, in order to identify temporal regulatory elements, and there may be yet more undiscovered elements that control temporal expression. For example, an element that confers cell-type-specific expression could also control onset of expression according to the availability of the relevant binding factor. Consistent with the idea of additional motifs, the synthetic promoters with two enhancer elements were functional but weak activators of expression compared to native promoters, which likely contain more regulatory motifs (e.g., myo-2 ). In addition to enhancers of pharyngeal expression, we identified one negative regulatory element, Late-1, that is dominant to PHA-4 in early pharynx development. The proximal PHA-4 consensus site in the R07B1.9 promoter is predicted to be a high-affinity binding site, suggesting that R07B1.9 should be expressed early in pharynx development, not late as is observed. Mutation of the Late-1 element enabled the R07B1.9 promoter to fire at earlier developmental stages. We hypothesize that some factor binds to Late-1 and represses expression in early pharynx development. Subsequently, the Late-1 factor is presumably inactivated or downregulated to permit gene expression. The elements characterized here can account for the temporal expression patterns of many but not all known pharyngeal genes. One possible reason for this is that we do not yet have sufficient information to identify all functionally relevant occurrences of these elements. For example, some occurrences of the temporal elements may not be biologically meaningful, thereby generating false positives, while others may be hard to identify because of sequence heterogeneity or placement within cis -regulatory sequences. Our analyses initially focused on regions within 500 bp of the predicted start codon of genes, but we further searched −501 to −1,000 upstream of the ATG for Ph-E and Ph-L genes whose expression could not be accounted for by elements in the region of −1 to −500. However, these extended searches did not find any conserved elements that could further account for onset of expression (data not shown). Another possibility is that regulatory elements may be found within introns or the 3′ UTR ( Shibata et al. 2000 ; Marshall and McGhee 2001 ; Gaudet and Mango 2002 ; Kirouac and Sternberg 2003 ). Consistent with this observation, we find that 40% of the Ph-E and Ph-L genes show significant conservation of intron sequence between C. elegans and C. briggsae (data not shown). Thus, some of the genes whose onset of expression cannot be accounted for in our analyses likely have larger and more complex regulatory regions than the 500-bp window we used. Nonetheless, this limited sequence window allowed us to account for the onset of expression of roughly half of the Ph-E and Ph-L genes. An interesting feature of the Early-1 element is that it is necessary for the expression of K07C11.4 in the proximal somatic gonad, as well as in the pharynx. Some, though not all, genes containing conserved Early-1 elements are expressed in the proximal somatic gonad (e.g., F30H5.3, F47D12.7, and ncr-1 ; Kohara 2001 ), consistent with Early-1 being necessary but not sufficient for gonadal expression. PHA-4 is also expressed in the somatic gonad, including the proximal region ( Azzaria et al. 1996 ; Kalb et al. 1998 ). The finding of multiple genes expressed in the two tissues under control of Early-1 and possibly PHA-4 suggests the presence of shared regulatory mechanisms in the two organs. Both the pharynx and proximal somatic gonad are epithelial tubes that connect to the external environment and as such may have conserved features that are regulated by some of the same factors. The role of PHA-4 in the gonad has not been carefully analyzed, but PHA-4 is critical for proper gonad development post-embryonically ( Ao et al. 2004 ). Candidate trans- Acting Factors The identification of new pharyngeal regulatory elements provides an entry point for identifying the relevant transcription factors that bind these sequences. We searched the TRANSFAC database and available literature for possible matches to the temporal elements identified here ( Knuppel et al. 1994 ; Matys et al. 2003 ). While we did not discover obvious candidate factors for the relevant transcription factors, we did find intriguing similarities between our elements and known transcription factor binding sites. The Early-1 element resembles the recognition sequence for Drosophila ZESTE and GAGA (YGAGYG and GAGAG, respectively; Benson and Pirrotta 1987 ; Omichinski et al. 1997 ). However, no clear ortholog of either gene exists in C. elegans . Early-2 resembles a hemi-site for nuclear hormone receptors, but the identification of specific candidate factors is complicated by the existence of 284 predicted nuclear-hormone-receptor-encoding genes in C. elegans ( Maglich et al. 2001 ). The Late-1 element is a GC-rich sequence that resembles a Sp-factor binding site and behaves as a negative regulator of early expression. C. elegans has four Sp-like homologs ( Zhao et al. 2002 ), one of which is a predicted pharyngeal gene (F45H11.1). However, F45H11.1(RNAi) did not affect expression of the Late-1-regulated gene R07B1.9 (data not shown). The Late-2 element (TTTTTCC) most closely resembles a Dorsal/Rel-homology domain binding site (KGGWWWWCCC; Matys et al. 2003 ), but there are no C. elegans Rel-homology domain proteins. Given the lack of other obvious candidate factors for the temporal elements, molecular or genetic screens will be required to identify the relevant transcription factors. Elements and Regulatory Modules Recent studies of transcription factor binding sites have revealed cases of multimers of a single site being important for expression ( Berman et al. 2002 ; Markstein et al. 2002 ; Yoo et al. 2004 ) and cases of entire modules of elements being conserved features of some promoters ( Senger et al. 2004 ). By contrast, we see little evidence of conserved spacing, order, or organization of our elements within pharyngeal promoters. Genomic searches for genes with multiple copies of a single element yielded few pharyngeal genes, suggesting that the temporal elements and PHA-4 sites typically act in single copy (data not shown). The analysis of PHA-4 targets suggests two strategies of transcriptional control. We propose that a minority of target genes respond consistently to the presence of PHA-4. For example, K07C11.4, T05E11.3, and M05B5.2 are active broadly throughout the pharynx and in other cells that express PHA-4, such as the gonad or rectum ( Gaudet and Mango 2002 ). These promoters contain four or more predicted PHA-4 binding sites, including at least one high-affinity site. The density of high-quality PHA-4 binding sites may promote activation of these genes whenever PHA-4 is present. This strategy may have been adopted by other transcription factors. For example, likely target genes of the Notch effector CSL (also known as CBF1/RBP-Jκ e , Su(H), and LAG-1) have been discovered in C. elegans and Drosophila ( Christensen et al. 1996 ; Rebeiz et al. 2002 ; Yoo et al. 2004 ). Many of these genes encode components of the Notch signaling pathway that function upstream of CSL and would therefore be expected to respond broadly to CSL as part of a regulatory feedback loop. Intriguingly, these genes contain multiple copies (e.g., 15–25) of the CSL binding site consensus, which may facilitate CSL-mediated activation ( Christensen et al. 1996 ). On the other hand, the majority of pharyngeal genes respond to PHA-4 in some cellular environments but not others ( Okkema and Fire 1994 ; Gaudet and Mango 2002 ). These genes appear to depend on a second regulatory strategy, in which combinations of elements synergize, with no individual element sufficient for transcription. These promoters typically carry one or two copies of any given element, as observed for the myo-2 promoter ( Okkema and Fire 1994 ; Thatcher et al. 2001 ; Gaudet and Mango 2002 ; Ao et al. 2004 ). Similarly, CSL factors have target genes that are activated in only a subset of tissues, and these contain only one or a few high-quality binding sites (for example, vestigial or D-pax2; Kim et al. 1996 ; Flores et al. 2000 ). The combinatorial mode of regulation relies on the relatively poor transcriptional activity of the individual factors. For example, a single Early-1 or PHA-4 binding site cannot activate expression of Δ pes-10 ::GFP. Moreover, PHA-4 can transform some cells towards a pharynx fate, but is not as potent as other developmental regulators such as end-1 ( Horner et al. 1998 ; Kalb et al. 1998 ; Zhu et al. 1998 ). Ectopically-expressed END-1 can transform the entire embryo into midgut ( Zhu et al. 1998 ). Accordingly, END-1 is expressed briefly within midgut precursors, where it likely activates a uniform panel of downstream targets ( Zhu et al. 1997 ). We suggest that the inherent activity of a transcriptional regulator coupled with promoter architecture defines the range of target genes available to a developmental transcription factor. The combinatorial mode of regulation exhibited by PHA-4 provides the organism with transcriptional flexibility in two ways. First, it provides a mechanism for selectivity for Fox transcription factors. The worm genome encodes fifteen Fox transcription factors, and these proteins regulate diverse biological activities such as cell fate specification, longevity, and cell migration ( Miller et al. 1993 ; Ogg et al. 1997 ; Horner et al. 1998 ; Kalb et al. 1998 ; Nash et al. 2000 ; Sarafi-Reinach and Sengupta 2000 ; Hope et al. 2003 ). Combinatorial regulation affords the animal with a means to distinguish different target genes for different Fox proteins, all of which share a similar DNA binding domain. Second, the combinatorial strategy enables PHA-4 to play a broad role in the pharynx. PHA-4 is activated at the earliest stages of organogenesis in all pharyngeal cells, where it is required to specify different pharyngeal cell types. PHA-4 continues to be expressed throughout the life of the animal, where it likely controls pharyngeal function and growth. These different functions presumably reflect different target genes activated in different cell types or during different developmental stages. FoxA factors in other animals exhibit long-term expression and, like PHA-4, are poor inducers of cell fate when expressed ectopically ( Sasaki and Hogan 1994 ). FoxA target genes such as albumin require additional factors for activation, and FoxA promoter association is not sufficient for transcription ( Zaret 1999 ). These data indicate that mammalian FoxA proteins likely rely on a transcriptional strategy similar to that of worms. Materials and Methods Identification of genes selectively expressed in the pharynx C. elegans strains KK822 ( par-1(zu310) IV; Kemphues et al. 1988 ) and EU1 ( skn-1(zu67)/DnT1 IV;V; Bowerman et al. 1992 ) were grown in liquid culture with OP50 as a food source, synchronized, and harvested. For KK822, we shifted synchronized homozygotes to the restrictive temperature (25 °C) and isolated embryos by hypochlorite treatment ( Sulston and Hodgkin 1988 ). For EU1, we grew synchronized animals and plated young adults. skn-1/DnT1 worms are uncoordinated, while skn-1 homozygotes are non-uncoordinated, allowing us to enrich for skn-1 homozygotes using a plate crawling assay. To synchronize, we performed a first round of hypochlorite embryo isolation and then allowed embryos to hatch overnight in liquid culture lacking food to obtain L1 larvae. We then added food to the cultures and grew the animals for 2–3 d to age them. For par-1, animals were aged to young adults, and embryos were collected from these by hypochlorite treatment. For the skn-1/DnT1 strain, animals were aged to L4 and then transferred to large plates for the crawling assay and later collected as young adults for embryo isolation. Collected embryos from either strain were aged another 3–6 h in small liquid cultures to ensure that par-1 and skn-1 embryos were at approximately the same stage of development, as determined by examining a sample of embryos under the light microscope. To extract total RNA from isolated C. elegans embryos, embryos pellets were frozen in microfuge tubes, crushed with a plastic pestle, and resuspended in RNA extraction buffer (1% lauroyl sarcosine, 0.1 M Tris base, 0.1 M NaCl, and 20 mM EDTA), followed by several rounds of phenol:chloroform extraction and ethanol precipitation ( Horner et al. 1998 ; Gaudet and Mango 2002 ). Once-selected poly-A+ RNA was purified using the PolyTract Isolation Kit (Promega, Madison, Wisconsin, United States). par-1 cDNAs were labeled with Cy3, and skn-1 cDNAs were labeled with Cy5. Labeled cDNA was prepared from 5 μg of poly-A+ RNA by the Huntsman Cancer Institute Microarray Core Facility. Construction and probing of the microarrays was as described by Reinke et al. under the auspices of the Kim lab ( Reinke et al. 2000 ). Analysis of microarray data We performed two microarray experiments using microarrays containing 62% of the C. elegans genome (“partial arrays,” experiments PS1 and PS2) and three experiments using microarrays containing 94% of the genome (“full arrays,” experiments PS3, PS4, and PS5). Our previous microarray experiments (PS1–PS3) detected 242 positives, corresponding to 227 genes ( Gaudet and Mango 2002 ). To extend the identification of candidate pharyngeal genes, we included data from PS4 and PS5. In this case, we selected genes that had an average log 2 ( par-1/skn-1 ) ≥ 1.00 in PS1–PS5 and were expressed above background in three of five experiments. In this selection, we observed considerable background of non-pharyngeal genes, primarily genes with maternally contributed transcripts or expression in pre-gastrulation embryos. We hypothesize that this background is the result of our par-1 embryos being harvested at an earlier stage than our skn-1 embryos in experiments PS4 and PS5. Because PS1–PS3 do not appear to exhibit this difference in staging, we applied an additional selection criterion to our data, requiring that all positives have a log 2 ( par-1/skn-1 ) ≥ 0.58 on PS1, PS2, or PS3. This threshold reduces the inclusion of non-pharyngeal genes identified by PS4 and PS5 and identifies a total of 241 microarray positives. We further subtracted probable maternal genes from this set of positives using the C. elegans expression map of Kim et al. (2001) , leaving us with 118 microarray positives corresponding to 112 new candidate pharyngeal genes. As a validation of these experiments, we note that among these 112 new genes are 44 genes with known expression patterns, and 33/44 (75%) of these are pharyngeally expressed. This fraction of pharyngeal genes is comparable to the fraction of pharyngeal genes present in our set of positives from PS1–PS3 (81/97 [84%]). Examining sequence conservation C. elegans and C. briggsae upstream and downstream sequences were extracted from the genome using the Ensembl EnsMart tool ( Kasprzyk et al. 2004 ). For four genes, ENSEMBL did not provide orthologous C. briggsae sequences. In these cases we used the Intronerator Tracks Display ( Kent and Zahler 2000 ) to obtain the C. briggsae sequence. Sequences were then aligned and visualized by VISTA ( Mayor et al. 2000 ). We scored as conserved those regions of DNA that had 75% or greater identity over 50 or more base pairs. Alignments in Figures 6 and 7 were obtained from the Tracks Display feature of Intronerator ( Kent and Zahler 2000 ). Motif searches using Improbizer We used the Improbizer program ( Ao et al. 2004 ; available at http://www.soe.ucsc.edu/approximately/kent/improbizer/ ), which employs a variation of the expectation maximization algorithm ( Bailey and Elkan 1994 ), to search for motifs. Improbizer was able to find expected regulatory elements (i.e., motifs that resemble PHA-4 and CEH-22 binding sites) in our promoter sequences, while other algorithms did not. Improbizer can be configured to simultaneously search for motifs on both strands of DNA, search for more than a single occurrence of a motif on each DNA sequence, and use a separate set of sequences for a background model. The source code for the Improbizer is freely available, and is the definitive reference for the details of the algorithm. In using Improbizer to search the Ph-E and Ph-L gene sets for possible regulatory motifs, we initially searched for motifs occurring once per sequence, with an initial scan through the first five sequences entered. For each gene set we ran searches three or more times, varying the order of input for genes in each run. Motifs presented here were obtained with searches for a motif size of six. Searches for motifs of larger sizes (8–20 bases) recurrently found variations of the motifs presented here. Other parameters of Improbizer were used at their default settings. For background sequences, we used three different sets of sequences (foreground sequence, and upstream sequences from each of two gene groups defined by Kim et al. [2001] : neuronal genes and carbohydrate metabolism genes) and obtained similar output matrices in all cases. All matrices presented here were obtained using the input sequence (foreground) as the background. This mixing was performed to prevent output being biased towards sequence motifs found in only the first few input genes. The motifs presented here were reproducibly obtained independent of the order of input sequences. As an initial screen for motifs that were over-represented in our test sets, we performed control runs in which the input gene sequence was randomized and searched. All motifs presented here obtained Improbizer scores greater than the scores of ten or more control runs. Identification of motifs using Cluster-Buster Cluster-Buster finds the best possible occurrence of a motif (or motifs) in a given sequence and therefore requires the establishment of a threshold score to determine which occurrences are likely to be meaningful ( Frith et al. 2003 ). We established threshold scores for our motifs that maximized the ratio of “hits” in a positive versus a negative group, with a “hit” defined as a gene that contained an occurrence of a motif above the threshold. For example, we chose a threshold score for Early-1 that gave the greatest ratio of Ph-E hits to Ph-L hits. These thresholds were then applied to searches of C. elegans Ph-E and Ph-L genes and their C. briggsae orthologs to determine which genes contained Early and Late elements in both C. elegans and C. briggsae . Position within the promoter was not required for a motif to be considered “conserved.” For Tables 3 and S4 , the following parameters were used: the cluster score threshold (C) and gap parameter (g) for all motifs were zero and 35, respectively. The motif threshold (m) used for Early-1, Early-2, E1var, and Late-1 was six, for Late-2 was seven, and for PHA-4 was five. The random gene set referred to in Table 3 was generated using a random number generator to select 200 genes from a complete list of all predicted C. elegans genes. Duplicates or splice isoforms of a gene were collapsed to a single selection, resulting in a total of 194 genes. For the genome searches, we searched for genes that contained a set of elements (e.g., Early-1 plus Early-2 plus PHA-4) within the first 500 bp of upstream sequence in both C. elegans and C. briggsae . We applied more stringent thresholds to the elements for these searches, to minimize the identification of non-pharyngeal genes. We optimized the Early element thresholds by searching the genome to identify known Ph-E and Ph-L genes and selecting the search parameters that maximized the ratio of Ph-E/Ph-L genes identified. The Cluster-Buster ( Frith et al. 2003 ) parameters for Early-1 plus Early-2 combined were C = 3.5, m = 6, and g = 35. The PHA-4 parameters were C = 1.9, m = 6, and g = 35. For E1var plus Early-2 combined the parameters were C = 1, m = 5.5, and g = 35. The PHA-4 parameters were C = 2, m = 6, and g = 35. Using these thresholds, our genomic searches identified four known Ph-E genes but no Ph-L genes. The same approach was used to optimize parameters for genome searches with the Late elements, maximizing the ratio of Ph-L/Ph-E genes identified. The parameters for Late-1 plus Late-2 combined were C = 2, m = 6, and g = 35. The PHA-4 parameters were C = 2.5, m = 6, and g = 35. Construction of plasmids To construct transcriptional reporters, we amplified promoter sequences from genomic N2 DNA using gene-specific primers that contained restriction endonuclease sites to facilitate cloning. PCR products were cloned into the vector pAP.10, which carries a GFP::HIS2B translational fusion, resulting in a nuclear-localized GFP ( Gaudet and Mango 2002 ). This cloning strategy removed the pes-10 promoter sequence present in pAP.10. Enhancer constructs and synthetic promoters were built using synthetic oligonucleotides that were cloned into pAP.10, upstream of the Δ pes-10 promoter fragment. Clones were verified by restriction digests and sequencing. For the triplicate enhancer sequences, we used the following insert sequences (sequences of the individual motifs are underlined; periods show spacing of elements in Late-1): Early-1, AGAGACGCAG ATT AGAGACGCAG ATT AGAGACGCAG ATT; Early-2, T A ACTGACCGT CTTA ACTGACCGT CTTA ACTGACCGT CT; Late-1, CTTGGCGGCGCC . CTTGGCGGCGCC . CTTGGCGGCGC C; Late-2, CTC TTTTTCC CACTC TTTTTCC CACTC TTTTTCC CA; Late-3, AC TCTCGGAA TCAC TCTCGGAA TCAC TCTCGGAA TC; P-1, T TGCTCACC TAAT TGCTCACC TAAT TGCTCACC TAA; P-2, TT TCTTCCAAA TTT TCTTCCAAA TTT TCTTCCAAA T; PHA-4 (high), CTAC TGTTTGC CCCTAC TGTTTGC CCCTAC TGTTTGC CC; and PHA-4 (low), CTAC TATTTGT CCCTAC TATTTGT CCCTAC TATTTGT CC. For the synthetic promoters, individual Early-1 or Late-2 sites were cloned in to the SphI and SalI sites of pAP.10, and individual PHA-4 sites were cloned in to the NheI and NsiI sites of pAP.10. Fragments from these single-site constructs were ligated to generate the constructs containing one temporal site and one PHA-4 site, with the sites separated by 95 bp of pAP.10 sequence. The sequences of the synthetic regions of these constructs were as follows (individual motifs are underlined): Early-1 plus PHA-4 (high), GCATGCTCG AGAGACGCAG ATTGTCGAC-(95-bp)-GCTAGCTAC TGTTTGC CCCCGGGATGCAT; Early-1 plus PHA-4 (low), GCATGCTCG AGAGACGCAG ATTGTCGAC-(95-bp)-GCTAGCTAC TATTTGT CCCCGGGATGCAT; Late-2 plus PHA-4 (high), GCATGCTCGAGCTC TTTTTCC CATCGAC-(95-bp)-GCTAGCTAC TGTTTGC CCCCGGGATGCAT; and Late-2 plus PHA-4 (low), GCATGCTCGAGCTC TTTTTCC CATCGAC-(95-bp)-GCTAGCTAC TATTTGT CCCCGGGATGCAT. Sequences chosen were the best match to the position weight matrix (PWM) generated by Improbizer, except Late-1 (which is based on the functional element in R07B1.9), P-1 (which is based on two overlapping PWMs), and the PHA-4 sites (which are based on a functional site in the Ce-pax-1 promoter; J. Stevenson and S. E. M., unpublished data). PWMs from Improbizer are listed in Dataset S1 . Complete details of all oligonucleotides and plasmids are available upon request. Construction of transgenic lines Several groups have reported artificial pharyngeal expression resulting from sequences present in the vector backbone of reporter constructs (e.g., Hope 1991 ). To minimize this effect, we routinely removed all vector sequence from our reporter constructs prior to injection into C. elegans . For our transcriptional fusions, we used a gene-specific oligonucleotide together with an unc-54 3′ oligonucleotide that anneals downstream of the unc-54 3′ cassette present in our plasmids ( Fire et al. 1990 ) to PCR-amplify linear fragments for injection. For our enhancer constructs, we used an oligonucleotide that anneals approximately 200 bp upstream of the MCS of pAP.10 ( Gaudet and Mango 2002 ) together with unc-54 3′ to amplify transgenes. We then digested the PCR products with either StuI or SphI to remove the remaining approximately 200 bp of vector sequence and gel-purified the desired fragment for microinjection. These adjustments ensured that there was no spurious expression in pharyngeal cells from Δ pes-10::GFP in the absence of an enhancer or when three copies of a random sequence (corresponding to the degenerate sequence CWNCAYKGA) were placed in front of the Δ pes-10 promoter ( Figure 4 ; data not shown). Linear transcriptional reporters were injected at 0.5–1.0 ng/μl together with 30 ng/μl pRF4 ( Mello et al. 1991 ) cut with EcoRI and 70 ng/μl sheared herring sperm DNA ( Kelly et al. 1997 ). In all cases where expression of transgenes was compared, injections were performed under the same conditions. To establish transgenic lines, we picked Roller animals from the F2 generation. For all transgenes, a minimum of two independent lines were analyzed. Estimating percent GFP expression The transgenic marker that we used, rol-6(su1006), which confers a Roller phenotype ( Kramer et al. 1990 ), does not allow us to identify transgenic embryos. Therefore, we estimated the fraction of transgenic embryos that express GFP as follows. Embryos from transgenic adults were collected and split into two samples. The first sample was scored for stage and GFP expression, while the second sample was allowed to develop and eventually scored for percent Roller animals. The percent Roller score was used as an estimate of the percent of animals that were transgenics. The percent transgenics with GFP expression was therefore estimated to be equal to (number of embryos expressing GFP) / ((total number of embryos scored) × (percent Roller)). Where reported in the text, numbers of transgenic embryos scored were estimated by this same approach. Supporting Information Dataset S1 Position Weight Matrices from Improbizer (14 KB PDF). Click here for additional data file. Table S1 The 339 Microarray Positives (57 KB PDF). Click here for additional data file. Table S2 List of 37 Ph-E Genes and 34 Ph-L Genes (14 KB PDF). Click here for additional data file. Table S3 Conservation of Non-Coding Sequences in Ph-E and Ph-L Genes (22 KB PDF). Click here for additional data file. Table S4 Occurrence of Conserved Temporal Elements and Predicted PHA-4 Sites in Ph-E and Ph-L Promoter Regions (18 KB PDF). Click here for additional data file. Table S5 List of Genes Containing Conserved Sites of Different Motifs Within 500 bp Upstream of Their Predicted Start Codons (30 KB PDF). Click here for additional data file. Accession Numbers The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink ) accession numbers for the genes and gene products discussed in this paper are act-1 (LocusID 179535), C49G7.4 (LocusID 178809), ceh-22 /Nkx 2–5 (LocusID 179485), Ce-pax-1 promoter (LocusID 187105), D-pax2 (LocusID 43825), end-1 (LocusID 179893), F30H5.3 (LocusID 175207), F47D12.7 (LocusID 175891), K07C11.4 (LocusID 179198), LAG-1 (LocusID 177373), M05B5.2 (LocusID 187451), myo-2 (LocusID 181404), ncr-1 (LocusID 180719), par-1 (LocusID 179912), pha-4 (LocusID 180357), R07B1.9 (LocusID 181201), rol-6(su1006) (LocusID 174397), skn-1 (LocusID 177343), T05E11.3 (LocusID 178014), vestigial (LocusID 36421), and Drosophila Su(H) (LocusID 34881).

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Nicotine as Therapy

In daily use for centuries by hundreds of millions of people, nicotine has only lately been investigated for its therapeutic potential in a long list of common ills

There's a cheap, common, and mostly safe drug, in daily use for centuries by hundreds of millions of people, that only lately has been investigated for its therapeutic potential for a long list of common ills. The list includes Alzheimer disease, Parkinson disease, depression and anxiety, schizophrenia, attention deficit hyperactivity disorder (ADHD), and even pain and obesity. Why has interest in this potential cure-all been slow to develop? One reason: in its current forms the drug offers pharmaceutical companies no possibility of substantial profit. Another, perhaps more important: the drug is reviled as the world's most addictive. The drug, of course, is nicotine. Nicotine is an alkaloid in the tobacco plant Nicotiana tabacum , which was smoked or chewed in the Americas for thousands of years before European invaders also succumbed to its pleasures and shipped it back to the Old World. Nicotine has always been regarded as medicinal and enjoyable at its usual low doses. Native Americans chewed tobacco to treat intestinal symptoms, and in 1560, Jean Nicot de Villemain sent tobacco seeds to the French court, claiming tobacco had medicinal properties and describing it as a panacea for many ailments. Higher doses are toxic, even lethal—which is why nicotine is used around the world as an insecticide. Yet few of the horrendous health effects of smoking are traceable to nicotine itself—cigarettes contain nearly 4,000 other compounds that play a role. Until recently, nicotine research has been driven primarily by nicotine's unparalleled power to keep people smoking, rather than its potential therapeutic uses. Nicotine locks on to one group of receptors that are normally targeted by the neurotransmitter acetylcholine. Nicotinic acetylcholine receptors (nAChRs) are ion channels threaded through cell membranes. When activated, either by acetylcholine or by nicotine, they allow selected ions to flow across the cell membrane. In vertebrates nAChRs are all over the autonomic and central nervous sytems and the neuromuscular junction. A nAChR is composed of five polypeptide subunits ( Figure 1 ), but there are many nAChR subtypes made of different subunit combinations, a diversity that helps explain why nicotine can have so many different physiological and cognitive effects. Figure 1 Schematic Illustration of an Acetylcholine Receptor (Illustration: Giovanni Maki) It is now conventional wisdom that acetylcholine and nicotine act at these receptors to alter electrochemical properties at a variety of synapses, which can in turn affect the release of several other neurotransmitters. This wisdom exists thanks in part to work by Lorna Role and her colleagues at Columbia University in New York City. “In 1995, we turned people's attention to how nicotine works as a modulator, tuning synapses and increasing the gain on transmitter release,” Role recalls. Although all nAChRs are activated by nicotine, other drugs could be found or designed that affect only a subset of these receptor types. “If you can dissect out the important players with respect to which nicotine receptors are tuning [a] particular set of synapses, then that provides another way to potentially target the therapeutics.” Nicotine and the Brain People with depressive-spectrum disorders, schizophrenia, and adult ADHD tend to smoke heavily, which suggested to researchers that nicotine may soothe their symptoms. Common to all these disorders is a failure of attention, an inability to concentrate on particular stimuli and screen out the rest. Nicotine helps. Researchers at the National Institute on Drug Abuse have shown via functional magnetic resonance imaging that nicotine activates specific brain areas during tasks that demand attention ( Box 1 ). This may be because of its effects, shared with many other addictive drugs, on the release of the neurotransmitter dopamine. “Schizophrenia is a disorder largely of the dopamine system,” says John Dani of the Baylor College of Medicine in Houston, Texas. Dopamine signals in the brain occur in two modes—a kind of background trickle, punctuated by brief bursts. “It's thought that schizophrenics have a hard time separating that background information from important bursts. We've shown that nicotine helps to normalize that signaling by depressing the background but letting the bursts through well,” he says. “I'll be surprised if there's not a co-therapy [to help schizophrenics] that takes advantage of nicotine systems in less than a decade.” Box 1. Nicotine's Effect on Attention Using functional magnetic resonance imaging, scientists at the National Institute on Drug Abuse provided the first evidence that nicotine-induced enhancement of parietal cortex activation is associated with improved attention. They compared brain activity during a task demanding sustained attention—rapid visual information processing (RVIP)—with that during an undemanding sensorimotor control task ( Figure 2 ). Group results from 15 smokers (right) illustrate the effects of nicotine and placebo patches in left and right parietal cortex (1 and 2) and left and right occipital cortex (3 and 4). Nicotine significantly increased activation in occipital cortex during both the control and rapid visual information processing tasks, suggesting a general modulation of attention. In contrast, nicotine increased activity in the parietal cortex only during rapid visual information processing, suggesting a specific modulation on task performance. Nicotine may be the link between two genes that appear to figure in schizophrenia. Sherry Leonard and Robert Freedman of the University of Colorado in Denver, Colorado, have shown that expression of the gene for the alpha 7 neuronal nicotinic receptor is reduced in schizophrenics, and have argued that alpha 7 abnormalities lead to attention problems. Researchers in Iceland and elsewhere have shown that a different gene, for the growth factor neuregulin, also appears to figure in the disease. Neuregulin, Role and her colleagues have shown, governs the expression of nAChRs in neurons and helps to stabilize the synapses where they are found. The researchers are currently studying interactions between neuregulin and alpha 7, which Role thinks will prove important. Smokers also have lower rates of neurodegenerative disorders, and nicotine improves cognitive and motor functioning in people with Alzheimer disease and Parkinson disease. The prevailing hypothesis is that nicotine increases release of neurotransmitters depleted in those diseases. Dani and his colleagues have recently shown that acetylcholinesterase inhibitors—which block the degradation of acetylcholine and hence prolong its action—used to treat Alzheimer disease also stimulate dopamine release. They suspect that malfunctioning of the dopamine system may be affecting noncognitive aspects of dementia such as depressed mood, and that this might be alleviated by nicotine. Paul Newhouse and his colleagues at the University of Vermont in Burlington, Vermont, are studying nicotine drugs as potential therapeutic agents for cognitive dysfunction. Newhouse, a long-time nicotine researcher, is heading the first study ever to examine the efficacy and safety of nicotine patches for treating mild cognitive impairment, thought to be a precursor of Alzheimer disease. The researchers hope to see a positive effect on attention and learning. Newhouse also heads two studies of nicotinic stimulation in ADHD, using the patch, nicotine blockers, and some novel drugs that activate nicotine receptors. Nicotine and Pain Nicotine's salutary effects in patients with neurodegenerative and mental disorders have been studied a lot and are fairly well known. Two much newer topics of academic research are nicotine's potential for pain relief and for treating obesity.Nicotine itself has provided modest pain relief in animal studies. Although the analgesic effect of drugs that mimic acetylcholine were originally attributed to a different class of receptors, it is now clear that nAChRs play an important role in the control of pain. For instance, epibatidine, a drug that is extracted from the skin of an Ecuadorian frog and that acts at nAChRs, has been shown to be 200 times more potent than morphine at blocking pain in animals. Current animal research is aimed at discovering just where, how, and which classes of nAChRs work against pain, with the aim of developing more selective drugs. Meanwhile, nicotine is also being investigated as an analgesic in humans. For example, Pamela Flood, an anesthesiologist at Columbia, is investigating nicotine's future as a postoperative analgesic. She recently completed a pilot study of 20 women undergoing gynecological surgery. All the women had access to unlimited morphine and also got either a single 3-mg dose of nicotine nasal spray or a placebo. The placebo group had peak pain scores of eight out of a possible ten in the first hour after surgery. Women who got nicotine averaged a pain score of five. Despite the small sample size, Flood says, the results were highly significant. “As far as I know this is the first clinical study to use nicotine for analgesia, and it was much more successful than I ever would have imagined.” “The nice thing about nicotine and drugs like nicotine is that they have opposite side effects to anesthetics. Instead of being respiratory depressants, they are respiratory stimulants. Instead of being sedating, they increase alertness. So theoretically this class of drugs is actually the perfect thing to add to an opioid regimen. The fact that they're synergistic was a fortuitous thing that we had never looked at, and neither had anybody else.” Nicotine and Weight Gain Nicotine may be the most effective drug around for weight control. As ex-smokers know, to their rue, one of the worst things about quitting cigarettes is putting on pounds—as much as 10% of body weight. “Something about being addicted to nicotine and then going off it causes massive increase in weight,” Role points out. Young-Hwan Jo in Role's lab is looking at a particular brain circuit involved in motivational behavior, especially feeding behavior. It is lodged primarily in the lateral hypothalamus but has projections all over the cortex, especially the nucleus accumbens, which is the center of reinforcement. “This is where information that has come in to the thalamus and the hypothalamus is relayed to cortical areas with some sense of salience or remembrance. It presumably is involved in changing perception and motivation for eating. It's not, ‘I have to eat this,’ it's, ‘I want to eat this,’” says Role. Jo has been comparing the synaptic effects of nicotine, which reduces appetite, to those of cannabinoids, which stimulate it. “Control of these projection neurons seems to be oppositely regulated by these two,” Role notes. “It doesn't necessarily mean we've found the root of the munchies, but it at least points to pathways that these things have in common.” Jo is also examining how nicotine and cannabinoids modulate these pathways in genetically obese mice, and also their interactions with leptins. Role says tuning these pathways up or down might be a reasonable aim. “If that could be done in a selective fashion, maybe that could be introduced in appetite control. Certainly I see…antagonism of some of these pathways that nicotine activates or the complementary activation of the cannabinoid pathways as very important targets for therapeutics with respect to the anorexia that's associated with chemotherapy.” Ming Li and his colleagues at the University of Texas in San Antonio, Texas, are studying nicotine's effects on weight and on expression of genes that nicotine upregulates orexin and neuropeptide Y and, more recently, that it also regulates leptin signaling. All three molecules regulate feeding behavior controlled by the hypothalamus. In the weight study, nicotine-treated rats not only lost weight, they lost about 20% of their body fat compared to saline-treated controls. The researchers suggest that, among its other effects, nicotine alters fat storage. The University of Texas researchers have scoured the literature for genes related to nicotine, and they are developing microarrays to study the expression of these genes ( Figure 3 ). While nicotine seems to affect all the molecules known to influence weight, Li says it's clear the story is even more complex. “That's the reason we keep looking at different molecules, to find key targets involved in this regulation.” The ultimate hope is to develop new drug applications. Figure 3 Microarray Showing Patterns of Gene Expression Influenced by Nicotine (Image: Ming Li, University of Texas Health Science Center at San Antonio) Dani predicts that weight control is likely to be one of the earliest nicotine-based therapies. “There's a very good chance that the first drug is unlikely to be…nicotine itself, but will take advantage of nicotinic receptors in the therapy,” he says. “I know there are drugs now being tested by drug companies just for that purpose.” Nicotine's Future Developing new drugs that selectively target specific subtypes of nicotine receptors is an expensive, albeit potentially lucrative, proposition. And therein lies a question. Will nicotine-based therapy consist mostly of costly new drugs from the pharmaceutical industry? Or can less expensive nicotine products like the patch, chewing gum, and nasal spray—which are generally intended for smoking cessation but widely available, usually without prescription—find their way into the world's medicine cabinets? “It's a little early to call whether nicotine will be used itself as a therapeutic agent or whether these more specific drugs that are being produced or maybe even used in combination with other drugs may be the most important way to go,” says Dani. But he doesn't see the medicinal use of plain nicotine as very likely. Dani points out that the body's own agent, acetylcholine, acts over milliseconds to activate nicotinic receptors, whereas nicotine itself stimulates these receptors for hours. That lengthy action means that, although nicotine activates the receptors, it then often turns particular receptor subtypes off again, a process called desensitization. “It's hard to predict inside of a body what you're getting. Am I getting an activation or am I turning the receptors off?” Yet much of the work to date showing nicotine's effectiveness on a huge range of disorders has involved products available at any drugstore and intended to help people quit smoking. Newhouse is using patches for mild cognitive impairment. Flood has demonstrated pain relief with nasal spray and will use patches in her next study. And Role feels that gum hasn't been adequately explored for its therapeutic potential. Nicotine gum, she notes, is a better imitator of smoking than the patch because it delivers brief hits rather than a steady supply. She's also uncertain whether natural nicotine has been studied enough. But Role also points out that nicotine has its serious problems—addictive potential, cardiovascular damage, and (especially when delivered through the mucosa) cancer. Dani says, “People are probably going to have to find creative ways to understand which subtypes of nicotinic receptors they're turning on and which ones they're desensitizing. Maybe drug delivery methods will matter. Maybe subtype specificity will matter. It's less than a decade that we've known how important nicotinic receptors are. Now we have to move forward from there.” “We've made an enormous amount of progress on understanding the biology of these receptor systems and how to target them. What has been trickier has been to develop an appropriate pharmacology that allows one to selectively target agents for particular therapeutic purposes with an adequate safety index,” Newhouse says. “But some of the drugs that are coming on in human trials now are very promising. So I'm cautiously optimistic that we're on the road to developing some useful nicotinic therapies.” Figure 2 The Brain on Nicotine (Image: Elliot Stein, National Institute on Drug Abuse)

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The effects of long-term total parenteral nutrition on gut mucosal immunity in children with short bowel syndrome: a systematic review

Background Short bowel syndrome (SBS) is defined as the malabsorptive state that often follows massive resection of the small intestine. Most cases originate in the newborn period and result from congenital anomalies. It is associated with a high morbidity, is potentially lethal and often requires months, sometimes years, in the hospital and home on total parenteral nutrition (TPN). Long-term survival without parenteral nutrition depends upon establishing enteral nutrition and the process of intestinal adaptation through which the remaining small bowel gradually increases its absorptive capacity. The purpose of this article is to perform a descriptive systematic review of the published articles on the effects of TPN on the intestinal immune system investigating whether long-term TPN induces bacterial translocation, decreases secretory immunoglobulin A (S-IgA), impairs intestinal immunity, and changes mucosal architecture in children with SBS. Methods The databases of OVID, such as MEDLINE and CINAHL, Cochran Library, and Evidence-Based Medicine were searched for articles published from 1990 to 2001. Search terms were total parenteral nutrition, children, bacterial translocation, small bowel syndrome, short gut syndrome, intestinal immunity, gut permeability, sepsis, hyperglycemia, immunonutrition, glutamine, enteral tube feeding, and systematic reviews. The goal was to include all clinical studies conducted in children directly addressing the effects of TPN on gut immunity. Results A total of 13 studies were identified. These 13 studies included a total of 414 infants and children between the ages approximately 4 months to 17 years old, and 16 healthy adults as controls; and they varied in design and were conducted in several disciplines. The results were integrated into common themes. Five themes were identified: 1) sepsis, 2) impaired immune functions: In vitro studies, 3) mortality, 4) villous atrophy, 5) duration of dependency on TPN after bowel resection. Conclusion Based on this exhaustive literature review, there is no direct evidence suggesting that TPN promotes bacterial overgrowth, impairs neutrophil functions, inhibits blood's bactericidal effect, causes villous atrophy, or causes to death in human model. The hypothesis relating negative effects of TPN on gut immunity remains attractive, but unproven. Enteral nutrition is cheaper, but no safer than TPN. Based on the current evidence, TPN seems to be safe and a life saving solution.

Background In the late 1960's, the introduction of total parenteral nutrition (TPN) as an alternative nutrition provided a life saving solution to children with chronic bowel obstructions, fistulas, loss of mucosal body surfaces, short bowel syndrome, and other clinical problems that precluded enteral diet by mouth or tube feeding for long periods of time. Intravenous administration of TPN became an essential fluid to meet nutritional needs and to avoid progressive starvation-induced malnutrition, which changed the outcome of patients from dying [ 1 ]. Since then, TPN has been a gold standard practice in treatment and a panacea for infants and children who are unable to eat or to absorb enterally provided nutrients [ 1 - 4 ]. As a result, the prognosis for patients with SBS has changed dramatically and the management with the expected survival for infants with congenital gastrointestinal anomalies and gut failure have improved significantly [ 5 , 6 ]. However, its use has been shown to associate with an increased incidence of infection [ 7 ]. A number of independent experimental studies have been carried out shown that intravenous TPN negatively influences gut barrier functions and mucosal immunity while withholding nutrients by mouth or enteral tube feeding, after the resection of small intestine. These studies demonstrated that TPN is associated with: 1) increases in intestinal permeability, bacterial overgrowth, and bacterial translocation, 2) rapid changes in gut-associated lymphoid tissue (GALT) T cells, B cell, and secretory immunoglobulin A (S-IgA) levels, 3) impairment in IgA-mediated mucosal immunity defenses in the respiratory tract, 4) impairment in neutrophil function, 5) alteration in gastrointestinal (GI) architecture or mucosal atrophy [ 8 - 14 ]. This paper presents a descriptive systematic review of published research articles on the effects of the long-term TPN on gut mucosal immunity in children with SBS; specifically, it addresses whether TPN: 1) promotes bacterial translocation, 2) impairs intestinal mucosal immunity by decreasing S-IgA levels, 3) inhibits neutrophil and cytokine functions in blood, 4) promotes atrophy of the mucosal villi, 5) hyperglycemia, and 6) causes death. It is hoped that these findings will expand the knowledge of pediatric nurses, and have an impact on clinical practice by being included in the pediatric parenteral nutritional guidelines. Since the review of literature did not reveal any systematic reviews of TPN and mucosal immunity on children with SBS, the aim of this descriptive systematic review of individually published scientific studies is to impart a better understanding of the effects of TPN on gut mucosal defense and barrier function. Short bowel syndrome, one of the major indications for diagnostic categories using long-term TPN [ 15 ], is a clinically complex disorder resulting from multiple alterations of normal intestinal anatomy and physiology and producing a variety of nutritional, infectious, and metabolic complications because of impairment caused by vascular disease, intestinal volvulus, ischemic bowel, inflammatory bowel, and necrotizing enterocolitis (NEC) [ 16 , 17 ]. SBS is also described as, " the malabsorptive state" that mostly follows massive resection of the small intestine [ 18 , 19 ]. After resection, the residual or remaining small bowel undergoes intestinal adaptation, a process characterized by mucosal hyperplasia, villus lengthening, increased crypt depth (intestinal gland), and bowel dilation. The process of adaptation is complex and includes both structural and functional changes. The earliest sign can be detected within 24-48 hours, and process may continue for months, possibly years. In the early phase, mucosal hyperplasia and villus hypertrophy occur. Oral nutrients and hormones stimulate this intestinal adaptation. The main clinical challenge in SBS lies in managing the many nutritional problems that occur as a result of malabsorption secondary to the reduced absorptive surface area [ 20 , 21 ]. Anatomy and physiology of the gastrointestinal immune system The normal length of intestine in full-term newborn is estimated at 200 cm-250 cm [ 22 ], and the normal adult small intestine is about 400 cm [ 23 ]. The small intestine extends from the pylorus to the ileocecal valve. It is functionally divided into three segments: the duodenum , the jejunum , and the ileum . The duodenum begins at the pylorus and ends where it joins the jejunum. The end of jejunum and beginning of the ileum are not distinguished by an anatomic marker[ 24 ]. The ileocecal valve functions as a barrier to prevent both reflux of colonic contents into small intestine and rapid passage of contents through the ileum [ 25 ]. The primary function of the small intestine is to absorb nutrients into the circulation [ 26 ]. During the course of this activity the intestine is exposed to a wide variety of antigens derived from foods, resident bacteria, and invading microorganisms. All products of carbohydrate, protein, and fat digestion, as well as most of the ingested electrolytes, vitamins, and water are normally absorbed by small intestine indiscriminately. Normally, nutrient digestion and absorption occur predominantly in the upper intestine. The jejunum has a larger absorptive surface due to longer villi, and a higher concentration of mucosal digestive enzymes and transport proteins, whereas the ileum has shorter villi. Very little absorption occurs in the ileum, not because the ileum does not have absorptive capacity but because most absorption has been already accomplished before the intestinal contents reach the ileum[ 21 , 25 , 27 ]. The tight junctions of the jejunum are highly porous, so that exposure of the mucosa to hyperosmolar nutrient solutions leads to efflux or flow of water and electrolytes into the lumen (interior space of intestine). This fluid is then reabsorbed in both the ileum and colon. The distal ileum has specialized transport carriers for the absorption of bile salts and vitamin B 12. The ileocecal valve forms an important barrier, slowing transit from the small intestine to the colon and limiting bacterial colonization of the small intestine [ 21 ]. Ileocecal valve prevents retrograde colonization of distal small bowel to a significant extent. In the absence of the valve, however, free reflux of right-sided liquid colonic content occurs, and the total load of colonic bacteria exposed to the distal intestine increases greatly [ 28 ]. Histological organization of the intestinal wall is divided into four major layers. These major layers include: (1) the mucosa , the innermost layer, which comprises epithelium , lamina propria , and muscularis layers; (2) The submucosa consists largely of loose connective tissue with collagen and elastin fibrils. Glands, nerve trunks and blood vessels are also present in the submucosa; (3) The next layer, the muscularis , typically consists of two substantial layers of smooth muscle cells; an inner circular layer and an outer longitudinal layer; and (4) The serosa is the outermost layer of intestinal tract [ 25 , 29 - 31 ]. The mucosa is considered an "architectural marvel." The epithelial layer of mucosa regulates absorptive, secretory, and protective barrier functions. This thin epithelial layer composed of columnar absorptive cells, goblet cells, undifferentiated crypt epithelial cells, panet cells, enteroendocrine cells, tuft cells, cup cells, and intraepithelial lymphocytes [ 32 ]. This mucosal layer covers the villus, a finger like projection that is made up of epithelial cells called enterocytes and its crypt or submucosal gland, and is responsible for absorption of electrolytes, water, and nutrition. The enterocyte surface contains special luminal projections called microvilli, which provide an increased surface area that is referred to as brush border membrane. Although not part of the epithelium, mucus on the surface of the mucosa shields the mucosal epithelial cells from direct contact with the intestinal luminal environment [ 29 , 32 , 33 ]. Beneath the mucosal epithelium is the connective and supportive tissue called the lamina propria. The lamina propria contains various immunocompetent cells, including plasma cells, mast cells, macrophages, and lymphocytes that produce not only immunoglobulins but also cytokine mediators [ 32 ]. Mucosal defense system Nonimmune antibacterial factors A complex intestinal mucosal defense system plays an important role to prevent invasion of gut bacteria or absorption of endotoxin. Peristaltic waves and contractions sweep the intestinal contents in a steady distal flow toward the colon, which is a major importance in reducing the growth of bacteria in the proximal intestine [ 28 ]. During fasting, motilin, a putative regulatory peptide, is believed to be one mechanism that initiates the cyclic motility pattern termed the migrating motor complex (MMC), which functions as a gastrointestinal "housekeeper" by sweeping lumenal content and bacteria from stomach and small bowel into colon[ 34 ]. Mucus, an epithelial secretion, also helps reducing bacterial overgrowth. Mucus has special properties that enable to trap bacteria in an intraluminal location while moving organisms distally in bolus fashion. Carbohydrate serves as a nutrient for some bacteria, thus attracting them to a mobile colonization site that is continuously replaced. Enzymatic digestion also significantly reduces bacterial overgrowth. Pancreatic juice has antibacterial activity. Intestinal secretions from the stomach, intestine, and pancreas are a significant deterrent to growth due to their ability to dilute the bacterial mass [ 28 , 32 ]. Gastric acidity acts as an initial line of defense against ingested bacteria. Gastric juice with pH less than 4.0 is bactericidal for most organisms, although not immediately [ 28 ]. In one in vitro study, bacteria instilled into an intact lumen of a normal human stomach were promptly killed in less than 15 minutes at a pH of less than 3.0, but remained viable in the achlorhydric stomach for at least 1 hour [ 35 ]. Chronic inhibition of gastric acid secretion by histamine 2 (H 2 ) receptor blockade in healthy adults, however, has been shown to increase the number of gastric bacteria [ 36 ]. The intestinal tight junctions between epithelial cells and permeability have been reviewed [ 37 ]. Permeability refers to the ability of small molecules to penetrate the gastrointestinal mucosa. The tight junctions are an important part of the intestinal barrier. Tight junctions are dynamic structures, and their barrier function may be modulated by nutrients such as glucose and amino acids as well as bacterial toxins and chemotaxins. Loss of intestinal barrier is associated with translocation of enteric bacteria in animal modal, but in humans bacterial translocation is not associated with increased intestinal permeability or villous atrophy. Glutamine, a nonessential amino acid, is considered to be the principal respiratory fuel for enterocytes. A lack of glutamine promotes mucosal atrophy and increases intestinal permeability [ 37 - 39 ]. Thomson and colleagues [ 40 - 42 ], citing various sources, suggest that enterocytes function as "nonclassical" immune cells, which play a major role as a source of proinflammatory cytokines and cytotoxins. A key proinflammatory mediator produced in intestinal mucosa is the free radical nitric oxide. Nitric oxide (NO), a pluripotent signaling and effector molecule, is increased with mild acidosis and enhances intestinal permeability. Nitric oxide is produced as a result of conversion of arginine to citrulline by the enzyme NO synthase (NOS). Nitric oxide has potent bactericidal effects against a wide variety of micro-organisms, including the majority of the intestinal microflora. Furthermore, arginine supplementation has been shown to improve survival in a guinea-pig model of peritonitis. Inhibition of NO synthesis has been shown to increase intestinal permeability via mast cells, which suggests that NO may regulate intestinal barrier function [ 43 , 44 ]. The permeability of the intestine can be increased by variety of factors, such as psychological stress, fasting, and certain drugs. During fasting or malnutrition, intestinal secretion of ions and fluid increased, permeability to ions and macromolecules is increased and associated mucosal atrophy reduces intestinal absorption of nutrients. Some drugs also alter intestinal permeability. Nonsteroidal anti-inflammatory drugs (NSAIDs) increase intestinal permeability in both animals and humans. Glutamine, the major source of the small intestinal mucosa, may be partially normalize this NSAID-induced permeability [ 37 , 45 ]. Immunological factors The gastrointestinal track or gut associated lymphoid tissue (GALT) has an important role in gut immunity. The gut is considered the largest lymphoid organ in the body housing more than 10 12 lymhocytes and more antibody (secretory IgA) than any other site in the body including spleen [ 46 ]. The lamina propria plasma cells produce secretory immunoglobulin A (S-IgA) in response to food antigens and microorganisms. Secretory IgA coats the lumen or interior surface of small intestine and prevents any microbial pathogens or viruses penetrating the epithelial layer and passing into the other organs [ 29 , 47 ]. S-IgA contains antibodies directed to biologically active antigens such as viruses, bacteria, enterotoxins, and enzymes normally restricted to the intestinal tract. A reduction in S-IgA level results in a greater frequency of GI infections and impaired reticuloendothelial or macrophage function, which predispose the child to systemic bacteremia [ 12 , 46 , 48 ]. The effects of surgery or trauma on mucosal immunity After surgical intervention, sympathetic response is activated creating a positive relationship between the severity of injury and the plasma concentration of adrenalin, noradrenalin and dopamine [ 49 ]. Following injury, the inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) are also released, which act only locally [ 39 ]. Cytokines are hormonelike peptides or intracellular signaling proteins released from white blood cells and all nucleated cells. They function by serving as chemical messengers within the immune system, and also communicate with certain cells in the nervous system. They have immune modulating effects, in which they work in parallel with other signal arising from direct cell-to-cell contact, providing a communication network involved in everyday function of the immune response [ 50 - 52 ]. In the systemic level, counter-regulatory hormones, such as adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), catecholamines and stress hormone, cortisol are released. These cytokines and systemic hormones working together cause the multiorgan system failure. They also induce hypercatabolism, which is characterized by protein breakdown within skeletal muscle, accelerated breakdown of branched-chain amino acids and increased release of glutamine and alanine into systemic amino-acid pool. Glutamine, conditionally essential amino acid, is critical as an energy source for enterocytes, immune cells, and rapidly growing tissues. Overall, physiological stress response results with increased intestinal permeability and bacterial translocation, which promotes sepsis or multiorgan failure and potantiate hypercatabolism and protein-calorie malnutrition [ 39 ]. During illness, stress increases the concentration of counterregulatory hormones (glucagons, epinephrine, cortisol, and growth hormone) and cytokines. Counterregulatory hormones increase serum blood sugars by increasing hepatic glucose production and by decreasing peripheral glucose uptake [ 53 ]. Glucose turnover is increased in sepsis and trauma, but glucose is oxidized with reduced efficiency. Fat becomes a preferred energy substrates in septic patients. In critical surgical illness the rates of both protein synthesis and catabolism are increased. However, the increase in catabolism is of greater magnitude resulting in a net breakdown of protein and, if prolonged, immune compromise [ 49 ]. Overall, this leads to prominent metabolic derangements composed of high release and low use of glucose, amino acids, and free fatty acids (FFA), resulting in increased blood levels of these substrates. Increased levels of glucose and FFA have stimulating effects on inflammatory signaling leading to additional release of proinflammatory mediators and endothelial and neutrophil dysfunction. Insulin has the inherent capability to counteract the metabolic changes in septic patients [ 54 ]. Complications of SBS The most important ongoing complications in children with SBS reported in the literature are: recurrent sepsis, catheter related sepsis, TPN-associated cholestasis, metabolic disturbances, hyperglycemia, electrolyte imbalances, hypertriglyceridemia, gastric hypersecretion, diarrhea, and organ dysfunction [ 15 , 55 - 57 ]. Bacterial overgrowth Bacterial overgrowth is defined as the presence of potentially pathogenic microorganism (PPM) in high concentration (≥ 10 5 colony forming units/mL) [ 58 ]; that is, increased numbers and species of bacteria in the small intestine. Bacterial growth in the normal bowel is controlled by gastric acid, pancreatic enzyme activity, enterocyte turnover, normal peristaltic activity in the small intestine, and ileocecal valve [ 59 ]. Bacterial overgrowth is found in children who have no ileacecal valve, the primary means for preventing reflux of bacteria from the colon into the small intestine. Progressive dilation of the small intestine as part of the adaptation response limits the efficacy of peristalsis in ridding the small intestine of bacteria. The diagnosis of bacterial overgrowth is made by culture of jejunal aspirate or by breath hydrogen testing. Bacterial translocation is a phenomenon where intestinal pathogenic microorganisms, which are normally resident within the lumen of the intestinal tract, travel from the gut lumen into local mesenteric lymph nodes, and from there to distant sites, such as upper gastrointestinal tract, thereby causing sepsis and septicemia [ 8 , 60 - 62 ]. Sepsis in infants and children is defined as the systemic response to a possible infection. Evidence of bacteremia or an infectious focus is not required. The term septicemia is used when organisms or their toxic products are identified in the bloodstream, in other words, a pathogen is recovered from blood cultures or identified [ 63 ]. Most studies of the pathogenesis of sepsis in animals have been carried out shown that gut mucosa is particularly susceptible to injury through a variety of mechanisms, such as decreased mucosal blood flow, increased oxygen demand, decreased oxygen delivery, and reperfusion injury [ 64 ]. Sepsis is also associated with metabolic and inflammatory response to trauma or surgical illness. It has been suggested that the neuroendocrine response to sepsis and trauma results in metabolic changes; and inflammatory mediators released from wound itself or from a septic focus may play a part in these changes. It is also widely believed that these infectious complications are related to the central venous access devices because the most recurring infection was reported as line infection [ 8 , 58 , 60 , 65 , 66 ]. However, it has been suggested that lipid emulsions of TPN may impair host defense, and in particular the bactericidal and migratory functions of the neutrophil polymorhonuclear (PMN) granulocyte in infants receiving TPN [ 7 , 51 , 60 , 67 - 69 ]. A large case-control study [ 70 ] involving 882 infants in two neonatal intensive care units, and one experimental animal study [ 71 ] conducted suggest that administration of lipids in parenteral nutrition regimens may cause phagocyte dysfunction, resulting in infectious complications such as bacteremia, pneumonia, and wound abscesses. In addition, these studies also suggest that the lipid component of TPN, which constitutes only long-chain triglycerides, possesses immunosuppressive properties that interfere with binding of interleukin 2 (IL-2), a product of activated T (helper) cell or cytokine, to its cellular receptor and impairs host defense, in particular the bactericidal and migratory functions of the neutrophil polymorphonuclear (PMN) granulocyte [ 50 , 69 - 72 ]. However, three randomized control clinical trials [ 73 - 75 ], and a case study with 4 patients [ 76 ] did not show that intralipid in TPN is associated with any immunosuppression. Interestingly, in contrast to others, one of the randomized control trials [ 73 ] showed that, rather than being immunosuppressive, lipid emulsions of TPN had immunostimulatory properties. D-lactate acidosis Bacterial overgrowth frequently complicates, especially when the ileocecal valve is absent and the dysmotility is present in the remaining bowel loops. In the colon, unabsorbed carbohydrates undergo bacterial fermentation to D-lactate, short-chain fatty acids (acetate, propionate and butyrate), which can not be metabolized by D-lactate dehydrogenase; and these small molecules are absorbed from colon, "carbohydrate salvage" [ 21 , 77 ]. Absorption of this metabolite (D-lactate) may cause neurological symptoms, metabolic acidosis with increased anion gap in these patients [ 55 ]. It is called, " D-lactic acidosis Syndrome." The episode of this syndrome causes patients develop slurred speech, ataxia, and altered affect. These patients appear "drunk." This condition is resolved after placing patient on a diet with restricted carbohydrate intake and metronidazole [ 23 ]. Methods In this review, a descriptive systematic, non-quantitative literature review was conducted using methodological guidelines [ 78 - 80 ]. A systematic review is a technique that uses systematically guided strategies to locate, select and critically appraise relevant original studies for its subjects, and summarizes the results that address a specific clinical question from studies that are included in the review. It is either descriptive (non-quantitative) when results of primary studies are summarized, but not statistically combined, or quantitative, in which statistical methods are used to combine results, also known as meta-analysis [ 80 - 83 ]. A descriptive systematic review is also called a synthesis of literature . This form of literature synthesis involves tabulation of study characteristics and results to summarize their findings in an area, and it also leads to new conclusions and knowledge as a result of systematically pulling together the fragmented results from single studies[ 80 , 84 ]. Selecting and appraising studies for systematic review To select and assess the methodological quality of the research studies to be included in this review, the author used a list of standardized criteria or the guidelines (Table 1 ) proposed by a group of researchers [ 78 , 82 , 85 - 89 ]. Studies were excluded if their published method section was not clear or absent; TPN was not used; study outcomes were not mucosal immunity related; or participants were not children. Table 1 Study selection criteria Review Question: To what extent does long-term TPN affect the intestinal immune system of infants who undergo bowel resection with no enteral nutritional support? Population Were study patients pediatric age groups between newborn to 17 years old? Did study patients have intestinal resection prior to TPN given? Did study patients have documented bacterial infection after TPN started? Did study patients have documented impaired mucosal immunity after TPN started? Study Intervention Did at least one study group received Intravenous (IV-TPN? Did study group received IV-TPN more than10 days? Control Intervention Did one study group receive enteral feeding, but no IV-TPN? Outcomes Was one of the measured outcomes documented as bacterial translocation, villi atrophy, impaired immuno-function, and death? Note. Modified Table from "Selecting and appraising studies for systematic review," by M. O. Meade and W. S. Richardson, 1997, Annals of internal medicine , 127 (7), p. 531-537 [86]. Copyright 2002 by the American College of Physicians-American Society of Internal Medicine. Adapted with permission. Research question To what extent does long-term TPN affect the intestinal immune system of infants who undergo bowel resection with no enteral nutritional support? Search strategies For the purpose of this paper, searches of the following resources were used to do a comprehensive and exhaustive literature search: 1) Electronic bibliographic databases, 2) Reference lists from relevant primary and review articles, 3) The Internet, 4) Hand-searched journals, and 5) Unpublished studies, such as Thesis and Dissertation Abstracts [ 80 ]. A literature search was conducted using the following online databases: OVID Databases (MEDLINE, BIOSIS, CINAHL, Current Contents , HealthStar, OVID Full text journal articles), Cochran Library, Dissertation Abstracts, Thesis, PubMed, Web of Science, Evidence-Based Medicine, Evidence-Based Nursing, Journal Citation Reports, MD Consult, Academic Search, CancerLit, and Journal Collection Databases (Academic/IDEAL, Blackwell Science, Elsevier, and SpringerLink). The newer articles provided a reference list of citations of previous articles. The research covered the years from 1990 through 2001 for original articles published in English. The following subject heading terms were included: Total parenteral nutrition, bacterial translocation, immunity, infant, children, short bowel-syndrome, short gut syndrome, intestinal immunity, epithelial permeability, immunonutrition, hyperglycemia, and sepsis. To prevent this systematic review from publication bias, unpublished studies were searched via Dissertation Abstract Online (FirstSearch) database, and one unpublished thesis relevant to this review was included [ 90 ]. Sample In this systematic review, 12 published studies using quantitative methods met the clinical trials criteria. In addition, one unpublished study was included in this review. These 13 studies included a total of 414 infants and children between the ages approximately 4 months to 17 years old, and 16 healthy adults as controls. One of the 13 clinical trial samples was not counted because the same sample was used for secondary analysis [ 58 , 60 ]. Although high quality, well-designed study, or the preferred research design for a review would be the one that randomly allocates (concealing the assignment code) the participants with the condition of interest to alternative therapeutic interventions, but when prospective randomized clinical trials (PRCT) are not possible to find or not available, NHS Center for Review and Dissemination Guidelines suggest that the next best available evidence should be considered based on the hierarchy of study designs, which are from highest to lowest level: 1) prospective experimental studies (e.g. randomized control trial with concealed allocation), 2) experimental study without randomization (quasi-experimental), 3) observational study with control group, a) cohort, b) case-control studies, 4) observational studies without control group, a) cross-sectional, b) before-and-after study, c) case series, and 5) expert opinion [ 80 , 91 , 92 ]. In this review, the following available research designs are included: two prospective experimental-control clinical trials [ 90 , 93 ], one case-control [ 94 ], two cohort studies [ 58 , 60 ], three in-vitro studies [ 7 , 51 , 67 ], one case study [ 95 ], and four retrospective medical chart reviews [ 5 , 96 - 98 ]. The studies were published between 1993 and 2001 in pediatric journals, annals of surgery, nutritional science journals, and immunology. The descriptive methodological and characteristics of the samples are given in Table 2 . Table 2 Methodological Characteristics of the Clinical Trials Included in This Systematic Review Studies Discipline Year Publication Country Funding Trial Type (Setting) Participants Andorsky et al. [96] Surgery 2001 J. Pediatrics USA Yes Retrospective (Hospital) Neonates Okada, Klein, Pierro, et al. [20] Pediatric Surgery 1999 J Pediatric Surgery UK Yes In Vitro, (Hospital) Infants and adults Okada, Klein, van Saene, et al. [7] Pediatrics 2000 Annals of Surgery UK Yes In Vitro, (Hospital) Infants Okada, Papp, et al. [19] Pediatric Surgery, Immunobiology 1999 J Pediatric Surgery UK Yes In Vitro (Hospital) Infants and adults Bines et al. [95] Gastroenterology & Clinical Nutrition 1998 J. Pediatric Gastroenterology and Nutrition Australia Yes Case study (Hospital) Infants and children Sondheimer et al [97] Pediatrics 1998 J. Pediatrics USA No Retrospective (Hospital) Neonates Kaufman et al. [98] Pediatric Gastroenterology & Ped. Surgery 1997 J. Pediatrics USA Yes Retrospective (Hospital) Infants and children Pierro, van Saene, Donnel, et al. [17] Pediatric Surgery 1996 Archives of Surgery UK No Cohort study (Hospital) Infants Pierro, van Saene, Jones, et al [18] Pediatric Surgery 1998 Annals of Surgery UK No Cohort study (Hospital) Infants Weber [94] Pediatric Surgery 1995 J Pediatric Surgery USA No Case-control (Hospital) Infants Chaet et al. [5] Pediatric Surgery 1994 J Pediatric Gastroenterology & Nutrition USA No Retrospective (Hospital) Children Rossi et al. [93] Pediatric Gastroenterology 1993 Digestive Disease and Sciences USA No Experimental-Control (Hospital) Infants and children Dahlstrom [90] Pediatrics 1988 Unpublished Thesis Sweden Yes Experimental-control (Hospital) Infants and children Data analysis To collate and present the extracted data, a coding sheet was used to collect information or study characteristics that included both demographic and methodological from the retrieved primary studies [ 79 , 99 ]. The results of the retrieved primary studies were reviewed and summarized in a coherent manner by using the NHS Center for Reviews and Dissemination [ 80 ] non-quantitative data synthesis guidelines, and classifying and structuring processes [ 100 ]. The data synthesis processes involve categorizing and classifying extracted data results, tabulation of each characteristic of the study and the major findings related to TPN, and then drawing a conclusion or making a coherent integrated report from the individual studies. Once the results were extracted, they were organized, and then categorized into similar themes. For example, the results related to sepsis, septicemia, bacterial overgrowth and bacterial translocation are grouped under sepsis. Five major themes were found and organized based on the most frequent reports. Results Five themes were identified: 1) sepsis, 2) impaired immune functions: In vitro studies, 3) mortality, 4) villous atrophy, 5) duration of dependency on TPN after bowel resection. Table 3 provides the demographic characteristics of the studies included in this systematic review. Table 3 Demographic characteristics of individual clinical trials included in this review Studies Participants (Age) Interventions Results Pierro, van Saene, Jones et al. (1998) [18] 94 infants on PN (median age 37 weeks) 94 infants were on TPN. Throat and rectal swabs (surveillance cultures) were obtained before and twice a week after TPN started. Cefotaxime and metronidazole were given for prophylaxis, then blind therapy with a combination of Gentamicin and teicoplanic was given at the onset of sepsis. Blood cultures (central/peripheral) were sent. 41 patients (44%) on PN for 30 days, developed abnormal carriage. Among these carriers, 2 infants developed oropharyngeal E. Coli followed by Klebsiella spp, enterobacter spp, and Pseudomonas aeruginosa. 9 infants had blood cultures positive with enterococci, E. Coli, Klebsiella, Candida, and coagulase (-) staphylococci. Pierro, van Saene, Donnell et al. (1996) [17] 94 infants, median gestation was 37 weeks Surveillance cultures of oropharynx and gut were obtained at the start of TPN and thereafter twice a week. Blood cultures (central/peripheral) were sent. 15 infants developed sepsis. 10 patients experienced septicemia. 6 patients had bacterial translocation, and overgrowth of bacteria observed in 9 patients. Andorsky et al. (2001) [96] Total of 30 infants with SBS (age <30 days) NEC = 13, Intestinal atresia = 9, Gastroschisis = 5, Malrotation/volvulus = 3 Median residual small bowel length was 61 cm. All infants were on TPN. The shortest duration of PN use was 101 days, and the longest was 3287 days, and median was 245 days. Of the 30 patients in the study, 20 (67%) were weaned from PN; 9 of the 10 TPN-dependent infants died from infection, cardiac arrest while receiving TPN. Okada, Klein, van saene et al. (2000) [7] 41 babies enrolled (<4 mos. old). Gastroschisis = 11, (NEC) = 7, intestinal atresia = 3, diaphragmatic hernia = 2, esophageal atresia = 2, infant with no GI problems = 11, control infants = 5, and healthy adult volunteers (control) = 5. 5 infants receiving TPN for more than 10 days. 5 infants on normal enteral diet. Coagulase-negative staphylococci were added to whole blood from control patients receiving TPN. Body weight was significantly lower in patients receiving TPN. The blood from control group killed 65% of the coag-neg. Staph, while the blood from long term TPN group failed to kill this organism. Okada, Klein, Pierro et al. (1999) [20] Surgical infants (NEC, gastroschisis)= 5, infants (control)= 5, healthy adults (control)= 5. 5 surgical infants on long term TPN (>10 days), 5 infants on normal enteral diet, 5 healthy adults. The percentage of bacteria killed by the neutrophils increased with time. However, the ability of killing was significantly lower in infants on TPN. Okada, Papp, et al. (1999) [19] 5 enterally fed infants (age<6 mos), and 6 healthy adults Fasting blood samples: A) 10 ml normal saline (N/S) B) 0.1 ml TPN in 9.9 ml N/S C) 1 ml TPN in 9 ml N/S D) 10 ml TPN In infants, 1 ml of TPN in 1 ml blood produced a significant decrease in TNF-α production. Weber (1995) [94] 21 infants and children with short bowel length (<80 cm) on TPN through central line. 20 infants without SBS (13 NEC, 4 atresia, 1 gastroschisis, 2 volvulus) had surgery No enteral feeding for 7 to 14 days during the post-op period. Blood cultures from central line and peripheral line were sent to identify the organism 6 patients had 8 separate episodes of sepsis before enteral feeding was began. After enteral feeding started, 16 patients had 67 episodes of bacteremia. Chaet et al. (1994) [5] 32 children with SBS Gastroschisis = 3 Volvulus = 5, NEC = 8, Atresia = 8, Hirschprung's = 5 32 children with residual small bowel length <100 cm (range 14-94, median 40). All patients required TPN support for minimum 2 months. 10 patients were on TPN for>3 years. 4 patients died. Two of these deaths were from complications of TPN, and other 2 had pneumonia and respiratory failure secondary to broncho-pulmonary dysplasia. All four patients were TPN dependent up to the time of their deaths. One of these death patients bowel length was 30 cm and ICV was intact while the other did not (bowel length, 15 cm). Kaufman et al. (1997). [98] 49 neonates with SBS, NEC = 20, Atresia = 12, Gastroschisis = 9, Volvulus = 8. Infants with SBS required TPN for more than 3 months after initial surgery. Oral feeding was permitted in small volumes. Patients went home with TPN. 42 patients were able to wean completely from TPN. Bacterial overgrowth was diagnosed in all 7 children who were receiving TPN. Occurrence of bacterial growth was related to small bowel length. 6 of them died. Sondheimer et al. (1998) [97] 44 infants NEC = 14, Atresia = 6, Gastroschisis = 4 Volvulus = 2, unknown = 10 Almost half of 32 infants had 50% or more of the estimated intestinal length resection. The remaining 12 infants had 10-50% of bowel resection. Of the 44 patients, four patients have died from liver failure while on TPN. Seven patients depended on TPN from 40 to 129 months. The rest, 27 patients were off TPN after 36 months of age. Outcome of 10 patients unknown. Bines et al. (1998). [95] 4 patients with SBS (6 months to 5 yrs), NEC = 2, Volvulus = 1 Hirschprung = 1 All patients had central line. Patients received pregestemil formula via continues GT, while on TPN, then study formula (Neocate) was given All patients were able to discontinued TPN within 15 months of initiating the study formula. After tolerating enteral study formula, morbidity and hospitalization reduced dramatically. Rossi et al. (1993) [93] 7 children, ages 9 months to 17 years. 3 children with inflammatory bowel disease (IBD) were on TPN for one month. 4 children with SBS on TPN for 7 to 54 months. 22 infants (control) on normal diet. All patients while on TPN for one month, underwent to intestinal endoscopy and biopsy. Of these patients: 7 on TPN, and had upper intestinal biopsy. 4 patients required TPN for >9 months Biopsies from patients in the IBD group didn't show atrophy. 3 patients on long-term TPN for SBS had very mild (grade I) villus atrophy. Dahlstrome (unpublished, 1988) [90] 29 children (4–111 month) SBS = 11/9 on TPN/ PPN (small bowel<25 cm) pseudoobstruction = 7 on PPN immunodeficiency = 1 radiation enteritis = 1 28 (control) healthy children (10 USA and 18 Swedish the same age group) Group I children absorb <5% of their daily caloric intake; Group II children was 30-70%. Home-TPN was given each night, and children were encouraged to eat daytime as much as possible for an average of two years. After two years of long-term TPN, children had abnormal lymphocyte count, low levels of serum albumin and protein in-group I. Four children developed selenium deficiency, and 15 children on PN for 3 yrs had significantly low Hb and Hct compared to controls. Eleven of 29 children died from low lymphocyte count. Seven died (5 from SBS, 1 from pseudoobstruction, 1 from immune deficiency), 4 from TPN induced cholesistatic liver disease and from bacterial septicemia. PPP:Partial parenteral nutrition, PN: Parenteral nutrition, Hb: Hemoglobin, Hct: Hematocrit Sepsis Six of the 13 trials reported bacterial overgrowth, sepsis, or septicemia [ 58 , 60 , 90 , 94 , 96 , 98 ]. In the first study, [ 96 ] investigators reported that two children died from gram-positive central venous catheter infections. In the second study [ 98 ], researchers compared a total of 49 neonates with 7 SBS who were on TPN and 42 weaned children. It was ascertained that the occurrence of bacterial overgrowth was related to small bowel length. Eleven TPN-weaned subjects who had bacterial overgrowth had a mean bowel length of 54 cm as compared to 105 cm in those without bacterial growth. In Pierro and colleagues' study [ 58 ], 41 children whose surveillance cultures developed abnormal carriage. These investigators stated that infants receiving TPN are at a very low risk of developing sepsis and septicemia as long as their surveillance cultures reveal normal flora only. Conversely, the presence of abnormal PPM in the throat and/or the gut increases the risk of sepsis and septicemia to about 25% of the population art risk. They have also stated that the sicker and more physiologically stressed infants would not regain normal gut function as fast as neonates who were doing clinically better. In the third study [ 60 ], the purpose of the investigation was to demonstrate an association between microorganisms that were carried in the digestive tract and present in the blood of infants with sepsis who were receiving TPN. Sepsis occurred in 15 patients (43 episodes) and 10 patients experienced septicemia (24 episodes). Six infants had 15 episodes of bacterial translocation due to E. coli , Klebsiella , Candida species and enterococci. Eight patients had nine episodes of septicemia caused by coagulase-negative staphylococci. The researchers concluded that there was an association between septicemia and elevated serum bilirubin level, indicating TPN related cholestasis. Dahlstrome [ 90 ] investigated a total of 29 children, most of whom had SBS and had been on TPN for 2 years. Children receiving TPN for an average of 2 years developed low biochemistry levels. The children were divided into two groups. The children in group I were estimated to have absorbed less than 5% of their daily caloric intake from the intestinal tract, while intake in the children in group II was 30-70%. Based on previous animal studies, the investigator predicted that the low lymphocyte count was related to extensive bowel resection, (the average bowel length of group I was<25 cm). Eleven of these children eventually died from TPN related sepsis, septicemia, central line infections, or cholestatic liver diseases. Impaired immune functions: in vitro studies In three in-vitro control studies, investigators elucidated that long-term TPN in infants suppresses specific mechanisms of immune functions [ 7 , 51 , 67 ]. In the first study, Okada and colleagues (1999) investigated the effects of TPN solution on neutrophil phagocytosis and whole-blood cytokine production in response to coagulase-negative staphylococci in an in vitro challenge in five enterally fed infants (age<6 months) and 6 healthy adults. They found that in infants, after 2 hours of incubation with a physiological dose of TPN (1 μ of TPN in 1 ml of blood) there was a significant decrease ( P < 0.05) in tumor necrosis factor alpha (TNF-α) production. The other two in vitro controlled-clinical trials focused on the cellular mechanism of neutrophil dysfunction, and the whole blood bactericidal activity against coagulase negative staphylococci in the infants receiving TPN [ 7 , 67 ]. These investigators used an in vitro -controlled whole blood model to measure the host bactericidal activity against coagulase-negative staphylococci. When researchers added coagulase-negative staphylococci to whole blood drawn from control patients receiving enteral feeding, the median killing of coagulase-negative staphylococci was 65%. In contrast, blood from infants receiving long-term TPN failed to kill this organism as effectively. Infants on long-term TPN had the lowest levels of both whole blood and intracellular killing of coagulase-negative staphylococci due to a defect in neutrophil function. These investigators reported that there was a negative linear correlation between the duration of TPN in days and killing of coagulase-negative staphylococci (P = 0.002). For every additional week of TPN, there was a 7% reduction in killing of coagulase-negative staphylococci. These investigators also noted that there was a significant positive correlation between neutrophil count and killing of coagulase-negative staphylococci (r = 0.80, P = 0.001). Mortality A total of 34 TPN complicated deaths were reported in five of the 13 clinical trials. Of these 34 deaths, 7 children died from SBS, 12 from sepsis, respiratory infections, and low immune deficiency, and other 15 from liver failure [ 5 , 90 , 96 - 98 ]. Duration of dependency on TPN after bowel resection There were only three studies, retrospective and case study that showed the dependency on TPN in infants who had undergone resection of the small intestine [ 95 - 97 ]. As reported in these studies, there were only 17 children who were totally dependent on TPN, and nine of them died while on TPN. In one of these studies, investigators also noticed that no child successfully discontinued TPN after 36 months of age [ 97 ]. They concluded that children dependent on TPN at or more than 36 months of age are permanently dependent on TPN. Andorsky and his co-workers [ 96 ] also emphasized that residual bowel length was highly correlated with duration of TPN. Their conclusion was that remaining functional small bowel length was an independent predictor of successful weaning. Although most children had difficulty being weaned from TPN in other studies, Bines and her co-workers [ 95 ] were able to wean all four children from TPN within 15 months of initiation of a study formula. They used an amino acid-based complete infant formula (Neocate, SHS Inc, Rockville, MD, USA) as enteral feeding. Small bowel length of these children was documented between 40 cm to 80 cm with no ileocecal, and in only one child had 13 cm with ileocecal present. All patients had repeated esophagogastroduodenoscopy and colonoscopy or jejunoscopy with findings of mild, nonspecific neutrophilic inflammation involving areas from biopsy specimens from upper and lower intestinal tract. In addition, two other studies used enteral formulas while children were on TPN. Andorsky and colleagues [ 96 ] fed the patients with continuous breast milk or protein hydrolysate formula. Of the 30 patients in the study, 20 were weaned from TPN. Based on their report, median residual bowel length was 61 cm, and ileocecal valve was preserved in 57% of the patients. There was a significant high correlation between use of breast milk and shorter the duration of TPN. Infants who received breast milk were weaned from TPN in 290 days versus 720 days in non-breast-fed infants. Villous atrophy In two separate clinical trials, researchers [ 93 , 95 ] examined whether the effects of TPN on the intestines of children are similar to those reported in animals. In the Rosi and colleagues' study, a total of 32 children (ages 9 months to 19 years) were involved. Based on the biopsy results, the investigators reported that 3 of the 4 patients receiving long-term parenteral nutrition for short-bowel syndrome (TPN >7 months) had mild villous atrophy. Biopsies from 3 patients in the inflammatory bowel disease (IBD) group (TPN = 1 month) did not exhibit atrophy. Bines and colleagues [ 95 ] also reported similar results; in their study only one of four children (bowel length, 40 cm) had partial villous atrophy of the small intestinal mucosa. Discussion Systematic reviews focus on empirical studies and seek to summarize past research by drawing overall conclusions from many separate investigations that address related or identical hypotheses [ 101 ]. The purpose of this descriptive systematic review was to assess the literature documenting whether TPN negatively influences gut barrier function and is associated with increases in intestinal bacterial overgrowth, bacterial translocation, increases in mucosal permeability, decreases in S-IgA levels, and changes in mucosal architecture in infants and children after small bowel resection. Thirteen clinical trials in children from 1990 to 2001 examined the effects of TPN on infection, mortality, impaired intracellular immune functions, villous atrophy, and long-term TPN dependency. These studies were conducted in four countries from various disciplines; they ranged in size from 4 to 94 children, and age from approximately 4 months to 17 years old, with the majority of children spending time in hospital. Numerous experimental studies suggest that long-term TPN has harmful effect. Evidence in experimental studies about TPN's effects on mucosal immunity and villous atrophy is very convincing [ 9 , 14 ]; however, findings in human subjects were inconsistent. Although animal models provide us wealth of evidence and continue to offer valuable information to apply to clinical conditions, Rossi and coworkers [ 93 ] argue that extrapolating these results to apply to the human model is inappropriate. Their study results showed that infants with SBS on TPN for more than 9 months had only minimal grade villi atrophy. They concluded that humans are more resistant to hypoplastic intestinal changes induced by TPN; and effects in humans seem to require longer periods of TPN. In adults, 203 surgical patients who had at least 10 days of preoperative TPN without enteral nutrients, no significant decrease in villous height or increase in bacterial translocation were noted compared with those on enterally fed controls [ 102 ]. Guedon and colleagues [ 103 ] performed biopsies in the duodenum of seven adults (all with inflammatory bowel disease) before TPN, after about 3 weeks of TPN, and after discontinuing TPN and restarting oral feedings. They noted no change in gross villous morphology with only moderate decrease in microvillus height after 21 days of TPN. Buchman and colleagues [ 3 ], however, found a significant decrease in villous height after 2 weeks of TPN study involving eight healthy volunteers. Although mucosal thickness decreased significantly, in contrary to animal studies, villous architecture was preserved after TPN; and five days of enteral refeeding was sufficient to reverse the intestinal morphologic changes. Pironi and colleagues [ 104 ] performed endoscopic biopsies in 2 adult patients who underwent long-term TPN for the treatment of a postoperative enterocuteneous fistula, and their results showed changes in villous height and crypth depth after 2 and 3 months of TPN; two months after oral refeeding, the values of morphometric parameters were significantly greater than those observed after TPN and were similar to those controls. Groos and colleagues [ 105 ] also conducted a study involving 20 adults to investigate whether TPN causes morphological changes in intestinal mucosa of human adults similar to those observed in animals experiments. They found jejunal mucosal atrophy, and a remodeling of luminal surface architecture with disappearance of cell shedding. Bacterial translocation In this review, bacterial translocation was reported only in one study [ 60 ]; it stated that 10 children experienced 24 episodes of septicemia, but only 6 out of 94 infants had bacterial translocation. Andorsky [ 96 ] reported that 9 of 10 children died from infection while on TPN, but no statement has been found whether those children died from infection or from other causes. In a large review, Lipman [ 106 ] critically assessed the effects of TPN and enteral nutrition, and did not find any convincing evidence that TPN promotes bacterial translocation or enteral nutrition prevents bacterial translocation in humans. In a large critical review, Lipman [ 107 ] examined the existence of bacterial translocation and the effects of enteral nutrition on bacterial location using both animal and clinical trials. He did not find any evidence supporting that enteral nutrition preserves gut barrier functions and prevents bacterial translocation. He concluded that bacterial translocation is an independent of intestinal structure; and also the villous atrophy seen may be species specific. Pierro and colleagues [ 58 ] used throat and rectal swabs as surveillance culture samples twice a week to investigate whether carriage of abnormal flora was associated with increased risk of sepsis and septicemia in children receiving TPN; and they concluded that infants receiving TPN are at very low risk of developing sepsis or septicemia as long as their surveillance cultures reveal normal flora only. However, the presence of abnormal PPM in the throat or the gut increases the risk of sepsis and septicemia by 25 % [ 58 ]. Surveillance samples are defined as specimens obtained from body sites were PPM are normally carried by the digestive tract. IgA The importance of enteral stimulation on the mucosa-associated lymphoid tissue (MALT) system was studied in neonates who died soon after birth. Histological examination of the biopsies showed that infants who received enteral stimulation showed clear evidence of B cells and T cells within the mucosa; whereas, parenterally fed infants who died had a villous atrophy. Gut and bronchus samples were obtained and related to time of death of infants who died of sudden infant death syndrome between two weeks and 90 moths after birth. IgA plasma cells first appeared in the gut and later in the bronchi as the system matured. Plasma cells increased rapidly over time as IgA plasma cells predominated by three weeks in the gut and six weeks within the bronchi [ 108 ]. In this review, none of these studies have tested whether TPN decreases S-IgA levels in intestinal mucosa causing increased bacterial translocation. Although there was only one study focused on the changes in IgA production during TPN in 8 healthy human volunteers [ 109 ], the results of this study did not show significant differences in the number of immunoglobulin-containing cells. Intestinal immune function was not affected by 2 weeks of TPN. Woodcock and colleagues [ 61 ] also investigated whether bacterial translocation is associated with changes in gut immune function in 22 adult patients (11 of whom were positive bacterial translocation and 11 negative). However, these patients were not on TPN. The study results showed a significant increase in immune functions, especially higher numbers of plasma cells and IgA and IgM values in small bowel mucosa of patients in whom bacterial translocation has been positive. Hyperglycemia In this review in vitro studies on infants and children demonstrate that TPN inhibits functions of neutrophils, cytokines, and bactericidal activity of phagocytosis [ 7 , 51 , 67 ]. Investigators found that cytokine production after bacterial challenge was directly impaired by addition of TPN solution. In these patients calorie intake varied from 100 to 130 kcal/kg/day; and TPN emulsion consisted of carbohydrate 15 to 18 g/kg/day, amino acids 2-3 g/kg/day, and fat 3 to 4 g/kg/day. Although these studies do not state whether these children's serum glucose levels were high, perhaps TPN-induced hyperglycemia may have also contributed to dysfunction of neutrophils. It has been hypothesized that the elevation of blood glucose in patients receiving TPN may be associated with complications, such as immunosuppression [ 110 ]. Glucose concentrations above 220 mg/dL have been shown to glycosylated immunoglobulins, causing a significant reduction in opsonic activity, which adversely affects wound healing and immunity [ 111 ]. There is a large body of evidence demonstrates that there is a positive correlation between high serum glucose levels and increased infection rates in acutely ill patients in critical care medicine. Insulin therapy seems to be beneficial in sepsis patients [ 54 ]. Rassias and colleagues recently performed two comparative randomized control clinical trials one involving 30 non-diabetic adult cardiac patients (15 in each group) [ 112 ], and the second clinical trial involving 26 diabetic adult cardiac patients (13 in each group) [ 113 ] who were scheduled for elective cardiac surgery with cardiopulmonary bypass surgery. The experimental group received intensive insulin treatment while the control group received standard insulin therapy. They found a significant increased in neutrophil count and neutrophil functions in intensive insulin therapy group. They suggest that hyperglycemia leads to increased endothelial adherence of neutrophils and may lead to lower white blood cells in hyperglycemic patients. Since its introduction, TPN has been a life saving nutrient to severely malnourished patients. It was believed that "if some nutrition is good, more is better" [ 114 ]. Even in treating diabetic patients in the hospital, for decades, an oral tradition was passed down from clinician to clinician, "keep the patient a little sweet" [ 115 ]. It has been recently recognized that in the presence of sepsis an increased intake of energy, or overfeeding the patients with carbohydrates or fats, increases the risk of complications, which contributes to hyperglycemia and sepsis. This TPN-induced and/or physiological stress-induced hyperglycemia may have also contributed to increased rates of sepsis [ 39 , 114 ]. In a careful analysis of stress-induced hyperglycemia in 102 non-diabetic patients receiving TPN, subjects who received dextrose at >5 mg/kg/min, had 50% chance of developing hyperglycemia [ 116 ]. In contrast, dextrose infusion at < or = 4 mg/kg/min, risk was substantially reduced. Therefore, to prevent hyperglycemia and infection complications in hospitalized patients, TPN dextrose infusion rates should be at < or = 4 mg/kg/min [ 117 ]. Two randomized prospective clinical trials [ 118 , 119 ] of aggressive insulin therapy now have revolutionized our current philosophy about treating critically ill hospitalized patients [ 53 ]. In the first study, a total of 620 patients (306 patients randomized to treatment with insulin-glucose infusion followed by multidose subcutaneous insulin for ≥ 3 months and 314 to conventional therapy) [ 119 ]. In the second study, Van den Berghe and coworkers [ 118 ] conducted a large prospective, nonblinded, randomized clinical trial (a total of 1548 surgical patients in ICU) of intensive glycemic control (glycemic goal of 80-110 mg/mL [4.4-6.1 mmol/L] compared with conventional treatment (maintenance of blood glucose at a level between 180 and 200 mg/dL). At the end of the study, results showed that intensive insulin treatment reduced episode of septicemia by 46%, and overall mortality rate by 32% during their stay in ICU. Hyperglycemia is commonly seen in stressed patients during administration of TPN or other glucose-containing solutions. Stress may also induce insulin resistance in adipose tissue, liver and heart [ 120 ]. However, the treatment of hyperglycemia starts only after glucose levels have exceed 200 to 250 mg/dL (11–14 mmol/L) because it was believed that avoidance of hypoglycemia and its potential consequences is more important than glycemic control while patients are hospitalized. The most recent target range for plasma glucose in the hospital are: preprandial = <110 mg/dl; peak postprandial = <180 mg/dl; and for critically ill patients = 80-110 mg/dl [ 53 ]. Some studies reported that the complications during TPN were associated with the residual or the remaining bowel length in patients with SBS. The longer the residual small bowel, the shorter the duration of TPN, and the fewer infections [ 5 , 96 ]. The results of Dahlstrome's [ 90 ] study revealed that children whose bowel length was <25 cm developed very low levels of circulating lymphocytes causing immuno-competence due to very short bowel length, and not ingesting the food antigens which are the main stimulants of lymphocytic proliferation and immunoglobulin (IgG, IgA) synthesis. Dahlstrome predicted that lack of ingested antigens might have caused the low circulating lymphoctes, which resulted in infection. He found that enteral support in combination with TPN did improve, but not normalize the plasma amino acid concentrations in the children investigated. Enteral versus parenteral nutrition It has been suggested that enteral stimulation is a required component to protect gastrointestinal and respiratory immunity via increased levels of mucosal IgA [ 15 , 121 ]. Enteral nutrition refers to nutrition either ingested orally or delivered to the stomach or intestine by tube via nasogastric (NG) tube, gastrostomy tube (GT), nasojejunal (NJ) tube, or jejunostomy tube (JT). Evidence showed that enteral nutrition is less expensive, but not safer, or more physiologic than TPN [ 106 ]. In a prospective observational nursing study [ 122 ], 64 elderly patients who were fed by an NG tube in an internal medicine. The type of formula was not stated in the study, but based on this report; most patients had electrolyte imbalances, tube dislodgements, hyperglycemia, diarrhea, pulmonary aspirations, and nasal ulcers. In another prospective observational study [ 123 ], a total of 153 critically ill patients also were fed with NG tube. Before each feeding, gastric residual volume was checked by using 50-ml syringe by aspirating the tube. The study results showed that there was a negative relationship between high gastric residual volume and days in the hospital stay. High gastric aspirate volume was associated with a higher incidence of nasocomial pneumonia, a longer length of hospital stay, and higher mortality. Effects of early enteral feeding on bacterial translocation In Weber's study [ 94 ], 21 infants and children with SBS were begun with enteral feedings via continuous drip technique with an elemental formula (Pregestemil:Mead-Johnson, Evansville, IN), after 3 weeks of postoperative period. Results showed that after enteral feeding began, 76 % of children had 67 episodes of bacteremia. The investigator suggests that early enteral feeding increases infection. However, Marik and Zaloga [ 124 ] performed a systematic review of 15 PRCT included 603 patients to evaluate the effect of early enteral nutrition on outcome of critically ill and injured patients. The results of this study showed that there was a significantly lower risk of infection in 19 % of the patients who received early enteral diet compared with 41 % in the delayed group. This study concluded that early feeding decreases infectious complications and length of hospital stay. In a large review, by pooling data from four PRCT involving 142 patients who had gastrointestinal cancer, Klein et al. [ 125 ] compared early postoperative JT feeding with the usual advancement of oral diet as tolerated. Based on the aggregated data, there were no significant differences in postoperative morbidity or mortality. However, Daly et al. [ 126 ] performed a PRCT involving 85 adult the patients (41 on supplemented diet, 44 on standard diet) with cancer of upper gastrointestinal tracts. The patients who were fed early postoperatively [on the first postoperative day] via JT with a formula supplemented with arginine, ribonucleic acids, and ω-3 fatty acids had fever infections, minimal wound complications, and a shorter duration of hospitalization compared to those who received standard formula. Braga at al. [ 127 ] also performed a PRCT involving 166 patients undergoing gastrointestinal surgery to evaluate the impact of the route of administration of artificial diet, IMPACT ® (Novartis Nutrition, Bern, Switzerland) supplemented with arginine, ribonucleic acid, and omega-3 fatty acids versus standard diet on outcome. Their results showed that early postoperative enteral infusion of nutrients is safe and well tolerated and stimulates an early return of bowel function. Immunonutrition There has been a considerable amount of interest in recent years in the use of alternative specific gut substrates. The term immunonutrition has been coined to describe molecular compound that, while being dietary components, such as glutamine, arginine, ω-3 fatty acids, ribonucleic acid, also influence immunologic response mechanisms when added to standard TPN solutions or enteral nutrition [ 128 - 130 ]. Glutamine, a nonessential amino acid, is considered to be the principal respiratory fuel for enterocytes, colonocytes, lymphocytes, and macrophages, and is a precursor for nucleotides synthesis and for glutathione, an important antioxidant that may be protective in a variety of circumstances. It has been hypothesized that during the stress, glutamine becomes conditionally essential amino acid. In the late 1980s, glutamine has been the most extensively studied substrate in animal research, especially in rat TPN model. It has been suggested that parenteral or enteral glutamine supplements could dramatically effect on the maintenance of GALT; and glutamine-enriched TPN significantly attenuates the mucosal hypoplasia associated with prolonged TPN following massive bowel resection [ 131 - 134 ]. Therefore, it was hypothesized that glutamine supplementation in human modal would have similar effects [ 77 ]. Encouraged by findings in animal investigations, in 1991 Darmaun and colleagues [ 135 ] first time reported a reduced rate of glutamine turnover in short bowel syndrome [adult] patients, and in 1994 in pediatric patients [ 136 ]. They suggested that the small intestine was a target organ for glutamine utilization in both adults and children. C.K. Ogle (unpublished, cited in Alexander, 1993) [ 137 ] also has shown that excess glutamine added to culture media can improve antimicrobial killing by neutrophils isolated from blood of burn patients and normal controls. Alexander also argues that infection can be reduced 75% and hospital stay by 20% by immune enhancement via enteral nutrition. In the following years, many controlled trials and case series were conducted to demonstrate the benefits of immune enhancing substrates to the intestinal adaptation process in patients with SBS during long-term TPN [ 138 - 147 ]. The results of these published studies have been conflicting. Although glutamine supplementation has been shown to be value in pediatric patients [ 38 , 148 ], Duggan and colleagues [ 149 ] conducted a randomized double-blinded, controlled clinical trial of enteral glutamine supplementation in 20 infants with SBS. At the end of study, there was no significant difference in weight and length gain between two groups. Two of nine infants in the glutamine group developed urinary and bloodstream infections. Overall, this study results showed that glutamine supplementation was safe, but had no effect on duration of TPN, tolerance of enteral feeds, or intestinal absorption or barrier function. However, in a PRCT pilot study Barbosa and colleagues [ 150 ] examined the tolerance and efficiency of glutamine in a pediatric intensive care unit setting with nine mechanically ventilated infants (treatment group = 5, and control group = 4). Their results showed that tolerance to glutamine-supplemented diet was good. Bacterial infections reported in 75% of the placebo population and 20% of the treated infants. There were two deaths from microbial infection in control group, suggesting a beneficial effect of glutamine. However, Scolapio and colleagues [ 142 , 151 ] conducted a series of double-blind, 6-week, placebo-controlled, crossover trial in 8 adult patients with SBS to assess: 1) the effects of growth hormone, glutamine, and high-carbohydrate-low-fat (HCLF) diet on body composition, and 2) to evaluate the potential mechanisms that might explain the beneficial effects of immunonutrition treatment. Active treatment consisted of subcutaneous recombinant human growth hormone and oral L-glutamine. In this study, two patients developed carpal tunnel syndrome that resolved 5 days after discontinuation of active treatment. Two patients noted sleep disturbances while on active treatment. Patients developed peripheral leg edema and weight gain; this combination therapy resulted in a significant increase in sodium and potassium absorption. Two studies [ 145 , 152 ] also reported similar findings in addition to severe hand pain, and gynaecomastia in a male patient. Two systematic reviews [ 138 , 153 ], and two large critical reviews [ 77 , 125 ] conclude that glutamine and growth hormone at present time can not be recommended in short bowel syndrome; more large-scale studies, or multicentered clinical trials are required. Currently, two commercially prepared immune-enhancing enteral formulas are available in the market, IMPACT ® (Novartis Nutrition, Bern, Switzerland) containing glutamine, arginine, dietary nucleotides and fish oil; and Immune-Aid (McGaw, Irvine, CA) containing glutamine, arginine, nucleic acids, and omega-3 fatty acids. Some studies indicate that these immune-enhancing diets demonstrated successful results or benefits in studies of sepsis, immunity, altered intestinal permeability, inflammation, including reduced mortality and the length of hospital stay [ 154 - 156 ]. Some studies also show that the use of breast milk in SBS contains high levels of immunoglobulin A (IgA), nucleotides, leukocytes, and other components that bolster the neonate's immune system[ 20 ]. In a study, a continuous infusion of small amounts of breast milk in infants with SBS is also resulted in a shorter duration of TPN compared to control group [ 96 ]. Overall, findings are inconclusive. Immunonutrition may be species or patient specific. Implications for evidence-based nursing practice Multidisciplinary nutritional team The successful management of infants and children with SBS is demanding and requires a multidisciplinary approach. The care of patients with SBS can become so complex that many patients with SBS almost lose their primary physician and become patients of multiple specialists. Long-term care is, therefore, best delivered by a variety of healthcare professionals, including the gastroenterologists, surgeon, nurses, nutritional support team, care coordinators, microbiologist or infection control team, clinical chemist, pharmacists, social workers, and psychologist with expertise in infant feeding difficulties. Patients cared for in this way have better outcomes [ 25 , 148 , 157 ]. Infants go through a critical developmental phase in feeding at about 6–12 months, and those who do not receive oral feeds at that time will loose the window of opportunities for sucking and swallowing coordination, and will have long-term eating difficulties [ 21 , 158 ]. In addition, children fed enterally or parenterally may have forgotten or never learned the association of with hunger, oral feeding, and satiety; they develop "defensive oral behaviors," such as gagging, choking, and vomiting. For a sick child, if this important developmental phase is ignored during the first year of life, flavor, texture, odor, and extreme temperature can be overwhelming. It is important that the pediatric nurses should consult pediatric occupational therapist and speech therapist, and behavioral psychologist to work with these children [ 159 ]. Clinical management The management of neonates and children with SBS continues to provide a major challenge for practitioners [ 160 ]. A group of pediatric surgeons critiqued the current clinical management of SBS in children, which is mostly based on "trial and error" regimen. They strongly recommended that prospective, randomized controlled trials should be established, and an evidence-based rather than a "gut-feeling" -based approach to be used in SBS children, such as intestinal permeability and sugar absorption tests, evaluation of adaptation by gut hormone production, immunohistochemistry, and other new techniques which are still in experimental stage [ 154 ]. Pediatric nurses should, therefore, provide an evidenced-based nursing care to these children with the support of APRN. Hypersecretion is seen in infants and children during the initial first 6 months after surgery and a start of enteral feeding. The H 2 blockers and proton pump inhibitors are effective for reducing gastric fluid secretion, and therefore, will also reduce fluid losses during this period [ 77 , 161 ]. The diagnosis of bacterial overgrowth is determined by aspiration of the jejunum and demonstration of increased bacterial contents by culture. Since this procedure is invasive and infeasible in some sick infants, breath hydrogen test can be used easily as noninvasive diagnostic test [ 162 ]. The breath hydrogen test is an oral test that uses the measurement of hydrogen in the breath to evaluate carbohydrate malabsorption and bacterial overgrowth in small intestine. Hydrogen gas is produced by bacterial fermentation of undigested carbohydrate that reaches the colon, enters the portal and systemic venous return, and is then released in the breath. After fasting 4-6 hours, the child ingests a load of carbohydrate (12 g/kg, maximum 50 g), and the end-expired air is collected in sealed plastic bags by aspirating 5 mL of air after each breath to total of 20-30 mL via nasal prong attached to a face mask at timed intervals up to 2 hr after ingestion. Malabsorption of any carbohydrate can be evaluated. The child should not be taking antibiotics at the time of the study because these drugs alter the colon flora and suppress hydrogen gas production [ 163 - 165 ]. In one of the clinical studies, investigators used 50 mg 13 C-xylose in children ages 3 to 12 years for breath hydrogen test [ 166 ]. 13 C-xylose is a safe, nonradiactive isotope that has recently been developed (Martek, Columbia, MD). This study results showed that all patients with bacterial overgrowth had positive breath test results (100% sensitivity). For infants prone to overgrowth, routine scheduled antibiotic treatment may be useful. D-Lactic acidosis has been reported in children with bacterial overgrowth, causing metabolic acidosis, drowsiness, and confusion. This diagnosis should be considered in a child with SBS who presents with metabolic acidosis, high serum anion gap, normal lactate level, and without urinary ketones [ 161 ]. Treatment for this syndrome is to start oral metronidazole, neomycin, vancomycin, and avoidance of "refined" carbohydrates [ 77 ]. Dietary management Optimal timing of the initiation of enteral nutrition has not been established [ 161 ]. Patients who undergone massive bowel resection require TPN for the first 7-10 days. American Gastroenterological Association (AGA), based on the current literature review, suggests that nutritional therapy should not be introduced until the patient is hemodynamically stable and fluid management issues are relatively stable. Although the optimal formula for feeding infants and children with SBS is not well established, elemental amino acid based hydrolyzed formulas are suggested by some clinicians and have been shown to be well tolerated [ 95 ]. Dietary protein is first digested, and then absorbed as dipeptides and tripeptides. Therefore, it was reasoned that dietary protein provided in a predigested form would be more readily absorbed [ 77 ]. A prospective, randomized, cross-over trial compared two protein hydrolysate formulas given by continuous nasogastric infusion to six malnourished infants with SBS aged 1-13 months [ 167 ]. Although there was good tolerance for both formulas and satisfactory weight gain, and also energy absorption was the same, but differences in the amount of malabsorbed carbohydrate existed. These researchers suggested that protein hydrolysate formulas should be reformulated with a lower concentration of carbohydrates and a higher one of fat. A controlled trial compared two feeding regimens, continuous intragastric feedings and intermittent oral feeding, in nine infants with protracted diarrhea and malnutrition and two infants with surgically created short bowel [ 168 ]. Continuous nasogastric feeding caused significant increases in enteral balance of the major nutrients, whereas intermittent feedings resulted in negative or only slightly positive enteral balance. There was also a significant increase in body weight during the continuous feeding as compared to the intermittent feeding. The authors suggest that improved enteral balance can be achieved with continuous feeding in infants with short bowel disease [ 168 ]. In infants under 1 year of age with SBS who might have dilated gut, poor motility, or bacterial overgrowth, increased epithelial permeability to food antigens occurs frequently and may result in the development of allergic reaction to any protein in the formula. In order to reduce the risk of allergic injury to the gut, a hypoallergenic formula, such as Nutramigen, Pregestemil(Mead Johnson Laboratories, Evansville, IN), and Alimentum (Ross laboratories, Columbus, OH), should be used during the first year of life. To further decrease the risk of allergic reaction in highly susceptible infants, an amino acid based formula should be used. Neocate (SHS Inc, Rockville, MD, USA) is the only one listed in this category for infants. Elecare (Ross Laboratories), which is quite similar to Neocate, was formulated for use in children over 1 year of age [ 169 ]. Initially slow introduction of continuous enteral feeding via a nasogastric or gastrostomy tube feeding is beneficial to reduce emesis, diarrhea, and maximally saturate carrier proteins. Rationales for this that constant saturation of mucosal digestive enzymes and transport carriers should be optimize absorption. Regardless of the length of small intestine, some oral, even if only 1 or 2 ml per day, and a few ml in continuous fashion should be offered from an early stage [ 158 ]. A diluted infusion is initiated continuously at low volumes and increased to 0.67 cal/mL for infants less than 1 year of age or to 1.0 cal/mL in older children. Once the final concentration is achieved in low volumes, enteral feeding rates are advanced, and TPN rates are decreased isocalorically every 1 to 3 days as tolerated [ 170 ]. A marked increase in stool loss by 50 % is usually contraindication to advancing enteral feedings. There is no agreement on the amount of stool output that should be accepted. Limits should be imposed on feeding once the stool output exceeds 45 ml/kg per day [ 161 ]. However, a higher volume of stool output may be acceptable by some clinicians [ 171 ]. If stool losses greater than 45 ml/kg/day or ostomy output strongly positive for reducing substances suggest that enteral feeding advancement should be slowed. Careful fluid and electrolyte management is essential as dehydration can occur rapidly [ 20 , 158 , 161 , 170 ]. Monitoring enteral feedings and verifying tube location Monitoring patients on enteral feeding is required routine assessment of gastrointestinal, metabolic, mechanical, and growth parameters [ 172 ]. Tolerance of enteral feeding is assessed by noting the presence or absence of vomiting, retching, abdominal distention, and diarrhea. For patients receiving gastric feedings, checking for residual formula for every 4 hrs is required to evaluate feeding tolerance or delayed gastric emptying [ 173 ]. During continuous feeding, if a single high gastric residual volume is greater than 1.5 times the hourly feeding of formula infusion, this indicates a sign of intolerance. Feeding should be stopped, and clinician should be notified. Gastric residual should be rechecked every 1-2 hours until the residual drops below the greater than or equal to 50% of the hourly rate mark [ 174 ]. Decreasing the rate by half for a few hours, rechecking residuals, and slowly advancing is recommended to achieve feeding tolerance. Changing the tube feeding might be beneficial. If this is unsuccessful, child can be placed in the right lateral decubitus position and i.v. metaclopromide (Reglan) can be started to enhance gastric emptying [ 172 , 175 ]. A literature review conducted by Metheny and colleagues [ 176 ] found that there was a confusion as how gastric residuals should be handled. Fifty percent of the respondents (registered nurses) from a survey (Mateo, 1996, cited in Metheny et al., 2004) [ 176 ] reported discarding the gastric contents and 49% reported re-administering them. Only one study was located that dealt with outcomes of discarding or returning gastric residuals to the patients (Brooker et al., 2000, cited in Metheny et al., 2004) [ 176 ]. In this study 35 subjects receiving enteral feedings were randomized to either a discard group or a return group. Repeated measures analysis of variance found no significant differences between the two groups in the body weight, serum electrolyte levels, tube clogging, nausea, and the feeding delays. In the absence of more convincing evidence, Metheny and colleagues [ 176 ] suggest for returning gastric residuals less than 500 mL to the patients. Ensuring correct placement of the feeding tube is also necessary. Although auscultation is the most common method of confirming placement, it has been found unreliable. The best evidence for confirming correct tube placement is X-ray [ 177 ]. Metheny and colleagues [ 177 , 178 ] provide evidence-based nonradiographic methods to check the tube location. There are two methods currently available at bedside to nurses to test tube placement during continuous feedings include (1) observing the appearance of fluid aspirated from the tube, and (2) measuring the fluid's pH. Gastric pH usually falls in the range of 1 to 5, whereas intestinal or respiratory pH is usually 7 or higher. If a pH ≤ 6 is a significant indicator of the gastric placement, whereas a pH >6 is an indicator of intestinal placement. Metheny and Stewart [ 178 ] also provide additional methods for checking the position of the tube, such as capnography, spring-gauge pressure manometer, enzyme tests (pepsin and trypsin), and bilirubin analysis; however, the most reliable method of tube placement assessment is the radiograph [ 172 ]. Monitoring TPN complications According to American Gastroenterological Association (AGA) [ 77 ], and American society for parenteral and enteral nutrition (ASPEN) guidelines [ 161 ], TPN should be infused via single lumen catheter with its tip positioned in either the superior vena cava or inferior vena cava to decrease the risk of infection and trombosis. Tunnelled catheters, implantable port, or percutaneously inserted central catheters (PICCs) should be used at home. Peripheral infusion of parenteral formulations is limited to dextrose concentrations of less than 12.5% [ 161 ]. ASPEN suggests that initially, TPN should be started with low dextrose infusion rate [ 161 ]. Starting rate is usually half of the hourly rate for the first hour, and then rate is increased to full rate after checking the blood sugar. Blood glucose should be monitored at least 4 times a day, and should be <180-200 mg/dl [ 77 ]. Glucose intolerance is the major adverse effect seen during the initial infusion period [ 179 ]. Urine sugar and acetone or ketone levels should be less than 2+. ASPEN suggests that dextrose infusion rates in infants and neonates should be 10 to 14 mg/kg per minutes[ 161 ]. Continuous insulin infusion (0.01-0.1 unit/kg per hour) has been shown to be safe and effective in managing hyperglycaemia in the neonate. Glucose concentration should be monitored at least every 2 hours, aiming for blood glucose concentration between 100 and 150 mg/dL. In children less than 2 years of age, hypoglycaemia develops rapidly if feedings are delayed or interrupted. ASPEN recommends that TPN be tapered over 1 to 2 hours in infants before discontinuation to avoid hypoglycaemia [ 161 ]. Some iv medications need to be administered over 30 to 60 minutes. If the patient has only one line which TPN is running, the nurse should communicate with the paediatric pharmacist to reconstitute the drug with 5% or 10% dextrose if that drug is incompatible with TPN. This will also prevent hypoglycaemia if TPN is temporarily interrupted for iv medication. Four major categories of complications exist: (1) mechanical or technical; (2) infectious; (3) metabolic; and (4) nutritional [ 161 ]. Mechanical or technical complications are usually related to catheter placement, such as pneumothorax, hemothorax, cardiac tamponade, or equipment malfunction. Catheter thrombosis is a significant problem of all central lines. A potential early sign of catheter thrombosis is progressively sluggish or absent blood return on catheter aspiration. Thrombolytic agents are effectively used to dissolve thrombi. Catheter related infections are the most common complications associated with central line TPN [ 161 ]. Localized infections are, such as erythema, tenderness, induration, or purulence that occurs at the exit-site or along the tunnel. Pocket infections occur only with implantable ports. Systemic infections, formerly called catheter sepsis or bacteraemia, are defined as positive culture of the catheter tip or a positive pathogen isolated from both blood drawn through the catheter and peripherally. Staphylococcus epidermidis , Staphylococcus aureus , and other skin flora are the most common pathogens isolated in patients with systemic infections. Enterococcus and enteric flora are the next most frequently isolated organisms. Catheter-related infections are treated based on the type of infection and pathogen [ 161 ]. Conclusion TPN is a life saving alternative nutritional support to severely malnourished surgical patients. Its use is indicated to prevent the adverse effects of malnutrition in patients who are unable to tolerate nutrients by oral or enteral routes. Evidence from a large systematic reviews involving 82 randomized control clinical trials on both adult and paediatric population showed that: 1) TPN did not have a significant impact on survival, 2) TPN did not affect either the total or infectious complication rates, 3) TPN had no effect on the duration of hospitalization of surgical patients [ 180 ]. Based on the current evidence, TPN seems to be safe and a life saving solution. List of abbreviations used Short bowel syndrome (SBS) Total parenteral nutrition (TPN). Immunoglobulin A (IgA) Secretory immunoglobulin A (S-IgA), Gut-associated lymphoid tissue (GALT) Gastrointestinal (GI) Mucosa-associated lymphoid tissue (MALT) Necrotizing enterocolitis (NEC) Migrating motor complex (MMC), Histamine2 (H 2 ) Nitric oxide (NO), Nitric oxide synthase (NOS Nonsteroidal anti-inflammatory drugs (NSAIDs) Tumor necrosis factor (TNF) Interleukin-1 (IL-1) Interleukin-6 (IL-6) Adrenocorticotropic hormone (ACTH) Antidiuretic hormone (ADH), Free fatty acids (FFA), Pathogenic microorganism (PPM) Neutrophil polymorhonuclear (PMN) Prospective randomized clinical trials (PRCT) Intensive Care Unit (ICU) Nasogastric (NG) Gastrostomy tube (GT) Nasojejunal (NJ) Jejunostomy tube (JT) American Gastroenterological Association (AGA) American society for parenteral and enteral nutrition (ASPEN) Percutaneously inserted central catheters (PICCs) Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here:

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523837

Assessment of Volume Depletion in Children with Malaria

ABSTRACT Background The degree of volume depletion in severe malaria is currently unknown, although knowledge of fluid compartment volumes can guide therapy. To assist management of severely ill children, and to test the hypothesis that volume changes in fluid compartments reflect disease severity, we measured body compartment volumes in Gabonese children with malaria. Methods and Findings Total body water volume (TBW) and extracellular water volume (ECW) were estimated in children with severe or moderate malaria and in convalescence by tracer dilution with heavy water and bromide, respectively. Intracellular water volume (ICW) was derived from these parameters. Bioelectrical impedance analysis estimates of TBW and ECW were calibrated against dilution methods, and bioelectrical impedance analysis measurements were taken daily until discharge. Sixteen children had severe and 19 moderate malaria. Severe childhood malaria was associated with depletion of TBW (mean [SD] of 37 [ 33 ] ml/kg, or 6.7% [6.0%]) relative to measurement at discharge. This is defined as mild dehydration in other conditions. ECW measurements were normal on admission in children with severe malaria and did not rise in the first few days of admission. Volumes in different compartments (TBW, ECW, and ICW) were not related to hyperlactataemia or other clinical and laboratory markers of disease severity. Moderate malaria was not associated with a depletion of TBW. Conclusions Significant hypovolaemia does not exacerbate complications of severe or moderate malaria. As rapid rehydration of children with malaria may have risks, we suggest that fluid replacement regimens should aim to correct fluid losses over 12–24 h.

Introduction Malaria claims one million lives annually, with more than 90% of these being those of children in sub-Saharan Africa [ 1 ]. Most deaths of hospitalised children occur in the first 24 h after admission. Even modest improvements in management during this time may improve survival [ 2 ]. There is considerable disagreement about the degree to which children with severe malaria become hypovolemic. In east African studies, clinical signs of severe malaria (such as tachycardia, prolonged capillary refill times, and decreased urine volume) have been interpreted as evidence for volume depletion [ 2 , 3 , 4 , 5 , 6 ]. However, determining fluid compartment volumes is the first and most critical step in optimising fluid replacement therapy for children with malaria because clinical assessment of fluid status is difficult and imprecise [ 7 ]. Our study was designed to measure total body water volume (TBW) and extracellular water volume (ECW) using nonradioactive tracer dilution techniques and to derive intracellular water volume (ICW). Bromide distributes in the extracellular space so that concentrations measured 2–4 h after administration safely and reliably estimate ECW. Heavy water ( 2 H 2 O) space represents TBW. ICW is calculated by subtraction of the ECW from the TBW. Tracer dilution methods are expensive and invasive and cannot be repeated at short intervals. Therefore, we simultaneously calibrated a noninvasive technique of bioelectrical impedance analysis (BIA) [ 8 ] to estimate the fluid volumes. BIA measures the opposition (impedance) of the body to the flow of a small alternating current between electrodes placed on the hand and the foot, and then estimates TBW and ECW using regression equations derived by calibration against ‘gold standard' tracer measurements of fluid volumes. We hypothesised that volume changes in fluid compartments would reflect disease severity in malaria and that these changes would be related to established markers of disease severity [ 9 ]. We also calibrated BIA assessments in children with moderate and severe malaria with direct measurements of volume of TBW and ECW. Methods The study was conducted at the Albert Schweitzer Hospital, Lambaréné, Gabon, and Centre Hospitalier de Libreville, Gabon. It was approved by the ethics committees of the International Foundation of the Albert Schweitzer Hospital, the Gabonese Ministry of Health, and the University of Tübingen. Children (aged 1 to 10 y, inclusive) admitted with suspected severe or moderate malaria were referred to the study team, who assessed them within 15 min, and the children were admitted to the study once informed consent had been obtained from the parents. Malaria was defined as the presence of asexual forms of Plasmodium falciparum in thick or thin blood films. Severe malaria was malaria with one or more of the following features: blood lactate ≥ 5 mmol/l, blood glucose ≤ 2.2 mmol/l, Blantyre coma score ≤ 2, or repeated, observed seizures [ 2 ]. Moderate malaria was malaria without any of the features of severe malaria but with a requirement for parenteral treatment because of one or more of the following: a history of frequent (> 2) and recent vomiting (within 12 h), drowsiness, obtundation, or prostration [ 2 ]. Alternative diagnoses were excluded clinically. Assessment and Management On admission children were weighed (undressed) with pediatric scales accurate to within 100 g (Seca, Birmingham, United Kingdom). A history was taken from parents, and the child was examined with particular attention to signs of dehydration, including: capillary refill time, skin turgor, sunken eyes, dry mucous membranes, and absence of tears. Vital signs, blood glucose and lactate concentrations, and hematocrit and parasitaemia were measured every 4 h for the first 24 h and then every 6 h until recovery. If peripheral venous access was impracticable, then a femoral central venous catheter was inserted, and the central venous pressure was measured every 4 h with a manometer zeroed at the midaxillary line. Children were managed in a standard manner as previously described [ 10 , 11 , 12 ]. All received intravenous quinine (20 mg/kg salt intravenously as a loading dose given over 4 h, then 10 mg/kg intravenously every 12 h until able to take oral medication) (Quinimax, Sanofi Synthelabo, Paris, France). Hypoglycaemia (blood glucose ≤ 2.2 mmol/l) was treated with 25% glucose (2 ml/kg). Convulsions were treated with diazepam (0.3 mg/kg intravenously or 0.5 mg/kg intrarectally) (Roche, Basel, Switzerland), and repeated convulsions were treated with phenobarbital (7.5 mg/kg intramuscularly). In addition to a 5% or 10% dextrose infusion (at least 3 mg/kg/min, i.e., 1.6 or 3.2 ml/kg/h), physicians were free to give any fluid replacement regimen as clinically indicated, including boluses of saline or blood. A strict fluid input/output chart was kept for each child. A blood transfusion of 20 ml/kg of cross-matched whole blood tested for blood-borne pathogens was given over 4 h if the hematocrit fell below 15%. Measurement of TBW and ECW A sterile standard dosing solution was prepared by adding 119 ml of 2 H 2 O per litre of 2.315% sodium bromide solution. A baseline sample of blood (3 ml) was drawn, and the plasma frozen at −70 °C for bromide and 2 H 2 O assays. At the start of the study, 2.8 ml/kg of the dosing solution was administered intravenously over 20 min. Four hours after injection a second blood sample was drawn (1.5 ml) for the determination of blood bromide and 2 H 2 O concentrations. Parents were asked to return with their children 28 d after admission; children were examined, and measurement of TBW and ECW repeated. 2 H enrichment was measured in duplicate by isotope ratio mass spectrometry, using a Sira 10 instrument (Micromass, Cheshire, United Kingdom) as described [ 13 ]. The precision of the TBW determination was estimated at 0.3% of the value obtained. Batch analysis of bromide enrichment in plasma was performed by high-performance anion-exchange liquid chromatography as described [ 14 ]. The intra-assay coefficient of variation for bromide was better than 1.5%. BIA was performed using a SEAC SFB3 multifrequency bioimpedance meter (Impedimed, Brisbane, Australia). An alternating electrical current of 200 μAmp was applied between 2 Ag/AgCl electrodes at the right hand and right foot. Whole body and segmental impedance were measured by rotating the sensing Ag/AgCl electrodes between four sites on the ankles or wrists as described [ 13 ]. Each set of measurements was taken at 496 frequencies between 4 kHz and 1012 kHz. Measurements were taken at 0 h, 4 h, 12 h, 24 h, and discharge. The process took about 2 min to perform and was simpler than obtaining an electrocardiogram. Data were analysed with software (Bioimp, version 1.1.0, Impedimed) that uses nonlinear regression to fit measured data to semicircular Cole-Cole plots [ 15 ] of reactance against resistance. Resistance and reactance values, obtained from the Cole-Cole plot for specific frequencies (0, 4, 50, and 100; characteristic and infinite kHz) were used in further analysis [ 13 ]. Plasma electrolytes were measured (Beckman Coulter, Allendale, New Jersey, United States), and osmolality was calculated using freezing point depression on a Micro Osmometer (Vitech Scientific, West Sussex, United Kingdom). Osmol gap was calculated as described [ 16 ]: where OG is osmol gap, MO is measured osmolality, [Na + ] is plasma sodium concentration (millimoles/litre), [glucose] is plasma glucose concentration (millimoles/litre), [urea] is plasma urea concentration (millimoles/litre), 1.86 is a correction factor as sodium chloride is only 93% dissociated, and 0.93 is the assumed proportion of water in plasma. Statistical Methods Statistical analyses were carried out using Stata Statistical Software (Releases 6.0–8.0, College Station, Texas, United States). After checking distributions with the Shapiro-Wilks W test, and transforming data logarithmically if necessary, we analysed normally distributed data by two-tailed Student's t test and nonparametric data with the Wilcoxon sign-rank test. Proportions were compared with Fisher's exact test, and correlations were assessed by linear regression analysis of Pearson or Spearman. Predictive values for TBW and ECW from impedance measurements were obtained by multivariate analysis in a backward elimination process with p < 0.05 for entry and p < 0.10 for exit, confirmed by forward selection. This analysis was based on the first pair of BIA and isotope dilution measurements after admission. In a second step, whole body BIA models were replaced with segmental BIA models as previously described [ 13 ] and compared with whole body estimates. Errors in the BIA estimate and isotope dilution methods were compared. TBW estimates were corrected for fluid input and output during the period of measurement. Sample size was calculated from previously published values [ 17 , 18 ] assuming a mean (SD) for convalescent TBW of 586 (30) ml/kg; we wished to detect a 5% difference in TBW between severe and moderate cases with a power of 90%. Results Between October 1999 and March 2000, 205 children who were judged ill enough to be hospitalised were referred to the study team. One hundred and thirty-two children had malaria, 20 with severe and 35 with moderate disease. Ten children with moderate and two with severe malaria were ineligible for study because of their age, and for seven children (one with severe malaria) consent could not be obtained. One child died before inclusion into the study, leaving nineteen children with moderate and sixteen with severe malaria admitted to this study. The median (interquartile range [IQR]) time from admission to administration of tracers was 54 (37–84) min, during which complications such as convulsions or hypoglycaemia were treated. The baseline characteristics of children are given in Table 1 . Those with severe malaria had significantly higher pulse rates, mean arterial pressure and blood lactate concentrations, and a longer capillary refill time ( p < 0.001), compared to children with moderate malaria. Capillary refill time and blood lactate concentrations were correlated with each other (adjusted r 2 [adj r 2 ] = 0.25, p = 0.031). Table 1 Baseline Characteristics of Children with Malaria Mean (SD) / median (IQR) shown a Parasitaemia values are geometric mean (range) The volume of fluid (including blood) given in the first 4 h of the study was similar in children with severe (median [IQR, range] of 3.7 [2.2–5.8, 2.1–11.4] ml/kg/h) and moderate (median [IQR, range] of 3.3 [2.2–4.2, 1.2–15.1] ml/kg/h) malaria ( p > 0.5). There was rapid correction of vital signs in all children ( Figure 1 ), and vital signs were similar in both study groups by 8 h. There were two deaths (4.5 and 6 h after admission), and two children with severe malaria had major persistent neurological deficits (4/16 [25%] with adverse outcomes). Central venous pressure (CVP) measurements were obtained in six children with severe malaria. The median (IQR) CVP on admission was +6.5 (3–7.5) cm H 2 O with no significant rise in the first 24 h. Figure 1 Vital Signs of Children during the First 24 h after Admission Mean and 95% confidence interval shown. Red circles, severe malaria; blue triangles, moderate malaria. (A) Pulse (per minute), (B) mean arterial pressure (millimetres Hg), (C) respiratory rate (per minute), and (D) blood lactate concentration (millimoles/litre). Fluid Volumes Volume determinations using isotope dilution were available at baseline in all but one of the children with severe malaria. The values for the TBW, ECW, and ICW are given in Table 2 . The TBW was significantly lower at admission compared with day 28 for the severe cases ( p = 0.028) but not for moderate cases ( p = 0.109). The mean (SD) TBW was lower in severe than moderate malaria at admission: 524 (44) ml/kg versus 555 (50) ml/kg ( p = 0.052). The mean (SD) change in TBW between admission and follow up was 48 (42) ml/kg and 12 (37) ml/kg for severe and moderate cases, respectively. Individual data from the children with severe malaria are shown in Table 3 . Table 2 Volumes Determined by Tracer Dilution and Electrolyte Measurements Mean (SD) / median (IQR) shown a Change in volume as measured by isotope dilution between admission and day 28 b Measurements of ECW and ICW were available at follow up in three cases with severe and ten with moderate malaria (discrepancy due to difficulties obtaining adequate sample volumes) Table 3 Individual Details of Severely Ill Children a Missing values because discharge weights unobtainable Bromide space (ECW) measurements were available in all but two children with severe malaria and in all but one of the children with moderate malaria. At baseline, the mean (SD) ECW was significantly lower in children with moderate than in those with severe malaria ( p = 0.045). Admission ICW measurements were significantly lower in children with severe malaria than in those with moderate malaria ( p < 0.001). Bioelectrical Impedance Analysis As predicted by theory [ 8 ], there was a strong correlation between height 2 divided by impedance at 50 kHz ( H 2 / Z 50 ) and measured TBW. The ‘best fit' regression equation to predict TBW from BIA was derived using the variables age, weight, and H 2 / Z 50 (standard error of the estimate [SEE] = 0.435, adj r 2 = 0.975): where A is age (months), H is height (centimetres), W is weight (kilograms), and Z 50 is impedance at 50 KHz frequency. Disease severity and gender did not contribute significantly to this model, allowing data from admission to be pooled in this prediction equation. Repeating the analysis using impedances measured at other frequencies (4–1012 kHz) did not show a clear advantage, so all data for TBW prediction are given for measurement at 50 kHz. By contrast, ECW was not significantly associated with age or weight, and in agreement with previous studies in babies [ 19 ] H 2 / R 0 emerged as the strongest predictive term (SEE = 0.584, adj r 2 = 0.753): where H is height (centimetres), and R 0 is resistance at zero frequency. Again, disease severity and gender did not contribute significantly to this model. Prediction equations based on segmental BIA data for ECW and TBW were inferior to whole body BIA models and are therefore not shown here. Errors in BIA estimates of TBW and ECW compared to values measured by isotope dilution are displayed with 95% limits of agreement in Figure 2 A and 2 B, respectively. Figure 2 Plots of TBW and ECW Estimates from Isotope Dilution and BIA Calculation with Measured Values Filled circles, admission value; open circles, day 28 follow up value; dotted lines, 95% confidence intervals for values. (A) TBW and (B) ECW. D 2 O, heavy water. Fluid Volumes from BIA Fluid volume estimates were available from BIA in 14 children with moderate and 11 with severe malaria on admission and 16 with moderate and 12 with severe malaria at discharge. Values for the TBW, ECW, and ICW are shown in Figure 3 . Figure 3 Body Fluid Compartment Volumes Derived from BIA on Admission and Discharge Red circles, severe malaria; blue triangles, moderate malaria. (A) TBW (litres/kilogram), (B) ECW (litres/kilogram), and (C) ICW (litres/kilogram). BIA-determined TBW was significantly lower in those with severe compared with moderate malaria (mean [SD] of 539 [ 32 ] versus 562 [ 30 ] ml/kg, p = 0.034). TBW increased from admission to discharge in children with severe malaria by 37 (33) ml/kg or 6.7% (6.0%) (paired t test, p = 0.012), but there were no significant changes in TBW for children with moderate malaria between admission and discharge. ECW did not differ between the severe and moderate groups on admission and did not change significantly between admission and discharge. Serial measurements of ECW did not show any rise during the first 4 d after admission, at discharge, or at day 28 in children with severe or moderate malaria (data not shown). ICW at admission was significantly lower in the severe malaria group than in the moderate malaria group, with a mean (SD) of 293 (17) ml/kg versus 325 (28) ml/kg ( p = 0.002). ICW remained unchanged between admission and discharge in children with moderate malaria. In the severe malaria group mean (SD) of ICW at admission was 40 (22) ml/kg, or 11.7% (11.0%) lower at admission than at discharge ( p = 0.002). There were no differences in the fluid volume measurements of the four children with adverse and those with good outcomes. There were no relationships found between TBW, ECW, or ICW, or changes in TBW, ECW, and ICW between baseline and discharge, and any of the following markers of severity in malaria: blood lactate concentration, coma score, plasma creatinine concentration, peripheral parasitaemia, blood glucose, coma recovery time, time to walk, time to eat, time to drink, length of hospital stay, or having a history of diarrhoea or vomiting. Admission weight relative to the weight at discharge showed a mean (SD)/median (IQR, range) percentage deficit of −4.3% (4.4%)/−4.0% (−1.3% to −6.1%, −0.1% to −11.0%) for the children with severe malaria on admission ( p = 0.002). For those with moderate malaria the deficit was not significant: −0.3% (4.6%) /−0.9% (1.6% to −1.5%, 7.0% to −8.1%) ( p = 0.41). As predicted, the deficits in weight and TBW were correlated (adj r 2 = 0.67, p < 0.001). Electrolyte Measurements The concentrations of plasma electrolytes are shown in Table 2 . The mean (SD) concentrations of sodium were significantly lower for the children with severe malaria than for those with moderate malaria ( p = 0.036). Plasma potassium concentration and osmolality were higher in children with severe than in those with moderate malaria ( p = 0.039 and p = 0.021, respectively). Plasma urea and creatinine were significantly higher in those with severe malaria. Osmol gap was significantly higher in children with severe than in those with moderate malaria ( p < 0.001), with 15/16 children in the severe group having an osmol gap greater than 8.2 mOsm (considered high in United States children [ 20 ]). Discussion We have shown that severe childhood malaria is associated with mild dehydration in most cases, with a mean (SD) depletion of TBW of 37 (33) ml/kg, or 6.7% (6.0%). Only 3/16 children (19%) in our study had moderate volume depletion (> 60–90 ml/kg), and none were severely dehydrated (> 100 ml/kg) [ 21 ]. Moderate malaria was not associated with any significant changes in TBW. Consistent with a lower TBW in severe disease, ICW was depleted in children with severe malaria by a mean (SD) of 40 (22) ml/kg, an 11.7% (11.0%) difference. However, we found no relationship between TBW, ECW, and ICW and clinical and laboratory markers of disease severity, in particular the two most important prognostic indicators of fatal outcome: hyperlactataemia and Blantyre coma score [ 9 ]. Our findings suggest that the degree of dehydration in children with severe malaria is unlikely to be a primary pathological process in the evolution of the disease. These findings are also consistent with our previous suggestion that hyperlactataemia arises from tissue hypoxia resulting from microvascular obstruction by infected erythrocytes [ 12 , 22 ] rather than gross hypovolaemia. ECW did not change significantly during hospitalisation. Furthermore, our fluid replacement regimen (median [IQR] 3.7 [2.2–5.8] ml/kg/h) normalised vital signs and blood lactate within 8 to 12 h. Our estimates of fluid compartment volumes in children after recovery from malaria are entirely consistent with previous work in children (range: TBW, 540–640 ml/kg; ECW, 250–320 ml/kg; ICW, 260–340 ml/kg) [ 17 ], although our study is the first that we know of to examine fluid status in childhood malaria. Studies in adults with uncomplicated or moderate malaria have given conflicting results [ 23 , 24 , 25 , 26 , 27 ]. In sepsis, ECW increases by up to 50% of TBW because capillary permeability increases by up to 300% of normal. There are no significant increases in ECW in children with severe malaria, confirming earlier studies that indicate that sepsis and malaria syndromes result from different pathophysiological processes [ 9 , 28 ]. Studies on fluorescein angiography in children and adults also confirm there is no increased capillary permeability in severe malaria [ 23 , 29 ]. Furthermore, CVP measurements in a subgroup (6/16) of children were not low, and did not change significantly after 24 h of intravenous fluid replacement. Taken together, these findings do not suggest that volume depletion or increased capillary permeability are important to the pathophysiology of malaria in our population. Hyponatraemia [ 30 ] has been attributed to high and possibly inappropriate arginine vasopressin secretion [ 31 ] in severe malaria. Our findings (high osmol gap, low ICW, and normal ECW) are more in keeping with sick cell syndrome than with inappropriate arginine vasopressin secretion [ 32 , 33 ]. To conclude that arginine vasopressin is inappropriately elevated, renal function must be normal and volume depletion excluded. No severely ill child in this study fulfilled these criteria. What are the implications of our findings for optimal fluid replacement therapy in malaria? We cannot answer precisely on the basis of measuring fluid compartment volumes because regimens are sometimes designed not only to correct existing fluid deficits and to provide maintenance requirements, but also to rehydrate more vigorously to maintain circulating volume. Such approaches are advocated by others for different populations of children with severe malaria, for example, in a series of studies published from Kilifi, Kenya [ 4 , 5 , 34 ]. However, estimates of fluid requirements for children with severe malaria have been based upon indirect measurements (such as monitoring vital signs and degree of acidosis) that are potentially misleading because they do not relate to the degree of fluid loss that we measured. Furthermore, adequately powered controlled studies aimed at defining appropriate fluid regimens for severe malaria are lacking, but should take into account our findings as well as the BIA methodology that we have now calibrated to measure fluid compartment volumes in malaria. Indeed, a fluid (0.9% saline) replacement rate of up to 20 ml/kg in 1 h is a considerably faster rate than we can advocate on the basis of our findings. In any case, BIA can now be used (equations 2 and 3 ) to measure ECW and TBW noninvasively to guide treatment in patients with severe malaria. There are risks to over-vigorous fluid administration just as there are with inadequate fluid replacement, particularly in hospitals where measuring plasma electrolyte concentrations and providing assisted ventilation are difficult. These risks include pulmonary [ 3 ] and cerebral oedema [ 6 ] (which occur in adults and children, respectively) and dangerously rapid changes in plasma electrolyte concentrations. Children with severe malaria have a low ICW and are at risk of hypokalaemia if ICW is restored rapidly (< 4 h), particularly when there is relative hyperinsulinaemia due to quinine administration [ 12 ]. A correlation between capillary refill time and blood lactate concentrations but none with fluid volume status suggests that prolongation of capillary refill time may be due to the common underlying process of microvascular obstruction. Our findings do not support the widespread use of aggressive fluid volume replacement in children with severe malaria. Clearly, volume depletion indicated by hypotension or by CVP measurements requires more aggressive therapy, but wherever possible plasma electrolyte concentrations should be closely monitored. Because of elevated requirements for glucose in childhood severe malaria (3–6 mg/kg/min) [ 35 ], there is a need to provide a maintenance fluid replacement rate of about 3 ml/kg/h [ 7 , 36 ]. In addition, fluid regimens should aim to replace mild fluid deficit within the first 12 to 24 h of admission. The tracer dilution techniques that we have used are expensive and time-consuming and consequently not amenable to large-scale deployment. We took this opportunity to calibrate a much simpler methodology (BIA) to derive TBW and ECW and validated BIA estimations in this population. We are now using BIA measurements to assess much larger numbers of patients (J. Jarvis and S. K., unpublished data). BIA is an excellent noninvasive screening tool that should detect subgroups of children with severe malaria who may be severely volume depleted. Patient Summary Background Although we have known for many years what causes malaria, how it is passed from person to person by mosquito, and how to treat the infection, more than a million people still die of malaria every year, mostly children under five years living in Africa. Children are affected most because they have not had the chance to develop the resistance to malaria that normally builds up over a lifetime when living in places with malaria. As well as being given specific drug treatments against the malaria, children are also often given fluids into their veins, as they appear dehydrated. What Did the Researchers Find? The researchers studied children who were sick with malaria and measured how dehydrated the children were. To do the measurements, they used an accepted technique that required injections into the vein, and also a newer, simpler method that used electrodes. Neither technique suggested that any of the children were severely dehydrated. What Does This Mean for Patients? The study suggests that severe dehydration isn't a big problem in children with severe malaria. So it may not be necessary to give lots of fluids into the vein. (While treatment of malaria is still being worked out, malaria can, of course, often be prevented by using insecticide-treated bednets.) Are There Any Problems with the Study? The new technique for measuring dehydration will need to be assessed in larger studies: this study is small, so the results may not be entirely accurate. Where Can I Get More Information? The World Health Organization is coordinating many of the initiatives to combat malaria ( http://www.who.int/topics/malaria/en/ ). Medicines for Malaria Venture is trying to develop new affordable antimalarial drugs ( http://www.mmv.org/pages/page_main.htm ).

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515375

A Novel Pathway for a Tumor Suppressor

Millions of different proteins exist in nature, each with a unique structure that determines its function. Proteins can have different effects depending on where they are in the cell and which proteins or pathways they associate with. The number of proteins produced by a cell varies, but scientists estimate that the human genome produces some 100,000 proteins, and with thousands of proteins likely to be active in a single cell, it's inevitable that the molecular components of cellular pathways overlap. It is thought that this may be the case for a tumor suppressor named VHL (after its role in von Hippel-Lindau disease, an inherited cancer syndrome that predisposes affected individuals to kidney and vascular tumors). Genetic evidence for HIF-independent VHL-regulated pathways The broad strokes of VHL action have been outlined: VHL is a ubiquitin ligase, an enzyme that targets proteins for destruction. VHL's best characterized target is a transcription factor called hypoxia-inducible factor 1 (HIF-1). When oxygen levels drop below normal—a condition called hypoxia—HIF-1 proteins are not degraded and may enter the nucleus, where they trigger the transcription of roughly 100 genes whose proteins either increase oxygen delivery or engage metabolic pathways that help the cell adapt to hypoxia. Scientists have long suspected that VHL has other targets, yet only HIF-1 has been clearly established. Now Peter Ratcliffe and colleagues use genetic methods in the nematode Caenorhabditis elegans to provide direct evidence of a HIF-1-independent function of VHL. Previous studies have shown that when cells lack VHL proteins, HIF-1 is not degraded, resulting in the overexpression of HIF target genes. VHL-defective cells also show abnormalities in the extracellular matrix, the structural scaffolding that surrounds the cell. But it has not been clear whether these effects stem from HIF-1 dysregulation or something else. To disentangle the actions of the two proteins, Ratcliffe and colleagues compared the consequences of VHL protein inactivation in a variety of genetic backgrounds in C. elegans . Then they analyzed the gene expression profiles of each of these mutant strains to identify pathways that required VHL but not HIF-1. To their surprise, the authors found, “all of the VHL-regulated genes fell into one of two patterns.” Their expression was either independent of HIF-1 and dependent on a range of genes associated with the extracellular matrix, or vice versa. What's more, these gene sets fell into distinct categories based on their chromosomal location, predicted functional similarities, and pattern of dysregulation. These results, Ratcliffe and colleagues conclude, reflect the disruption of “two discrete aspects of VHL function.” One depends on HIF-1—inhibiting the transcription factor when oxygen concentrations are normal—and one doesn't; disruption of this HIF-1-independent function produces defects similar to those seen in mutants with defects in extracellular matrix assembly. These results also fall in line with other studies that have linked VHL deficiency to extracellular matrix defects, though the precise link remains unclear. It's also not clear whether this HIF-1-independent function means that VHL is still functioning as a ubiquitin ligase but targeting a different substrate or whether it represents a completely different function of VHL. For now, direct evidence of a HIF-1-independent pathway for VHL charts a clear path for researchers interested in pinning down the functions of this undoubtedly multidimensional ubiquitin ligase. It also gives VHL syndrome researchers—who have long suspected that other functions of the VHL tumor suppressor play a role in the onset of the disease—a promising lead to explore.

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524180

Mapping medical careers: Questionnaire assessment of career preferences in medical school applicants and final-year students

Background The medical specialities chosen by doctors for their careers play an important part in the workforce planning of health-care services. However, there is little theoretical understanding of how different medical specialities are perceived or how choices are made, despite there being much work in general on this topic in occupational psychology, which is influenced by Holland's RIASEC (Realistic-Investigative-Artistic-Social-Enterprising-Conventional) typology of careers, and Gottfredson's model of circumscription and compromise. In this study, we use three large-scale cohorts of medical students to produce maps of medical careers. Methods Information on between 24 and 28 specialities was collected in three UK cohorts of medical students (1981, 1986 and 1991 entry), in applicants (1981 and 1986 cohorts, N = 1135 and 2032) or entrants (1991 cohort, N = 2973) and in final-year students (N = 330, 376, and 1437). Mapping used Individual Differences Scaling (INDSCAL) on sub-groups broken down by age and sex. The method was validated in a population sample using a full range of careers, and demonstrating that the RIASEC structure could be extracted. Results Medical specialities in each cohort, at application and in the final-year, were well represented by a two-dimensional space. The representations showed a close similarity to Holland's RIASEC typology, with the main orthogonal dimensions appearing similar to Prediger's derived orthogonal dimensions of 'Things-People' and 'Data-Ideas'. Conclusions There are close parallels between Holland's general typology of careers, and the structure we have found in medical careers. Medical specialities typical of Holland's six RIASEC categories are Surgery (Realistic), Hospital Medicine (Investigative), Psychiatry (Artistic), Public Health (Social), Administrative Medicine (Enterprising), and Laboratory Medicine (Conventional). The homology between medical careers and RIASEC may mean that the map can be used as the basis for understanding career choice, and for providing career counselling.

Background Medical careers begin as undifferentiated, and postgraduate training ends with most doctors specialised for a specific area of practice. Relatively little is known about the transition from the medical student, who can be seen as a relatively undifferentiated, totipotent 'stem doctor' [ 1 , 2 ], potentially capable of entering any speciality, through to the final, fully-differentiated specialist who is almost entirely restricted to one specialised area of medical work. Although medical career specialisation has been subject to a moderate amount of research (for reviews see e.g. [ 3 , 4 ]), some of it going back over half a century (e.g. [ 5 ]), much of that research has concentrated on the personal characteristics of individuals choosing particular careers (e.g. [ 6 - 8 ], on background factors in childhood influencing career choice (e.g. [ 8 - 10 ]), on associations with particular personality types (e.g. [ 11 ]), on the careers of specific groups, such as women doctors (e.g. [ 12 ]), on attitudes towards specific specialities, such as psychiatry (e.g. [ 13 , 14 ]) or anaesthetics (e.g. [ 15 , 16 ]), or has concentrated on the basic statistics necessary for workforce planning (e.g. [ 17 , 18 ]). There is, however, a lack of any broad theoretical framework in which to place career choice and specialisation. UK medical education requires undergraduates to study a wide range of medical specialities, and most students will have sampled many of the broad areas of practice by the time they qualify. As a result, it is often assumed that students do not make their career choices until after they have finished at medical school, remaining agnostic about their final speciality choice until that time. However, not only medical school entrants (e.g. [ 19 ]), but even medical school applicants, a year or so earlier, at the typical age of about seventeen, often have surprisingly strong preferences for, and particularly, against, some medical careers (e.g. [ 20 ]). There is strong evidence, therefore, that career choice can be determined during or even before medical school ([ 21 , 22 ])). Thus, it makes sense to try and understand those preferences, which probably underpin eventual career choice. Much research into medical careers does not take into account the broader research literature on non-medical careers (see [ 23 - 25 ]), or on socio-psychological models of the theoretical underpinnings of career choice (e.g. [ 26 ], [ 27 ], [ 28 , 29 ]). Consequently, medical careers research often fails to provide any broader theoretical framework or conceptualisation within which the empirical findings may be explained or which allow generalisations beyond the immediate data collected in the study (although there are exceptions, e.g. [ 30 , 31 ]). The present study takes its origins in three separate sets of theoretical approaches, each of which examines different aspects of careers. None of these approaches, however, concerns medical careers specifically. Neither are they restricted to career choice in adulthood. Furthermore, at least one of them is specifically developmental, emphasising the processes by which career choice occurs and changes. The best place to begin this brief theoretical review is with the work of Gottfredson [ 27 ], who identifies the distinct processes of circumscription and compromise in career choice. Careers differ in their demands, requiring different amounts of intellectual ability, manual skill, long-term commitment, or willingness to work in particular environments, and can be better suited to particular personalities, aptitudes, and physical dispositions. Individuals also differ, having different aptitudes, interests and abilities. Career choice therefore involves people considering the entire range of careers and then circumscribing those which they regard as broadly acceptable, making their eventual choices within that subset. An important practical point highlighted by studies such as Gottfredson's is that choices tend to be negative , meaning that careers are rejected because they do not have attributes which are consonant with the person making the choice, rather than positively chosen for their special suitability. Once circumscription has taken place, a number of possible careers still remain. The second stage of choice is compromise . Because of various practical constraints, certain careers are restricted in the number of people they can accommodate or they are unsuitable in other terms, such as their geographical location or the remuneration they can provide. The eventual career chosen is one that 'satisfices,' [ 32 ] being realistically good, though not optimal. The applications of this theory to medical careers are self-evident and describe many of the problems facing medical students and junior doctors. Implicit in Gottfredson's conceptualisation is the concept of a map of careers. In her 1981 paper she provides an example a two-dimensional representation of 129 occupations which have been scored in terms of 'Prestige level' (high vs low) and 'sextype rating' (masculine vs feminine). When careers are mapped into this space, the process of circumscription involves drawing an area within which careers are acceptable to a person, being neither too masculine nor too feminine, nor being too high in terms of their prestige and hence effort required, nor too low, and hence insufficiently rewarding. A primary concern of the present study is the nature of the map underlying medical careers, and on which circumscription eventually takes place. Perhaps the most influential study of the structure of career preferences is that of Holland [ 26 ], an overview and critical analysis of which can be found in the special issue of the Journal of Vocational Behavior published in 2000 (e.g. [ 25 ]; see also [ 33 ] and [ 34 ]). Holland's theory suggests that careers can be organised into six broad types, which can be represented around a hexagon (see figure 1 ), and which are often known by the acronym RIASEC, standing for Realistic, Investigative, Artistic, Social, Enterprising and Conventional. In Holland's original conceptualisation the specific orientation of the hexagon is arbitrary to rotation, but subsequent analyses have suggested that the hexagonal structure can be reduced to two dimensions [ 35 , 36 ]. One dimension runs from Realistic to Social, involving careers that are primarily Things-oriented rather than People-oriented. The second orthogonal dimension runs from midway between Enterprising and Conventional to midway between Artistic and Investigative, and involves careers varying from those that are primarily Data-oriented to those that are primarily Ideas-oriented. Holland's RIASEC model provides an appropriate two-dimensional space in which Gottfredson's circumscription model can apply [ 37 ]. Figure 1 The hexagon of Holland's RIASEC typology, along with the Things-People and Ideas-Data dimensions proposed by Prediger (1982). Although Holland's work suggests how careers might be mapped, and Gottfredson's work suggests how career choices might take place within the space underlying those careers, a missing link in the overall picture concerns how individuals choose within the space. This is a significant question because individuals are expected to circumscribe in different ways according to their particular personalities and abilities. Ackerman [ 28 , 29 ] has described how intellectual ability and personality relate to Holland's RIASEC model. Measures of intellectual ability primarily correlate with interest in the Realistic, Investigative and Artistic careers, people with higher verbal abilities preferring careers in Artistic and Investigative careers, and people with higher spatial and mathematical abilities preferring Realistic and Investigative careers. In contrast, measures of personality mainly correlate with the SEC components of RIASEC. Ackerman uses the Big Five typology of personality (see [ 38 ], [ 39 ]), and shows that Extraversion primarily correlates with an interest in Social and Enterprising careers, whereas Conscientiousness correlates with an interest in Conventional and Enterprising careers. The personality dimension of Openness to Experience is to some extent a hybrid between intellectual ability and personality, and tends to correlate positively with Artistic, Investigative and Realistic careers, and negatively with Conventional careers. This pattern is similar to that which Zhang has reported in which the RIA cluster of careers relates to a deep approach to learning [ 40 , 41 ], whereas the SEC cluster relates to a strategic approach to learning [ 42 ]. Between them, the models of Holland, Ackerman and Gottfredson provide, respectively, a good conceptualisation of i) the structure of careers and career preferences, ii) the correlations of careers with ability and personality, and iii) the developmental processes by which career choices are made. The question for medical education is the extent to which these approaches are appropriate for understanding medical career choice. If they are valid, then that will allow the much broader research literature from career choice in general to inform the more specific area of medical career choice. Underpinning the models of both Ackerman and Gottfredson is Holland's picture of a relatively simple, two-dimensional career map, onto which ability and personality can project, and on the basis of which career choices can develop. We therefore have two main objectives in this paper; firstly, to use data on career preferences from three separate cohorts of medical students, both at the time of application and in their final year at medical school, in order to derive a map of medical careers. And second, to assess the extent to which this specific map of medical careers is homologous to Holland's more general map of a broad range of careers. The data collected in our studies consist of ratings of attractiveness of different medical careers on a five-point scale, ranging from 'Definite intention to go into this' through to 'Definite intention not to go into this'. However, our primary interest for the purpose of deriving a map of careers is not in career preference , but rather in career similarity. If a student has a preference for career A and career B, but has no interest in career C and D, it follows that career A is probably relatively close to career B on the map, and career C is relatively close to career D, whereas careers A and B are likely to be more distant from careers C and D. A matrix of similarities between all possible pairs of a large number of careers from a large number of students then allows one to construct the underlying map (just as, in a classic example, a knowledge of the geographical closeness, or the drive-time, between many pairs of towns in a country allows one to reconstruct a map of the country [ 43 , 44 ]). The statistical technique is known as multi-dimensional scaling (MDS). Although conventional MDS can reconstruct the underlying map showing the relations between a number of objects, the map itself is arbitrary to rotation. Turning the map through any angle does not change any of the distances between pairs of objects, and therefore the axes of the map cannot be known – in the case of a geographical map, there is no indication of the north-south and east-west axes. The problem of the arbitrariness of dimensions can be circumvented by means of a variant of MDS known as INDSCAL ( In dividual D ifferences Scal ing) [ 44 , 45 ]. This method analyses the similarity matrices either of individual subjects or of groups of subjects who are likely to differ, so that, for instance, one might have groups based on sex and age, the presumption being that older students may have different career preferences from their younger peers, and female students may have different preferences from their male peers. INDSCAL then allows the assignment of axes, it being likely that the grouping variables will mainly affect one rather than all of the dimensions on which the map is represented. An example in the case of geographical distance might be to examine the time of travel between pairs of towns in winter and summer. Inclement winter weather will increase the time of travel in the more northerly towns, but the dimension of east-west will have little impact on the measures. In this paper, we use INDSCAL to construct our maps of medical careers, so that the axes are identified and not arbitrary to rotation. Methods Two different types of data have been used in the present study. The bulk of the analysis looks at data collected during studies of medical student selection and training, and can be used to map medical careers. A subsidiary, but important, analysis looks at a large convenience sample of people of different ages who were taking part in a survey about careers in general, and were asked about their interest in a range of careers, of which only a few were medical. These latter data allowed us both to calibrate specific medical careers in the context of the general Holland typology, and to validate the INDSCAL methodology for deriving a map of careers. Medical student data The data were collected during three longitudinal studies of medical student selection, the first of which began in the autumn of 1980, looking at students who had applied for entry to medical school in 1981 [ 46 , 47 ], the second began in the autumn of 1985, studying applicants for entry to medical school in 1986 [ 48 , 49 ], and the third began in 1990, studying applicants for entry to medical school in 1991 [ 50 , 51 ]. The 1981 and 1986 cohort studies were restricted to students applying for entry to St. Mary's Hospital Medical School in London, although since applicants had each applied to five or six medical schools, many students entered schools other than St. Mary's. The 1991 cohort study looked at applicants to five different English medical schools, and because each applicant applied to several schools, these applicants represented 70% of all applicants and entrants to UK medical schools in that year. In each survey, applicants were sent questionnaires as soon as possible after UCCA, the central universities admission system, had received their application and entered their names had been onto the computer database. In general this was many weeks or even months before applicants were asked to come for interview, or were sent decisions on whether they had been accepted or rejected. The data are therefore to a great extent properly prospective. For the present paper, the analysis of the 1981 and 1986 cohort data considers data on all applicants who replied to our questionnaires, whereas the 1991 cohort, which was very much larger, considers only those questionnaire respondents who entered medical school (although we will generally refer to this group as 'applicants' since that reflects the time at which the questionnaire was completed). In additional file 1 we present separate information for the entire 1991 cohort which shows that there are unlikely to be response biasses, either due to differences between accepted and rejected applicants, or due to not all entrants responding to the final-year questionnaire. Response rates in the 1981, 1986 and 1991 applicants surveys were 85%, 93% and 93% [ 46 , 48 , 50 ]. Students who entered medical schools in 1981, 1986 or 1991 (or in a few cases due to deferred or repeated entry, in 1982, 1987 or 1992) were followed up as final-year students in 1986 (or 1987), in 1991 (or 1992) and 1996 (or 1997). Students still in medical school were identified through their medical schools, and questionnaires sent to those medical schools. Response rates were 65%, 50%, and 56% in the follow-up of the 1981, 1986 and 1996 cohorts of students in their final year [ 49 , 51 ]. The questionnaires used in the study, both at application and in the final year, were detailed, typically covering 16 sides of A4, and the results reported here concern only one of the questions asked. Career preferences were assessed by a question which used the rubric, "Below is a detailed list of specialities in which a medical career can be pursued. Please indicate your attitude towards each speciality as a possible career. If you either know nothing about a speciality, or have no opinions about it at all, simply leave that answer blank". A list of specialities followed, each of which was rated on a five-point scale, for which the categories were, "Definite intention to go into this", "Very attractive", "Moderately attractive", "Not very attractive", and "Definite intention not to go into this". The list of specialities varied a little over the different surveys, becoming slightly more extensive as the years passed. The original list was based on the questionnaire distributed as part of the Royal Commission on Medical Education of 1968 [ 52 ] (The Todd Report). The questionnaire for 1981 applicants had 24 questions. The final-year questionnaire for the 1981 applicants had 26 questions, the two new categories being "Pre-clinical teaching" and "Geriatric Medicine". The questionnaire for the 1986 applicants was the same as that for the 1981 applicants except that it had 25 specialities, "Geriatric medicine" having been added. The final-year questionnaire for the 1986 cohort had 27 questions, the 25 used for applicants, with the addition of "Genito-Urinary Medicine" and "Infectious Diseases". The applicant questionnaire for the 1991 cohort had the same 27 questions as did the final-year questionnaire for the 1986 cohort. The final-year questionnaire for the 1991 cohort was similar to that for the applicants except that it had 28 questions, "Radiology/Radiotherapy" having been split into two separate specialities. In the present study all of the questionnaires have been used in the form in which they were originally administered, the only omission being the speciality "Pre-clinical teaching", which was used in one survey only and is of little interest. The general population sample This questionnaire was completed by a sample of 1026 subjects, stratified by age using a median split (≥ 42; <42) and by sex. It asked about the suitability of twenty-four different careers for the person. Twenty careers were derived, as far as possible, from Holland's RIASEC classification, with at least three in each of the six categories. In addition there were four categories which were medical (Anaesthetist, Hospital Doctor, Psychiatrist and Surgeon). The rubric was, "Below is a list of careers. Please indicate for each one how much you think it might have been suitable for you as a career". Each career was rated on a five-point scale, ranging from 'Extremely suitable', through 'Very suitable' and 'Quite suitable', to 'Not very suitable' and 'Completely unsuitable'. The subjects were a convenience sample obtained from amongst friends and relations by a first year lab class at University College London, each student being responsible for obtaining a group of twelve subjects, stratified by age and sex. Statistical analysis INDSCAL analysis was carried out using the ALSCAL program within SPSS 10.1. Data in each subset were broken down into four groups by age and sex, age referring to mature vs non-mature students in the medical student samples (≤ 21; >21) and to subjects aged <42 or ≥ 42 in the general population sample. The raw data, which were collected on a 5 point Likert scale, were transformed into Euclideanbased dissimilarities for all combinations of career pairs, using the PROXIMITIES program in SPSS. Four different dissimilarity matrices were produced (nonmature males, nonmature females, mature males, and mature females), and these matrices provided the basis for the INDSCAL analysis that involved minimisation, in Euclidean space, of the discrepancies between the career dissimilarities and the corresponding interpoint distances on the map. The loadings of each career on the two extracted dimensions were then plotted onto the figures to provide the maps. The dimensionality of MDS/INDSCAL analyses can be assessed, in a manner analogous to that used in factor analysis in which eigenvalues are plotted against components. In MDS one plots a measure of 'stress' (in effect, the opposite of goodness-of-fit) against the number of dimensions which have been extracted. If too few dimensions have been extracted then the stress is high, the model not accounting adequately for the richness of the data. The optimal number of dimensions is typically indicated by a sudden 'dog-leg' in the stress plot. Results We consider firstly the general population sample since it both validates the method which we will subsequently use for the medical student samples, and also helps calibrate the axes. The general population sample The questionnaire was completed by 1044 subjects, 49.2% of whom were male, and 46% aged over 42. Multidimensional scaling used the INDSCAL method, with the four groups comprising older and younger males and older and younger females. The stress plot indicated that there were two major underlying dimensions in the data, as Holland's typology would suggest (see also Prediger [ 53 , 35 ]). The locations of the different careers are shown in figure 2 . There is good evidence for Holland's RIASEC typology, and the letters R, I, A, S, E and C have been placed on the graph to clarify interpretation. Pilot and Engineer are typical of Realistic careers, Biologist of Investigative careers, Artist and Museum Curator of Artistic careers, Social Worker, Counsellor and Teacher of Social careers, Personnel Director and Lawyer of Enterprising careers, and Accountant and Computer Programmer of Conventional careers. For this non-medical group of subjects, the four medical careers are all placed in the top half of the figure, with surgeon and anaesthetist closest to Investigative, and Psychiatrist closest to Social. Figure 2 INDSCAL group space of the career preferences expressed by the general population sample. The locations of the labels R, I, A, S, E and C are approximate and are only for guidance and orientation. The four medical specialities are shown in blue so that they are more visible. Of some importance, given the arbitrariness of the Holland hexagon to rotation in conventional MDS, is that the INDSCAL analysis clearly sets one axis as running from R to S, with the other axis orthogonal to that, running from I and A to C and E. These are similar to the Things-People and Ideas-Data dimensions shown in figure 1 . The medical student samples Sample sizes for the medical student studies were 1135, 2032 and 2973 for the students in the 1981, 1986 and 1991 cohorts (and these samples consisted of all applicants in the 1981 and 1986 cohorts, and all entrants in the 1991 cohort), and were 330, 376 and 1437 for the final-year students in the 1981, 1986 and 1991 cohort studies. The INDSCAL analyses were restricted to those subjects for whom complete career information was available; this consisted of 538 applicants and 312 final-year students in the 1981 cohort, 1118 applicants and 301 final-year students in the 1986 cohort, and 1638 entrants and 1437 final-year students in the 1991 cohort. See additional file 1 for details of the breakdown of samples by sex and maturity. The dimensionality of the medical student samples was assessed by carrying out a standard multi-dimensional scaling analysis (i.e. MDS, not INDSCAL), separately for the combined applicant data and the combined final-year data from the three cohorts. The stress formula attempts to quantify the discrepancies between the fitted distances in the model and the observed dissimilarities among the career ratings, with larger values indicating poorer fit. It is obviously the case that the more dimensions are extracted, the better the fit of the model and, hence, the lower the stress value. However, it is also the case that a greater number of dimensions complicates interpretation and may lead to overfitted and unstable solutions. The stress levels with 1,2,3,4,5, and 6 dimensions were .352, .174, .112, .077, .059 and .048 for applicants, and .390, .174, .112, .082, .064 and .052 for final-year students. For the final-year students, it is clear that two dimensions are necessary, and that there is little advantage of adding extra dimensions. The applicant data are slightly less clear and although there is still no doubt that at least two dimensions are necessary there is a suggestion that a third dimension may be of value. Subsequent scrutiny of models with three dimensions suggested that the third dimension was contributed almost entirely by one or two specialities such as forensic medicine, which have a high public and media profile, but which form only a small proportion of medical personnel. It was, therefore, felt to be safe to extract two dimensions, particularly since Holland's typology provided an a priori expectation that there would be two dimensions. INDSCAL analyses Separate analyses were carried out for the applicant and final-year data in each of the three cohorts. In each case, data were broken down into sub-groups according to sex (male-female) and age (mature at entry to medical school, i.e. >21 yrs old; or typical post-school entry, at ≤ 21 years old). INDSCAL analyses can clarify the underlying dimensions within data as long as the sub-groups are likely to vary along those dimensions. It should be noted that many studies have found sex differences in medical career interest (e.g. [ 54 ]), and younger students are also likely to have different attitudes towards careers than their non-mature counter-parts [ 55 ]. Figures 3 , 4 and 5 show the group plots of the different specialities in applicants to medical school, and figures 6 , 7 and 8 show the group plots for the specialities in final-year medical students. In order to help interpret these plots, and for reasons which will become clearer later, we have joined together the data points for Surgery, Hospital Medicine, Psychiatry, Public Health, Administrative Medicine and Laboratory Medicine. For the applicants, it is now clear that these specialities are arranged approximately in the form of a hexagon, with Surgery at the extreme left and Administrative Medicine at the bottom right-hand corner. The pattern shown in the final-year students is similar, Surgery still being at the left-hand side, and Administrative Medicine at the bottom right. Although there are some minor differences between the three cohorts, the broad picture is of overall similarity in the structure of the maps. Figure 3 The INDSCAL group space for the medical specialities for the applicants in the 1981 cohort. For abbreviations see the Abbreviations section. Figure 4 The INDSCAL group space for the medical specialities for the applicants in the 1986 cohort. For abbreviations see the Abbreviations section. Figure 5 The INDSCAL group space for the medical specialities for the entrants in the 1991 cohort. For abbreviations see the Abbreviations section. Figure 6 The INDSCAL group space for the medical specialities for the final-year medical students in the 1981 cohort. For abbreviations see the Abbreviations section. Figure 7 The INDSCAL group space for the medical specialities for the final-year medical students in the 1986 cohort. For abbreviations see the Abbreviations section. Figure 8 The INDSCAL group space for the medical specialities for the final-year medical students in the 1991 cohort. For abbreviations see the Abbreviations section. The maps shown in figures 3 to 8 are, in INDSCAL terminology, group spaces [ 44 , 45 ]. They are, however, composed of several different sources, broken down by age and sex. Maps can also be produced of 'source space' which shows how the groups differ in their relative weighting of the two extracted dimensions. Figure 9 shows the source spaces for the applicant and final-year student data in the three cohorts. The vertical axis represents the relative importance of the Things-People dimension, whereas the horizontal dimension shows the importance of the Data-Ideas dimension. It should be noted that these axes do not mean that, say, People are more important than Things, but that the Things-People dimension is more differentiated than the Data-Ideas dimension (just as, say, in a map of Italy or Chile, there is far more north-south differentiation than east-west, as they are long-thin countries). In each of the six analyses, the male subjects put more emphasis on the Things-People dimension whereas the female subjects put more emphasis upon the Data-Ideas dimension (and hence the male subjects tend to be in the top left corner and the female subjects in the bottom-right). In the 1981 cohort there is also a suggestion that younger subjects put more emphasis on the Things-People dimension, and older subjects on the Data-Ideas dimension for differentiating careers, but the effect is smaller in the 1986 cohort, and barely visible in the 1991 cohort, suggesting a possible change in the way these groups perceive medical careers. In interpreting these analyses it should be noted that although the absolute size of the various groups was more than adequate for the INDSCAL analyses, the group weights for the mature candidates (male and female) in the 1981 and 1986 finalyear data were based on very small samples, ranging between 5 and 17 participants, and may, therefore, be somewhat unstable. Figure 9 INDSCAL source spaces for the applicants/entrants and final-year medical students in the 1981, 1986 and 1991 cohorts. Square symbols are for male subjects and circles for female subjects. Solid symbols are for younger students, whereas hatched symbols are for mature students. To help visualisation, the solid arrows connect from younger males to younger females, whereas dashed arrows connect from mature males to mature females. See text for further details of interpretation. Discussion The primary objectives of this study were to use the empirical method of individual differences scaling to derive maps of the underlying perceived structure of medical career specialities, and to assess the extent to which those maps are similar to those described by Holland in his hexagonal representation of the RIASEC groups of careers. That this method is a valid way of deriving Holland's structure in general is seen in figure 2 , in which a broad range of non-medical careers is assessed by non-medical individuals, and the RIASEC structure is readily derived. Of particular importance is that because the analysis used INDSCAL, the dimensions are not arbitrary to rotation, and that the R-S dimension (corresponding to the Things-People dimension) and the IA-EC dimension (corresponding to the Ideas-Data dimension) are the basic underlying structure, as shown by Prediger [ 35 , 53 ]. The "Things-People" dimension also bears a strong similarity to the Technique orientation and People orientation which has also been described in relation to medical specialities [ 56 ]. The general population sample also rated four medical specialities, with Surgery and Anaesthetics at one extreme, and Psychiatry at the other, and these medical specialities differed principally along the R-S dimension. That Surgery and Anaesthetics are more concerned with Things, and Psychiatry is more concerned with People fits well with the reduction of Holland's hexagon to the two dimensions of Things-People and Ideas-Data. It is also worth noting that all of the four medical specialities are seen by the general public as being primarily concerned with Ideas rather than with Data, as surely befits medical careers. The MDS analyses demonstrate that the representation of the various medical specialities by the medical student samples can be captured within a two-dimensional space, as Holland had suggested. The maps shown in figures 3 to 8 indicate that the structures are broadly similar across the three cohorts, and that although there are some minor differences between the applicants and the final-year students, it is the case that overall the similarities are more impressive than the differences. The crucial question therefore concerns whether the medical student maps are homologous to those of Holland's RIASEC typology. If there is a homology, then one may ask what are the Realistic, Investigative, Artistic, Social, Enterprising and Conventional specialities of medicine. From scrutinising figures 3 to 8 we suggest that the RIASEC structure of medicine is typified by the six prototypical specialities of Surgery, Hospital Medicine, Psychiatry, Public Health, Administrative Medicine and Laboratory Medicine. It should be emphasised that in suggesting this we are not implying a direct comparability in the posts, rather a formal similarity within the limits imposed by being within the domain of medicine, as opposed to that of careers in general. Surgery – Realistic Surgeons can be seen as the engineers of medicine, solving problems at high levels of mechanical and technical proficiency, with an emphasis upon practical skills, craftsmanship, and immediate and effective results. Hospital Medicine – Investigative The core of Hospital Medicine (Internal Medicine) is diagnosis, achieved by carrying out appropriate investigations. Physicians typify the model of the 'scientist-practitioner', investigating symptoms and signs and relating them to the underlying pathophysiology of the patient. Psychiatry – Artistic Psychiatrists, and also General Practitioners, have a more artistic approach to medicine, seeing, interpreting and responding imaginatively to a range of medical, social, ethical and other problems. The emphasis in many ways is on the uniqueness of the patient, the ideas that they are expressing, and the psycho-social theories and concepts which are necessary for interpreting the individual. Public Health – Social Although most medicine is concerned with individual patients, the remit of Public Health is primarily social in the sense of applying medicine to society as a whole, treating the 'body politic'. It is noteworthy that in the maps, Public Health is not only at the Social end, but also closer to Data than to Ideas. Public Health manages social and community health by the appropriate analysis of data. Administrative medicine – Enterprising The management of hospitals and health-care requires the creative skills of the business executive, the lawyer and the personnel director to achieve a smoothly running system. People, both patients and carers, are at the heart of any health-care system, and therefore administrative medicine is at the People end of the dimension. Laboratory Medicine – Conventional The running of efficient systems in haematology, histopathology or chemical pathology requires many of the attributes shared with the accountant or the banker, including the willingness to develop, implement and follow standard procedures within a complex system. The emphasis is inevitably upon the things that do the measurements, and upon the data collected, rather than the ideas or people behind the data and the technology. The analyses in this paper suggest that in our groups of students there is a broad similarity between preferences for medical careers and the typology found by Holland in careers in general, suggesting that the structures are homologous. Although our study has been restricted to medical students in the UK, our findings are likely to be generalisable, given that the patterns are found in three separate cohorts studied over a decade, and across medical school applicants and final-year students. Just as Holland's typology is found in most studies of careers, over a period of three decades and in many countries, despite a wide range of changes in society, in education, and in the nature of jobs and careers themselves, so we would predict that our typology of medical careers will be robust to such changes. To put it more strongly, we would predict that despite enormous changes in every aspect of medicine over two and a half millennia, just as Hippocrates recognised that surgery is different in many ways from other branches of medicine, and that not every doctor wishes or is able to be a surgeon, so the same applies today and will probably continue to apply as long as medicine is practised. That is likely to be so primarily, as Ackerman has suggested, because Holland's typology is underpinned by wide-ranging, broadly defined individual differences in aptitude and personality [ 36 ] which are also likely to be stable across time and cultures [ 57 ]. It may at first be felt that our approach to mapping careers is fundamentally different to that of Gale and Grant [ 58 , 59 ], who describe a questionnaire, the Sci-45, which has twelve sub-scales and allows discrimination between 45 different medical specialities as possible careers. However, the purposes of that instrument and our analyses are very different. Gale and Grant aimed at developing a practical instrument for counselling individuals, which would allow a detailed differentiation between careers. In contrast, we aimed at investigating and mapping the broad picture underlying careers. To use an analogy with geography, our map is primarily a large-scale representation of a region such as Britain, which lays out the main north-south and east-west axes and defines the broad regions of that map (Scotland, Wales, South of England, East Anglia), as well as placing the main cities, which are analogous to the specific careers. Gale and Grant in contrast are developing a method of differentiating between the various cities, particularly when, as say in the West Midlands conurbation, some cluster closely together within the map. We therefore expect that underlying the Gale and Grant questionnaire will be two broad dimensions equivalent to those which we have described. Abbreviations INDSCAL Individual differences scaling MDS Multidimensional scaling RIASEC Realistic-Investigative-Artistic-Social-Enterprising-Conventional Speciality abbreviations in figures 3 to 8 . ADM Administrative Medicine ANS Anaesthetics ARM Armed Forces BMS Basic Medical Sciences DRM Dermatology ENT Ear, Nose & Throat FRN Forensic GER Geriatrics GPlrg GP Large Group practice GPsml GP Small practice GPsng GP Single handed GUM Genito-urinary medicine IND Industrial Medicine INF Infectious diseases LAB Laboratory (Haematology, Clinical Chemistry, etc.) MED Internal Medicine O&G Obstetrics & Gynaecology O&T Orthopaedics & Trauma OPH Ophthalmology PED Paediatrics PHM Pharmaceutical Medicine PSY Psychiatry PTH Pathology PUB Public Health RAD Radiology/ Radiotherapy RDL Radiology RDT Radiotherapy RES Research SRG Surgery Competing interests The authors declare that they have no competing interests. Authors' contributions ICM had collected the data in the various surveys over a number of years. ICM and KVP jointly decided how to do the statistical analysis, and KVP was responsible for the programming and data analysis. ICM wrote the first draft of the paper, which was revised by KVP, with both authors being responsible for the final draft. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Additional analyses of data Click here for file

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Controversial significance of early S100B levels after cardiac surgery

Background The brain-derived protein S100B has been shown to be a useful marker of brain injury of different etiologies. Cognitive dysfunction after cardiac surgery using cardiopulmonary bypass has been reported to occur in up to 70% of patients. In this study we tried to evaluate S100B as a marker for cognitive dysfunction after coronary bypass surgery with cardiopulmonary bypass in a model where the inflow of S100B from shed mediastinal blood was corrected for. Methods 56 patients scheduled for coronary artery bypass grafting underwent prospective neuropsychological testing. The test scores were standardized and an impairment index was constructed. S100B was sampled at the end of surgery, hourly for the first 6 hours, and then 8, 10, 15, 24 and 48 hours after surgery. None of the patients received autotransfusion. Results In simple linear analysis, no significant relation was found between S100B levels and neuropsychological outcome. In a backwards stepwise regression analysis the three variables, S100B levels at the end of cardiopulmonary bypass, S100B levels 1 hour later and the age of the patients were found to explain part of the neuropsychological deterioration (r = 0.49, p < 0.005). Conclusions In this study we found that S100B levels 1 hour after surgery seem to be the most informative. Our attempt to control the increased levels of S100B caused by contamination from the surgical field did not yield different results. We conclude that the clinical value of S100B as a predictive measurement of postoperative cognitive dysfunction after cardiac surgery is limited.

Background Despite the fact that incidence figures between 4–79% have been reported for cognitive dysfunction after cardiac surgery [ 1 ], diagnostic steps are seldom taken to diagnose this impairment. The golden standard for detecting cognitive dysfunction is neuropsychological tests, which are complex and difficult to use as a routine procedure. Lately the brain derived protein S100B has been proposed as a simple method for detecting brain dysfunction after cardiac surgery [ 2 - 5 ]. The protein is a member of the larger S100 family, where S100B is one isoform, and considered to be brain specific, and the other is the S100A isoform [ 6 ]. The S100B chain is uniform in contrast to the S100A chain which exhibits several subgroups. The dimers of interest in studies concerning cerebral events are those containing S100B (S100BB and S100A1B). The physiological roles of S100B are pleiotropic including neurotrophic and neuroprotective functions, mediated by calcium dependent regulation of phosporylation, enzyme activation and proliferation [ 6 ]. On the other hand, high concentrations of S100B have been shown to be toxic and induce apoptosis in neuronal cell cultures [ 7 , 8 ]. At least five experimental studies have indicated a possible role of S100B in learning and memory function, three of which reported impaired memory and learning effects in transgenic S100B mice and two reported memory deficits after injection of S100B antiserum [ 9 - 13 ]. Several studies in humans suffering from stroke of different ethiologies, have shown a rather strong correlation between serum levels of S100B and size of lesion(s) as well as outcome [ 14 - 17 ]. Lately a number of studies have addressed the question whether S100B can be considered as a marker for cognitive dysfunction after cardiac surgery; however the conclusions presented are disparate. One major reason for this could be the fact that S100B is present in high concentrations in shed mediastinal blood that is retransfused to the patient by cardiotomy suction and autotransfusion, thus obscuring the measured levels of S100B early after surgery. To date, only one study have been published where this contamination was taken into account [ 18 ]. We recently reported the half life of S100B in serum to be 25 min [ 19 ]. With the present study we wanted to use our knowledge of inflow and elimination to obtain a more reliable measurement of cerebral release of S100B after cardiac surgery and correlate it with neuropsychological outcome. Methods Study design The study group comprised 56 patients who underwent coronary artery bypass graft (CABG) surgery at the Division of Cardiac Surgery, University Hospital MAS, Malmoe, Sweden and Department of Cardiothoracic Surgery, Lund University Hospital, Lund, Sweden. Only patients planned for elective CABG with cardiopulmonary bypass (CPB) as their sole procedure were included. The study protocol was approved by the local ethics committee, and patients gave a written informed consent before the study protocol was initiated. Patients with a history of stroke, transient ischemic attack (TIA), reversible neurological disorder (RIND), known carotid artery disease or other brain diseases were excluded. In order to avoid possible influence of renal disorder on the elimination of S100B, patients with known renal failure (Creatinin > 160 μmol/L) were excluded. The patients were examined for signs of neurological dysfunctions daily during the hospital stay by either experienced cardiac anesthesiologists or by experienced cardiac surgeons. Perioperative management Anesthesia was induced with midazolam 3–5 mg iv (Dormicum ® , Roche, Basel, Switzerland) or propofol (Diprivan ® , Zeneca Ltd, Cheshire, England) 10 mg/kg. It was subsequently maintained with fentanyl 10 μg/kg (Leptanal ® , Janssen Pharmaceutica, Beerse, Belgien), a continuous infusion of propofol 3–6 mg/kg/h or inhalation of isoflurane 0,5–1% (Forene ® , Abbott Laboratories). Nitrous oxide (Aga Industries, Stockholm, Sweden) was used before CPB but not during or after CPB. CABG surgery was performed during aortic cross clamping with the distal anastomosis preceding the proximal anastomosis. A tangential occluder replaced the cross-clamp during the proximal anastomosis. Antegrade cold S:t Thomas crystalloid cardioplegia was used (Cardioplegi ® , Pharmacia-Upjohn, Uppsala, Sweden) and administered in the ascending aorta and the anastomosed vein-grafts intermittently. Perfusion was performed with a roller pump (Cobe Industries, Denver, Colorado, USA). The perfusion catheters and circuit were made of polyvinylchloride in the line and silicon in the pumphead. The arterial cannulation was made in the ascending aorta and venous cannulation in the right atrium by a two-stage venous cannula. All circuits contained a heparin-coated 40 μm arterial filter (Cobe Sence, Cobe Industries) and a membrane oxygenator (Cobe Duo oxygenator, Cobe Industries). The circuit was primed with approximately 1000 ml of Ringer's lactate (Pharmacia-Upjohn), 250 ml 15% Mannitol (Pharmacia-Upjohn) and 75 mmol Addex tromethamine (Pharmacia-Upjohn). Perfusion flow was non-pulsatile with a flow rate of 2.4 l/min/m 2 at normothermia. The perfusate was cooled to approximately 32°C. Heparin (400 U/kg bodyweight) was given prior to cannulation and reversed with equal doses of protamine sulphate at decannulation. After surgery, the patients patient were transferred to the ICU for recovery and enrolled in the sampling scheme for S100B analysis. None of the patients received autotransfusion. S100-protein analysis and calculations Serum for S100B analysis was sampled before surgery, at the end of CPB, and then 1, 2, 3, 4, 5, 6, 8, 10, 15, 24 and 48 hours after surgery. The S100B levels at these time points will be referred to as T0, T1, T2....T48. Blood samples, both arterial and venous samples, were cooled and centrifuged within 5 hours. All samples were measured by a monoclonal two-site immunoluminometric assay (Sangtec 100, AB Sangtec Medical, Bromma, Sweden). S100B kinetic calculations Since early levels of S100B are contaminated by S100B from cardiotomy suction, an attempt was made to exclude this S100B from the levels measured one and two hours after surgery. Assuming that all of the measured S100B at the termination of CPB was a contamination, this non-cerebral S100B was eliminated with a half-life of 25 minutes, as illustrated in figure 1a and 1b . The estimated true levels cleansed from the contamination 1 and 2 hours after the end of surgery were thereby calculated by the formula: Figure 1 1a – Measured S100B release pattern in one patient and the calculated residual levels from the S100B from cardiotomy suction during surgery, a half-life of 25 minutes was used. 1b – Estimated true release, calculated by subtracting the residual levels (from 1a) from measured levels. 1c – Measured levels and estimated true release from one patient with high S100B at T0 and low estimated true release at T1. This patient also had a good neuropsychological outcome. 1d – Measured levels and estimated true release from one patient with low S100B at T0 and high estimated true release at T1. This patient had a bad neuropsychological outcome. where C e is the estimated true levels of S100B at time t, C t = the measured concentration of S100B at T1 or T2, C 0 the concentration at the end of CPB, t the time after T0 and t 1/2 the half-life of S100B. We have earlier suggested that the elimination rate could be used as another measure of cerebral release [ 5 ], and the elimination rates of S100B between the end of CPB (T0) and 1 hour later, between T1 and T2, and between T2 and T3 were calculated accordingly. The differences between measured S100B levels at T0 and T1, T0 and T2 were also calculated. Neuropsychological method The patients underwent neuropsychological testing by the same trained neuropsychologist 1–2 days before and 5–7 days after surgery. The tests used were: Mental Control, Figural Memory, Logical Memory (A/B), Visual Reproduction, Rey Auditory/Verbal Learning Test (RAVLT), Trail Making A, Trail Making B, Digit Symbol, Digit Span, Visual Memory Span, Visual Paired Associates II or Verbal Paired Associates I and RAVLT, Delayed Retention. The tests were chosen from the Wechsler Memory Scale-Revised (WMS-R), the Wechsler Adult Intelligence Scale (WAIS – R) and the Halsted-Reitan Neuropsychological Battery [ 20 , 21 ]. Differences for each sub-test were first calculated and then standardized to z-values. All sub-tests were then aggregated to create an impairment index [ 4 ]. The impairment index was a continuous variable, where a positive value reflected an improvement in neuropsychological test and a negative value reflected deterioration. The incidence of neuropsychological impairment in the study group was calculated according to the 1 standard deviation criterion (SD) as defined by Newman and the 20% criterion as defined by Stump [ 22 - 24 ]. Statistical analysis All results were analyzed with the Statistica version 5.0 for PC. Regression analysis was performed with the least square method with a casewise deletion of missing data. For the multiple regression a backwards stepwise regression was performed to determine which variables to include in the final analysis. The initial variables included in this analysis were age, gender, perfusion time, years of education, and S100B levels at all sampling times. A p-value < 0,05 was considered significant. Unless otherwise stated numerical values are presented as mean ± 1 standard deviation. Results Patient demographics are presented in table 1 . None of the patients suffered a clinically detectable stroke. The incidence of neuropsychological impairment was 37,5% (n = 21, 95% C.I 26,0–50,6%) according to the 1 standard deviation criterion, and 80,4% (n = 45, 95% C.I. 68,1–88,6%) according to the 20% criterion. Table 1 Demographics for the study group. Mean St. dev. Number 56 Sex (M/F) 47/9 Age (years) 60.4 9.0 Education (years) 9.6 3.1 Perfusion time (Minutes) 82.8 31.5 X-clamp (minutes) 53.9 23.5 The appearance of S100B protein followed the same pattern in all patients with high levels at the end of CPB and a decrease or slight increase the first hour (figure 2 ). Thereafter a subsequent decrease was observed during the rest of the study period, except in one patient in whom the concentration of S100B increased to 2,0 μg/L 48 hours after surgery. Mean estimated true release of S100B, calculated according to equation [ 1 ], was lower at T1 and T2 compared to measured S100B levels (1.43 ± 1.37 μg/L vs. 2.11 ± 1.81 μg/L and 1.22 ± 1.10 μg/L vs. 1.32 ± 1.17 μg/L). Three representative examples of this calculation are shown in figure 1b , 1c and 1d . Figure 2 S100B release pattern after cardiac surgery with cardiopulmonary bypass shown as a boxplot. We found a correlation between patient age and S100B levels measured up to 24 hours after bypass (Table 2 ). No significant correlation was found between measured S100B levels at the sampling times and neuropsychological impairment in simple regression analysis (Table 3 ). Neither did age, years of education and duration of perfusion correlate with neuropsychological impairment when tested in simple regression analysis (Table 3 ). Table 2 The relation between S100B levels and patient age tested in univariate linear regression analysis. r-value p-level S100B at T0 0.19 n. s S100B at T1 0.30 <0.05 S100B at T2 0.30 <0.05 S100B at T3 0.34 <0.05 S100B at T4 0.37 <0.05 S100B at T5 0.32 <0.05 S100B at T6 0.37 <0.05 S100B at T8 0.38 <0.01 S100B at T10 0.42 <0.005 S100B at T15 0.36 <0.05 S100B at T24 0.46 <0.005 S100B at T48 0.004 n.s Table 3 The relation between S100B levels, demographic variables and neuropsychological impairment index, expressed as r-value from linear regression analysis (* = p < 0.05). Mean (± SD) S100B levels are also presented. (Est. true release = estimated true release when residual levels from contamination have been excluded) Mean ± St.dev. r-value S100B at T0 3.63 ± 2.84 0.08 S100B at T1 2.11 ± 1.81 -0.16 S100B at T2 1.32 ± 1.17 -0.11 S100B at T3 0.87 ± 0.73 -0.12 S100B at T4 0.60 ± 0.42 -0.06 S100B at T5 0.49 ± 0.38 -0.12 S100B at T6 0.42 ± 0.33 -0.15 S100B at T8 0.38 ± 0.25 -0.12 S100B at T10 0.31 ± 0.21 -0.06 S100B at T15 0.25 ± 0.16 -0.09 S100B at T24 0.21 ± 0.11 -0.10 S100B at T48 0.21 ± 0.27 -0.09 Est. true release at T1 1.43 ± 1.37 -0.22 Est. true residual at T2 1.22 ± 1.10 -0.12 Elimination rate T0-T1 0.50 ± 0.46 0.26 Elimination rate T1-T2 0.48 ± 0.25 -0.05 Elimination rate T2-T3 0.68 ± 0.32 -0.12 Difference T0-T1 1.53 ± 1.66 0,30* Difference T0-T2 2.31 ± 2.01 0,18 Age 0.16 Education -0.06 Perfusion time 0.12 In multiple regression analysis, measured S100B levels at the end of CPB (T0), one hour later (T1) and age were found to explain part of the neuropsychological impairment (r = 0.49, p < 0.005, Table 4 ). It is worth noting that the correlation was positive at T0 and negative at T1, implicating a better outcome if S100B was high at T0 and worse if the levels were high at T1. Age had a negative correlation. Furthermore the combination of age, S100B at T0 and S100B at either T3 or T6 gave significant correlations in the same manner (r = 0.41, p < 0.05 and r = 0.42, p < 0.05 respectively). The difference between S100B levels at T0 and T1 was found to correlate positively with neuropsychological outcome (Table 3 ). No correlation was found when the estimated true release of S100B was tested against neuropsychological outcome, neither did the elimination rate fall out significantly in simple regression analysis (Table 3 ). Table 4 The results of the backwards stepwise multiple regression analysis model to explain the neuropsychological impairment index (r = 0.49, p < 0.005). Variable Partial correlation Beta p-value S100 at T0 0.45 0.845 <0.005 S100 at T1 -0.47 -0.939 <0.001 Age 0.31 0.307 <0.05 Regression <0.005 Discussion This study presents some interesting findings that may be relevant in clinical practice. There is a clear relation between patient age and release of S100B up to 24 hours after cardiac surgery. This correlation is strong and not influenced by the perioperative contamination of S100B caused by the use of coronary suction [ 4 ]. The univariate analysis did not provide a clear answer to the question whether there is a relationship between neuropsychological outcome and S100B release. However, from table 3 it is worth noting that the r-values in univariate regression analysis are negative at all sampling points except at T0 (immediately after bypass). The consistent finding of a negative correlation 1 hour and onwards after bypass is intriguing and warrants further exploration. The multiple regression analysis resulted in a significant correlation between neuropsychological deterioration and the three variables: S100B at T0, S100B at T1 and patient age. The r-value in the multiple regression analysis was 0.49, which evokes the question why the multiple regression is stronger than the univariate regression. One possible explanation could be that by including the S100B level at the end of CPB we compensate for some part of the contamination in the S100B levels one hour after surgery. Age correlated also with outcome. As expected, the older the patient the higher the risk for neuropsychological deterioration, which is in accordance to the fact that age is a risk factor for decline in neuropsychological tests after surgery [ 23 ]. Our results are to some extent in concordance with other reports in this field. Two groups have reported no relation between S100B and neuropsychological test results, [ 2 , 18 ] and two groups have found a relation using a composite S100B end-point [ 3 , 4 ]. Only one of these studies was designed to exclude the extracerebral inflow of S100B [ 18 ]. These contradicting findings support our notion that S100B kinetics after cardiac surgery are complicated. Interestingly, in the multiple regression analysis there was a positive correlation between S100B levels at T0 and outcome, a finding that stands in contrast to the hypothesis that S100B could be used as serum marker for cognitive dysfunction. This finding is especially interesting since most of the measure S100B at T0 is contamination. In an earlier study we found that approximately 80% of measured S100B at this point is from extracerebral sources in CABG patients [ 4 ]. We offer no explanation for this unexpected finding, but it is intriguing and calls for further investigations. When interpreting the results of our study, as well as comparing them with those of other reports, several issues need to be addressed. To begin with the origin of S100B measured after CPB is not exclusively cerebral. From our previous study as well as from reports by Anderson et al [ 25 ], it is clear that shed blood in the mediastinum contains very high levels of S100B. When this blood is retransfused either directly by cardiotomy suction or later in the course by autotransfusion during the postoperative care, the systemic levels of S100B are affected. The source of this S100B is non-cerebral, probably from S100B containing tissue such as fat, skin and bone marrow. No autotransfusion was used in this study, however reabsorbtion of S100B from injured fat tissue may contribute to the measured serum levels of S100B. Since we previously have been able to determine the half life of S100B in blood to be 25 min, it was possible to control for the peri-operative contribution from cardiotomy suctions by using the equation mentioned to obtain the estimated true release at T1 and T2. When these values were entered in simple regression analysis, no correlation was found to neuropsychological outcome. However, if multiple regression analysis was used, a positive correlation was found, these results were identical to those obtained with the measured S100B values, and therefore we can conclude that the use of the correction procedure represented by the equation does not contribute to the evaluation of the results. Moreover, we should be careful to overinterpret the significance of correlations in a study of relatively few patients and several end-points, i.e sampling times for S100B. There is always a risk of accepting a false hypothesis. Preferably, a study of this sort should contain a large number of patients and only one or few end-points. However, the aim of this study was to clarify the complex kinetics of early S100B release and its possible connection to a neuropsychological decline, not to determine the perfect use of S100b for detecting neuropsychological outcome after cardiac surgery. In the mathematical correction model suggested here, we assume that all S100B measured at T0 is of extrecerebral origin. This is of course not the case, but we do not know the relation between cerebral and extracerebral S100B at this time-point in each individual. By assuming that none of the S100B measured at T0 is of cerebral origin, we can be sure that our mathematical model does not include non-cerebral S100B at T1 and later. However, these levels might be lower than the true levels of cerebral S100B since we could have excluded cerebral S100B present at T0. This could also be an explanation that the multiple regression analysis showed stronger correlations. One important issue, which must be considered in this type of study, is the strength that can be expected in the correlation between two different measures of brain function. The functional domains in the brain covered by neuropsychological tests vary depending on the tests used and are by no means complete in any test battery. By the same reasoning, the magnitude of a possible cerebral release of S100B after an injury may vary according to the severity of the insult as well as the location of the insult, since glia dense areas express more S100B than others [ 26 ]. Conclusions In conclusion, with the present knowledge, a single S100B sampled in the postoperative course after cardiac surgery can not be of use in clinical practice in order to predict neuropsychological outcome with an acceptable sensitivity. However, utilizing a statistic model, an association between S100B levels the first hours after surgery and neuropsychological outcome was found, where the most informative time point seems to be 1 hour after the termination of CPB. The controversial significance of an increased S100B immediately after surgery is indeed intriguing and it inspires further studies of the mechanisms of S100B release after cardiac surgery as well as other fields of brain damage. Competing interests The author(s) declare that they have no competing interests. Authors' contributions HJ: Principal investigator recruited, enrolled patients analyzed and wrote the paper PJ: Had an integral role in the planning and the analysis, also help with writing MB: Designed neuropsychological test battery. Analyzed neuropsychological data CB: Performed all neuropsychological testing with patients CA: Did the S100B-analyzes SB: Mentor an principal leader of the project, who also helped with the writing. Pre-publication history The pre-publication history for this paper can be accessed here:

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514719

Anti-ischemic therapy and stress testing: pathophysiologic, diagnostic and prognostic implications

Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. In the present review, the pathophysiological mechanisms of ischemia protection by antianginal therapy are described. Not all stressors are created equal in front of the different classes of antianginal drugs and on their turn the different classes of drugs exert different levels of protection on inducible ischemia. Several clinical implications can be drawn: From the diagnostic viewpoint antianginal therapy decreases test sensitivity, offsetting the real ischemic burden for a too high percentage of false negative tests. From the prognostic viewpoint test positivity in medical therapy identifies a group of subjects at higher risk of experiencing cardiac death and positivity on medical therapy can be considered a parameter of ischemia severity. Nonetheless in patients with known coronary artery disease the ability of antianginal therapy to modify the ischemic threshold at stress testing represent a powerful means to assess therapy efficacy. From a practical viewpoint, the use of antianginal therapy at time of testing has advantages and disadvantages which are largely dependent on the purpose a test is performed: if the purpose of testing is to diagnose ischemia, it should be performed in the absence of antianginal medications. If the purpose of testing is to assess the protective effects of antianginal therapy, the test should be performed on medications.

Background Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. The diagnostic and prognostic impact of anti-ischemic therapy on stress testing is largely ignored but not negligible. The issue raises several questions: How to evaluate patients at time of testing for myocardial ischemia? How to interpret a stress test performed on anti-ischemic therapy? Are the stressors employed for the detection of myocardial ischemia created equal in relation to the different classes of drugs used in clinical practice? Is stress testing able to assess the efficacy of medical therapy in patients with known coronary artery disease? Has the protection of anti-ischemic therapy on inducible myocardial ischemia any impact on long-term survival? Pathophysiologic implications of anti-ischemic therapy during stress testing The answer to all these issues relies on the mechanism through which myocardial ischemia is induced by the different stressors (exercise or pharmacologic such as dipyridamole and dobutamine) employed during stress testing. Test exploring organic coronary artery stenosis can induce ischemia by two basic mechanisms: 1. an increase in oxygen demand, exceeding the fixed supply and 2. flow maldistribution due to inappropriate coronary arteriolar triggered by a metabolic/pharmacologic stimulus [ 1 ]. The mechanism of increased demand can be easily fitted into the familiar concept framework of ischemia as a supply-demand mismatch, deriving from an increase in oxygen requirements in the presence of a fixed reduction in coronary flow reserve. The different stresses can determine increases in demand through different mechanisms (Fig. 1 ). In resting conditions, myocardial oxygen consumption is dependent mainly upon heart rate, inotropic state, and the left ventricular wall stress (which is proportional to the systolic blood pressure) [ 2 ]. Following dipyridamole or adenosine administration, a slight increase in myocardial function, a modest decrease in blood pressure, and mild tachycardia can be observed, overall determining only a trivial increase in myocardial oxygen demand [ 3 ]. During exercise, the increase in heart rate, blood pressure, and inotropic state accounts for the overall increase in myocardial oxygen consumption [ 4 ]. Pacing and dobutamine also increase – to a lesser degree – myocardial oxygen demand [ 5 ]. During pacing, the increase is mainly due to the increased heart rate. Dobutamine markedly increases contractility and heart rate. Further augment in myocardial oxygen consumption for heart rate increase occurs with the co-administration of atropine with dobutamine [ 6 ]. and dipyridamole [ 7 ]. (Fig. 2 ). Figure 1 Major determinants of myocardial oxygen consumption in resting conditions (left) and during stress commonly employed with echocardiography. Figure 2 Conceptual allocation of tests employed in combination with echocardiography to detect coronary artery disease stenosis inducing ischemia via steal effect (left) or increased myocardial oxygen demand (right), or both mechanisms. In the presence of coronary atherosclerosis, appropriate arteriolar dilation can paradoxically exert detrimental effects on regional myocardial perfusion, causing overperfusion of myocardial layers or regions already well perfused in resting conditions at the expense of regions or layers with a precarious flow balance in resting conditions [ 8 ]. Anti-ischemic therapy can interfere with all the above mentioned mechanisms of ischemia induction, although in a very different fashion. The mechanism of action of anti-ischemic drugs is easily fitted within the familiar framework of supply-demand mismatch. In particular, beta-blockers are credited with reducing exercise-induced ischemia by decreasing myocardial oxygen demand and possibly by increasing supply through a reduction in extravascular forces [ 9 ]. Experimental studies demonstrated that beta-blockers reduce dipyridamole-induced ischemia by an alteration of regional myocardial blood flow [ 10 ]. However, this straightforward explanation seems inadequate in justifying the protective effects of beta-blockers on dipyridamole-induced ischemia. Experimental data show that beta-blockers do not affect the dipyiridamole-induced increase in flow [ 12 ]. On the other hand, the increase in myocardial oxygen consumption does not play any significant role in the induction of dipyridamole-induced ischemia, which is due to an absolute reduction in subendocardial flow (tightly linked to regional wall thickening) mostly for "vertical" and "horizontal" steal phenomena. However, experimental studies on the model of the exercising dog have shown that beta-blockers protect myocardium from stress-induced myocardial blood flow-function relation: for a given transmural flow, there is a rise of subendocardial and a fall of subepicardial flow, with an improved regional performance [ 10 ]. This same mechanism has also been documented with some calcium antagonists, such as diltiazem and may explain, in part, the beneficial effects of this class of drugs on dipyridamole-induced ischemia. Calcium antagonists can effectively prevent ischemia provoked by dipyridamole also through other mechanisms, which they share with nitrates, and they tend to increase the coronary flow supply during stress. In this case, the prevention of steal phenomena may be due to the increase in collateral flow (which has been shown with nitrates and, to a much lesser extent, with some calcium antagonists) [ 12 ]. and to the dilation of epicardial coronary lumen size. The pronounced increase in collateral flow can prevent horizontal steal phenomena due to dipyridamole, wheras even a small increase of the coronary diameter can dramatically reduce the blood pressure drop across the stenosis, therefore preventing vertical steal phenomena. Beta-blockers exert a direct and competitive action on beta-1 receptors, as they are employed as specific antagonists of dobutamine-induced ischemia. Dobutamine, through its beta-1 agonist action determines the increase in oxygen consumption, but it induces flow maldistribution through beta-2 arteriolar receptors. Diagnostic implications of the use of anti-ischemic therapy during stress testing On the basis of these premises and taking into consideration that the markers of inducible myocardial ischemia (electrocardiogram, perfusion, wall motion) are very different therefore expressing a different sensitivity to the action of anti-ischemic therapy at time of testing, it is clear that medical therapy affects test results (see table 1 . In fact, the AHA/ACC Guidelines on Chronic stable angina state that [ 13 ]. "whenever possible, it is recommended that beta-blockers (and other anti-ischemic drugs) be withheld for four to five half-lives (usually about 48 h) before exercise imaging studies for the diagnosis and initial risk stratification of patients with suspected CAD". Ideally, these drugs should be withdrawn gradually to avoid a withdrawal phenomenon that may precipitate events. When beta-blockers cannot be stopped, stress testing may detect myocardial ischemia less reliably, but it usually will still be positive in patients at the highest risk. The same recommendations apply to imaging stress testing. Nonetheless, in patients who exercise to a submaximal level because of the effect of drugs, perfusion or echocardiographic imaging still affords higher sensitivity than the exercise ECG alone [ 14 ]. On the basis of these recommendations patients undergoing a stress testing for diagnostic purposes should be evaluated off therapy not to offset test results. Exercise imaging stress (nuclear perfusion or ultrasound) testing have a lower sensitivity when performed on anti-ischemic therapy [ 14 - 19 ]. due to the limited increase in heart rate and blood pressure which determine oxygen consumption. Antianginal therapy lowers the sensitivity of exercise echocardiography as it does with vasodilator stress testing [ 19 , 20 ]. Antianginal therapy with beta-blockers, calcium-antagonists or nitrates in various combinations prevent dipyridamole-induced ischemia by delaying the appearance of the transient dyssynergy [ 21 ]. this variation on dipyridamole time parallels variations in exercise time at exercise stress testing [ 21 ]. (Fig. 3 ). Dipyridamole stress sensitivity was 91% off therapy and fell to 65% under therapy in various combinations (beta-blockers and/or calcium antagonists and/or nitrates). The same reduction of dipyridamole test sensitivity is obtained with monotherapy with beta-blockers at time of testing (100% off therapy vs. 38% on therapy) [ 22 , 23 ]. (Fig. 4 ). Interestingly, the positive effects of beta-blockers on dipyridamole stress are largely independent of the effect on heart rate, possibly involving a direct anti-steal effect [ 21 - 23 ]. Angiotensin-converting enzyme inhibitors have no effect on dipyridamole stress echocardiography results [ 24 ]. The sensitivity of dobutamine is heavily affected by concomitant beta-blocker therapy. Beta-blockers effect a rightward shift in the dose-response curve to dobutamine and sharply lower test sensitivity, unless atropine is used [ 25 ]. (Fig. 5 ). Calcium antagonists and/or nitrates only mildly reduce dobutamine stress sensitivity (100% off therapy vs.88% on therapy, p=ns) (Fig. 6 ). Non-beta-blocker antianginal therapy reduces the severity of dobutamine-induced ischemia by reducing the value of peak wall motion score index and time of ischemia appearance. However, these changes are not correlated to variations in exercise tolerance [ 26 ]. Figure 3 Correlation between the therapy-induced variations in dipyridamole and exercise time in the 38 patients with positivity of both tests off treatment (Modified from 21). Figure 4 Dipyridamole test sensitivity on and off beta-blocking therapy (Modified from 23). Test sensitivity is significantly reduced in patients studied on beta-blocking therapy. Figure 5 Heart rate during dobutamine-atropine stress testing on and off beta-blockers (Modified from 25). Figure 6 Dobutamine-atropine stress echocardiography test sensitivity in patients on and off non-beta blocking therapy (Modified from 26). Dipyridamole stress nuclear imaging techniques do not seem to be influenced by anti-ischemic therapy [ 27 ]. However, it has been recently demonstrated that acute administration of beta-blockers in patients with known coronary artery disease reduces dipyridamole SPECT sensitivity from 69% with placebo to 52% (p = 0.039) with 10 or 20 mg of metoprolol in a per-vessel analysis, but not overall sensitivity [ 28 ]. The reason for this difference between stress echocardiography and nuclear imaging is likely to be related to the different markers of ischemia, i.e. wall motion abnormalities vs. perfusion, in the face of the same pathophysiologic mechanism of ischemia: the reduction of coronary reserve. In fact, in the presence of a coronary stenosis, during stress, flow remains elevated in the subepicardial layer but falls in the subepicardium. This selective stress-induced hypoperfusion is important for stress echocardiography, since the regional systolic thickening is linearly and closely related to subendocardial perfusion and only loosely related to subepicardial perfusion [ 29 , 30 ]. (Fig. 7 ). Figure 7 Schematic illustration of the principle underlying the impact of antianginal therapy on different markers of ischemia: regional function and perfusion imaging. At rest, perfusion is homogeneously distributed between endocardial and epicardial layers. In the presence of a significant coronary stenosis, vasodilation induced by pharmacologic stress, provokes a subendocardial underperfusion with a relative epicardial overperfusion which is translated into an impairment of function (echocardiographic dyssynergy) and perfusion (reversible defect at scintigraphy). Medical therapy at time of testing re-equilibrates the imbalance between subendocardial and subepicardial layers, but it affects only function. Figure 8 Kaplan-Meier survival curves (considering total mortality as an endpoint) in patients stratified according to presence (DET +) or absence (DET -) of myocardial ischemia at pharmacological stress echocardiography on and off antianginal medical therapy. The best survival is observed in patients with no inducible ischemia off therapy; the worst survival in patients with inducible ischemia on therapy (Positive DET vs. Negative DET off antianginal medical therapy, p < 0.000; Positive DET vs. Negative DET on antianginal medical therapy, p < 0.074) (Modified from 31). Prognostic implications of anti-ischemic therapy during stress testing The protective effect of anti-ischemic therapy on inducible myocardial ischemia might exert a powerful impact on prognosis. From the EPIC-EDIC Data bank, it has been analyzed the prognostic impact of antianginal therapy at time of testing in 7333 patients with suspected or known coronary artery disease undergoing pharmacologic stress echocardiography with either dipyridamole or dobutamine. The results show that a positive test on medical therapy is an additional marker of ischemia severity at stress testing whereas a negative test on medical therapy is less prognostically benign, being a false negative result [ 31 ]. (Fig. 8 ). No prognostic difference was found among the various forms of anti-ischemic drugs at time of testing, but the presence per se of antinaginal therapy at time of testing is an independent predictor of death. It is worth noting that in the study only a very low percentage of patients was taking beta-blockers: if on one side this aspect represent a clear lack of adherence to recommendations [ 13 ]., on the other it is an observed pattern of prescription in our data base, which simply reflected the clinical practice and the lack of a universally accepted policy of testing regarding concomitant therapy [ 32 , 33 ]. Marwick et al. [ 34 ]. have demonstrated a protective effect on mortality of beta-blocker therapy in patients with a negative exercise echocardiography whereas specificity and negative predictive value is increased for the prediction of cardiac events (cardiac death, myocardial infarction and unstable angina) during exercise scintigraphy in patients evaluated off medical therapy at time of testing [ 35 ]. The clinical implications of these results are far-reaching. Inducible myocardial ischemia during pharmacological stress testing on medical therapy identifies the subset of patients at highest risk of death. On these patients an aggressive approach has to be undertaken in order to change the natural history of coronary artery disease. On the far opposite end the incidence of death in patients with a negative pharmacologic test off therapy is so low that no intervention could lower the spontaneous rate of death any further. At intermediate risk are those patients with a negative test on medical therapy or with a positive test off medical therapy. Different clinical scenarios can be foreseen on the basis of the present results: 1) A negative test on medical therapy might represent a false negative result, therefore it is advisable to repeat the test off therapy in order to assess the real ischemic burden through the conventional stress echocardiographic parameters [ 36 , 37 ]. – i.e. number of ischemic segments, severity of induced dysfunction, (both expressed by peak wall motion score index), pharmacologic load and time of onset of ischemia. This is in line with the recommendations of the American Heart Association in patients with stable angina [ 13 ].; 2) In the case of a positive test off medical therapy, the effect of therapy can be assessed with the advantage of using an objective, primary ischemic end point such as changes in wall motion during stress. Conclusions Patients may be undergoing various forms of antianginal therapy at the time of testing, both an advantage and a disadvantage for stress echocardiography testing. The disadvantage is that antianginal therapy reduces sensitivity, since stress-induced wall motion abnormalities are caused by the development of obligatory myocardial ischemia. The advantage is that the effect of therapy can be assessed using an objective, primary ischemic end-point such as changes in stress-induced wall motion abnormalities. The presence of ischemia can be titrated on the basis of the ischemic-free stress time and the extent and severity of the induced dyssynergy. The various forms of stress are differently affected by various forms of therapy. In patients with known or suspected coronary artery disease, ongoing anti-ischemic therapy at the time of testing heavily modulates the prognostic value of pharmacological stress echo. In presence of concomitant anti-ischemic therapy, a positive test is more prognostically malignant and a negative test less prognostically benign. However, the decision to remove a patient from beta-blocker therapy for stress testing should be made on an individual basis and should be done carefully to avoid a potential hemodynamic rebound effect, which can lead to accelerated angina or hypertension [ 38 ]. No major side effects were recognized when medical therapy was withdrawn in large series of consecutive patients undergoing pharmacologic stress echocardiography [ 39 ]. In practical terms, when a test is performed for diagnostic purposes it should be done off medical therapy in order to avoid the influence of medical therapy (in case of hypertensive patients it is possible to prescribe ACE-inhibitors or Angiotensin II receptor blockers that do not exert any protective effect on myocardial ischemia). In patients with known coronary artery disease the decision to suspend medical therapy should be taken on an individual basis in view also of the fact that pharmacologic stress echocardiography is a versatile tool that can assess medical therapy efficacy in the long term prognosis [ 31 ]. Table 1 Effects of oral therapy on stress testing sensitivity STRESS Exercise Dipyridamole Dobutamine Beta-blockers ↓ ↓ ↓↓ Calcium channel blockers ↓ ↓ ↓↔ Nitrates ↓ ↓ ↓↔ ACE-inhibitors ↔ ↔ ↔ Aminophylline ↓↔ ↓↓ ↔ ACE, angiotensin-converting enzyme; ↓, decreased sensitivity; ↓↓ markedly decreased sensitivity; ↔, no effect on sensitivity; ↓↔, mild decrease in sensitivity.

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535927

4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin – poor tracks

Background The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei. Results We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 μm – wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data. Conclusions Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

Background Interphase nuclei are structurally highly organized with chromosomes restricted to defined territories[ 1 ]. The movement of large complexes or nuclear bodies such as Cajal bodies or PML bodies in the nucleus has been described by various groups [ 2 - 4 ]. This type of organization of interphase chromosomes indicates that the resulting interchromatin compartment provides a so-called interchromosomal domain (ICD) space that differs significantly from that occupied by chromatin [ 5 ]. It was shown that nuclear bodies as well as specific RNA are excluded from the chromosome territories but reside in an interchromatin compartment [ 5 - 7 ]. Investigation of the diffusional accessibility of the nucleus for microinjected DNA and dextrans of varying molecular weight by fluorescent recovery after photobleaching (FRAP) methods revealed significant differences in mobility according to size. FITC-dextrans of molecular sizes up to 580 kDa were demonstrated to be fully mobile, whereas DNA fragments were nearly immobile [ 8 ]. Furthermore, a size- and electrical charge-dependent exclusion for macromolecules is encountered for chromatin regions [ 9 ]. In contrast, poly(A) RNA has been shown to move freely throughout the interchromatin space of the nucleus with properties characteristic of diffusion [ 10 ]. Moreover, the large ribosomal subunits seem to exhibit a random movement in a Gaussian manner without evidence for any direct path on their way from the nucleolus to the nuclear pores [ 11 ]. Recently, microinjection of fluorescent nanospheres has been used to track the movement of such particles under distinct experimental conditions [ 12 ]. The authors employed a silicon – intensifier target camera coupled to an epifluorescence microscope in combination with a 2-D particle – nanotracking routine implemented in the Metamorph / Metaview image processing system [ 13 , 14 ]. In particular, tracking of nanospheres within the nucleus revealed that the particles diffused freely in restricted "cages", eventually translocating into another "cage". These studies, however, did not reveal any information on the 3-D movement of particles in comparison with local chromatin density. Such a study requires recording of simultaneous time-lapse recording of three-dimensional image stacks of particles and chromatin using a confocal laser scanning microscope as described in the present study. Kinetic analysis of nuclear bodies requires spatio-temporal microscopic imaging of live cells generating a huge amount of data that is only difficult or impossible to analyze in a standardized way without computational support. The present developments of an Open Microscopy Environment (OME) aims at providing a standardized informatics solution for the storage, management and analysis of light microscopic image data [ 15 ]. For quantitative analysis of complex data from live cell experiments a variety of systems have been developed (for review see [ 16 ]). An integrated image analysis solution should include tools for all steps in the image processing chain, i.e. image preprocessing and segmentation of objects, registration of moving and deforming cells, tracking of objects over time, and multi-dimensional visualization and kinetic analyzes of dynamic objects. Only with the availability of quantitative kinetic data it is possible to obtain insight into the underlying mechanisms of biological processes such as those involved in the functional and spatial organization of the cell nucleus. In the present study we describe a combined computational and experimental approach to study the dynamic behaviour of nuclear body-like particles formed by GFP-NLS-vimentin [ 17 ] in response to different cellular inhibitors and, most importantly, in relation to the chromatin structure of the nucleus. This has been compared with the motion of polystyrene particles of similar size. Since both kinds of "bodies" display identical movement, the vimentin bodies can be regarded and hence employed as entities supposedly "biologically inert" for the nucleus. Using our novel image processing platform TIKAL we show that the kinetics of nuclear particles are influenced by various cellular inhibitors. Furthermore we show that the kinetics of nuclear bodies is directly influenced by local restructuring of chromatin domains. Results An experimental system for probing particle kinetics in the cell We used fast 3-D time-lapse confocal laser scanning microscopy to analyze the mobility of Xenopus laevis GFP-NLS-vimentin and synthetic particles (polystyrene microspheres) within the nucleoplasm. GFP-NLS-vimentin is deposited at 37°C within the nucleus of stably transfected SW13 cells in multiple discrete bodies (8 – 40). On average the cells contain bodies that are nearly 1 μm in diameter as observed in the light microscope (Figure 1a ). This corresponds to a particle diameter of about 200 – 500 nm in fixed cells as prepared for conventional electron microscopy (data not shown). From correlative light and electron microscopy studies we know that nuclear vimentin particles are excluded from dense chromatin regions (Richter et al., submitted). In contrast, transfection of human vimentin-free SW13 cells with an expression plasmid encoding GFP-vimentin without the engineered NLS sequence leads to the formation of many cytoplasmic particles (> 100) of very similar size (Figure 1c ). Figure 1 A) Nucleus of a SW13 cell stably transfected with an expression plasmid encoding for GFP-NLS-vimentin. B) SW13 cell nucleus containing microinjected 100 nm-microspheres. C) SW13 cell transfected with GFP-vimentin showing cytoplasmic particle formation. A', B', C'): Corresponding Hoechst 33342 chromatin stain, A", B", C") superimposed images. To study the nucleoplasmic mobility of particles of a defined size we microinjected unloaded polystyrene beads into the nucleus of living cells. We used orange fluorescent 100 nm-beads that resemble in size authentic nuclear bodies such as PML- or Cajal bodies [ 3 , 18 - 20 ] on the light microscopic level. Thereby we attempted to find out how the mobility of the ectopically expressed nuclear vimentin particles related to polystyrene particles [ 17 , 21 ]. In the course of these studies we found that a system based on an ectopically expressed protein has several clear advantages compared to the microinjection of beads. First, the expression efficiency of the GFP-vimentin construct is very high. More than 50% of the cell nuclei show formation of nuclear vimentin bodies. Since the cells are stably transfected they reflect a normal physiological state. In contrast, for microinjection only approximately 10% – 20% of injected the cells survived over night culture (n = 300). Additionally, the artificial microspheres have to undergo tedious processing steps such as sonification and centrifugation prior to injection to avoid the formation of aggregates. A computational system for tracking nuclear particles on the background of moving cells For the analysis of complex data derived from spatio-temporal imaging of trafficking particles we developed a proprietary image processing platform, TIKAL (see Methods). The platform allows to directly and easily handle complex microscopic data and to dynamically interact with the data set throughout the whole quantitative data analysis steps. The image processing pipeline is initiated by image pre-processing steps including noise reduction followed by object segmentation (for details see Methods). In many cases, cells move and also change their morphology during the observation period. Global movements include translocation and rotation, whereas morphological changes are either caused by global changes in size (affine transformation) or by local deformations. Since any of these transformations overlay the actual movement of nuclear particles within the cell, we corrected for them by rigid transformations (translocation and rotation), affine transformations (scaling) and by thin-plate spline models (local deformations; [ 22 , 23 ]; for details see Methods). These transformations allow a direct measurement of nuclear particle movements without any bias induced by external forces and cellular movements. For quantitative evaluation of kinetics of moving particles we extended our single particle tracking approach formerly developed for two-dimensional time series [ 24 ] to automatically track objects in 3-D time series. The automatically computed 4-D tracks are visualized together with a surface rendered 3-D reconstruction of segmented nuclear particles in a multi-dimensional scene viewer (Figure 2 ). By interacting with the automatically computed trajectories the user is able to interactively control and correct for possible artefacts during the tracking procedure, e.g. deriving from noisy images. Applying TIKAL, we rapidly reconstructed, visualized and analyzed the trajectories of 1131 particles in more than 50 cells. Figure 2 Screen shot of the image processing platform TIKAL. (top) Image shows a sample two-dimensional section through a nucleus with binarized nuclear particles (red) counterstained with Hoechst 33342 stain (green). Pull down menu exemplifies different tools for quantitative analysis integrated into TIKAL. Numbers indicate different nuclear particles reconstructed by 3D isosurface reconstruction (bottom). Computed tracks of nuclear bodies over time are displayed as spheres on a string in the multi-dimensional scene viewer. In vivo observation of microspheres We imaged the microinjected microspheres and the GFP-NLS-vimentin particles in SW13 cells over a time interval of 20 min (Figure 1a and 1b ). After image processing a qualitative analysis of the trajectories of 154 microspheres visualized in 12 cells suggested the same kind of mobility for both the 100 nm-beads and the GFP-NLS-vimentin bodies (Figure 3a ). For a more rigorous quantitative comparison the mean square displacement (MSD) was calculated for each individual particle as well as its anomalous diffusion coefficient α. Based on α, the analyzed particles were classified into four arbitrary groups of mobility using the theoretical framework from previous studies [ 25 , 26 ]: (i) confined diffusion (α < 0.1), (ii) obstructed diffusion (0.1 ≤ α < 0.9), (iii) simple diffusion (0.9 ≤ α < 1.1) and (iv) directed motion (α ≥ 1.1) (for sample trajectories see Figure 4 ). The comparison of the calculated anomalous diffusion coefficients of the GFP-NLS-vimentin bodies with those of the 100 nm-microspheres revealed no significant changes in the distribution (p = 0.126; compare Figure 5a and 5b . For statistical significance of interexperimental differences of distribution patterns for anomalous diffusion coefficients refer to Table 1 ). Figure 3 4-D visualization of vimentin particle trajectories a) in the nucleus and b) the cytoplasm. Each color represents an individual vimentin body. The respective centers of masses are indicated as spheres. Tracks are symbolized by interconnecting lines. Arrows indicate the different kinds of diffusion (see Figure 4a). Major types of movement are indicated: I) confined diffusion; II) obstructed diffusion; III) normal diffusion; IV) directed motion. Figure 4 Different classes of mean square displacement of tracked nuclear vimentin particles. a) Mean squared displacement (MSD) of four representative modes of particle mobility (x-axis: acquisition time in seconds; y-axis: MSD). Roman numbers: I) confined diffusion; II) obstructed diffusion; III) normal diffusion; IV) directed motion (The numeration refers to indicated trajectories in Figure 3a). b) The four different mobility classes are represented by particle trajectories. The trajectories correlate to the numbers indicated by arrows in Figure 3a. Figure 5 Classification of particles into four groups of diffusional motion according to cellular localization and their calculated anomalous diffusion coefficient. a) nuclear vimentin particles, (b) microinjected nuclear 100 nm microspheres, (c) cytoplasmic vimentin particles. Table 1 Significance analysis of interexperimental differences in distribution of anomalous diffusion coefficients by Kolmogorov – Smirnov test Statistical significance analysis for interexperimental differences in cumulative distribution of anomalous diffusion coefficients (for corresponding distribution plots see Figure 5 and Figure 9) was performed by the Kolmogorov – Smirnov test [45]. The different experiments are depicted as followed: control = vimentin particles in the nucleus; beads = sepharose microspheres (100 nm) in the nucleus; cytoplasm = vimentin particles in the cytoplasm; Vimentin particles in the nucleus with drug treatment: azide / deoxyglucose (10 mM / 50 mM); cytochalasin D (1 μg/ml); nocodazole (0.04 μg/ml); sorbitol (600 mM). Experiment 1 Experiment 2 p-value control beads 0.1264 control cytoplasm 1.11E-15 beads cytoplasm 5.22E-08 control azide / deoxyglucose control cytochalasin D 0.01643 control nocodazole 9.70E-05 control sorbitol 1.78E-14 azide / deoxyglucose cytochalasin D 0.1095 azide / deoxyglucose nocodazole 0.004754 azide / deoxyglucose sorbitol 8.45E-11 cytochalasin D nocodazole 0.7306 cytochalasin D sorbitol 2.00E-09 nocodazole sorbitol 7.96E-09 Next, we were interested in the differences of the kinetic behaviour of the Xenopus GFP-vimentin particles in the nucleoplasm as compared to that in the cytoplasm. Transfected SW13 cells lack endogenous vimentin and therefore do not have intermediate filaments. Instead, only small spherical aggregates of the temperature-sensitive amphibian protein were deposited throughout the cytoplasm (Figure 1c ; [ 27 , 28 ]). When directly comparing the particle trajectories of the nucleoplasmic vimentin bodies to the cytoplasmic vimentin bodies striking differences were found. Nuclear-targeted vimentin particles displayed a spatially restricted movement within distinct corrals. However, on occasion they were able to move spontaneously to an adjacent corral (Figure 3a ). The maximum distance that a NLS-vimentin particle moved was 4 μm within the observation time of 20 min. This corresponds on average to a speed of 0.2 μm / min. Most strikingly, we never encountered crossing nuclear trajectories. In contrast, cytoplasmic vimentin particles moved along more extended trajectories and did hardly ever exhibit corralling events (Figure 3b ). Moreover, the cytoplasmic bodies moved three times as fast, i.e. up to 12 μm in distance within 20 minutes. A comparison of the overall kinetic characteristics of nuclear vimentin bodies versus sepharose beads revealed that in the nucleus obstructed diffusion is the major type of movement whereas in the cytoplasm directed motion is observed to a similar extent, both accounting for approximately 40 %. Notably, confined diffusion is very rarely found in the cytoplasm whereas in the nucleus 11.4 % of the movement can be accounted for it (Figure 5 , Table 1 ). Chromatin remodelling directly effects mobility of nuclear particles In the next step we analyzed the influence of chromatin density on mobility of nuclear particles. Upon inspection of corralled versus highly mobile nuclear particles (Figure 6 ) we frequently observed a correlation between chromatin density in the neighbourhood of particles and their degree of motility. For a more rigorous quantitative analysis the mean grey value in a neighbourhood of 9 × 9 pixels was measured for each particle over an observation time of 20 min with a time-lapse of Δt = 10 seconds (Figure 7 ). Evidently, there is a strong correlation between chromatin density and particle velocity. Particles with high velocities were exclusively formed in areas of very low chromatin density. An increase in chromatin density directly led to a decrease of particle velocity (Figure 8a and 8b ). A similar reverse effect was detected in cases where particles had very low velocities. After release of a body from a dense chromatin cluster a sudden increase of its mobility could be observed. In this case a high chromatin density was measured during the resting phase of the body, whereas a decrease of chromatin density was detected before the particle started to increase its velocity (Figure 8c ). This phenomenon was prominently encountered with particles showing a high frequency of changes in corralled and more directed movement (Figure 8a,8b,8c ). For particles with minimal changes in distance and velocities, a constant chromatin density with low fluctuation in measured grey values was observed (Figure 8d ). Figure 6 4-D – tracking of vimentin particles in the nucleus. (a, b) particles with restricted movement (confined diffusion) c) corralled particle and corresponding trajectories. (red = vimentin bodies; green = Hoechst 33342 staining). Figure 7 Chromatin intensity analysis showing the preprocessing steps for the Hoechst 33342 images: a) unprocessed original image; b) Gaussian smoothing; c) image classified into 8 regions of grey values; d) localization of particles in the cell nucleus (red); e) measuring the mean grey value intensity around the center of mass for each individual vimentin particle (see Methods for a complete description). Figure 8 Correlation plots between mean grey values (green line) of chromatin densities and particle velocity (blue line) over a time range of 20 minutes. Arrows indicate significant changes in intensity and particle velocity. Red: NLS-GFP-vimentin; green: Hoechst 33342 stain. Influence of inhibitors on the mobility of nuclear vimentin bodies In order to investigate the contribution of structural elements of the cytoplasm to nuclear body mobility in living cells, inhibitors of cellular energy as well as drugs that lead to the depolymerization of cytoskeletal systems were employed. In particular, we inhibited cellular ATP production and incubated cells with agents that depolymerize microtubules or microfilaments, both of which are tracks for molecular motors in the cytoplasm. Cells were imaged prior to addition of the inhibiting substance for 10 minutes with image stacks acquired every Δt = 10 seconds. In a first step, the dependency of nuclear vimentin particles on energy-dependent mechanisms as investigated by depletion of ATP through incubation with 10 mM azide and 50 mM deoxyglucose followed by live cell imaging over a time interval of another 10 minutes. For more than 140 bodies in eight cells the diffusion coefficients were calculated. Compared to the control group (Figure 5a , 9a and Table 1 ) an absolute increase of 5.7 % for confined diffusion, an absolute increase of 0.6 % for obstructed diffusion, an absolute decrease of 1.7 % for simple diffusion and an absolute decrease of 4.6 % for directed motion were observed (Figure 9b , Table 1 ). Interestingly, after addition of azide / deoxyglucose a rapid condensation of chromatin was observed. Chromatin condensation was reversed after removal of the inhibitor as also reported recently [ 29 ], [ 46 ]. Figure 9 Mobility of nuclear vimentin bodies under the influence of inhibitors. Classification was performed according to the diffusion coefficient. a) control; b) azide / deoxyglucose (10 mM / 50 mM); c) cytochalasin D (1 μg/ml); d) nocodazole (0.04 μg/ml); c) sorbitol (600 mM). Secondly, the impact of the presence of a functional actin cytoskeleton on vimentin body movement was tested using the actin polymerization inhibitor cytochalasin D at 1 μg/ml (Figure 9c , Table 1 ). Five cells were imaged after addition of cytochalasin D every Δt = 10 seconds for 10 minutes. A quantitative analysis of 121 bodies revealed an absolute increase of 5.3 % and 8.6 % for confined and obstructed diffusion, respectively, and an absolute decrease of 3.9 % for simple diffusion and a decrease of 10.0 % for directed motion compared to the control group. To study the role for microtubule structures on particle mobility we used the microtubule polymerization inhibitor nocodazole (Figure 9d , Table 1 ). The effect of nocodazole treatment (0.04 μg/ml) was also imaged in 5 cells with image stacks every Δ t = 10 seconds for 10 minutes. By analysing 119 bodies, an absolute increase of 8.7 % and 9.9 % was detected for confined and obstructed diffusion while an absolute decrease of 7.7 % and 10.9 % compared to the control groups was observed for simple diffusion and directed motion, respectively. Particle movement in dehydrated cells Finally the dependency of the GFP-NLS-vimentin mobility on availability of water in the nucleus was tested by treating the cells with sorbitol (600 mM) [ 30 ] (Figure 9e , Table 1 ). In contrast to the previous inhibitors, we observed a dramatic change in particle mobility. Calculation of diffusion coefficients for 101 particles in five cells revealed the total loss of simple diffusion and directed motion activities. Accordingly, 79.1 % of all particles were found in the confined diffusion and 20.9 % in the obstructed diffusion group. In summary kinetic changes were most prominent for the directed motion mode. ATP depletion decreased directed motion about 30 % relative to the control group. Treatments with cytochalasin D and nocodazole even showed a 70 % decrease in directed motion relative to the control group. The most striking effect was encountered by treating the cells with sorbitol. Simple diffusion and directed motion were totally abolished whereas the number of particles exhibiting confined diffusion increased by a factor of 7 relative to the control group. Discussion In this study we developed comprehensive bioinformatics tools to analyze the kinetic behaviour of small particles in the cell nucleus. For this purpose, fast time-lapse confocal laser scanning microscopy was used to record fluorescent particles in their chromatin environment. Automated image processing algorithms such as image registration and single particle tracking were instrumental to analyze the resulting complex data sets in a most efficient way. Usually, sophisticated image processing methods are widely not accessible for cell biology laboratories working with multi-dimensional data sets. A qualitative, interactive analysis of complex processes in living cells can yield interesting results. However, a quantitative insight into the underlying mechanisms can only be achieved by a rigorous computational analysis. While computational systems have been provided for estimating diffusion and binding constants based on photobleaching experiments of populations of small proteins [ 23 , 31 - 33 ], integrated software packages for single particle tracking of nuclear bodies on the background of moving and shape changing objects have not been provided yet to the community. Here, our system TIKAL closes an important gap. For an automated analysis of even larger sets of spatio-temporal data as in this study any software system needs to be adapted to data storage systems that are devoted for handling such huge data sets [ 15 ]. At the same time image analysis workflows have to be deployed onto computing clusters or the GRID ([ 34 ], accepted). Both developments are underway in our laboratory. We visualized the different kinetic behaviours of nucleus-injected 100 nm polystyrene microspheres. Furthermore, a stably transfected cell line expressing GFP-NLS-vimentin, which forms nuclear particles in the same size range as microspheres, was used. The majority of nuclear particles moved with obstructed diffusion within distinct corralled regions. This kind of movement was essentially found also for microinjected polystyrene beads. The obstructed diffusion behaviour supports the notion that these particles can diffuse within corrals restricted by dense chromatin regions. Upon chromatin remodelling distinct less dense chromatin regions are formed and enable the particle to move to an adjacent corralled region. We were able to quantitatively assess this phenomenon by measuring the chromatin intensity around an individual particle. Our data show that chromatin intensity decreases prior to a global velocity increase of the particle. Therefore we conclude that the particles do not actively push their way through the chromatin. Moreover, the chromatin itself is able to support or induce the movement of individual particles. The ability of the particle to move from one corral to the next is restricted and regulated by the surrounding chromatin remodelling activities. However, whether local chromatin regions can actively influence the destination of small nuclear particle movement has to be resolved in future investigations. With the present assay we cannot discern whether changes in the velocity of a body simply correlate with the entry of a body into a domain or whether the changes are caused by interaction between a body and the surrounding chromatin domain surfaces. The addition of cellular inhibitors caused significant changes in the diffusional behaviour of nuclear particles. In all treatments a reduction of active transport processes were observed. This suggests that the coordination of nuclear processes such as chromatin remodelling is not solely dependent on single factors such as ATP, i.e. ATP consuming enzymes. Moreover, chromatin regions in interphase nuclei apparently move in a diffusional way [ 19 ], while other factors such as cytoplasmic microtubules and actin filaments attached to the nuclear periphery possibly account for large-scale spatial chromatin rearrangements [ 35 ]. The phenomenon of energy dependent nuclear body movement has been also described in other studies where an anomalous diffusion behaviour and an ATP- and transcription-dependent association of Cajal bodies with chromatin was reported [ 2 ]. Further, upon ATP depletion in BHK cells rapid and large-scale movement of PML bodies stopped, whereas small localized movements of PML bodies were still observed [ 3 ]. A recent examination of the dynamic behaviour of PML nuclear bodies showed their fission to microstructures after different physiological stresses, and their fusion upon recovery [ 36 ]. Moreover it has been shown that movements of PML and other nuclear bodies can be described by diffusion of the individual body within a chromatin corral and its translocation resulting from chromatin diffusion [ 46 ]. However, future systematic studies will help to reveal the influence of drug treatments and cellular inhibitors on the dynamic behaviour of those and other nuclear bodies. A further interesting observation was the reversible formation of chromatin dense regions upon energy depletion. The general effect of this reorganization seems to influence the mobility distribution pattern of the particles only slightly. However, we could observe a significant decrease in directed motion of up to 30 % upon inhibition of energy-depended processes. Furthermore, in order to evaluate the degree of nuclear particle movement with respect to cytoplasmic dynamics, we used the vimentin system to analyze cytoplasmic particle mobility. Mostly active transport processes were observed. Two possible explanations for this phenomenon are suitable. Vimentin, which belongs to the group of intermediate filaments, forms crossbridges to other cellular structures. Since the SW13 cells lack endogenous expression of intermediate filament proteins such as cytokeratins and vimentin, possible interactions with these cytoplasmic intermediate filaments can be omitted. Specific interactions with dynein have been described [ 37 , 38 ]. Hence, newly synthesized vimentin is subjected to active transport processes and "guided" to cellular locations for the establishment of vimentin networks. Another explanation could assume that the filaments do not bind any cytoplasmic structure. In this less likely scenario the active transport of vimentin particles would result from the densely packed cytoplasm and the resulting pushing and pulling of adjacent actively transported molecules. From our data we conclude that the NLS-vimentin system is very suitable for further studies of nuclear architecture. Though we obtained the same results with microinjection assays, the GFP-NLS-vimentin system has significant advantages such as the higher expression efficiency and the fact that the experiments can be performed in a cell system with normal proliferation characteristics. Conclusions We presented a novel image analysis platform TIKAL that for the first time allows the 4-D tracking of nuclear particles on the background of moving and shape changing objects. TIKAL is complementary to other software systems designed for diffusion studies based on photobleaching experiments. Applying TIKAL we were able to analyze the dynamics of nuclear bodies under various different conditions and thus demonstrated that local chromatin remodelling accounts to a large extent for changes in the dynamics of individual nuclear bodies. Methods Expression plasmids and construction The cloning of the Xenopus laevi GFP-NLS-vimentin expression plasmid has been described previously [ 17 ]. For the generation of the N-terminally tagged GFP-vimentin construct the vimentin cDNA was modified at the 5' – end to contain a BspE I – site followed by a Nde I – site containing the start methionine in frame subcloned into pBlueScript (Stratagene). The BspE I / BspE I fragment was then subcloned into pEGFP – C 1 and the orientation verified by DNA sequencing. Cell culture and transfection SW13 lacking endogenous vimentin [ 27 ] were usually grown in DMEM (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (Seromed, Berlin, Germany), 20 mM glutamine and 100 μg/ml penicillin/streptomycin (Invitrogen) at 37°C and 5% CO 2 . For live cell imaging purposes the cells were resuspended in complete DMEM without Phenol Red (Invitrogen) and grown in 2- or 4-well Lab-Tek ® II chambers (Nalge Nunc International, Rochester, USA). Transient transfections were carried out using the FuGene 6 transfection reagent according to the manufacture's protocol (Roche, Mannheim, Germany). Immediately before imaging, chromatin counter stain was obtained by incubating the cells with 1 μg/ml Hoechst 33342 in complete DMEM without Phenol Red for 20 min followed by three times washing with DMEM without Phenol Red. Cells were then kept in complete DMEM supplemented with 20 mM Hepes without Phenol Red. For tracking of nuclear particles we stably transfected SW13 cells with the expression plasmid encoding Xenopus laevis GFP-NLS-vimentin. To track vimentin particles in the cytoplasm, SW13 cells were transiently transfected with the Xenopus laevis GFP-vimentin cDNA construct described above. Mircroinjection of fluorescent polystyrene microspheres SW13 cells were cultured in P35G-1.5-7-C-Grid cell locate culture dishes (MatTek Corporation, Ashland, MA) in complete DMEM medium for 1 day after plating. Carboxylate-modified 0.1 μm microspheres (FluoSpheres, #F8800, Molecular Probes, Leiden, NL) were obtained as 2 % solids in solution and further diluted to 0.04 % solution in 1 M BSA in PBS. Before microinjection, the microspheres were sonificated for 30 seconds to avoid aggregation. The AIS 2 system (Cell Biology Trading, Hamburg, Germany) was used for microinjection. Injection needles were drawn from borosilicate glass capillaries GC120TF-10 (Harvard Apparatus, Edenbridge, UK) using a Flaming Brown micropipette puller P-97 (Sutter Instruments, Novato, CA). The injection pressure was adjusted to 20–250 kPa in the different experiments. For evaluation of cell viability microinjected cells were grown at 37°C with 5% CO 2 overnight and examined the next day. Imaging Live cell imaging was carried out on a confocal laser scanning microscope TCS SP2 AOBS (Leica Microsystems, Wetzlar, Germany) using a 63x oil immersion objective with 1.4 optical apperture (HCX PL APO lbd.BL 63x / 1.4, #506192, Leica Microsystems). The microscope was further equipped with a 29 mm objective heater (#0280.010, Leica Microsystems) with temperature – controlled device (#0504.000, Leica Microsystems) and a temperature – controlled fan (ASI 400E, Nevtek, Burnsville, VA, USA). A diode laser (λ = 405 nm) was used for excitation of Hoechst 33342. An argon (λ = 488 nm) and a helium/neon laser (λ = 543 nm) was used for EGFP and fluorescent microsphere excitation, respectively. 3-D image stacks, each consisting of 17 2-D-images, of GFP-vimentin particles and Hoechst 33342 stained chromatin were acquired in parallel at the maximum scanning speed of 1400 Hz (i.e. scan lines per second) with a constant Δt = 10 seconds. Imaging format was set to 256 × 256 pixel, voxel sizes were generally between 0,093 μm × 0,093 μm × 0,325 μm and 0,093 μm × 0,093 μm × 0,450 μm. The laser intensity was adjusted to a minimum to avoid photo damage during imaging. For this purpose the acousto-optical beam splitter (AOBS) were set between 2 – 5% for both lasers with photo multiplier (PMT) settings of 715.7 Volt for the diode laser and 717.4 Volt for the argon and helium/neon lasers. All the image processing steps were carried out in TIKAL (see below). Drug treatment Before drug treatment, we acquired 3-D time series of cells with a time-lapse of Δt = 10 seconds for 10 minutes. Immediately afterwards the medium was exchanged for one of the following solutions: i) 600 mM sorbitol; ii) 20 mM azide / 50 mM deoxyglucose; iii) 0.04 μg/ml nocodazole; and iv) 1 μg/ml cytochalasin D – all in complete DMEM without Phenol Red except for ii), which was applied in PBS [ 39 ]. After change of medium, we immediately acquired further 3-D time series images for another 10 min. Thereafter, the drug containing medium was replaced by complete DMEM without Phenol Red. In order to document the recovery and viability of cells, images were acquired for another 10 minutes with the same microscope settings. Image registration 4-D image registration [ 40 , 41 ] of consecutively captured three-dimensional images of cell nuclei counterstained with Hoechst 33342 was performed for correcting for global movement of cell nuclei. In order to reduce alignment artifacts due to acquisition noise all three-dimensional image stacks were preprocessed using a 3-D median filter and a 3-D automatic gamma correction for maximum gray value range. Image registration was then performed using an implementation of an automated image registration algorithm [ 40 ] running on a high-performance computing cluster. In this study, we only applied rigid and affine transformation since we did not observe drastic local deformations in cellular shape which would require correction by our non-rigid transformation method [ 23 ]. Rigid and affine transformation matrices were computed in a two-step process for optimal 4-D object alignment. First, objects at time point t + 1 were consecutively aligned with 3-D objects at time point t providing a pre-registered image stack at each time point. Secondly, each pre-registered image stack was aligned with respect to the initial image stack at time point t = 0. Transformation matrices calculated for the chromatin stained images were applied to all corresponding image stacks in the other color channels. Tracking beads and vimentin particles Image processing was carried out using our in-house developed image analysis platform. The analysis chain consisted of three major modules: image preprocessing and segmentation, 4-D tracking and quantification of dynamics, 4-D visualization and user interaction with 4-D data sets. To reduce noise in the vimentin channel, images were subjected to 3-D diffusion filtering [ 24 ] followed by segmentation with a pyramid linking algorithm [ 42 ]. Particle tracking was performed by extending our already implemented single particle tracking algorithm from 2-D + time to 3-D + time [ 43 , 44 ]. The tracking algorithm uses parameters such as the individual center of masses, volumes, total grey value intensities, velocities and accelerations. To control possible tracking and segmentation artifacts we visualized 4-D tracks of beads and vimentin particles, respectively, together with their isosurface reconstruction at each time point. Correlation analysis of chromatin density and tracked nuclear particle Chromatin images were preprocessed by a 2-D Gaussian smoothing filter to reduce noise. Additionally a gamma filter was applied to use the full grey value range of 8 bits. For additional noise reduction the 256 different grey values were divided into eight grey value classes ranging from 0 – 32, 33 – 65, etc. The centre of mass for each individual polystyrene bead or nuclear vimentin body was calculated and tracked in 3-D over time. For each time point the corresponding binned mean grey value intensities of a 9 × 9 pixel area around the centre of mass was determined. Calculation of mean squared displacements and anomalous diffusion coefficients The mean square displacement (MSD) was calculated for each particle and time point of the data set according to <Δd2> = < [d(t) - d(t + Δt)] 2> and plotted as < d 2 > (μm 2 ) versus Δ t (s) using Matlab (The MathWorks, Inc., Novi, MI). Further evaluation of anomalous diffusion (α) [ 25 , 26 ] was determined by using the regression curve fitting functions implemented in Matlab. TIKAL image processing platform TIKAL is an in-house developed platform for multi – purpose image processing (executable code can be requested at: ). The program consists of a graphical user interface for easy access of the underlying algorithms. The software is written in C/C++ and can be deployed both on Linux and Windows systems. The main components of the program are separated into several main modules, namely a filter, registration, tracking, visualization and data handling module. The filter module contains 2-D and 3-D image processing algorithms such as gamma correction, median, gaussian, anisotropic diffusion filters [ 24 ]. Segmentation filters range from simple threshold operations to complex pyramid linking segmentation [ 42 ]. The registration module is capable of handling single 3-D image stacks as well as 4-D time-lapse series of image stacks. Image stacks can be corrected by a combination of rigid and affine transformations as well as non-linear correction of local deformations by registration based on a thin plate spline model [ 22 , 23 ]. The tracking [ 44 ] and visualization part can be combined to allow the user a visual inspection of the tracking result and enable a correction of falsely assigned trajectories. The tracking and visualization module contains functions for calculation of quantitative parameters such as distances, velocities and mean squared displacement (MSD). The software is capable to import both the proprietary Leica and the commonly used Tiff file format. Currently we are working on an implementation to integrate and combine our software with the Open Microscopy Environment, OME [ 15 ]. This is of particular importance when working with huge data sets, when either computer memory or disk space become limiting factors in the analysis stream. List of abbreviations Expression plasmids and construction NLS – Nuclear localization sequence GFP – green fluorescent protein Authors' contributions CPB wrote the program for 4-D data analysis (TIKAL). With this platform he was able to perform data analysis and data evaluation. Further, microinjection of cells and nuclei and their respective imaging was carried out by CPB. In additions he assisted MR with imaging and cell culture of the vimentin transfected SW13 cells. MR performed the transfection, culturing, imaging and inhibitor treatment of SW13 cells. Additionally she contributed to the interactive 4-D tracking analysis. CA was involved in planning of this study at an earlier stage. HH supervised the experimental part, whereas RE was responsible for the entire setup and planning of the computational part of this study.

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544927

A Functional Neuroimaging Study of Sound Localization: Visual Cortex Activity Predicts Performance in Early-Blind Individuals

Blind individuals often demonstrate enhanced nonvisual perceptual abilities. However, the neural substrate that underlies this improved performance remains to be fully understood. An earlier behavioral study demonstrated that some early-blind people localize sounds more accurately than sighted controls using monaural cues. In order to investigate the neural basis of these behavioral differences in humans, we carried out functional imaging studies using positron emission tomography and a speaker array that permitted pseudo-free-field presentations within the scanner. During binaural sound localization, a sighted control group showed decreased cerebral blood flow in the occipital lobe, which was not seen in early-blind individuals. During monaural sound localization (one ear plugged), the subgroup of early-blind subjects who were behaviorally superior at sound localization displayed two activation foci in the occipital cortex. This effect was not seen in blind persons who did not have superior monaural sound localization abilities, nor in sighted individuals. The degree of activation of one of these foci was strongly correlated with sound localization accuracy across the entire group of blind subjects. The results show that those blind persons who perform better than sighted persons recruit occipital areas to carry out auditory localization under monaural conditions. We therefore conclude that computations carried out in the occipital cortex specifically underlie the enhanced capacity to use monaural cues. Our findings shed light not only on intermodal compensatory mechanisms, but also on individual differences in these mechanisms and on inhibitory patterns that differ between sighted individuals and those deprived of vision early in life.

Introduction Animals and humans deprived of vision have been shown to have enhanced nonvisual perceptual abilities. Indeed, many blind individuals are extremely efficient in tactile processing, including Braille reading; the talent manifested by some well-known blind musicians, singers, and even piano tuners is often in part attributed to the fact that they were blind since their youth. One may conclude that blind persons should be better in nonvisual tasks since they compensate for their lack of vision by focusing on their remaining modalities. Many studies have in fact shown that some early-blind human subjects outperform sighted persons in nonvisual tasks, such as speech perception [ 1 , 2 , 3 ], unfamiliar voice recognition [ 4 ], verbal memory [ 5 , 6 , 7 ], and musical abilities [ 8 , 9 , 10 ]. Of particular relevance to the present study are data suggesting that some blind individuals show better auditory spatial discrimination [ 11 ] or localization of sound sources than sighted subjects [ 2 , 12 , 13 ]; however, other studies have failed to show this advantage [ 14 , 15 ], raising the question of what may underlie individual differences. In general, the nature of any behavioral enhancement, its extent, and its neural bases are still matters of considerable debate. Animal studies provide some insight as to the neural substrates underlying such enhanced capacities (reviewed in [ 16 ]). For instance, in cats that had been visually deprived for several years by eyelid suture shortly after birth, the auditory cortical representation expanded into visual areas [ 17 ], and auditory spatial tuning was sharpened in the auditory cortex [ 18 ]. Similarly, in neonatally enucleated rats, electrophysiological recordings showed somatosensory responses in the visual cortex [ 19 ], and the somatosensory cortex showed an enlargement of receptive fields of the cells in some barrels together with an increase of angular sensitivity for deflection in another barrel [ 20 ]. Thus, those experiments indicate a recruitment of the visual cortex for nonvisual tasks, but do not conclusively prove that the enhanced perceptions of the blind rely on the visual cortex. Several studies using neuroimaging techniques have also established that posterior visual areas in blind individuals may be active during the performance of nonvisual tasks such as Braille reading [ 21 , 22 ], memory retrieval [ 7 ], and auditory localization [ 23 , 24 ] as well as other auditory functions [ 25 , 26 , 27 , 28 , 29 ]. It remains to be established whether recruitment of visual cortices necessarily reflects functional reorganization, or whether it indicates a nonspecific or even pathological response. Indeed, despite numerous studies showing activation in visual areas during nonvisual tasks, the functional significance of this phenomenon has been questioned by some investigators who suggest that the occipital cortex might be nonspecifically coactivated [ 30 ]. If the visual cortex participates in nonvisual functions in the blind, then its activity level should be related to individual differences in behavior, and in effect predict behavioral outcome. Localization of sound, a very important function for the blind, is one domain in which it is particularly useful to study the cross-modal interactions that may occur following visual deprivation. This task entails integration of binaural and monaural cues to derive spatial information. In accordance with the idea that nonvisual processing can be enhanced in the blind, a prior study demonstrated that a subset of early-blind subjects was more accurate than sighted controls (SIG) at localizing sound sources, specifically when using monaural cues [ 12 ]. These findings provide a clear opportunity to study the nature of visual cortical recruitment, and the extent to which it relates to behavioral improvements. Thus, in the present study, subjects were first studied in an anechoic chamber using binaural and monaural sound localization tasks. Depending on their performance at the monaural task, they were divided into three groups: (i) early-blind participants who could localize the sounds more accurately than control subjects (early blind with superior performance [EBSP]); (ii) early-blind participants who were unable to localize the sounds any more accurately than controls (early blind with normal performance [EBNP]); and (iii) SIG. The same localization task was next adapted so that it could be carried out within the positron emission tomography (PET) apparatus, using a speaker array which permitted pseudo-free-field presentations [ 31 ]. Two control conditions, for monaural sound localization (MSL) and binaural sound localization (BSL), were used to control for the auditory input and motor responses. The hypothesis tested was that blind persons showing supranormal performance do so because they recruit visual cortical areas to carry out the task. We therefore predicted that they would show activation in visual areas specifically during the MSL task, and not during the binaural or control tasks. The other blind group, which does not have enhanced MSL ability, should not show this activation pattern. We further hypothesized that the degree of visual cortical activity would be predictive of individual differences in the behavioral performance of the monaural task. Results Anechoic Chamber Experiments In the anechoic chamber experiments, only five of the 12 early-blind subjects could accurately localize the sounds monaurally, whereas most of the sighted subjects could not ( Figure 1 ). In order to differentiate two groups of early-blind participants on the basis of their performance at this monaural task, we computed their mean absolute error score and set the cut-off at a score of 45° (see Materials and Methods ). Individuals with scores below this value formed the EBSP group, while those above it constituted the EBNP group. The results of the former group are illustrated on the right side of Figure 1 B. As can be seen, in these subjects the regression line was very close to the dashed line representing ideal performance. By contrast, the other subgroup of early blind subjects, EBNP, and the SIG could not localize the sounds correctly, and the regression line is quite distant from that representing ideal performance. To confirm this localization performance, the mean absolute error score was compared for the three groups; a group X position interaction was observed (F 30, 225 = 6.299, p < 0.01), localization being more accurate in the EBSP group, especially on the side of the obstructed ear. The performance of the EBNP did not differ from that of the SIG. These findings confirm the previous study of Lessard et al. [ 12 ] with a different group of early-blind participants. Figure 1 Monaural Sound Localization in PET Experiments Performed in the Three Groups of Subjects (A) CBF increases. Activations of the right striate and extrastriate cortices are observed in EBSP but not in the two other groups for the contrast of MSL minus its control task. Upper image series, sagittal slices; lower image series, coronal slices. X and Y coordinates refer to standardized stereotaxic space. (B) Behavioral data. Behavioral results in MSL task (with SE bars). The dashed lines represent the ideal performance, whereas the solid lines indicate the best linear fit to the observed localization performance. Negative angles on the abscissa correspond to the obstructed ear, while positive angles correspond to the unobstructed ear. Note the better performance of the EBSP group compared to the EBNP and SIG. In contrast, all 19 subjects tested, whether sighted or early blind, could correctly localize sounds binaurally ( Figure 2 ). Despite an overall good performance, as measured by the absolute error score, the EBNP group was somewhat less efficient than the other two groups, especially for more lateral positions (group X position: F 30,225 = 4.058, p < 0.01; see Figure 2 C). The latter finding is reminiscent of the undershooting performance also found in visually deprived cats [ 32 ]. Figure 2 Binaural Sound Localization in PET Experiments Performed in the Three Groups of Subjects (A) CBF decreases. In the sagittal (upper image series) and coronal (lower image series) slices, a decreased CBF is observed in the visual cortex of SIG (striate and extrastriate cortices), for the contrast of BSL minus its control task. X and Y coordinates refer to standardized stereotaxic space. (B) CBF increases. In the sagittal (upper image series) and coronal (lower image series) images, a CBF activation peak is seen in the right ventral extrastriate cortex for the EBSP group, but not for the other two groups, for the contrast of BSL minus its control task. (C) Behavioral data. Behavioral results in the BSL task are presented (with SE bars). The dashed lines represent the ideal performance, and the solid lines indicate the best linear fit to the observed localization performance. All three groups were able to localize sounds accurately. PET Scanner Experiments Binaural sound localization During binaural localization of the sound, when compared to the control task ( Figure 2 A and Table 1 ), cerebral blood flow (CBF) decreased in the extrastriate and striate cortex of SIG, suggesting inhibition between visual and auditory areas. However, neither group of early-blind subjects showed this deactivation. A further confirmation of these results was obtained by carrying out a direct intergroup comparison of the activation of each of the blind groups to that of the sighted one. These differences are presented in Figure 3 . The two blind groups show what appears to be an increase in CBF relative to the SIG, confirming that the CBF response of these groups differ in this region. However, given the deactivation observed within this region in the SIG in the first analysis, as opposed to the lack of CBF difference in the blind, the more likely interpretation is that this effect reflects a decrease in CBF in the sighted. Figure 3 Intergroup Contrasts in Binaural Sound Localization Minus Control Task Sagittal (top) and coronal (bottom) images showing the contrasts between EBNP (left) compared to SIG, and EBSP (right) compared to SIG. These contrasts confirmed the differences in occipital areas between the SIG and the two other groups, which are likely attributable to a decrease in CBF activity in the sighted relative to the control task (see Figure 2 ). X and Y coordinates refer to standardized stereotaxic space. Table 1 Stereotaxic Coordinates and t Values of Activation and Deactivation Foci in Occipital Areas Empty cells indicate that there is no activation above the threshold for this group. Coordinates x, y, and z refer to standardized stereotaxic space [ 75 ]. See Table S1 for complete list of foci Another finding from the binaural versus control task contrast was a small region of activation in the visual cortex (ventral extrastriate) of the EBSP group, but not in the other two groups ( Figure 2 B and Table 1 ). Finally, all three groups also showed activation in several other cortical regions (see Table S1 for further details); among the most relevant of these is a focus in right inferior parietal cortex ( Figure S1 ). Monaural sound localization. Of greatest relevance to our hypotheses, during monaural stimulation (one ear plugged), as compared to control task, right-hemisphere striate and ventral extrastriate areas showed increased CBF only in the EBSP subset of blind subjects ( Figure 1 A and Table 1 ). For the EBNP subset, occipital activation was not significant, as was also the case with the SIG. Once again, direct intergroup contrasts confirmed the differences in activation in occipital areas during MSL between the EBSP group and the two other groups ( Figure 4 ). Figure 4 Intergroup Contrasts in Monaural Sound Localization Minus Control Task Sagittal (top) and coronal (bottom) images showing contrasts between the EBSP and EBNP (left), and between the EBSP and SIG (right). These contrasts confirmed the differences in occipital areas between the EBSP group and the two other groups. X and Y coordinates refer to standardized stereotaxic space. While parietal and frontal activations were also seen in the three groups, temporal activations were not found for monaural stimulation, the EBNP group showing even some deactivation in temporal areas. The EBSP group also showed some activation in the right cerebellum (see Table S1 for further details). Significant differences between groups were not observed in temporal and parietal cortices, however. Correlation analysis In order to assess whether occipital activations have a functional role in auditory localization, independent voxel-wise covariation analyses were carried out across the entire group of blind individuals. Irrespective of the group to which they had been assigned, the individual absolute error score was entered as a regressor in the analysis examining covariation with CBF change between overall accuracy at the localization tasks and activation across the entire brain volume, following the procedure outlined by Paus et al. [ 33 ]. For MSL, a negative and significant correlation was observed between the absolute error score and CBF in some areas of the visual cortex (especially extrastriate but also striate [ Figure 5 and Table 1 ]). It follows from these results that the degree of activation (percent CBF change) predicted behavioral performance in MSL. The highest correlation observed was in the right ventral extrastriate cortex (lingual gyrus; Brodmann area [BA]18, r = –0.81, p < 0.01) but two other significant foci were found in right dorsal extrastriate cortex (superior occipital gyrus; BA19, r = –0.77, p < 0.01), and striate cortex (BA17, r = –0.68, p < 0.05). Two of these foci are close to the ones identified in the analyses presented for MSL. These findings support the hypothesis that the visual cortex is directly involved in localizing a sound stimulus in the monaural condition. Figure 5 Correlational Analysis for Monaural Sound Localization in Blind Persons These data show the correlational analysis between performance (mean absolute error) in pointing task to monaurally presented sounds and CBF in a group of blind subjects. The two columns of brain images (left image series, sagittal sections; right image series, coronal sections) illustrate the statistical parametric map of the correlation, which is maximal in the ventral extrastriate cortex (A) but also significant in dorsal extrastriate (B) and striate (C) cortices. The red arrows in the coronal slices indicate the focus selected for the respective sagittal slices. The scattergram shows the individual values extracted from each of these regions; closed circles indicate blind subjects; open circles indicate SIG. The dotted vertical line represents the cutoff in performance for the a priori classification of blind subjects into those with low error rates (EBSP) and those who do not show the enhancement (EBNP). X and Y coordinates refer to standardized stereotaxic space. Discussion The imaging results of this study support our hypothesis that blind persons recruit occipital areas in the context of auditory localization and, more importantly, the correlation observed with MSL performance strongly suggests that individual differences in reorganization of the occipital cortex have behavioral consequences. Hence, this relationship does not support the possibility that the recruitment is a nonspecific coactivation or a pathological response. Instead, these results suggest that visual cortex is specifically recruited to process subtle monaural cues more effectively. Functional activation of the visual cortex by nonvisual stimulation in the blind has already been shown in several previous studies. Activation of primary and secondary visual areas was observed during Braille reading and other tactile discrimination tasks in early-blind persons [ 21 ]. This tactile-induced activation in the occipital cortex was also confirmed by a series of subsequent studies [ 7 , 22 , 34 ]. The hypothesis proposing a functional role for this activation in the visual cortex was supported by the study of Cohen et al. [ 35 ] who showed, using transcranial magnetic stimulation, that this occipital area is required for Braille reading in blind subjects. This phenomenon was further illustrated in the case of a proficient Braille reader, blind since birth, who became unable to read Braille, despite normal somatosensory perception, after bilateral occipital damage resulting from an ischemic stroke [ 36 ]. Moreover, in a speech processing study in the blind, it was shown that occipital activity (striate and extrastriate) varied as a function of semantic or syntactic content [ 29 ]. Activation of area V1 has also been found to correlate with performance in memory tasks [ 7 ]. Of greatest relevance for the present findings are the results of Weeks et al. [ 24 ] in which CBF was measured during BSL in blind and sighted individuals, and reported activation in posterior parietal areas and also in association areas in the right occipital cortex only in the blind. In addition, interregional covariations observed between the right parietal and occipital (ventral, dorsal, and parieto-occipital) cortices were interpreted as reflecting parts of a functional network for auditory localization [ 24 ]. These data and the present findings converge on the conclusion that the visual cortex is recruited during auditory localization in the blind. However, whereas the study of Weeks et al. [ 24 ] reports extensive recruitment of visual areas during binaural processing, we observed only a small area of activation in the ventral visual area in the binaural condition. Instead, our data point to the importance of visual regions in successful localization under monaural conditions, which we interpret as reflecting a recruitment of these areas for processing of spectral cues. One possible interpretation of the discrepancy is that the BSL task of Weeks et al. [ 24 ] may have involved spectral cues, provided by the head-related transfer functions used in that study to simulate extrapersonal space. Functional Significance of the Recruitment of Visual Areas in the Blind: Better Use of Spectral Cues? It is interesting to consider the function of the right occipital areas, which seem to be important for MSL. Monaural cues (spectral cues and head shadow effect) are involved in the localization of sounds when one ear is obstructed, or in unilaterally deaf subjects [ 37 ]. However, spectral cues also contribute to BSL, particularly for vertical and front-back discrimination [ 38 ], but also for azimuth localization [ 39 , 40 ]. Moreover, some authors suggest that spectral cues based on head-related transfer function templates are sensitive to experience [ 41 , 42 ]. In this vein, Doucet and coworkers [ 43 ] showed that the supranormal performance of early-blind persons in MSL was decreased by occlusion of the pinna or by high-pass and low-pass filtering of the stimuli, again suggesting that use of spectral cues is important for this task. However, because performance was not completely abolished, head shadowing cues [ 37 ] might also have operated. Similarly, the study by Röder and collaborators [ 13 ] suggests that blind persons might be more sensitive to spectral cues, since their blind participants were better at localizing at lateral positions. Finally, a recent study showed superior binaural spatial discrimination performance in both early- and late-blind subjects compared to sighted subjects, when the stimuli were presented in peripheral field [ 44 ] Combination of Intramodal and Cross-Modal Plasticity? Cross-modal plasticity may not necessarily be the only mechanism to explain the present results. Some studies have also shown intramodal plasticity in auditory cortex of the blind. For example, the tonotopic region of area A1 in blind persons seems to be enlarged compared to that of sighted subjects [ 45 ], presumably reflecting greater use of auditory cues by the blind. Enhanced recruitment and sharpening of spatial tuning of auditory cortical neurons has also been found in binocularly deprived cats [ 17 , 18 ]. Thus, a combination of intramodal plasticity in auditory cortex and cross-modal plasticity involving visual cortex may have contributed to the superior performance seen in our early-blind subjects. In the present study, a significant difference in activation in auditory cortical areas was not observed among the three groups. However, we cannot exclude the possibility of plasticity at this level, as CBF responses might not be sensitive to effects such as better spatial tuning properties of auditory neurons. Experience-driven improvements in auditory localization can occur without necessarily invoking cross-modal recruitment. Indeed, some studies have shown that in MSL tasks, practice may lead to increased performance in the case of unilaterally deaf patients [ 46 ] or even normal subjects [ 47 ]. Excellent performance in MSL was also observed in juvenile ferrets when they were raised with one ear plugged. Even in adult ferrets, changes were seen in the performance during MSL after regular practice [ 48 ]. Moreover, adult humans seem to be able to calibrate auditory cues after their pinnae were modified with moulds, showing good performance with their “new ears” after a few weeks [ 49 ]. These results would favor the hypothesis that, instead of becoming supranormal in their remaining senses, blind persons may use them more efficiently within normal limits [ 50 ]. Nonetheless, our data suggest that this efficiency gain in the blind is achieved at least in part via recruitment of visual cortical areas. Why do some blind persons and not others acquire superior monaural sound localization skills? It may be that these changes are entirely experience-driven. That is, some blind persons may have had more practice navigating or using auditory cues to explore their environments. On the other hand, the individual differences we observed in the degree of cross-modal plasticity could reflect innate factors that remain to be identified. An additional explanation may be that the blind persons who are not better than normal at sound localization may be superior in other nonvisual tasks, such as Braille reading or other somatosensory discrimination, because the visual cortex was preferentially recruited to carry out these tasks instead of auditory ones. If this is the case, it is possible that cross-modal plasticity is limited to a certain extent, such that recruitment of these areas by one modality inhibits recruitment by another. These speculations will have to be explored systematically in future studies. How Does the Visual Cortex of the Blind Process Auditory Information? What is the nature of the mechanism implied in the processing of auditory stimuli by visual cortical areas? The specific areas of visual cortex recruited may provide a clue. The analyses yielded one peak in the right V1 area. V1 has already been shown to be activated in other studies examining Braille reading [ 21 , 22 , 34 ], verbal memory [ 7 ], verb generation [ 7 , 28 ], and speech processing [ 29 ]. These findings therefore suggest that V1 may play a very general role in a variety of nonvisual tasks in the blind. However, a right lingual gyrus peak was the main focus revealed by the analyses in the present study. This cortical region is known to form part of the ventral visual pathway, which is important for identifying visual objects [ 51 ]. If this region is important for the processing of visual object features, such as contour or texture [ 52 , 53 ], we may speculate that the same area is possibly used in the blind to process analogous features for auditory stimuli such as spectral contour. These cues to auditory object identity, which are normally processed in anteroventral regions of the auditory cortex [ 54 ], might be processed in the occipital ventral stream in the blind when they are relevant for spatial position. Contribution of Parietal Cortex to Binaural Sound Localization The parietal activation observed in all groups during BSL suggests that these areas are important when carrying out the task used in the present experiment (see Figure S1 ). This finding agrees with a previous study, which reported a right-sided inferior parietal activation that positively correlated with absolute error score in normal sighted subjects with the same procedure as in our study [ 31 ]. Parietal activation of both hemispheres, or a right hemisphere advantage, has been shown in several other neuroimaging studies of auditory localization and spatial discrimination experiments with sighted subjects [ 24 , 55 , 56 , 57 , 58 , 59 , 60 ]. In the study of Weeks et al. [ 24 ], a strong right-hemisphere recruitment of parietal and occipital regions was shown for blind subjects. Our findings therefore agree with these studies and with the well-known right-hemisphere advantage for spatial processing. However, we did not observe preferential activation in this parietal region in the blind as compared to the sighted, nor did CBF correlate with behavioral performance in this region. Based on those findings, we conclude that parietal area activation is related to the sensory-motor integration and spatial coordinate transformation required by the pointing task [ 31 , 61 ] at some stage after sensory processing has occurred. Thus, we propose that in those blind subjects who have specifically learned to use monaural cues, parietal regions receive additional input from the ventral visual cortex, but that no reorganization within the parietal cortex itself has occurred. Different Inhibitory Patterns for the Visual Cortex in Blind and Sighted Persons During BSL, the sighted control group (SIG) showed a deactivation in both extrastriate and striate areas of the occipital lobe, a phenomenon that was not observed in either subset of early-blind individuals. Many previous studies with sighted subjects have shown that following stimulation in one modality, cross-modal inhibition occurs in the unattended modalities [ 62 , 63 , 64 , 65 , 66 ] or even in some areas within the same modality [ 66 , 67 ]. Interestingly, an imaging study with sighted subjects carried out by Zatorre and coworkers [ 57 ] reported a visual deactivation in tasks of pitch and location discrimination. Because deactivation is not seen in all studies, the phenomenon may be related to the nature of the task [ 64 , 65 , 68 ]. Deactivation of primary visual areas has also been seen in sighted subjects during a tactile discrimination task, whereas in blind subjects activation was shown in the same area [ 21 ]. Within the context of auditory localization, Weeks and coworkers [ 24 ] also reported some occipital deactivation in sighted subjects, while the blind showed activation in the same area. All these results suggest that cross-modal inhibitory processes could be different in blind and sighted subjects, at least under some experimental conditions. Blind subjects might not have to inhibit the normally competing visual cortex when they perform some of the same nonvisual tasks as sighted people do. By contrast, the specific recruitment of the same cortex in order to complete a difficult task might permit them to compensate for their handicap. Conclusion The present study establishes for the first time in certain early-blind persons a clear relationship between monaural sound localization performance and increased CBF in occipital areas. Indeed, some of the blind persons showed occipital activation that appeared to be functional, since this phenomenon was correlated with a supranormal performance in MSL. This finding suggests that visual deprivation from an early age could lead to important cross-modal plasticity and give blind persons an advantage in using spectral cues to carry out a crucial everyday task, sound localization. Moreover, we report that inhibitory patterns differ between early-blind and sighted individuals. Under binaural conditions, the SIG seemed to inhibit part of the occipital areas when localizing sounds, but this was not the case for either group of blind persons. This differential pattern may provide clues as to how different parts of the brain normally interact during unimodal stimulation, and further suggests that these interactions may be modified in the absence of a sensory modality. It may also be important in future studies to investigate whether blind persons can recruit visual areas in other auditory tasks, for example in a task in which spectral and level cues are relevant but in a nonspatial context. Along the same lines, a spatial discrimination task not requiring the explicit localization of the sounds may also be of interest. One can thus verify that this special competence of some blind persons can be generalized in different auditory contexts other than MSL. Indeed, it would be interesting to know whether this ability is related to more complex tasks such as navigation, obstacle detection, or analysis of sound flow, for example when the subject moves or objects move around the subject. Similarly, it may be pertinent to investigate whether special training or substitution devices, frequently described in the literature, not only improve the relevant behavior but also facilitate cross-modal plasticity. Materials and Methods Subjects. The participants were seven healthy sighted volunteers and 12 early-blind subjects who had lost their vision before puberty, most of them in the first few years of life (see Table 2 ). In each case, the visual deficit was of peripheral origin and led to total blindness except for some light perception in a few subjects (categories 4 and 5, according to the World Health Organization classification [ 69 ]). All participants underwent audiometric testing to ensure good hearing, equal in both ears. They gave their written informed consent in accordance with guidelines approved by the Ethics and Research Committees of the Montreal Neurological Institute and the Nazareth and Louis Braille Institute for the Blind. Table 2 Characteristics of Blind and Sighted Subjects Anechoic chamber experiments. Subjects were asked to localize sounds binaurally or monaurally while they were seated in the anechoic room. The acoustic apparatus used to test sound localization, previously described in detail [ 70 ], consisted of 16 loudspeakers mounted on a graduated semicircular perimeter with a radius of 50 cm (positions: ±5°, ±16°, ±26°, ±37°, ±47°, ±58°, ±68°, and ±78°). The subject was seated in the center of the perimeter, the head placed on a headrest attached to the chair with the speakers positioned at ear level. The stimuli were broadband noise bursts that lasted 30 ms (10-ms rise and fall times, and a 10-ms plateau). The sound pressure level (SPL) was maintained at 40 dB. A stimulus was delivered through a randomly selected loudspeaker and repeated five times for each position. A buzzer warned the subjects that a sound was about to be presented and that they should maintain a stable head position and fixate straight ahead. Compliance with all instructions was ascertained by an experimenter remaining in the chamber behind the subject. The response consisted of pointing with the dominant hand toward the apparent source of stimulation. Lines graduated in 1° steps were drawn on the perimeter, and the response of the subject was recorded by the experimenter. For monaural testing, one ear was plugged with a combination of an ear plug (mean attenuation = 37.5 dB SPL) and a hearing protection muff (mean attenuation = 29 dB SPL). In order to compare the overall accuracy in localization between the subjects, the absolute error score was utilized in both the anechoic chamber and scanner experiments. This value is the average of the difference (in absolute value) between the correct position and the response for each trial. To allow combining of data from subjects with left or right ear plugged, the behavioral results were transformed such that the left side was arbitrarily assigned to correspond to the obstructed ear. Thus, in the behavioral data presented, negative angles on the abscissa correspond to the obstructed side, while positive angles correspond to the unobstructed side. Scanner experiments. In this part of the experiment, subjects were asked to localize sounds binaurally or monaurally while they were lying within the scanner. Monaural testing was carried out using the same ear attenuation procedure as used in the anechoic chamber. All conditions, localization tasks, and their specific control task, were part of a larger study. These conditions were counterbalanced across subjects for the order of scan conditions. Approximately half of the subjects within each group received the ear plug in the left and the other half in the right ear during the monaural part of the scanning session. Auditory stimuli were presented using a circular array of nine speakers, positioned 15° apart from ±60°, and having a radius of 24 cm [ 31 ]. The array was placed inside the PET scanner such that the head was in the center of the array, with speakers positioned on the horizontal plane relative to the subject's head, at the level of the ears. In order to ensure stable head position, the head was maintained by a Velcro band, and its position was checked frequently by means of three laser pointers included in the scanner. Background noise in the scanner room was 56 dB SPL. The stimuli were two broadband noise bursts that lasted 30 ms and were separated by a 0.5-s intrapair interval while the intertrial interval was 2.5 s. Each pair of stimuli was presented from a single speaker at 60 dB SPL, as measured at the center of the array. Each of the nine speakers was utilized 12 times in random order for a total of 108 trials for each condition. The behavioral tasks were started around 15 s before the beginning of data acquisition with the scanner. The response consisted of pointing with a joystick, placed at the subject's side, to the apparent source of stimulation. It was ascertained before the experiments that all subjects were familiar with the use of the joystick and with the task requirements. In a series of preliminary experiments, it was verified that subjects ( n = 6) wearing binaural earplugs and ear muffs could not localize any of the stimuli from the speakers. The control task consisted of pointing in alternation to the left and right (−90°, +90°, −90°, and so on), after hearing a stimulus pair presented always in the frontal (0°) position. Two control tasks were tested, one binaural and one monaural; in the monaural case the same ear was plugged as was used for the localization task for that individual. Thus, four conditions were tested in all subjects:BSL, binaural control task, MSL, and monaural control task. PET scans were obtained with a Siemens Exact HR+ tomograph (Forchheim, Germany) operating in three-dimensional acquisition mode. The distribution of CBF was measured during each 60-s scan using the H 2 O 15 water bolus method [ 71 ]. MRI scans (160 1-mm slices) were also obtained for each subject with a 1.5T Philips ACS system (Andover, Massachusetts, United States) to provide anatomical detail. CBF images were reconstructed using a 14-mm Hanning filter, normalized for differences in global CBF, and co-registered with the individual MRI data [ 72 ]. Each matched MRI/PET dataset was then linearly resampled into a standardized stereotaxic coordinate system based on the MNI305 target (a sample of 305 normal subjects) via an automated feature-matching algorithm [ 73 ], resulting in a normalized brain space similar to the Talairach and Tournoux atlas (for additional information, see: http://www.mrc-cbu.cam.ac.uk/Imaging/ ). Statistical analysis was performed applying the method described by Worsley et al. [ 74 ]; covariation analysis followed the procedure outlined by Paus et al. [ 33 ]. A t value of 3.5 was used for significant changes in CBF during exploratory searches. However, a t value of 3.0 was used for a priori regions of interest, such as occipital areas. Supporting Information Figure S1 Parietal Activation Foci in Binaural Sound Localization Task Sagittal and coronal images contrasting BSL to the control task. All three groups showed increased CBF in the right inferior parietal lobe (as shown by the red arrows), consistent with other neuroimaging studies of auditory localization. X and Y coordinates refer to standardized stereotaxic space. (2.2 MB TIF). Click here for additional data file. Table S1 Stereotaxic Coordinates and t Values of Activation and Deactivation Foci (35 KB DOC). Click here for additional data file.

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A murine model of ulcerative colitis: induced with sinusitis-derived superantigen and food allergen

Background The etiology of ulcerative colitis (UC) is to be understood. The basic pathological feature of UC is intestinal chronic inflammation. Superantigen, such as Staphylococcus enterotoxin B (SEB), is reported to compromise intestinal barrier function by increasing epithelial permeability and initiate inflammation in the intestinal mucosa. Inasmuch as anatomic position of the sinus, chronic sinusitis-derived SEB may follow the secretion and to be swallowed down to the gastrointestinal tract and induce lesions to the intestinal mucosa. Methods Sinus wash fluid (SWF, containing SEB) was collected from a group of patients with both chronic sinusitis (CS) and UC. A group of mice were sensitized to ovalbumin (OVA) in the presence of SWF. The sensitized mice were challenged with the specific antigen OVA. The inflammatory status of the colonic tissue was determined with histology, serology and electron microscopy. Using horseradish peroxidase (HRP) as a tracer, another group of mice was stimulated with SWF for 2 hours. The HRP activity was detected in the colonic tissue with enzymatic approaches and electron microscopy. Results Epithelial hyperpermeability in colonic epithelium was induced by stimulating with SWF. The HRP activity in the colonic mucosa was almost 11 times more in the SWF treated group (3.2 ± 0.6 μg/g tissue) than the control group (0.3 ± 0.1 μg/g tissue). Mice were sensitized using a mixture of SWF and OVA (serum OVA-specific IgE was detected with a highest titer as 1:64). Challenge with OVA induced extensive inflammation in the colonic mucosa by showing (1) marked degranulation in mast cells (MC, 46.3 ± 4.5%) and eosinophils (Eo, 55.7 ± 4.2%); (2) inflammatory cell infiltration (MC = 145.2 ± 11.4; Eo = 215.8 ± 12.5; mononuclear cell = 258.4 ± 15.3/mm 2 tissue); (3) increased MPO activity (12.9 ± 3.2 U/g tissue) and inflammatory scores (1.8 ± 0.3); (4) mucosal surface ulcers; (5) edema in the lamina propria; (6) bacterial translocation and abscess formation in the subepithelial region. Conclusion Introducing Sinusitis-derived SEB-containing SWF to the gastrointestinal tract compromised colonic mucosal barrier function increasing epithelial permeability to luminal macromolecular protein in mice. The SWF facilitated colonic mucosal sensitization to luminal antigen. Multiple challenging the sensitized colonic mucosa with specific antigen OVA induced inflammation, induced a condition similar to human ulcerative colitis.

Background Ulcerative colitis (UC) is a disease characterized by inflammation and ulcers in the mucosa of the large intestine with unknown etiology. The inflammation usually occurs in the rectum and lower part of the colon, but it may affect the entire colon. The inflammation speeds up colonic motility and causes diarrhea. Ulcers form in places where the inflammation has killed the cells lining the colon; the ulcers bleed and produce pus. Theories about what causes ulcerative colitis abound, but none have been proven. The most popular theory is that the body's immune system reacts to a virus or a bacterium causing ongoing inflammation in the intestinal mucosa; others include genetic predisposition, autoimmune disorders and impaired immune regulation [ 1 - 3 ]. over the past 15 years, more than 2000 patients with chronic sinusitis (CS) including some patients with both CS and UC visited our clinic and were treated with different remedies, including medical treatment and functional sinus endoscopic surgery (FESS). Apart from improvement of chronic sinusitis, those patients with both CS and UC showed great improvement of UC as well (data not shown) that couldn't be explained with the specific treatment alone. Therefore, we postulated that there might be an association between CS and UC in these patients. Microbial infection is the most common cause of CS. The microbial products, such as lipopolysaccharide (LPS), staphylococcus aureus enterotoxin B (SEB) [ 4 , 5 ] can be discharged into the nasal cavity through the natural ostia going backward to the pharynx, and then be swallowed, entering the gastrointestinal tract to affect mucosal physiological functions [ 6 , 7 ]. Staphylococcus enterotoxin B is an extracellular toxin produced by certain strains of Staphylococcus aureus ( S. aureus ) [ 8 ]. Many cases of food poisoning worldwide involve S. aureus enterotoxins [ 9 ]. In addition, enterotoxins can be found in cases of toxic shock syndrome [ 10 ] and have been implicated in the autoimmune disease rheumatoid arthritis [ 11 ]. SEB is synthesized as a precursor protein of 266 amino acids. This precursor is then activated during excretion by cleavage of the N-terminal portion of the molecule. The active enterotoxin B is a single 239 amino acid chain of molecular weight 28,000 daltons and isoelectric point of 8.6 [ 12 ]. SEB is a superantigen and possesses powerful immune regulatory capability that results in increased T cell activation and proliferation. SEB-treated Balb/c mice display a dose-dependent colonic inflammation [ 13 ]. SEB can also induce colonic epithelial barrier dysfunction [ 14 ] that may promote uptake of exogenous antigens, microbial products and other noxious substances into the intestinal tissue to contact immune cells and initiate inappropriate immune reactions. The nasal cavity and sinus are primary sites of colonization by S. aureus , and a quantity of SEB was detected in the nasal cavity of the patients with allergic rhinitis [ 15 , 16 ]. Based on clinical observations, we hypothesized that sinusitis-derived SEB plays a certain role in the pathogenesis of chronic inflammation in the intestinal mucosa via impairing the epithelial barrier function and inducing inappropriate immune reactions. In this study, we aimed to investigate if (1) sinusitis-derived SEB increases intestinal permeability in the mice; (2) the presence of sinusitis-derived SEB facilitates intestinal sensitization to luminal antigen; (3) oral challenge with specific antigen induces intestinal allergic inflammation in the sensitized animals. Accordingly, we developed a murine model of ulcerative colitis with oral allergen in the presence of SEB-containing SWF. The animal model showed intestinal inflammation associated with intestinal mucosal eosinophilia, mastocytosis, acute diarrhea, bacterial translocation and micro-ulcer formation on the surface of the colonic mucosa. Methods Sinus-wash fluids collected from the patients with CS and UC Sinus-wash fluids (SWF) were collected from 32 patients with both CS and UC (18 male and 14 female; aged from 26 to 58, with an average of 35.82). We diagnosed CS in the patients as an inflammation of the sinus mucosa with a persistent mucoid or mucopurulent nasal discharge for longer than 3 months that resisted antimicrobial therapy and antral irrigation. Diagnosis was made on the basis of clinical history, rhinoscopic findings, and computed tomographic scan of paranasal sinuses. CS was confirmed by computed tomographic examination that showed diffuse mucosal thickening in the ethmoid or/and maxillary sinuses bilaterally with scores higher than 12 by the Lund-Mackay staging system [ 17 ]. Twenty-five healthy medical students were enrolled in this study, their nasal wash fluids were used as controls. The diagnosis of UC was based on clinical history, colonoscopy and histology. The history included persistent bloody diarrhea, rectal urgency, or tenesmus. Examinations and sigmoidoscopy and biopsy were performed to confirm the presence of colitis and to exclude the presence of infectious etiologies. Every patient underwent maxillary sinus puncturing and washing. The SWF was collected prior to other procedures. Five ml saline was injected into the sinus cavities and re-collected and stored at -70°C for further use. SEB content in SWF was evaluated with ELISA (All the reagents used in this study were purchased from Sigma unless otherwise mentioned). None of the subjects had recent upper respiratory acute infections. This study was approved by the Ethical Committee of the First Hospital of Shanxi Medical University. Animals For the purpose of verifying our hypothesis of an association between sinus pathology and colitis, an animal model of ulcerative colitis was developed. Mice were sensitized to a model food antigen, ovalbumin (OVA) with or without the presence of SEB-containing SWF. Animal experiments were approved by Animal Use and Care Committee at Shanxi Medical University. Male Balb/c mice (10 week old) were purchased from Beijing Animal Research Institute and maintained in the animal center at Shanxi Medical University. All mice were housed according to guidelines of the animal center. Water was available continuously through automatic ports, and a commercial mouse diet was provided ad libitum. Effect of SEB containing SWF on colonic epithelial permeability in the mice A group of mice was introduced 0.2 ml SWF (containing SEB 50 μg and 10 mg horseradish peroxidase, HRP. SWF was concentrated with the method of ammonium sulfate precipitation for higher content of SEB) via intragastric gavage under a light anesthetization. Mice were killed by cervical dislocation 2 hours later (based on preliminary results; data not shown). Control groups were designed as: a naïve control group, treated with HRP 10 mg in 0.2 ml PBS via intragastric gavage; an inactivated-SWF control group, the SWF was pretreated with anti-SEB (100 μg in 0.2 ml) for 30 min, then introduced to the mice in gavage with HRP. Colon was removed; a piece of colon (3 cm) was opened; the contents were collected and dissolved in 1 ml PBS for HRP assay; the colon tissue was snap frozen and stored at -70°C for HRP assay; another piece of colon (2 × 2 × 4 mm) was fixed with 2.5% glutaraldehyde for 2 hours; then rinsed in sodium cacodylate buffer, incubated in 3,3'-diaminobenzidine tetrahydrochlorine and H 2 O 2 (pH 7.6) for 30 min, and postfixed with 2% osmium tetroxide for 60 minutes, followed by staining en bloc with 4% uranyl acetate for 30 minutes. Tissue samples were dehydrated through a graded series of ethanol, cleared in propylene oxide, and embedded in Epoxy embedding medium. Thin sections were prepared and stained with 4% uranyl acetate for 5 min and subsequently with 2.5% lead citrate for 2 min and examined at 80 kV with a JEM 1200 electron microscope. HRP containing endosomes were photographed randomly for further analysis. Frozen colon tissue was weighed and immersed into lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 (sodium ortho-vanadate), 1 μg/ml leupeptin) and homogenized on ice; the homogenates and colon content were centrifuged at 1,500 g for 20 minutes at 4°C respectively, the supernatants were collected for HRP assay. HRP amount in colon content and colon tissue was determined by assaying enzyme activity in the collected supernatant according to the previous report [ 18 ]. Briefly, a 0.2-ml sample of supernatant was mixed with 2.8 ml phosphate buffer (0.1 M, pH 6.0) containing 0.003% H 2 O 2 and 0.025 ml of a solution of o-dianisidine di-HCl (10 mg/ml). The optical density was determined at 460 nm with a spectrophotometer. Intestinal sensitization to luminal OVA with the presence of SEB-containing SWF Ten mice were used for each group. A group of mice was sensitized by means of intragastric gavage with 50 μg OVA in 0.2 ml alum-SWF (the adjuvant, prepared with SWF instead of distilled water that contains 50 μg SEB). Another group was also treated with the same protocol, but the SWF in the adjuvant was pretreated with an anti-SEB antibody (100 μg in 0.2 ml). Control groups were designed as: mice were only exposed to OVA without the presence of SWF, or were only exposed to SWF without OVA; naïve controls were treated with 0.2 ml saline. Each mouse was injected with 50 ng pertussis toxin peritoneally. Mice were challenged with specific antigen OVA From the 14th day after sensitization, all mice were challenged with 50 mg OVA in 0.3 ml saline by means of intragastric gavage 3 times, 48 h apart. Diarrhea was determined by visually monitoring mice for up to 1 hour following intragastric challenge. Mice demonstrating profuse liquid stool were recorded as diarrhea-positive animals. Two days after the last challenge, mice were sacrificed by decapitation; blood samples were collected, serum was separated and stored at -70°C for further analysis. Colon was removed, one piece was fixed with 4% paraformaldehyde for histology; one piece was snap frozen for MPO analysis; one piece was fixed with Carnoy solution for mast cell count; one piece was fixed with 2% glutaraldehyde for electron microscopy. Myeloperoxidase activity evaluation Myeloperoxidase (MPO) activity was determined according to the method of Bradley et al [ 19 ]. Tissue samples were homogenized in hexadecyltrimethylammonium bromide buffer in a glass homogenizer on ice. The homogenates were centrifuged and MPO activity in the supernatants was determined. One unit of MPO activity was defined as the amount required to degrade 1 mM H 2 O 2 in 1 minute at 25°C. Colonic mucosal inflammatory score The morphology of the epithelium, villi, and subepithelial layer of colon mucosa were assessed, and the numbers of eosinophils and mononuclear cells were counted in 10 randomly selected fields (magnification, ×400) for each mouse (100/group). To determine mast cell numbers, tissues were fixed in Carnoy's fixative and paraffin sections were stained with 0.5% toluidine blue. Mast cells were counted in 10 fields for each mouse (100/group). Cell numbers were expressed per mm 2 of mucosa. All sections were coded to avoid observer bias. The degree of inflammation on microscopic tissue sections was scored as follows: (from 0 to 4, 0 indicates normal, 4 indicates severe condition): 0, no leukocyte infiltration; 1, low level of leukocyte infiltration; 2, moderate level of leukocyte infiltration; 3, high vascular density and thickening of the colon wall; and 4, transmural leukocyte infiltration, loss of goblet cells, high vascular density, and thickening of the colon wall. Grading was done in a blinded manner [ 20 ]. Observation of mast cell and eosinophil activation Colon tissue was processed with routine procedures and observed with an electron microscope. Activation of mast cell and eosinophil in colonic mucosa was determined by the phenomenon of degranulation [ 21 ]. Serum specific IgE measurement Passive cutaneous anaphylaxis (PCA) was employed to determine serum OVA-specific IgE level. The serum was diluted in phosphate-buffered saline (PBS) from 1:4 to 1:128. The mice were injected intradermally with 50 μl serum into each of 4 dorsal shaved skin sites. The sites were outlined with a water-insoluble red marker. Forty-eight hours later each mouse received an injection of 0.1 ml OVA (1 mg/ml) containing 4% Evans blue via the tail vein. The results were recorded thirty minutes after the challenge; the diameter of the blue spot on the injection sites larger than 6 mm was recorded as positive reaction. Statistics Data were expressed as mean ± SD; Student t test was used to compare the difference between groups. p < 0.05 was accepted as significant criteria. Results Staphylococcus enterotoxin B was detected in SWF from the patients with both CS and UC The content of SEB was significantly higher in the SWF of patients with both UC and CS (from 30.5 to 565.6, with an average of 154.5 ± 81.7 pg/ml) than in the nasal wash fluids (from 0 to 18.6, with an average of 8.5 ± 4.3 pg/ml) of healthy control subjects. Stimulation with SWF increased mouse colonic mucosal permeability to luminal macromolecular protein HRP Horseradish peroxisase activity was detected in colonic content in both control mice (group of HRP-only and group of anti-SEB treated SWF) and SWF treated mice (Fig 1A ). The HRP activity was significantly higher in the colonic tissue of the mice treated with SWF compared to controls (Fig 1B ). Observation with EM revealed that few HRP endosomes of small size were observed in the upper region (above the nucleus) of cytoplasm in the colonic epithelium of control mice. Large size HRP endosomes were observed in both upper and lower (below the upper edge of the nucleus) regions of the mice treated SWF. Image analysis of HRP endosome showed that the HRP endosome area was significantly larger in the mice treated with SWF comparing with those control mice (Fig 2 ). Figure 1 Mouse colonic epithelial permeability increased by stimulation of SWF . Bars stand for HRP activity in colonic content (Fig 1A) and colonic tissue (Fig 1B). Each group consists of 10 mice. *, p < 0.05, compared with controls. Figure 2 HRP endosomes in the colonic mucosa . Representative EM photomicrographs were taken from colonic mucosa of control (Fig 2A) and sensitized mice (Fig 2B). Bars stand for HRP endosome area in 300 μm 2 cell area (Fig 2C). *, p < 0.05, compared with controls. Serum specific anti-OVA IgE was detected in the mice treated with SWF and luminal OVA Passive cutaneous anaphylaxis (PCA) is the gold standard method to measure allergen-specific IgE antibody levels in mouse models of allergy. The present study demonstrated that only the group treated with both SWF and OVA showed positive results with the highest titer 1:64. No specific IgE was detected in the serum of mice in control groups. Activation of mast cells and eosinophils in the colonic mucosa of the sensitized mice after challenging with specific antigen Activation of mast cells and eosinophils was observed as degranulation of the granules in the cytoplasm. Piecemeal type degranulation was observed more often than the anaphylactic type in the mast cells in the colonic mucosa of the sensitized mice with EM. Crystal core degranulation and matrix degranulation were observed in eosinophils of the sensitized mice. Granules were categorized intact or degranulated for each mast cell or eosinophil. Each type granule was numerated with EM. After challenge with specific antigen OVA, degranulation was observed in both mast cells and eosinophils of the sensitized mice. The ratio of degranulation was much higher in the sensitized mice compared with controls (Fig 3 ). Figure 3 Activation of mast cell and eosinophil in the colonic mucosa . Representative photomicrographs (10 for each mouse) were taken from colonic mucosa of naïve mice (Fig 3A, 3C) and sensitized mice (Fig 3B, 3D). Bars stand for ratio of degranulation that was calculated with the numbers of degranulated granules divided by the numbers of total granules of mast cells (Fig 3E) and eosinophils (Fig 3F). *, p < 0.05, compared with naïve controls. Histopathology of colonic mucosa of the sensitized mice after challenge with specific antigen Challenge with specific antigen OVA resulted in inflammatory cell infiltration in the lamina propria and subepithelial regions. The infiltrate consisted of mainly mast cells, eosinophils and mononuclear cells. Compared with control, the sensitized group showed significantly more inflammatory cell infiltration, most of them might be activated immune cells (Fig 4 ). Capillary or small vein dilation was observed in the subepithelial region of the intestinal mucosa after OVA challenge; profound edema in the tissue of the same area was frequently observed (Fig 6B , 6F ). Figure 4 Inflammatory cell infiltration in the colonic mucosa. Bars stand for numbers of mast cell (Fig 4A), eosinophil (Fig 4B) and mononuclear cell (Fig 4C). Cell numbers are expressed as cells/mm 2 tissue. *, p < 0.05, compared with naïve controls. Figure 6 Ultrapathology of the colonic mucosa of the sensitized mice after challenge with OVA. Representative EM photomicrographs are taken from the colonic mucosa of the sensitized mice after challenge with OVA and show (A) epithelial cell (epi) necrosis (EN) and dilation of the blood vessel (BV) in the subepithelial region (×3,000); (B) epithelium destruction, basement membrane (BM) hyperplasia and blood vessel dilation (×2,000); (C) bacteria (arrows) adhering to and penetrating the epithelial cells (×3,000); (D) abscess (Ab) formation in subepithelial region with a colony of bacteria (arrows) and a red blood cell (RBC) in it (×2,000); (E) micro-ulcer (empty arrow) formation on the surface of colonic mucosa with bacteria (arrow) adherence (×2,500); (F) edema and blood vessel (BV) dilation in the lamina propria (LP) (×2,500). Inflammatory status in the colonic mucosa was assessed by colonic tissue MPO measurement, inflammatory cell infiltration and mucosal surface condition observation. MPO is a critical parameter that indicates activation of neutrophils. We observed a significant increase in MPO and inflammatory score in colonic mucosa after challenge with OVA in the mice treated with SWF and OVA (Fig 5 ). Figure 5 MPO activity and inflammatory scores of the colonic mucosa. Colonic tissues show an increased MPO activity (A) and increased inflammatory scores (B) after challenge with OVA. *, p < 0.05, compared with naïve controls. Electron microscopy revealed ultrapathology in the sensitized and OVA-challenged colonic mucosa that included: (i) epithelial cell destruction (Fig 6A, B ); (ii) bacteria adherent to or penetrating the epithelial cells; (iii) bacteria translocated to the subepithelial region and abscess formation (Fig 6C, D ); (iv) micro-ulcer formation on the surface of the colonic mucosa (Fig 6E ); (v) edema in lamina propria (Fig 6F ). Challenge with specific antigen OVA induced diarrhea in the sensitized mice Following the protocol of challenge with specific antigen OVA, the sensitized mice developed diarrhea 15–30 minutes after each challenge that lasted up to 1 hour. The number of diarrhea episodes (3 to 7 times) increased in parallel with the number of OVA challenges. No diarrhea was noted in control mice although those mice also received the same number of OVA challenges. Diarrhea was also noted by direct observation of the colon and cecum; the liquid stool observed following OVA challenge-induced diarrhea in the sensitized mice contrasts with the solid pellets seen in the distal colon of other mice in control groups. Discussion Our knowledge about the etiology of ulcerative colitis is still limited. Although some theories about its origins have been advanced, such as genetic predisposition, autoimmune disorders, infection, and so on [ 23 , 24 ], the precise pathogenesis needs to be further understood. In clinical practice, we noted a close association between CS and UC in some patients and their UC was significantly improved after having removed sinus pathology (data not shown). The results of animal experiments verified our speculation: superantigen SEB from sinusitis cooperated with ingested antigen to induce intestinal sensitization. Challenge with the obligate antigen initiated colonic mucosal inflammation as well as the clinical symptom diarrhea. Book DT et al [ 25 ] also noted the same phenomenon and suggested that IBD was more prevalent in those people with chronic sinusitis than in other populations. Rhinosinuses are empty cavities lined with mucosa. The anatomic feature, only having a small ostium, makes them very easily to be blocked and subsequently infected. Infection with S. aureus in sinuses is frequently encountered [ 4 , 5 ]. Thus, chronically infected sinuses may be a source of SEB that is released to nasal cavity frequently. A mucus blanket on the surface of nasal mucosa naturally traps small particles from air and the secretions from sinuses and removes them subsequently. Since the direction of the locomotion of the mucus blanket is backward, people sometimes swallow the secretions into the gastrointestinal tract (e.g., during sleep). There are many toxic substances in the secretions from chronic sinusitis. SEB is one that has been well characterized. The unique feature of SEB is that it can down regulate intestinal barrier function [ 6 , 13 ], activate T lymphocytes without the help from antigen presenting cells to activate T cells. Superantigens bind directly to MHC class II molecules and to a subset of T-cell receptor (TCR) Vβ chains [ 26 , 27 ]. Unlike conventional antigens, superantigens do not require processing by antigen-presenting cells to activate immune cells [ 31 ]. Administration of superantigen results in initial selective expansion of T cells that bear specific Vβ chains that recognize the superantigen, followed by their deletion [ 29 ]. Another unique feature of superantigen is that it mutes T suppression cell function and promotes Th1/Th2 skewing [ 30 ]. It primes an environment to develop sensitization in local tissue. The results in the present animal experiments are consistent with previous studies. Mice treated with SEB-containing SWF and OVA developed intestinal sensitization, but not in those mice treated with only OVA, or SEB-depleted SWF plus OVA. This finding demonstrates that SEB plays a crucial role in the sensitization of the intestinal mucosa to luminal antigen in these mice. Louini D et al [ 31 ] reported that SEB also directly sensitized skin and caused Th2 pattern inflammation in the local skin. Intestinal epithelial cells form a barrier between the luminal contents and the subepithelial region. The barrier restricts substances to be absorbed. It only allows some small molecules such as water to pass it freely. Antigens are macromolecular proteins that are not allowed to be absorbed before being digested to small peptides or amino acids under normal physiological conditions. But in reality, intact antigens do pass the intestinal barrier to reach lamina propria to induce inappropriate immune reactions under certain circumstance. How antigens cross the intestinal epithelial barrier is still a mystery. By introducing both SWF and HRP to mouse gastrointestinal tract, intact HRP in the colonic tissue was increased nearly 11 times compared to control. The results implicate that superantigen SEB is one of the factors that facilitate antigens to be absorbed without destroying their antigenicity. Lu et al also reported that SEB significantly increased colonic mucosal permeability in a mouse study [ 13 ]. We have begun to appreciate that food allergy plays a role in the inflammation of intestinal mucosa [ 32 ]. The results of this animal model support that inappropriate immune reactions initiate intestinal inflammation. Simply delivering OVA to gastrointestinal tract did not show sensitization in the mice while the combination of SEB-containing SWF and OVA induced sensitization in the colonic mucosa. Based on these data, we suggest that SEB facilitate sensitization of the intestinal mucosa to OVA. The mechanism behind this phenomenon might be that SEB increases permeability of the intestinal mucosa [ 6 , 13 ]. Thus OVA in the intestinal lumen can be transported to deep region of the mucosa. This exogenous protein then contacts the local immune cells to initiate inappropriate immune reactions and sensitizes the mucosa subsequently. The results of challenge with OVA show extensive inflammation in the colonic mucosa in this study. The inflammatory changes may be a result of local mast cell degranulation in response to OVA challenge. Mast cells release chemical mediators such as histamine that is able to increase vascular permeability and to induce edema in the tissue that was noted in the present study and also reported elsewhere [ 33 ]. In chronic allergic diseases such as asthma, during continuous antigen exposure, eosinophils are primed by IL-5 and attracted by chemokines, infiltrating the local tissue [ 34 ]. We also observed extensive eosinophil infiltration in the colonic tissue after three challenges with specific antigen OVA in the present study. These eosinophils are believed to be responsible for the late phase of the allergic reaction, producing the major basic protein which is toxic to the epithelium [ 34 ]. The micro-ulcers on the surface of colonic mucosa may be caused by eosinophil activation. Supportive evidence acquired from EM observation demonstrates that most eosinophils have been activated by showing extensive degranulation. We also noted this phenomenon in the jejunal mucosa in the late phase reaction in a rat model of food allergy [ 32 ]. Marked mast cell hyperplasia in the colonic tissue was observed in this study. In general, mast cell numbers in tissues are relatively constant, even though mast cell hyperplasia is observed in both the inflammatory and in repair/remodeling stage of various inflammatory disorders [ 35 ]. The functional significance of the accumulation of mast cells in these processes is largely unknown. In allergy, apart from their classical role in eliciting the early phase, mast cells also have an important role in late and chronic stages as we observed in a previous study [ 32 ]. In these stages they may interact with and be activated by infiltrated inflammatory cells and by resident structural cells such as epithelial cells, smooth muscle cells and fibroblasts. In the case of allergic reaction, mast cells are mainly activated by the mechanism of IgE mediated FcεRI bridging that accounts for the mast cell activation in the present study. The roles of mast cells in the late phase reactions may be amplified by eosinophils, platelets and neutrophils [ 36 ]. If a sensitized patient frequently ingests an obligate antigen unconsciously whereas the allergic reaction only reaches subclinical level, an early inflammation may progress without being noticed until reaching the advanced stage. The data also show that the degranulation type of mast cells and eosinophils in this study is mainly piecemeal. It indicates that the nature of the degranulation type belongs to a chronic process [ 37 ]. The local inflammation may progress to chronic status in the tissue without any further medical intervention. Striking epithelial damage of colonic mucosa of the sensitized mice after challenge with OVA was observed in this study. This phenomenon has been well-documented in airway allergy [ 38 ]. Although ulcers are one of the main clinical signs in inflammatory bowel diseases, the etiology is not clear. There have not been many studies considering an association between the ulcers and allergic reactions in the gastrointestinal tract. Eosinophil released major basic protein is suggested to be the major offender to cause epithelial cell exfoliation in the airway mucosa [ 38 ]. The erosion and prominent damage to the epithelial barrier can explain the phenomenon of bacterial adhering to and penetrating to colonic mucosa in this study. These bacteria are commensal bacteria. They usually are not considered pathogenic. The damaged epithelium may provide an entry port for the colonizing bacteria to invade the colonic mucosa and to establish an infection. Conclusion In summary, we reported a murine model of ulcerative colitis in this paper that was induced with sinusitis-derived SEB and OVA sensitization followed by repeat challenges with specific antigen OVA. The histopathology of the colonic mucosa included inflammatory cell infiltration, activation of mast cell/eosinophil, epithelial barrier damage, micro-ulcer formation on the surface of colonic mucosa, bacteria translocation and abscesses formation in the subepithelial region. List of abbreviations UC, ulcerative colitis; CS, chronic sinusitis; FESS: functional endoscopic sinus surgery; OVA, ovalbumin; SEB, Staphylococcus enterotoxin B; MPO, myeloperoxidase; HRP, horseradish peroxidase. Competing interests The author(s) declare that they have no competing interests. Authors' contributions PCY was involved in study design, histology, EM observation, data analysis and manuscript preparation; CSW and AZY were involved in SWF collection and animal model. Pre-publication history The pre-publication history for this paper can be accessed here:

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526754

Review on ''Bioinformatics, Biocomputing and Perl'' by Michael Moorhouse and Paul Barry

The book " Bioinformatics , Biocomputing and Perl " [ 1 ] attempts to encompass those numerous volumes which most bioinformaticians keep on their office bookshelves and which are often entitled as "S omething in a Nutshell ". The book aims at both biology- and computation-oriented audiences and is designed as a number of 'crash-courses' quickly updating the reader on the basics of bioinformatics. It starts with a preface outlining main biological and technological concepts of the modern computational biology. The rest is organized into four sections consisting of 18 chapters elaborating on essential bioinformatics tools and skills. The section 'Working with Perl' presents an extended tutorial with practical tips and useful references for Perl beginners. Following this is 'Working with Data', which familiarizes the reader with some public genomic and proteomic databases and discusses important subjects of database formats, non-redundancy, cross-referencing and programmable access, etc . By working through the section, the reader acquires basic skills for mySQL database use and DBI Perl programming. Next, the authors offer Perl-based solutions for remote database access and for creation of WWW-based bioinformatics services using Perl functionalities in 'Working with the Web'. The final topic of the book, 'Working with Applications', features basic tools for sequence alignment, protein homology modeling and data visualization, all commonly used in bioinformatics practice. The section also offers recent and relevant examples of BioPerl applications. In general, the book reflects the state of bioinformatics field with its strengths and weaknesses. Many Perl chapters, such as Perl regular expressions, modular organization, DBI-programming, BioPerl and web-automation, are excellent. The presented material is rather comprehensive and yet easy to read – the authors spent appreciative efforts to make the book interesting and enjoyable. The authors also acknowledge the open-source nature of Perl and the bioinformatics community and offer on-line support and direct feedback to the readers. There are also certain aspects, in which the book could be further improved. Several sections may be too advanced for the beginner level (such as Perl basics and database downloading), while others may contain too excessive details (the Protein Databank section). In addition, it may be of advantage to mention AcePerl [ 2 ], Perl-programmable access to the SRS as well as XML- [ 3 ] and distributed data processing by Perl. The book would greatly benefit from color illustrations. Several figures in the 'biological' sections are not very informative or readable (such as Figure 10.5), and one contains a critical error (Figure 1.1). A very useful feature of the book is the use of maxims that highlight key points throughout the text. The authors also provide helpful technical comments where necessary and offer practical exercises at the end of each chapter. The book is concluded with six appendices covering the Linux basics, Perl installation, operators, on-line support and suggested reading materials which, in my mind, benefit the book tremendously. Thus, the overall product, the " Bioinformatics , Biocomputing and Perl ", serves well its purpose as an introductory textbook and a resource of reference materials for bioinformaticians. List of Abbreviations used AcePerl – is a Perl interface for the AceDB – a popular object-oriented bioinformatics database. DBI Perl – the primary interface for database programming by Perl. BioPerl – a collection of Perl modules specifically designed for several most common bioinformatics tasks. XML – Extensible Markup Language – a popular standard for documents containing structured information. SRS – the Sequence Retrieval System – a popular relational database for bioinformatics.

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529249

Management of multifactorial idiopathic epilepsy in EL mice with caloric restriction and the ketogenic diet: role of glucose and ketone bodies

Background The high fat, low carbohydrate ketogenic diet (KD) was developed as an alternative to fasting for seizure management. While the mechanisms by which fasting and the KD inhibit seizures remain speculative, alterations in brain energy metabolism are likely involved. We previously showed that caloric restriction (CR) inhibits seizure susceptibility by reducing blood glucose in the epileptic EL mouse, a natural model for human multifactorial idiopathic epilepsy. In this study, we compared the antiepileptic and anticonvulsant efficacy of the KD with that of CR in adult EL mice with active epilepsy. EL mice that experienced at least 15 recurrent complex partial seizures were fed either a standard diet unrestricted (SD-UR) or restricted (SD-R), and either a KD unrestricted (KD-UR) or restricted (KD-R). All mice were fasted for 14 hrs prior to diet initiation. A new experimental design was used where each mouse in the diet-restricted groups served as its own control to achieve a 20–23% body weight reduction. Seizure susceptibility, body weights, and the levels of plasma glucose and β-hydroxybutyrate were measured once/week over a nine-week treatment period. Results Body weights and blood glucose levels remained high over the testing period in the SD-UR and the KD-UR groups, but were significantly (p < 0.001) reduced in the SD-R and KD-R groups. Plasma β-hydroxybutyrate levels were significantly (p < 0.001) increased in the SD-R and KD-R groups compared to their respective UR groups. Seizure susceptibility remained high in both UR-fed groups throughout the study, but was significantly reduced after three weeks in both R-fed groups. Conclusions The results indicate that seizure susceptibility in EL mice is dependent on plasma glucose levels and that seizure control is more associated with the amount than with the origin of dietary calories. Also, CR underlies the antiepileptic and anticonvulsant action of the KD in EL mice. A transition from glucose to ketone bodies for energy is predicted to manage EL epileptic seizures through multiple integrated changes of inhibitory and excitatory neural systems.

Background Epilepsy is a neurological disorder involving recurrent abnormal discharges of neurons that produce epileptic seizures [ 1 ]. With the exception of stroke, epilepsy is one of the most prevalent human neurological afflictions affecting about 1% of the US population [ 2 , 3 ]. Many persons with epilepsy manifest partial or generalized seizures without symptoms of brain abnormality, i.e., idiopathic epilepsy [ 1 , 4 , 5 ]. In contrast to idiopathic epilepsy, symptomatic or acquired epilepsy often accompanies brain trauma, injury, or neurostructural defects. While some idiopathic epilepsies are inherited as simple Mendelian traits, most are multifactorial where more than one gene together with environmental factors contribute to the disease phenotype [ 6 , 7 ]. Epilepsy animal models are used widely to test the influence of environmental and genetic factors on seizure mechanisms. The epileptic EL mouse is a natural model for human multifactorial idiopathic epilepsy and was first discovered in 1954 in an outbred DDY mouse colony [ 6 , 8 - 10 ]. EL mice experience complex partial seizures with secondary generalization similar to those seen in humans [ 6 , 10 ]. Seizures in EL mice commence with the onset of puberty (50–60 days), originate in or near the parietal lobe, and then spread to the hippocampus and to other brain regions [ 6 , 11 - 13 ]. The seizures are accompanied by electroencephalographic abnormalities, vocalization, incontinence, loss of postural equilibrium, excessive salivation, and head, limb, and chewing automatisms [ 10 , 12 , 14 - 17 ]. A reactive gliosis accompanies seizure progression in adult EL mice involving both astrocytes and microglia [ 18 , 19 ]. Epileptic seizures in EL mice also model Gowers' dictum, where each seizure increases the likelihood of recurrent seizures [ 6 ]. Seizure susceptibility can be managed with phenytoin and phenobarbital as well as with diet therapies to include the ketogenic diet and caloric restriction [ 20 - 22 ]. Gene-environmental interactions play a significant role in the determination of seizure frequency and onset in EL mice as with multifactorial human idiopathic epilepsies [ 6 , 9 , 23 ]. Despite intensive antiepileptic drug (AED) research and development, seizures remain unmanageable or refractory in many persons with epilepsy [ 24 - 26 ]. As an alternative to AEDs diet therapies can be effective in the management or control of epilepsy. Fasting has long been recognized as an effective antiepileptic therapy for a broad range of seizure disorders [ 27 - 29 ]. Since fasting produces ketonemia, it was originally thought that ketone bodies (β-hydroxybutyrate and acetoacetate) might underlie the antiepileptic effects of fasting [ 27 , 30 ]. Consequently, high fat, low protein, low carbohydrate KDs were developed to mimic the physiological effects of fasting [ 25 , 27 , 31 , 32 ]. Although the KD significantly elevates circulating ketone body levels, later studies showed that ketone bodies alone were unable to account for the antiepileptic and anticonvulsant effects of the KD in humans or in animal epilepsy models [ 20 , 31 , 33 - 38 ]. Since the KD manages epilepsy best when administered in restricted amounts and since fasting lowers blood glucose levels, Seyfried and co-workers suggested that caloric restriction might contribute to the antiepileptic and anticonvulsant effects of the KD [ 21 , 29 , 38 ]. CR is a natural dietary therapy that improves health, extends longevity, and reduces the effects of neuroinflammatory diseases in rodents and humans [ 21 , 29 , 39 , 40 ]. CR is produced from a total dietary restriction and differs from acute fasting or starvation in that CR reduces total caloric energy intake without causing anorexia or deficiencies of any specific nutrients [ 38 ]. In other words, CR extends the health benefits of fasting while avoiding starvation. Besides improving health, CR has both antiepileptic and anticonvulsant effects in EL mice and in other animal epilepsy models [ 20 , 21 , 41 ]. A reduction in blood glucose with a corresponding elevation in blood ketone bodies is thought to underlie the antiepileptic and anticonvulsant effects of CR [ 21 , 29 , 38 ]. Glucose uptake and metabolism increases more during epileptic seizures than during most other brain activities [ 42 - 44 ]. Blood glucose also positively correlates with flurothyl-induced seizures in rats and high glucose may exacerbate human seizure disorders [ 45 ]. Neuronal excitability and epileptic seizures are directly related to rapid glucose utilization and glycolysis [ 42 , 43 , 45 - 51 ]. It is not yet clear, however, to what extent enhanced glycolysis is related to the cause or effects of seizure activity [ 29 ]. Nevertheless, a transition in brain energy metabolism from glucose utilization to ketone body utilization reduces neural excitation and increases neural inhibition through multiple integrated systems [ 29 , 38 ]. Based on these and other observations [ 50 , 52 - 54 ], we proposed that many epilepsies, regardless of etiology, might ultimately involve altered brain energy homeostasis [ 29 ]. In this study, we compared the antiepileptic and anticonvulsant effects of both the KD and CR in adult EL mice that experienced at least 15 recurrent complex partial seizures. The results show that seizure control in EL mice is more associated with the amount than with the origin of dietary calories, and that CR underlies the antiepileptic and anticonvulsant action of the KD in EL mice. A preliminary report of these findings was recently presented [ 55 ]. Results Diet composition and tolerance The composition of each diet is shown in Table 1 and in the Methods. No adverse effects of the diets were observed in either R-fed mouse group. Despite the 20–23% body weight reduction, mice in both R-fed groups appeared healthy and were more active than the mice in the UR-fed groups as assessed by ambulatory and grooming behavior. With the exception of oily fur, the KD-fed mice appeared active and healthy throughout the study as previously found [ 20 ]. No signs of vitamin or mineral deficiency were observed in the R-fed mice according to standard criteria for mice [ 56 ]. These findings are consistent with the well-recognized health benefits of mild to moderate caloric restriction in rodents [ 57 ], and support our previous findings that both the KD and a moderate CR are well tolerated by EL mice [ 20 , 21 ]. Table 1 Composition (%) of the Standard Diet and the Ketogenic Diet 1 Components Standard Diet (SD) Ketogenic Diet (KD) Carbohydrate 62 0 Fat 6 75 Protein 27 14 Fiber 5 12 Energy (Kcal/gr) 4.4 7.8 1 According to manufacturer's specifications (see Methods). Influence of caloric restriction on body weight All mice were matched for age (approximately 210 days) and body weight (approximately 31.0 ± 1.5 g) before the start of the dietary treatment (Fig. 1 ). All mice lost approximately 7–9% of their body weight during the 14 hr fast. Body weight remained relatively stable over the nine-week treatment period in both UR-fed mouse groups (Fig. 1 ). The 20–23% body weight reduction was achieved in the R-fed groups after about two weeks of gradual food restriction. However, more difficulty was encountered initially in maintaining a stable body weight reduction for the KD-R group than for the SD-R group. This difficulty may result from the high caloric content of the KD that produces greater body weight changes per calorie adjustment than the SD. We also estimated that the degree of CR necessary to maintain the 20–23% body weight reduction was about 38–45% for the SD and about 45–52% for the KD. Figure 1 Influence of diet on body weight in adult EL mice fed the SD (A) or the KD (B). Squares represent the pre-trial period when all mice were fed the SD-UR. Circles and triangles represent the UR-fed and R-fed groups, respectively. Values are expressed as the mean ± SEM (n = 6 mice per group). Arrow indicates initiation of CR. Influence of diets on seizure susceptibility in adult EL mice All mice had at least 15 recurrent seizures before the start of dietary treatment (arrow, Fig. 1 ). The seizures occurred occasionally during routine cage changing prior to the pre-trial period and regularly from handling during the pre-trial test period. Seizure susceptibility was analyzed in all mouse groups after the R-fed mice achieved a stable body weight reduction, i.e., week five of treatment (Figs. 1 and 2 ). Seizure susceptibility was high for both UR-fed groups throughout the study. In both R-fed groups, seizure susceptibility decreased from 1.0 to about 0.3 after two weeks and remained significantly lower than that of the UR-fed control groups from treatment weeks 5–12 (Fig. 2 ). Only a single mouse in the KD-R group had a break-through seizure on week 8. Taken together, our findings show that seizure management in EL mice is more associated with the amount than with the origin of dietary calories. Figure 2 Influence of diet on seizure susceptibility in adult EL mice. Asterisks indicate that seizure susceptibility was significantly lower (p < 0.001) in the R-fed groups than in their respective UR-fed groups. Values were pooled from treatment weeks 5–12 (see Fig. 1) and are expressed as the mean ± SEM (n = 6 mice per group). Influence of diets on plasma glucose and β-hydroxybutyrate levels Plasma glucose levels were analyzed in all mouse groups after the R-fed mice achieved a stable body weight reduction (Figs. 1 and 3 ). Glucose levels remained high for both UR-fed groups throughout the study and were stable over treatment weeks 5–12. However, plasma glucose levels were somewhat lower (about 8 mM) in both UR-fed groups between treatment weeks 3–5 compared to the pre-trial glucose levels (about 10 mM). This reduction might result from a combination of repetitive handling, seizures, blood collection, and the initial fast. In both R-fed mouse groups, the plasma glucose levels decreased from about 10 mM to about 5.0 mM after three weeks and remained significantly lower than those of their respective UR-fed control groups (Fig. 3 ). Figure 3 Influence of diet on plasma glucose levels in adult EL mice. Asterisks indicate that the plasma glucose levels were significantly lower (p < 0.001) in the R-fed groups than in their respective UR-fed groups. Other conditions are as in Figures 1 and 2. Plasma β-hydroxybutyrate levels were also analyzed in all mouse groups after the R-fed mice achieved a stable body weight reduction (Figs. 1 and 4 ). These levels remained low in the SD-UR group throughout the study and were stable for treatment weeks 5–12. β-hydroxybutyrate levels were significantly higher in the R-fed groups than in their respective UR-fed control groups (Fig. 4 ). These levels were also significantly higher in the KD-UR group than in the SD-UR group. The levels increased from about 0.4 mM to about 1.7 mM in the SD-R group and to about 3.0 mM in the KD-R group. These findings demonstrate that circulating β-hydroxybutyrate levels were inversely related to circulating glucose levels and that elevated β-hydroxybutyrate levels alone are not associated with seizure susceptibility. Figure 4 Influence of diet on plasma β-hydroxybutyrate levels in adult EL mice. Asterisks indicate that the plasma β-hydroxybutyrate levels were significantly higher (p < 0.001) in the R-fed groups than in their respective UR-fed groups. The cross indicates that the plasma β-hydroxybutyrate levels were significantly higher (p < 0.001) in the KD-UR group than in the SD-UR group. Other conditions are as in Figures 1 and 2. Statistical relationships among variables The relationship between body weight, food intake, plasma glucose levels, plasma β-hydroxybutyrate levels, and seizure susceptibility was determined using Pearson bivariate correlation analysis (Table 2 ). All variables were significantly (p < 0.01) correlated with each other. Positive correlations were found among body weight, food intake, glucose, and seizure susceptibility. On the other hand, β-hydroxybutyrate was negatively correlated with all variables. The correlations among glucose, β-hydroxybutyrate, and seizure susceptibility were also apparent from the data in Figures 2 , 3 , 4 . Plasma glucose was significantly (p < 0.001) associated with seizure susceptibility in the EL mouse, as determined by chi-square analysis (Fig. 5 ). These results support our previous findings that glucose levels are predictive of seizure susceptibility in adult EL mice [ 21 , 29 ]. Table 2 Pearson bivariate correlation of body weight, food intake, plasma glucose levels, plasma b-hydroxybutyrate levels, and seizure susceptibility in adult EL mice 1 Parameter Body weight (g) Food Intake (Kcal) Glucose (mM) Ketones (mM) Seizure Susceptibility Body weight (g) 1.000 Food Intake (Kcal) 0.488* 1.000 Glucose (mM) 0.509* 0.382* 1.000 Ketones (mM) -0.379* -0.379* -0.429* 1.000 Seizure Susceptibility 0.512* 0.464* 0.616* -0.510* 1.000 1 Data were obtained from all four dietary groups over the treatment weeks 3–12 for a total number of 210 seizure and glucose measurements (see figure 1). * All correlations were significant at the 0.01 level (2-tailed). Figure 5 Association of plasma glucose and seizure susceptibility in adult EL mice. Data were obtained from all four dietary groups over treatment weeks 3–12 for a total of 234 seizure and glucose measurements. Seizure frequency in the three plasma glucose groups (< 6.5 mmol, 6.5–8.5 mmol, and > 8.5 mmol/L) was 8/234, 44/234, and 70/234, respectively. The association between glucose and seizure susceptibility was highly significant as determined by Chi-square analysis (p < 0.001). Binary logistic regression was also used to determine the relationship between seizure susceptibility, plasma glucose, and plasma β-hydroxybutyrate levels when mice were fed either the SD and/or the KD. The data indicate that regardless of diet, glucose could predict seizure susceptibility with an approximate 75 to 78 % accuracy (Table 3 ). Although β-hydroxybutyrate could also predict seizure susceptibility, we previously showed that β-hydroxybutyrate levels were dependent on and were inversely related to plasma glucose levels [ 21 ]. Table 3 Binary logistic regression analysis of the maximum likelihood estimates between plasma glucose, and seizure susceptibility in adult EL mice fed either the SD or KD 1 Dietary groups Parameter Df 2 B 3 SEM 4 Wald x 2 5 p value 6 SD Glucose 1 0.774 0.139 30.962 0.01 Constant 1 -0.584 1.013 29.292 0.01 KD Glucose 1 0.787 0.157 25.033 0.01 Constant 1 -5.801 1.180 24.177 0.01 Both Diets Glucose 1 0.752 0.102 54.682 0.01 Constant 1 -5.507 0.759 52.625 0.01 1 Data were obtained from all four dietary groups over the treatment weeks 3–12 for a total number of 210 individual measurements of plasma glucose and seizure susceptibility. 2 Df, degrees of freedom. 3 B, Estimate of the association between glucose and seizure susceptibility. 4 The estimated error of the mathematical weighting, indicating the precision of the estimated coefficient. 5 The Wald test statistic was computed from the data compared by using x 2 distribution with 1 degree of freedom. The test statistic is used to determine the p value. 6 The probability of Type I error. Discussion We found that restriction of either a high carbohydrate low fat standard diet or a high fat low carbohydrate KD was equally effective in reducing seizure susceptibility in adult EL mice with active epilepsy. Moreover, seizure susceptibility remained similarly high in these mice when either diet was fed ad libitum or unrestricted. These findings indicate that the KD, when fed unrestricted, is unable to reduce seizure susceptibility in adult EL mice. Although the KD delays epileptogenesis in young seizure naïve EL mice when fed ad libitum , the effect is transient [ 20 ]. These findings are interesting since previous observations with children suggest that the antiepileptic and anticonvulsant effects of the KD are best when the diet is administered in restricted amounts [ 25 , 31 ]. Indeed, seizure protection is often less in children that gain weight than in those who maintain or reduce body weight on the KD (Freeman, personal communication). Previous studies also indicate that restriction of high carbohydrate diets elevate seizure threshold [ 58 ]. Our findings in EL mice support these observations and suggest that CR may be necessary for the antiepileptic and anticonvulsant effects of the KD. We previously showed that mild to moderate CR delayed epileptogenesis and reduced seizure susceptibility in seizure naïve juvenile and adult EL mice by reducing blood glucose and elevating ketone bodies [ 21 ]. Although our data show that circulating β-hydroxybutyrate levels are inversely related to circulating glucose levels, elevated ketone body levels are not directly associated with reduced seizure susceptibility in EL mice. This conclusion derives from the finding that seizure susceptibility is high in the KD-UR mice despite elevated β-hydroxybutyrate levels and from finding that seizure protection was similar in the SD-R and KD-R groups despite significantly higher β-hydroxybutyrate levels in the KD-R than in the SD-R group. These results are consistent with previous studies in EL mice and in non-genetic seizure models that elevated ketone bodies alone are unable to account for the antiepileptic or anticonvulsant action of the KD [ 20 , 31 , 33 - 38 ]. Under normal physiological conditions brain cells derive most of their energy from glucose or glucose-derived metabolites, e.g., lactate [ 46 , 59 , 60 ]. Also, brain glucose uptake is greater during epileptic seizures than during most other brain activities [ 43 ]. During fasting or caloric restriction, however, circulating glucose levels fall causing brain cells to rely more heavily for energy on ketone bodies that gradually increase with food restriction [ 29 , 61 ]. It is the transition from glucose to ketone bodies for brain energy that is thought to underlie the antiepileptic and anticonvulsant effects of caloric restriction [ 29 ]. Although the KD we used contained no carbohydrates, the mice eating this diet maintained high glucose levels and seizure susceptibility. The persistence of high glucose levels in the KD-UR group would prevent the transition to ketones for energy despite high levels of circulating ketone bodies. Our results show that circulating glucose levels accurately predict seizure susceptibility in EL mice regardless of diet composition or circulating ketone body levels. We used a new experimental design for caloric restriction in this study. Instead of restricting calories in the R-fed mice based on the average food consumption of the UR control mice as we did previously [ 21 ], each R-fed mouse served as its own control to achieve and maintain a 20–23% body weight reduction. We found in a pilot study that isocaloric restriction of the KD was unable to reduce body weight to the same degree as that observed for a similar restriction of the SD. The new experimental design reduces variability in body weights and in caloric intake among mice fed diets widely different in nutritional composition and caloric content. In using body weight, rather than caloric intake, as an independent variable we were able to more accurately measure the statistical associations among circulating energy metabolites and seizure susceptibility. Thus, this type of experimental design is recommended for those studies attempting to evaluate the relationships among nutrition, metabolism, and disease phenotype. We previously discussed the potential mechanisms by which CR might reduce seizure susceptibility [ 21 , 29 , 38 ]. Some of the cellular systems potentially modulated through CR that could influence brain excitability are illustrated in Fig. 6 . We suggest that the transition from glucose to ketone bodies as a major energy fuel for the brain produces multiple changes in gene-linked metabolic networks. It is these changes that gradually adjust neurotransmitter pools and membrane excitability to restore the physiological balance of excitation and inhibition [ 29 ]. CR could also influence seizure susceptibility through the neuroendocrine system involving leptin signaling and increased levels of neuropeptide-Y, a peptide with antiepileptic and anticonvulsant effects [ 62 - 65 ]. While the levels of γ-aminobutyric acid (GABA) are increased in synaptosomes via the increased action of glutamic acid decarboxylase during the metabolism of ketone bodies for energy, the levels of aspartate decrease due to the formation of glutamate [ 66 ]. In addition, ketone body metabolism could increase membrane ionic pump activity [ 67 , 68 ]. Increased pump activity could increase membrane potential in neurons while also increasing neurotransmitter uptake in glia [ 29 ]. We do not exclude the possibility that CR may reduce seizure susceptibility in EL mice through additional mechanisms [ 31 , 69 ]. It is our contention that CR reduces seizure susceptibility through multiple integrated systems providing a multifactorial therapy to a multifactorial disease. Further studies in the EL mouse and in other epilepsy models are needed to identify the exact mechanisms of CR action in managing epileptic events. Figure 6 Perspectives on the metabolic management of epilepsy through a dietary reduction of glucose and elevation of ketone bodies. A dietary reduction in blood glucose levels will increase ketone utilization for energy. This is expected to shift the neural environment from excitation to inhibition through multiple integrated systems. Abbreviations: GLUT-1 (glucose transporter), MCT (monocarboxylate transporter), PFK (phosphofructokinase), PDH (pyruvate dehydrogenase), SCOT (succinyl-CoA-acetoacetate-CoA transferase), β-OHB (β-hydroxybutyrate), β-HBDH (β-hydroxybutyrate dehydrogenase), NPY (Neuropeptide Y), GABA (gamma-aminobutyric acid). Modified from Seyfried et al., 2004 [38]. Conclusions We conclude that seizure susceptibility in EL mice is dependent on plasma glucose levels and that seizure control depends more on the amount than on the origin of dietary calories. Also, we found that CR underlies the antiepileptic action of the KD in EL mice. A transition from glucose to ketone bodies for energy is predicted to manage EL epileptic seizures through multiple integrated changes of inhibitory and excitatory neural systems. Methods Mice The inbred EL/Suz (EL) mice were originally obtained from J. Suzuki (Tokyo Institute of Psychiatry). The mice were maintained in the Boston College Animal Care Facility as an inbred strain by brother × sister mating. The mice were group housed (prior to initiation of study) in plastic cages with Sani-chip bedding (P.J. Murphy Forest Products Corp., Montville, N.J.) and kept on a 12-hr light/dark cycle at approximately 22°C. Cotton nesting pads were provided for warmth when animals were individually housed. All cages and water bottles were changed once per week. Only females were used for these studies as adult males die sporadically with age from acute uremia poisoning due to urinary retention [ 70 ]. The procedures for animal use were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee. Seizure Susceptibility and Seizure Testing Seizure onset in EL mice is generally between 60–70 days of age as previously described [ 6 ]. These seizures occur occasionally during routine cage changing. Our recently developed seizure handling protocol was used to regularly induce seizure susceptibility in EL mice [ 6 , 21 ]. Briefly, the testing procedure included repetitive handling and simulated the stress normally associated with weekly cage changing, i.e., picking the mouse up by the tail for short intervals and transferring it to a clean cage with fresh bedding. The test included two trials that were separated by 30 min. In each trial, a single mouse was held by the tail for 30 sec at approximately 10–15 cm above the bedding of its home cage. After 30 sec, the mouse was placed into a clean cage with fresh bedding for 2 min. The mouse was then held again for 15 sec before being returned to its home cage. Trial 2 was performed even if the mouse experienced a seizure in trial 1. The epileptic seizures commenced during holding or soon after the mice were placed on the clean bedding. Mice that developed an epileptic seizure while handling were placed immediately in either the clean cage or their home cage depending on the testing stage. Mice were tested each week for a total of 13 measurements over a 12-week period using this method. Mice were undisturbed between testing phases (no cage changing) and testing was performed between 12 to 3 pm. Seizure Phenotype Mice were designated seizure susceptible if they experienced a generalized seizure during seizure testing. Generalized seizures in EL mice involve loss of postural equilibrium and consciousness, together with excessive salivation, head, limb, and chewing/swallowing automatisms. An erect forward-arching Straub tail, indicative of spinal cord activation, was also seen in most mice having generalized seizures. Mice that displayed only vocalization and twitching without progression to generalized seizure were not considered seizure susceptible [ 6 , 21 ]. Seizure susceptibility scores were generated for each mouse according to the seizure severity scores previously described [ 6 ]. Mice having a score of 4 or 5 were assigned a susceptibility score of 1.0, whereas mice having a seizure severity score less than 4 were given a susceptibility score of 0. The seizure susceptibility for each mouse was then averaged over multiple tests and the mean seizure susceptibility for a mouse dietary group was determined. Diets All mice received PROLAB RMH3000 chow diet (LabDiet, Richmond, IN, USA) prior to the experiment. This is the standard food pellet diet (SD) and contained a balance of mouse nutritional ingredients. According to the manufacturer's specification, this diet delivers 4.4 Kcal/g gross energy, where fat, carbohydrate, protein, and fiber comprised 55 g, 520 g, 225 g, and 45 g/Kg of the diet, respectively. The ketogenic diet (KD) was obtained from the Zeigler Bros., Inc. (Gardners, PA, USA) in butter-like form and also contained a balance of mouse nutritional ingredients. According to the manufacturer's specification, the KD delivers 7.8 Kcal/g gross energy, where fat, carbohydrate, protein, and fiber comprised 700 g, 0 g, 128 g, and 109 g/Kg of the diet, respectively. The fat in this diet was derived from lard and the diet had a ketogenic ratio (fats: proteins + carbohydrates) of 5.48:1. The individual % composition of each dietary energy component for the SD and KD diet is shown on Table 1 . Pre-Trial Period Seizure susceptibility, body weight, and food intake was measured four times over a three-week period in 24 singly caged female EL mice (approximately 210 days of age). All mice received the SD during the pre-trial period and food intake was determined by subtracting the weight of food pellets remaining in the food hopper after one week from the initial amount given (200 g). The difference was then divided by seven to estimate the average daily food intake. Thus, all mice were highly seizure susceptible at the initiation of the diet therapy. Dietary Treatment After the three-week pre-trial period, the mice were placed into four groups (n = 6 mice/group) where the average body weight of each group was similar (about 31.0 ± 1.5 g) (Fig. 1 ). All mice were then fasted for 14 hr to establish a similar metabolic set point at the start of the experiment (arrow, Fig. 1 ). The mice in each group were then given one of four diets to include: 1) the standard diet fed ad libitum or unrestricted (SD-UR), 2) the KD fed ad libitum or unrestricted (KD-UR), 3) the SD restricted to achieve a 20–23% body weight reduction from the pre-trial weight (SD-R), and 4) the KD restricted to achieve a 20–23% body weight reduction from the pre-trial weight (KD-R). Each mouse in the two R groups served as its own control for body weight reduction. Based on food intake and body weight during the pre-trial period, food in the R-fed mouse groups was reduced until each mouse achieved the target weight reduction of a 20–23%. In other words, the daily amount of food given to each R mouse was reduced gradually until it reached 77–80% of its initial (pre-trial) body weight. The mice in the SD-UR group received 200 g of food in the hopper/week as in the pre-trial period. For mice in the SD-R group, weighed food pellets were dropped directly into each cage for easy access. The KD was administered to the mice in a modified plastic Falcon tissue culture dish (60 mm × 15 mm). The dish edges were shaved to reduce the height from 15 mm to about 6 mm. After placing about 5 g of KD in the dish for the KD-UR mice, the dish with the weighed KD was inverted and placed on the top of the food hopper. An empty water bottle was placed on top of the dish to prevent dish movement during animal feeding. The butter-like consistency adhered the KD to the inverted dish. This feeding apparatus allowed the mice easy access to the KD and prevented KD contact with bedding material. After about 24 hr, the amount of KD consumed was determined and another 5 grams of fresh KD were added to the dish. The KD was therefore given fresh every day without moving or disturbing the mice. The total amount of KD consumed per day was summed each week and divided by 7 to obtain the average weekly food intake of each mouse. For the KD-R mice, a calculated restricted amount of KD was placed directly on top of the food hopper bars for easy access. The R-fed mice licked the bars clean of the KD. Measurement of plasma glucose and β-hydroxybutyrate Blood was collected approximately 1 h after seizure testing except for the pre-trial period where blood was not collected. Blood was first collected from all mice about 24 hr prior to the initiation of the 14 hr fast (Fig. 1 ). Mice were anesthetized with isoflurane, USP (Halocarbon, River Edge, NJ, USA) and blood was collected in heparinized tubes by puncture of the retro-orbital sinus using a borosilated capillary tube (FHC, Bowdoinham, ME, USA). The blood was centrifuged at 6,000 × g for 10 min, the plasma was collected, and aliquots were stored at -80°C until analysis. Plasma glucose concentration was measured spectrophotometrically using the Trinder Assay (Sigma-Aldrich, St. Louis, MO, USA). Plasma β-hydroxybutyrate concentration was measured using either the Stanbio β-Hydroxybutyrate LiquiColor ® procedure (Stanbio, Boerne, TX, USA), or a modification of the Williamson et al procedure [ 71 ]. Statistical Analysis Both ANOVA and a two-tailed t test were used to evaluate the significance of differences of body weight, seizure susceptibility, plasma glucose levels, and plasma β-hydroxybutyrate levels between unrestricted and restricted groups. Chi-square analysis was performed on the association between glucose and seizures. Pearson bivariate correlation analysis (SPSS software) was used to determine the relationship between body weight, food intake, plasma glucose levels, plasma β-hydroxybutyrate levels, and seizure susceptibility. Binary logistic regression (SPSS) was used to determine the relationship between seizure susceptibility, plasma glucose, and β-hydroxybutyrate levels on mice fed either the SD or the KD. Differences were considered significant at p < 0.01. All values are expressed as mean ± SEM. All statistical data were presented according to the recommendations of Lang et al., [ 72 ]. Lists of abbreviations AED, antiepileptic drug; CR, caloric restriction; KD, ketogenic diet; R, restricted; SD, standard diet; UR, unrestricted. Competing interests The authors declare that they have no competing interests. Authors' contributions JGM carried out all described methods and drafted the manuscript. NAC helped with the blood assays, participated in the feeding of the mice and carried out the incorporation of the data in Excel spreadsheets. MTT helped with the design of the study. RM participated in the data discussion and the statistical analysis. TNS conceived the study, participated in its design and coordination and helped prepare the manuscript. All authors read and approved the final manuscript.

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544884

Hospitalisations for respiratory syncytial virus bronchiolitis in Akershus, Norway, 1993–2000: a population-based retrospective study

Background RSV is recognized as the most important cause of serious lower respiratory tract illness in infants and young children worldwide leading to hospitalisation in a great number of cases, especially in certain high-risk groups. The aims of the present study were to identify risk groups, outcome and incidences of hospitalisation for RSV bronchiolitis in Norwegian children under two years of age and to compare the results with other studies. Methods We performed a population-based retrospective survey for the period 1993–2000 in children under two years of age hospitalised for RSV bronchiolitis. Results 822 admissions from 764 patients were identified, 93% had one hospitalisation, while 7% had two or more hospitalisations. Mean annual hospitalisation incidences were 21.7 per 1.000 children under one year of age, 6.8 per 1.000 children at 1–2 years of age and 14.1 per 1.000 children under two years of age. 77 children (85 admissions) belonged to one or more high-risk groups such as preterm birth, trisomy 21 and congenital heart disease. For preterm children under one year of age, at 1–2 years of age and under two years of age hospitalisation incidences per 1.000 children were 23.5, 8.7 and 16.2 respectively. The incidence for children under two years of age with trisomy 21 was 153.8 per 1.000 children. Conclusion While the overall hospitalisation incidences and outcome of RSV bronchiolitis were in agreement with other studies, hospitalisation incidences for preterm children were lower than in many other studies. Age on admission for preterm children, when corrected for prematurity, was comparable to low-risk children. Length of hospitalisation and morbidity was high in both preterm children, children with a congenital heart disease and in children with trisomy 21, the last group being at particular high risk for severe disease.

Background Respiratory tract infections due to respiratory syncytial virus (RSV) are very common in young children worldwide. In temperate climates the infection occurs in yearly winter epidemics, and by two years of age most children have been infected [ 1 ]. Reinfections are common throughout life but the first infection is usually the most severe [ 2 ]. Symptoms vary from a mild upper respiratory tract infection to a severe bronchiolitis with hyperinflated lungs and hypoxemia [ 3 ]. Children in the first months of life, particularly those with preterm birth, underlying chronic lung disease (CLD), congenital heart disease (CHD), neuromuscular disease, airway malformations or impaired cellular immunity are at risk for severe disease [ 4 - 6 ]. Local epidemiological studies are important when considering new strategies for the prevention of RSV bronchiolitis. An example of such a strategy is the monthly injection of the humanised murine monoclonal antibody palivizumab to certain high-risk groups, which in many studies has shown a significant reduction in the incidence of hospitalisation for RSV bronchiolitis [ 7 ]. The aims of the present study are to identify risk groups, outcome and incidences of hospitalisation for RSV bronchiolitis in Norwegian children under two years of age, and to compare these results with other studies. Methods Background population Akershus is a large suburban and countryside region located around the capital of Oslo with approximately 10% of the total population in Norway. The Paediatric Department at Akershus University Hospital (Ahus) is the only hospital for children under 15 years of age living in the 20 northern, southern and eastern communities of the region and serves all kinds of hospital services, except for paediatric surgery. Deliveries have been rather stable at approximately 3.500 per year in the service area during the last decade. Due to a local agreement infants from approximately 500 unselected term deliveries from our service area are born at Rikshospitalet in Oslo each year, but these infants are admitted to our hospital in case of disease during childhood. The number of children under two years of age living in the service area at any time during the follow-up period were identified from official Norwegian Population Statistics . The number of children under two years of age born before completed 37 weeks of gestation in the service area during the study period and still alive after the neonatal period was identified from the Norwegian Medical Birth Registry . All children with trisomy 21 have a program for follow-up from birth in our hospital and the number of children under two years of age with trisomy 21 was therefore identified from in-patient and out-patient hospital records. The number of children in the general population with a CHD was not known in the present study and hospitalisation incidences could therefore not be estimated. Overall mortality after the neonatal period as well as the number of children moving out of the service area during the study period were low and have not been corrected for. Study population The present study is a population-based retrospective survey on children under two years of age admitted to the hospital with a diagnosis of bronchiolitis (ICD 9 & 10) during the period February 5 th 1993 to January 31 th 2000 and diagnosed as positive with RSV in nasopharyngeal aspirate (NPA). RSV was diagnosed by an enzyme-linked immunosorbent assay (ELISA) for RSV (Abbott TestPack RSV). The test is simple and easy to perform giving a result within 30 minutes. The sensitivity, specificity, positive and negative predictive value is stated to be 74–92%, 86–100%, 81% and 93% respectively [ 8 , 9 ]. 764 children who fulfilled the diagnostic criteria were identified. A total of 77 children belonged to one or more well known high-risk groups. Of these, 58 children had been born before completed 37 weeks of gestation, some with additional diseases such as CLD (12 children) and CHD (four children). 12 children had a diagnosis of CHD as the only additional disease and seven children had trisomy 21, four of these also diagnosed with CHD. These 77 children were regarded as a high-risk group for subgroup analysis. No children with other well-known risk-factors (neuromuscular disease, airway malformations, impaired cellular immunity or others) were identified. The remaining 687 children were born at term and healthy and regarded as a low-risk group for subgroup analysis. Statistics Data were analysed with the statistical programme SPSS, version 11. For analysis of gender we used the Chi square test and for comparisons between low-risk and high-risk groups we used the independent sample T-test. A p-value <0.05 was used as limit for statistical significance. Results Epidemiology During this seven years follow-up period 764 children (822 admissions) were hospitalised for RSV bronchiolitis. 93% (707 children) had only one hospitalisation and 7% (57 children) had two or more hospitalisations for RSV bronchiolitis during the first two years of life. The highest number of admissions was recorded during the winter months December-April, but cases were also found in late spring and early autumn. Both the number of admissions and peak-time varied between seasons but with no specific pattern of variability (figure 1 ). A significant male predominance was observed, with 517 (63%) of all admissions being boys (P < 0.001) Figure 1 Seasonal variations by month and year in hospitalisations for RSV bronchiolitis in children under two years of age in Akershus, Norway, February 1993 to January 2000 The majority of children (75% of all admissions) were hospitalised within the first year of life with children less than six months old being responsible for 45% of all admissions. Median age at hospitalisation was 6 months (range 0–23 months) and a median length of stay of 4 days (range 1–41 days) was observed (table 1 ). Table 1 Age on admission and length of stay in children under two years of age hospitalised for RSV bronchiolitis in Akershus, Norway, February 1993 through January 2000 Risk groups Children (no.) Adm (no.) Median age a P-value (age) Median stay b P-value (stay) All children 764 822 6.0 4.0 Premature c 58 64 8.0 NS e 8.0 <0.001 e Corrected age d 58 64 5.4 NS e Trisomy 21 7 8 9.0 NS e 7.5 <0.001 e CHD* 12 13 7.0 NS e 6.0 NS e All low-risk 687 737 6.0 4.0 All high-risk 77 85 8.0 0.045 e 8.0 <0.001 e Corrected age d 77 85 5.5 NS e a Age in months b Days including admission day c Born at <37 weeks of gestational age d Corrected for weeks of prematurity e Versus low-risk children During the study period nine children (1.2%) developed severe respiratory distress and needed mechanical ventilation and two of them (0.3%) died. Four of these children had no known risk-factors for severe disease. However, the two children who died were both considered as high-risk patients, one of them was diagnosed with CHD and the other with trisomy 21 as well as CHD. Of all RSV hospitalisations during the first two years of life 58 children (64 admissions) were born before completed 37 weeks of gestation with a median gestational age of 30 weeks (range 24–36 weeks). A significant male predominance of 68% was also observed in this group (P = 0.006). Median age on admission, after postnatal age, was 8 months (range 0–23 months). When corrected for prematurity (corrected age), median age on admission was 5.4 months (range -2.5–21.3 months). Also, a median length of hospitalisation of 8 days (range 2–27 days) was observed (table 1 ). Two of the preterm children (3.4%) needed mechanical ventilation and two were treated with inhaled nebulized ribavirin [ 10 ]. No preterm children in this study died from RSV bronchiolitis. Seven children (eight admissions) diagnosed with trisomy 21 were hospitalised for RSV bronchiolitis during this follow-up study. Four of them also had a CHD. Two children needed mechanical ventilation during hospitalisation and one of them subsequently died. A median age on admission of 9.0 months (range 1–20 months) and a median stay of 7.5 days (range 2–34 days) were recorded for children in this particular risk group (table 1 ). A total of 20 children had a CHD, four combined with prematurity, four combined with trisomy 21, and 12 with no other additional risk-factor for severe disease. In the group of children with only CHD as risk factor median age on admission was 7.0 months (range 0–23 months) and median stay of 6.0 days (range 2–14 days) was observed (table 1 ). One child needed mechanical ventilation and subsequently died. Hospitalisation incidences Overall hospitalisation incidences were calculated from the known number by official Norwegian statistics of 58.179 children under two years of age living in the service area for the whole study period as well as for each year as given in table 2 . As shown, the overall incidences for the whole study population was 21.7 admissions per 1.000 children under one year of age, 6.8 admissions per 1.000 children 1–2 years of age and 14.1 admissions per 1.000 children under two years of age, with some year to year variation (table 2 ). Table 2 Mean annual hospitalisation incidences per 1.000 children by age and risk-groups in children under two years of age with RSV bronchiolitis in Akershus, Norway, 1993–2000 Children <1 year Children 1–2 years Children 0–2 years Year a Pop d Adm Inc Pop d Adm Inc Pop d Adm Inc All 93–94 4.027 70 17.4 4.195 16 3.8 8.222 86 10.5 All 94–95 3.999 53 13.3 4.192 25 6.0 8.191 78 9.5 All 95–96 3.994 112 28.0 4.132 30 7.3 8.126 142 17.5 All 96–97 4.055 80 19.7 4.143 22 5.3 8.198 102 12.4 All 97–98 4.154 115 27.7 4.242 57 13.4 8.396 172 20.5 All 98–99 4.178 70 16.8 4.350 15 3.4 8.528 85 10.0 All 99–00 4.127 120 29.1 4.391 37 8.4 8.518 157 18.4 All 93–2000 28.534 620 21.7 29.645 202 6.8 58.179 822 14.1 Premature b 93–2000 1.999 c 47 23.5 1.955 c 17 8.7 3.954 c 64 16.2 Trisomy 21 93–2000 52 8 153.8 a Study period February 5th, 1993 to January 31th, 2000 b Born at <37 weeks of gestational age c Number of children born at <37 weeks of gestation and corrected for a mean infant mortality rate of 2.4% during the neonatal period d Background population for selected risk groups Data from the Norwegian Birth Registry identified that 1.999 infants born in the service area before completed 37 weeks of gestation were alive and below 1 year of age, and 1.955 infants were alive and between 1–2 years of age during the study period. As shown, the corresponding hospitalisation incidences for preterm children were 23.5, 8.7 and 16.2 per 1.000 children under one year of age, 1–2 years of age and <2 years of age respectively (table 2 ). From the hospital records 52 children under two years of age were diagnosed with trisomy 21 during the follow-up period. Among these seven children (eight admissions) were hospitalised for RSV bronchiolitis, giving a hospitalisation incidence under two years of 153.8 per 1.000 children. Discussion In this retrospective study, as in many other comparable epidemiological studies, we found that RSV bronchiolitis appears as an annual winter epidemic with relatively high hospitalisation incidences, predominance of boys, young age, short length of stay, few complications and low mortality [ 1 ]. The rather long study period makes the results less vulnerable for yearly variations in magnitude of the annual epidemic and in virulence of the microbe. By using NPA for microbiological diagnosis in all children with symptoms of a lower respiratory tract infection like tachypnoe, wheezing or apnoic spells we believe that the great majority of cases are included. However, the enzyme-linked immunosorbent assay used in the present study could give both false positive and false negative results [ 9 , 11 ]. Several international studies have shown an increased incidence of RSV-related hospitalisations over the last two decades. A Norwegian study for the period 1972–78 showed a mean hospitalisation incidence of 9.5 per 1.000 children for children under one year of age compared to our much higher incidence of 21.7 per 1.000 children for the period 1993–2000 in the same age group [ 12 ]. Also, for children 1–2 years old the incidence in our study was much higher. A large study from USA for the period 1980–96 showed an increased incidence rising from 12.9 per 1.000 children in 1980 to 31.2 per 1.000 children in 1996 for children under one year of age [ 13 ]. However, comparing historical data is difficult, and for the period 1993–2000 we observed no increase in the mean annual hospitalisation incidences of RSV bronchiolitis. Factors such as increased microbial virulence, increased day-care attendance, improved microbiological diagnosis and more precise ICD-coding might all be important factors to explain these historical differences. Number of hospitalisations are also related to the severity of the clinical symptoms. Thus, changes in hospital admission policies over time as well as changes in availability of hospital services might also influence hospitalisation incidences. A recent study from USA for the period 1997–99 showed that RSV bronchiolitis was the leading cause of hospital admissions of infants younger than one year of age with an associated hospitalisation incidence of 25.2 per 1.000 infants. This is in accordance with our findings [ 14 ]. Our study showed that only 1.2% of the children in the study population needed mechanical ventilation. This result is lower than in many other studies [ 15 ]. On the other hand, when children were in need of mechanical ventilation, the risk of severe outcome was in our study very high, with two out of nine mechanically ventilated children subsequently dying. A Danish study showed that only 0.6% of their children needed mechanical ventilation, however, 20% of all children with severe respiratory failure were given ventilatory support with nasal continuous positive airway pressure (N-CPAP) and some infants possibly avoided mechanical ventilation for that reason [ 16 ]. More controlled studies on different treatment strategies are very much needed, especially when considering how to avoid respiratory failure and mechanical ventilation [ 17 , 18 ]. Another observation in our study was that among the mechanically ventilated children 44% had no underlying risk-factors for severe disease. Further research into why some otherwise healthy children have a severe course of RSV bronchiolitis is therefore very important, not least when trying to define risk groups even better [ 19 ]. Research for a better understanding of the host immune response to RSV bronchiolitis the later years might be one important piece in this puzzle [ 20 - 22 ]. For the population at large our study showed, as in other studies, a low mortality. A total of two children (0.3%) died, one had trisomy 21 with an atrioventricular septum defect (AVSD) and the other had an underlying CHD. The number of high-risk children in our study is relatively small and the results should be interpreted with caution. The mean hospitalisation incidences for preterm children were lower than in many other studies, even though 20% of the preterm children suffered from CLD as well and had a low median weeks of gestational age (wGA) of 30. One possible explanation is that the population data for this particular risk group was not corrected for migration out of our service area and mortality after the neonatal period. However, it is unlikely that such corrections would significantly influence the results. Another explanation for the low incidence among preterm infants might be that most of them are hospitalised up to almost 40 wGA and are discharged only when the child has reached a weight of more than 2.000 g and have normal oral feedings. This means that they are more or less completely protected from RSV for a long period after birth. Our routines with focus on good parental information on how to protect the newborn infant from RSV after discharge from hospital might also be important. Median length of hospitalisation for preterm children was in our study significantly longer when compared to the low-risk children and is in accordance with results from many other larger studies. Children with CHD are a wellknown risk-group for severe RSV disease. Children with trisomy 21 with or without a concomitant CHD should also be considered a high-risk group. Our study showed a mean hospitalisation incidence per 1.000 children under two years of age with trisomy 21 more than 11 times the whole study population. Even more important, out of a total of seven children with trisomy 21, two needed mechanical ventilation and one child died. We would, however, recommend a larger study to meet these observations for children with trisomy 21. One of the findings in our study was the large proportion of otherwise healthy children needing mechanical ventilation. Our numbers are small, but when new policies to prevent severe RSV bronchiolitis are discussed this aspect should also be considered. The research for improved identification of risk-groups and development of an effective vaccine for immunization should therefore be given high priority. Conclusions While the overall hospitalisation incidences and outcome of RSV bronchiolitis were in agreement with other studies, hospitalisation incidences for preterm children were lower than in many other studies. Age on admission for preterm children, when corrected for prematurity, was comparable to low-risk children. Length of hospitalisation and morbidity was high in both preterm children, children with a congenital heart disease and in children with trisomy 21, the last group being at particular high risk for severe disease. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Our individual contributions to the study have been as following: HOF had primary responsibility for protocol development, outcome assessment, data acquisition and analysis and writing of the manuscript. TF participated in the development of the protocol and analytic framework of the study, and contributed to the writing of the manuscript. DB participated in the data analyses and the writing of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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545595

Alternative splicing of mouse transcription factors affects their DNA-binding domain architecture and is tissue specific

Splice variants of 461 transcription factor loci were analyzed using a new database of splice variants in the mouse transcriptome, MouSDB3, providing quantitative evidence that alternative splicing preferentially adds or deletes domains important to the DNA-binding function of the TFs.

Background Alternative splicing is a widespread mechanism involved in regulation of gene expression, which enables production of many structurally and functionally different forms of proteins from a single gene, adding to the complexity of the genomes [ 1 - 3 ]. Different mRNA transcripts of a gene can be expressed in different tissues or developmental stages or physiological conditions [ 4 , 5 ]. An expanding body of expressed sequence data from the human and mouse genomes indicates that alternative splicing is an important mechanism in creating protein diversity, and adds to functional complexity encoded in eukaryotic genomes. Earlier studies indicate that at least 50% of the genes in the human genome are alternatively spliced [ 6 ]. Examples include the vast majority of immune system and nervous system genes [ 7 ]. Comprehensive analysis of alternative splicing is essential to understand fully the proteomes of organisms [ 8 ]. Several reports have indicated that variant splice forms result in proteins with different functions. These can range from minimal changes in function to absolutely opposite functions. For example, the cAMP-response element modulator has three different isoforms with entirely different DNA-binding domains, which are all transcription activators. On the other hand, isoforms of the human transcription factor AML1 function both as positive and as negative regulators of transcription [ 9 ]. However, for the majority of genes, the functional significance of alternative splicing is still not known [ 8 ]. Transcription is a critical process that specifies the mRNAs and the proteins expressed within a cell. Expression of a given gene is dependent on the interactions of different transcription factors and their cofactors with the regulatory regions of that gene. These transcription factors are in turn regulated by processes that include interaction with other proteins and signaling cascades [ 9 ]. Alternative splicing is a mechanism that regulates transcription factor (TF) activity by generating a variety of protein isoforms from a single gene. As noted by Lopez, alternative splicing can affect TF structure in two primary ways [ 9 ]: alterations can be in the DNA-binding domains affecting their affinity or specificity; or alterations can modulate interactions of transcription factors with their cofactors. Such changes have been observed experimentally to alter specificity or binding strength or to switch between activator and repressor isoforms of the same TF [ 10 ]. TF isoforms can have stage-specific and tissue-specific expression patterns throughout the development of an organism [ 9 ]. Little is known about the tissue specificity of alternative splicing [ 11 ]. In this paper, we use an integrated approach to analyze DNA and protein sequence data jointly to determine the potential effect of alternative splicing on protein structure and function. We perform a detailed analysis of tissue-specific distribution of alternatively spliced mouse TFs to gain biologically meaningful insights into regulation of gene expression by alternative splicing. Results Definitions For our joint DNA-protein analysis described here, we developed MouSDB3 [ 12 ], which identifies, classifies, computes, stores and answers queries about splice variants within the mouse genome. As described in Materials and methods, MouSDB3 uses the mouse genome and expressed sequences in GenBank [ 13 ] and dbEST [ 14 ] to compute splice variants of mouse transcripts organized by genomic loci. This section provides definitions of terms used in MouSDB3 and in the joint DNA-protein analysis method described here. A 'transcript' is a sequence transcribed from the genomic DNA sequence. MouSDB3 is restricted to transcripts with at least one splice junction. A 'locus' is a genomic region that includes a set of overlapping transcripts mapped to the genome such that a transcript appears in only one locus and all transcripts whose genome coordinates overlap by at least one nucleotide are included in the locus. Within a locus, a 'cassette exon' is completely included in some transcripts and completely excluded in others. A 'length variant exon' has alternative 5' or 3' splice sites, or both, in different transcripts. An exon can be both length variant and cassette. A 'variant exon' is either cassette or length variant or both. We consider an exon whose number of nucleotides is a multiple of three and which starts at the first base of a codon to be an 'in-frame exon'. Such exons do not introduce an amino-acid substitution or a stop codon when skipped, unless they are terminal exons within the coding sequence. A 'genomic exon' is an uninterrupted series of nucleotides, each of which is mapped to a transcript. By this definition the genomic exon for a length variant exon reflects the outermost splice sites. A 'cluster' is the set of transcripts that map to a locus. A 'variant cluster' contains one or more variant exons. An 'invariant cluster' has no variant exons. MouSDB3 cluster analysis Our cluster analysis revealed that out of the 461 TF clusters, 62% are variant, compared to 29% of all genes in MouSDB3 (Table 1 ). The majority (62%) of the variation in TFs is due to cassette exons, which is comparable to cassette-exon distribution in the entire transcriptome (68% of the variant exons in all loci are cassette). As the majority of alternative splicing is due to cassette exons, we focus on these exons for our analyses. Cassette exon analysis We screened the 287 variant TF clusters for the presence of cassette exons within coding sequences. We categorized MouSDB3 transcripts into three categories with respect to each cassette exon within a cluster. Category 1 transcripts contain the exon and are referred to as 'long transcripts'. Category 2 transcripts skip the exon and are referred to as 'short transcripts'. Category 3 transcripts do not overlap with the cassette exon due to 5' or 3' truncations. In our structural analysis, we computationally delete in-frame cassette exons from Category 1 transcripts to produce an 'altered transcript'. Figure 1 displays a MouSDB3 cluster and illustrates these categories. The 287 variant TF loci contain 324 cassette exons of which 23% (76 exons) are in-frame. Only 11% of cassette exons are expected to be multiples of three and in codon position 1 randomly. The twofold difference between expected and observed numbers indicates a bias towards in-frame cassette exons. The exons which are a multiple of three and in codon position 2 and 3 comprise 10% and 7%, respectively. When deleted, these exons introduce an amino-acid substitution to the sequence. As exons which are a multiple of three starting at codon position 1 are enriched and do not introduce an amino-acid substitution when deleted, our study focuses on these exons only. As shown in Figure 2 , of the 76 in-frame cassette exons, 66 have domain architectures predicted by SMART. The remaining 10 exons are either from transcripts with too short sequences or these transcripts do not have any of the domains annotated in SMART. Of the 66 in-frame cassette exons, 80% (53) induce a domain-structure alteration to the protein when skipped. Of these 53 structure-altering exons, 68% are within coding regions for the domains that are important for TF activity, such as DNA-binding or activation domains. The remaining 32% (17) of exons are proximal to the computed domain boundaries; that is, the domain is coded by the upstream or the downstream neighboring exon of the cassette exon. When the cassette exon is removed, the sequence no longer meets the computational criteria for the domain (Figure 2 ). Assessing domain architecture alterations SMART [ 15 , 16 ] and Pfam [ 17 , 18 ] entries for the altered domains revealed that 75% of the domains affected by alternative splicing with known functions are DNA-binding domains. The names of all altered domains and links to their annotated biological functions are provided on our web page [ 19 ]. There we provide the 53 in-frame cassette exons (shown in Figure 2 ), which alter the domain architecture of their transcripts when skipped. Links to MouSDB3 clusters containing these transcripts and links to their GenBank entries are provided. In addition, we provide the names of the domains altered by these 53 exons as active links to their SMART and Pfam annotations. All sequences for long transcripts, altered transcripts and in-frame cassette exons are provided as links to fasta files on the same web page. Our domain-alteration results correlate with recent findings of Resch et al . [ 20 ], who show that alternative splicing preferentially removes certain domains more frequently. Tissue-distribution analysis Part two of our analysis assessed the tissue distribution of alternatively spliced transcription factors. We chose 18 tissues from the existing libraries in MouSDB3 on the basis of the fact that they contain both variant and invariant transcripts annotated as TFs. There are a total of 1,413 library names in MouSDB3 imported from expressed sequence records in GenBank and dbEST. Of these, 328 are ambiguous in that they list several different tissues or cell types for a single library, such as 'mixture of brain and testis' or no tissues at all, such as 'embryo or carcinoma'. For the work described here we did not include tissue information from such ambiguous libraries. There are a total of 95 libraries in MouSDB3 for which there are TF transcripts. In addition, to account for library ambiguities within these 95 libraries, we pooled different parts of a tissue into one library. For example, the term 'brain' corresponds to all parts of the brain found in MouSDB3, including cerebellum, thalamus, hippocampus and 16 other libraries. When analyzing the tissue distribution of all genes, only the libraries that contain TF transcripts have been used. Transcript counts within variant loci for 18 pooled libraries indicated that in 14 of the 18 analyzed tissues, the proportion of TFs that are variant is higher than the proportion of all genes that are variant (Figure 3a ). This finding, together with the observation that 62% of TF loci are variant, indicates the widespread impact of alternative splicing on regulation of gene expression via TFs. For each of the 18 tissues in Figure 3a , we compared the proportion of TFs that vary to the proportion of all genes that vary. As shown in Figure 3b , eight tissues exhibited more than twofold difference in variant TFs versus variant genes in total. (Note that values in Figure 3b are base 2 logarithms of the ratios. Tissues with twofold differences have log 2 values above 1 on the graph). In salivary gland, skeletal muscle, urinary bladder and testis, the fold-differences are 8.7, 5.6, 3.8 and 3.0-fold respectively. Spinal cord, liver, adipose tissue and eye also exhibit more than twofold differences. These values are independent of the sampling depth of the transcripts from these tissues, as illustrated in Figures 4a and 4b . Sampling depth is the number of transcripts sequenced per tissue (either a single library or a pooled library as in the case of 'brain'). Figure 4a displays absolute numbers of variant TF transcripts and Figure 4b displays absolute numbers of the entire variant transcripts of the transcriptome. In Figures 4a,b , tissues are presented along the x -axis as in Figure 3b for the reader's convenience. The correlation coefficient of the absolute numbers of TFs and the fold-differences between variant TFs and all genes is -0.13, indicating that they do not correlate. Likewise, the correlation coefficient of the absolute numbers of all genes and the fold-differences between variant TFs and all genes is -0.46. Additionally, the scatter-plots in Figures 4c,d show that there is no correlation between the fold-differences and sampling depth. The datasets used in calculating the correlation coefficients can be found on our web page [ 19 ]. Isoform heterogeneity We analyzed the presence of different isoforms of transcription factors within and across these 18 tissues. For this analysis we consider transcripts with coding sequence information only. We ignore variation due to 5' and 3' truncation of transcripts. We consider only cassette exons within coding sequences when assessing the differences between isoforms. Within a cluster we compute homogeneity and heterogeneity within a single tissue by checking for the transcripts from the same library and comparing the cassette exons within their coding sequences. If all transcripts from the same tissue contain the same cassette exons with same splice sites they are termed 'homogeneous within'. If the cassette exon distribution within the coding sequences of these transcripts differ, they are termed 'heterogeneous within'. We compute 'homogeneity across' and 'heterogeneity across' tissues in the same way by taking into account transcripts within the same clusters but from different libraries. As shown in Figure 5 , when heterogeneity to homogeneity ratios are compared within and across tissues, there is significantly more heterogeneity of isoforms across tissues than within a single tissue ( p -value = 0.04). This is true for both transcription factors and the rest of the genes in the mouse transcriptome. When single tissues are taken into account, TFs are more homogenous within each tissue analyzed. As shown in Figure 6 , heterogeneity to homogeneity ratios in all tissues are lower than 1, indicating that these tissues are more homogeneous in terms of TF isoforms. In fact, except for brain and thymus, all values for TFs are zero, hence the absence of blue bars from Figure 6 . When all genes are considered, heterogeneity to homogeneity ratios are also below 1, indicating homogeneity of isoforms of all genes within these tissues. However, there is still a significant difference in heterogeneity to homogeneity ratios between TF isoforms and isoforms of all genes: TFs are significantly more homogeneous within single tissues when compared to all genes ( p -value = 0.02). (The data used in calculating the homogeneity and heterogeneity values can be found on our web page [ 19 ].) Figures 5 and 6 show that the majority of TF isoforms and the isoforms of all alternatively spliced genes differ across tissues: within a given single tissue there generally is only one isoform. These data indicate the presence of tissue-specific alternative splicing throughout the mouse transcriptome. In addition, our findings indicate expression of different TF isoforms in different tissues. This implies contribution of alternative splicing to regulation of gene expression in a tissue-specific manner by controlling activation or repression of different sets of genes in different tissues via variant TF isoforms. These data have significant implications in further understanding the regulation of tissue-specific gene expression and control of transcription. Discussion Through integrated analyses of DNA and protein sequences for TF genes, we show that alternative splicing of TFs are more prevalent in the entire mouse transcriptome and in specific tissues when compared to alternatively spliced forms of all the genes. In 78% of the tissues analyzed, higher proportions of TFs exhibit alternative splicing compared to all the genes in the mouse transcriptome. This result, along with the finding that 62% of TF loci are variant, indicates the widespread impact of alternative splicing on regulation of TF function. We also show that alternative splicing changes TF structure by adding or deleting domains. This study reveals that 80% of alternatively spliced TFs have different domain architectures due to introduction of an in-frame cassette exon by alternative splicing. Of the altered domains, 75% have a role in DNA binding. These findings provide quantitative evidence for the role of alternative splicing in controlling the presence of domains in the proteins. They also suggest that alternative splicing might regulate TF activity by changing the architecture of the DNA-binding domains of these proteins. Our analyses revealed that within a single tissue there generally is only one TF isoform, and that across tissues, isoforms differ. This finding indicates tissue specificity of alternatively spliced TFs and suggests that TFs might regulate gene expression in a tissue-specific manner by having different isoforms in different tissues. These findings further indicate the role of alternative splicing in regulation of tissue-specific gene expression. Activation and repression of different sets of genes within different tissues can be regulated through variant TF isoforms created by alternative splicing. These findings will significantly aid further understanding of control of transcription and tissue-specific gene expression. In addition, our study shows that all variant loci in the mouse transcriptome display isoform homogeneity within single tissues and heterogeneity across tissues. This finding demonstrates the presence of tissue-specific alternative splicing across the mouse transcriptome and greatly expands the knowledge on the tissue specificity of alternatively spliced genes. Conclusions Overall, our study provides quantitative evidence for the effect of alternative splicing on protein structure and sheds light on how alternative splicing might regulate transcription factor function in a tissue-specific manner. This, in turn, reveals the contribution of alternative splicing to regulation of gene expression via tissue-specific TF isoforms. The work described here implies that future high-throughput screens of gene expression analyses should be sensitive to multiple alternatively spliced forms of TFs. Because gene-expression arrays are intended to measure transcription, the next generation of arrays should contain probes specific to all known isoforms of genes represented on the arrays. Given that alternatively spliced exons are highly conserved across species [ 21 , 22 ], it would be of further interest to extend this study to other organisms. Strong sequence homology between mouse, human and rat exons suggests that a comparative analysis of human, mouse and rat TF variations will be a natural extension of the studies described here. Materials and methods Development of the alternative splicing database MouSDB3 For this analysis, we constructed a database of alternatively spliced mouse transcripts called MouSDB3 [ 12 ], using the methods described in [ 23 ]. Briefly, full-length transcript nucleotide sequences were obtained by an Entrez query on 5 August 2003 from GenBank [ 24 ] with molecule selected as mRNA and limits used to exclude expressed sequence tags (ESTs), sequence-tagged sites (STSs), genome sequence survey (GSS), third-party annotation (TPA), working draft and patents. EST sequences were downloaded on 31 July 2003 from dbEST [ 25 ] by extracting only Mus musculus entries. All expressed sequences were mapped to a region of the University of California Santa Cruz (UCSC) February 2003 version mm3 of the mouse genome assembly using BLAT [ 26 ]. BLAT tools gfServer and gfClient were installed from jksrc444 dated 15 July 2002 [ 27 ]. This was followed by a careful alignment by SIM4 [ 28 ] version 3/3/2002 to establish splice sites of exons. A post-processing analysis computed genomic exons and determined types of variation for each exon, transcript and locus. Cassette exon analysis We identified in-frame cassette exons and extracted from MouSDB3 nucleotide and amino-acid sequences for transcripts containing these exons. The selected amino-acid sequences were then analyzed with SMART [ 29 , 30 ] to compute protein-domain architecture for each transcript within a cluster. Tissue distribution of alternatively spliced TFs From MouSDB3, we then extracted library information for the transcripts within clusters and their annotations. We used these data to compute the tissue distribution of variant transcripts as reported in Results. All scripts and README files used to carry out this data-gathering process are available upon request from the Laboratory of Computational Genomics of The Rockefeller University.

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423162

A Method for Studying Calcium Dynamics in Transgenic Mice

Calcium makes up just 2% of the human body, and 99% of it is sequestered in bones and teeth. The remainder exists within and around cells, influencing a variety of cellular processes, from transcription and cell survival to nerve signaling and muscle contraction. Calcium inhabits the intra- and extracellular space as a gradient, with extracellular concentrations some 1,000 times greater than those inside the cell. These gradients are maintained by calcium pumps. Calcium signaling operates mostly through voltage- and ligand-gated calcium channels (ligands are signaling molecules), both in the plasma membrane of the cell and in the membranes enclosing intracellular organelles. Signaling is typically initiated by an influx of calcium across the plasma membrane or by the release of calcium from an organelle, such as the endoplasmic reticulum. In neuron-to-neuron signaling, calcium signaling helps convert electrical signals into chemical signals in the form of neurotransmitters. The arrival of an electrical signal at a nerve terminal opens the many calcium channels in the nerve terminal, admitting a stream of calcium ions. The increased intracellular calcium concentration in turn releases the resident neurotransmitters accumulated in the nerve terminal, converting the electrical signal into a chemical signal. As neurotransmitters bind to their receptors in the next target neuron, they change the cell's membrane potential, prompting the neuron to generate an electrical signal, thereby converting the chemical signal back into an electrical one and completing the signaling circuit. Since calcium dynamics mediates most neuronal information flow, it can be used as a general measure of neural activity. Calcium-indicator-expressing mouse on a Ca 2+ activity odor map (Image by Rolf Sprengel) Through an elaborate network of electrical activity, the brain encodes, combines, and interprets signals to process information about the world. Simultaneous measurement of this activity in multiple brain locations has provided valuable insight into how neural networks function. But since electrical recordings can't pinpoint activity in the fine branches of individual neurons or pick up biochemical (nonelectrical) signals, researchers are increasingly turning to approaches that measure calcium concentrations as a proxy for neuron activity. Now Mazahir Hasan et al. have created mice engineered to stably express two different kinds of fluorescent calcium indicator proteins (FCIPs) in the brain (the fluorescence produced by these proteins can be seen when the brain is viewed with a two-photon microscope). Because the indicators are incorporated into the mouse genomes, this approach offers the possibility of targeting specific cells (by using promoters that specify which cells the genes should be activated in), allowing researchers to map the activity of select neuron populations. Fluorescent proteins can be incorporated into a gene of interest to help researchers track that gene's protein in living tissue. FCIPs report calcium concentrations by changing fluorescence when they bind to calcium. Introducing calcium indicators into neural tissues was largely impractical, often failing to target specific cell types, until these genetically engineered indicators were developed in the late 1990s, allowing the desired specificity. While FCIPs have been used to good effect in worms, fruitflies, and zebrafish—and just recently in mouse muscle—they had not been stably and functionally expressed in the mammalian brain until now. To deliver the FCIPs to the mouse brain, Hasan et al. used a regulatory promoter called Ptet (in the tetracycline system), which offers the possibility of targeting the expression of the FCIPs in different neural populations. To test the functionality of the proteins, they used fluorescence microscopy to analyze neurons from mouse lines and found high levels of FCIP expression. The real test, however, was whether the FCIPs could fulfill their promise as a probe for calcium activity. When the authors electrically stimulated brain slices from a mouse line expressing moderate to high levels of FCIP (electrical stimulation is known to increase intracellular calcium concentration), fluorescence increased rapidly following the stimulus. Significant changes in FCIP fluorescence were also observed when live mice responded to odor stimulation. That “fast and robust” FCIP signals were detected in live animals responding to sensory stimulation, the authors argue, proves the promise of FCIPs as a reporter on the activity of select neural populations in living systems. And since these indicator proteins retain stable functional expression over time (8- to 12-week-old mice continued to express the proteins), they could help researchers track neuronal activity over extended periods. While a variety of bugs remain to be worked out with FCIPs—it's unclear, for example, why only the Ptet promoter generates high levels of FCIP expression in the brain and why not all neurons in a given population express the proteins—Hasan et al. demonstrate that the tetracycline system supports stable expression of the calcium indicators. The FCIP approach avoids the complications of invasive techniques like surgically administering dyes and produces a more interpretable signal, since the cell populations are already known. Because FCIPS can be used in living animals, they can reveal where and when neurons are firing. And because FCIP mice can be crossed with mice containing mutations in genes important for neural function, this method could reveal how specific genes contribute to the construction of neural networks.

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526768

Family doctors' involvement with families in Estonia

Background Family doctors should care for individuals in the context of their family. Family has a powerful influence on health and illness and family interventions have been shown to improve health outcomes for a variety of health problems. The aim of the study was to investigate the Estonian family doctors' (FD) attitudes to the patients' family-related issues in their work: to explore the degree of FDs involvement in family matters, their preparedness for management of family-related issues and their self-assessment of the ability to manage different family-related problems. Methods A random sample (n = 236) of all FDs in Estonia was investigated using a postal questionnaire. Altogether 151 FDs responded to the questionnaire (response rate 64%), while five of them were excluded as they did not actually work as FDs. Results Of the respondents, 90% thought that in managing the health problems of patients FDs should communicate and cooperate with family members. Although most of the family doctors agreed that modifying of the health damaging risk factors (smoking, alcohol and drug abuse) of their patients and families is their task, one third of them felt that dealing with these problems is ineffective, or perceived themselves as poorly prepared or having too little time for such activities. Of the respondents, 58% (n = 83) were of the opinion that they could modify also relationship problems. Conclusions Estonian family doctors are favourably disposed to involvement in family-related problems, however, they need some additional training, especially in the field of relationship management.

Background There are significant differences in the way how primary health care is organised in Europe [ 1 ]. Estonia was one of the first Eastern European countries where modern general practice was implemented [ 2 ]. Previously, the primary health care system functioned according to the Soviet model, which was basically a specialist-oriented system [ 3 ]. In the 1990s, there occurred a transition to a more personal, comprehensive continuous care on the primary level. In 1991, the training of family doctors was launched, both 3-year postgraduate residence training, as well as the retraining of currently practising primary care physicians through attending different courses at the University of Tartu parallel with their everyday practice. The courses on family practice covered such topics as special features of family practice, common clinical problems in family practice, diagnostic strategies, teamwork, ethical issues, prevention and health promotion [ 3 ]. For the population, the most important change was the introduction of the patient's list system for FD's: persons choose their own FD by registering in a patient list. Currently, all primary health care physicians in Estonia are trained family doctors who are able to provide a wide scope of medical services for their patients and fulfilling gate-keeping function for specialized medical care. Irrespective of the health system, general practitioners/family doctors should care for individuals in the context of their family [ 1 ]. Family has a powerful influence on health and illness and family interventions have been shown to improve health outcomes for a variety of health problems [ 4 ]. However, there are differences in the physicians' involvement with families. Doherty and Baird have described five levels of physician involvement with families [ 5 ]. In Estonia, it has been aimed that a family doctor should work at least at level three, i.e. he or she has to communicate appropriate medical information and advice to family members, to be aware of gross family dysfunctions and to deal with the family members' feelings and concerns related to the condition of the patient [ 5 , 6 ]. It may differ in different countries what is expected and valued in general practice care [ 7 ]. What constitutes good medical care is determined culturally within a specific historical and geographic context [ 8 ]. In Estonia, a decade has passed after the new speciality, family doctor, was introduced into the health care system. The aim of the present study was to investigate the attitude of FDs in Estonia to family-oriented general practice [ 9 ]: FDs' awareness of various family-related matters of their patients, FDs' preparedness for management of family-related issues and FDs' self-assessment of the ability to manage different problems (substance abuse, relationship problems) in the family. Methods A 21-item questionnaire was designed for the study. The items were developed by researchers considering the aims of the study. First, the FDs were asked whether their patients have registered on the list by families or not and whether they regard it as appropriate that the family should be cared by one doctor, or whether they think that children should have a separate primary care physician. The FDs rated their opinion on the degree of involvement with various problems in the family: they should deal only with the treatment and counselling of a particular patient, or to cooperate also with family members, in addition to treatment of a particular patient, or they should deal also with emotional and relationship problems of the family members. The questions about FDs' awareness of various issues related to their patients' families such as familial diseases and diseases of family members, financial coping, relationships in the family, living conditions (overcrowding), drug addiction, alcohol abuse, smoking and leisure activities were inquired on a three-step scale : yes, in the case of each patient; yes, in certain cases; no, it is not necessary. FDs' were asked to self-estimate their ability to manage problems in families such as substance abuse and relationship problems. Questions about the FDs' ability to manage problems in families were open-ended, for example: how do you assess your possibilities as a FD to influence relationship problems in the family? If you assess that you cannot influence them, please specify why? If you assess you can influence them, please specify how? Also, the FDs were asked to estimate whether their professional training for dealing with the problems of families is adequate or inadequate. Several questions were related to sociodemographic characteristics (sex, age) and professional history (character and size of practices and length of service in primary health care). The questionnaire was piloted for clarity and relevance in a group of five FDs, and minor changes were introduced. In February 2002, the questionnaire was mailed to 236 FDs. a random sample of family doctors of Estonia. The random sample of FDs was formed by choosing the name of every 3 rd doctor, in alphabetical order, from the list of doctors who had passed residency or retraining courses in family medicine by that time in Estonia (n = 715). A note of reminder and a new questionnaire was sent to the non-responders 4 weeks after the first mailing. Of the 236 mailed questionnaires, 151 were returned after two mailings (64%), five of them were excluded as the respondents did not actually work as FDs. Thus, altogether 146 questionnaires were included in the study, of these 124 were fully completed, while in 22 cases some of the answers (1 to 3 per questionnaire) were missing. The data were analysed using SPSS for the Windows version 10. The chi-square test was used to test the differences in the proportions, all p-values calculated were two-tailed, the p-values higher than 0.05 were considered non-significant (NS). All open questions were analysed as follows: all statements expressing motivation for or indicating problems with dealing with family issues were marked. Further, all similar expressions were grouped under one category. Proceeding from this, the key problems relevant to the study were identified [ 10 ]. Results Respondents' characteristics The mean age of the respondents was 46 (± 8) years, the mean length of the period during which they had worked in primary health care was 18 (± 9) years and the mean size of the list was 1800 (± 513) patients. Of the respondents 55% worked in urban areas, 40% in rural areas, and 5% worked in both areas. The majority of the doctors (92%) were female. The age and sex distribution of the respondents and non-respondents did not differ significantly. Individual versus family registration in patient lists A total of 90 (62%) FDs were of the opinion that it was good to have the same FD for the whole family, 31 (21%) responded that it was preferable that every family member chooses a FD on the basis of personal preference and 25 (17%) thought that children should have a primary health care physician other than adults of the same family. Of the FDs, 119 (82%) responded that most patients were registered in their lists by families. The degree of involvement of FDs in family matters Of the respondents 15 (10%) were of the opinion that FDs should deal only with the health problems of concrete patients without involvement of family members, 94 (65%) responded that, besides managing the health problems of patients, FDs should communicate and cooperate with family members, and 36 (25%) thought that apart from the previously mentioned issues, FDs should deal with the family members' emotional and relationship problems. FDs' belief about the necessity for awareness of different family matters Over 70% of Estonian FDs agreed that in the case of all patients, it is necessary to be aware of drug addiction in the family, diseases of family members, living conditions and alcohol abuse in the family, while the remainder believed that they should be aware of these issues on certain occasions. Of the respondents, over a third thought that the FD should always be aware of relationships in the family and 12% thought that the FD should always be aware of leisure activities of their patients. However, between 60 to 75% believed that FDs should be aware of these issues in certain occasions (Figure 1 ). Very few FDs responded that patients had never actively sought FDs to discuss family relations (4 out of 143) or health risks in the family (5 out of 145), while 44 (30%) of the respondents stated that patients commonly addressed them to discuss family relations, and 34 (23%) reported that it was common to discuss the health risks associated with the familial diseases. Figure 1 Percentage distribution of the FDs' answers to the question: "Is it necessary to be aware of the following issues related to their patients' families?" Preparedness for management of family-related issues The respondents valued highly their preparedness to counsel for harmful habits: 104 (71%) of the respondents felt that their preparedness was adequate. Regarding the other issues, less than half of the respondents considered their training adequate (Table 1 ). Table 1 FDs' self-assessment of their preparedness for management of different family related issues Issues Preparedness adequate n (%) Preparedness inadequate n (%) Training in counselling for harmful habits 104 (71%) 42 (29%) Training in counselling for the health risks associated with hereditary diseases 63 (43%) 82 (57%) Training in relationships counselling 39 (27%) 106 (73%) p < 0.0001 FDs' self-assessement of the ability to manage different problems (substance abuse, relationship problems) in family Altogether 142 FDs responded to the question about their ability to reduce the use of harmful substances (alcohol, tobacco, drugs) in families. One hundred (70%) of the respondents reported that this was within the scope of their ability, while the majority (n = 71) stated that the methods used were advice and counselling, but also referral to specialists, use of specific medications and suggestions regarding appropriate reading material. However, 16 FDs admitted that the efficacy of their work in this field was low. Nearly one-third of the respondents (30%) estimated that they were not able to reduce the use of harmful substances in their patients' families. The analysis of the open-ended questions identifed some key problems: • Low motivation of patients. • Socio-economic reasons for substance abuse. • Limited time for consultation. • Inadequate preparedness for management of these issues. One hundred and forty-three FDs responded to the question about their influence on relationship problems in their patients' families, 83 (58%) of them were of the opinion that they were able to modify these problems. In most cases, FDs used advice and counselling (n = 52), but they also cooperated with specialists as the psychotherapist, family therapist, psychiatrist or social worker. Of the family doctors 60 (42%) thought that they were not able to influence the patients' relationship problems. In the analysis of the open questions, the doctors identified several key problems: • Limited time. • Lack of special training. • Patients do not address FDs with their problem. • Patients themselves deny the existence of the problem. • These are the patients' private issues in which physicians could not intervene. The FDs who were sure that they were able to modify the patients' harmful habits as well as family relationships were more likely to estimate their preparedness for the management of these issues as adequate. Among the doctors who reported that their preparedness for counselling for lifestyle issues was adequate, 76% (n = 76) believed that they were able to treat harmful habits, versus 57% (n = 24) of those who reported that their preparedness for such issues was not adequate (p < 0.05); in the case of relationship problems, the respective percentages were 41% (n = 34) versus 9% (n = 5) (p < 0.0001). There were found no other significant determinants among the sociodemographic or work related factors. Discussion The present study addressed the family doctors' opinions about their involvement in the patients' family issues. This is the first study of this kind conducted in Estonia, a country where family doctors were introduced into the health care system ten years ago. There is yet no definite agreement as to what are the appropriate, ideal or minimal levels of family orientation that family doctors should have [ 9 ]. Several studies have shown that the frequency of discussing family issues varies significantly [ 11 - 13 ]. The limitation of the study was that the response rate was quite low, 64%. However, the age and sex distribution of the respondents corresponds to that of the Estonian family doctors in general [ 14 ]. Our study revealed that care of patients in the context of the family is an important issue for FDs and that Estonian family doctors have good possibilities to take care of the whole family. Although all patients have the right to choose an individual family doctor, the FDs who responded to the questionnaire were sure that family members mostly have one and the same family doctor. This is concordant with the results of a recent survey among patients according to which 74% of the respondents reported that they had one and the same family physician for the whole family [ 15 ]. In Estonia, similar with the other Eastern Europe countries shift from separate pediatric and adult primary care system to family doctors system occurred in the 1990s [ 16 ]. Only 17% of the FDs in our study were of the opinion that children and adults should have different primary care doctors. In our study, altogether 90% of the family doctors thought that they should communicate and cooperate with family members in management of the health problems of patients. It is a good result considering that family medicine is a new and developing speciality in Estonia. At the same time, most family doctors are not yet ready to deal with the family members' emotional and relationship problems. Primary care is an important early intervention site of most serious relationship issues, domestic violence, etc [ 17 ]. However, studies conducted in other communities have identified that physicians need more continuing education concerning these topics [ 18 , 19 ]. Currently, a two-day course on domestic violence and child abuse, which are serious problems also in Estonia [ 20 ], is included in the postgraduate residence training curriculum. Concerning the family doctors' attitudes to the importance of awareness of their patients' family-related issues, the results indicated that the awareness of drug addiction, diseases of family members, living conditions of the family and alcohol problems in the family were considered the most essential. The awareness of the patients' relationship problems, economic problems and leisure activities was not so highly valued. A recent survey among patients in Estonia revealed that they were also more disposed to involve the family physician in such problems as harmful habits and diseases in the family, but they were less willing to share the relationship problems [ 15 ]. This can reflect the current situation in Estonia where the number of drug users as well as alcohol users has significantly increased during the last five years [ 21 ]. Lately, much attention has been paid to the problem by politicians, doctors and the mass media. The low willingness to be aware of the patients' relationship problems is partly related to insufficient preparedness in this field, as the doctors who considered themselves to be adequately prepared to tackle relationship issues were also more often willing to do this. From another point of view, family issues, especially relationships and economic situation, are always delicate topics and require consideration of the patient's attitude to corresponding activities. It has been shown that patients vary considerably in their preferences for physician inquiries into such problems as social functioning, psychosocial issues and health risks. Also, it may reflect cultural differences: in the Nordic countries biomedical talk is more common, while in the southern regions psychosocial dialogue is prevalent [ 22 ]. Although most of the family doctors agreed that modifying of the health damaging risk factors (smoking, alcohol and drug abuse) of their patients was their task, they also felt that management of these problems was ineffective, or they perceived themselves as poorly prepared, or had lack of time for such activities. This shows that despite the fact that family doctors are becoming increasingly more aware of their role, there exists the actual need to improve their instruments for handling lifestyle related and psychosocial problems. In practice, both individual and family–centered working methods are needed, while the choice depends on the patient's problems and needs [ 23 ]. Management of such issues requires development of new interviewing strategies and different ways to use the visit time more effectively [ 24 ]. Finnish experience shows that after completing an education programme, the family doctors' became more family-oriented and family doctors satisfaction with their work was also increased [ 25 ]. Conclusions The results of the present study allow to conclude that Estonian family practitioners are favourably disposed to involvement in family-related problems, but they need additional training especially in the field of relationship management. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors participated in the design of the study. MO and RK carried out the data collection, performed the data analyses and drafted the manuscript. All authors participated in the discussion of the drafts. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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212696

Neuroscience Networks

To study the brain from molecules to behaviour, neuroscientists face the challenge of communicating an emerging wealth of information in coherent accessible forms

The completion of the human genome project has ushered in a new era in which biology has become an information science. In this new era, sharing of information is quickly becoming a critical aspect of scientific discovery. As directors of National Institutes of Health (NIH) institutes dedicated to neuroscience, we recognize several areas of research where sharing of primary data will be necessary for us to reach our scientific goals, including brain-mapping, genetics, and clinical trials. Progress in each of these areas will require not only new tools for sharing information but a change in our scientific culture. Here we describe some of the recent progress in efforts to map the brain as an example of the potential and the challenge of sharing data in an era when neurobiology, like genomics, is becoming an information science. In parallel to the worldwide effort to map the human genome, investigators in neuroscience have used a range of techniques to map the brain. The efforts share some superficial similarities: the genome has 3 × 10 9 bases and the human brain has roughly 100 × 10 9 neurons; both the genome and the brain have embedded modules of functional units (genes versus circuits) that can be mapped in space; and localization of both genes and circuits requires computational power that can be distributed across laboratories. But the analogy breaks down quickly. Whereas fundamental genome data can be addressed as unidimensional text of four letters in varying order, a comprehensive map of the brain includes molecular, cellular, system, and behavioral data—all of which are dynamic, interacting, and interdependent. For example, brain circuitry is organized in three-dimensional space constantly changing in time, with each neuron having 10 3 –10 4 synapses and with many of those synapses capable of plasticity that may, in turn, have significant functional consequences. As we emerge from the “decade of the brain,” we are entering a decade for which data-sharing will be the currency for progress in neuroscience. As a testament to the complexity of brain data, a century after the classic age of neurohistology, there are continuing arguments about the taxonomy of neurons, depending on location, morphology, neurochemistry, or RNA profile. For instance, a population of neurons in a small region of the brain, the dorsal raphe, is the main source of the neurochemical serotonin that has been implicated in stress responsiveness and mood disorders. These serotonergic neurons can be subdivided according to rostralcaudal location, axon thickness, or projections ( Mamounas et al. 1991 ; Lowry 2002 ). However, what we recognize by immunochemical stain as a single shared phenotype in an anatomically distinct region may consist of a heterogeneous population of cells with diverse RNA profiles. In this sense, the strategy for brain-mapping might borrow a page from astronomy, with its maps of galaxies with mixed elements, as well as from the experience of the genome project. Indeed, advances in human brain-mapping, like discoveries in astronomy, have until recently largely depended on the tools available. The postmortem studies of the early 20th century provided delineation of cortical areas through light microscopic histology and gross connectional information. Neurochemical techniques in the last three decades yielded maps with cellular and subcellular resolution, identifying populations of cells usually by one or two neurochemical phenotypes. During the same period, electrophysiological approaches revealed the exquisite distribution of function across the brain, within particular brain subdivisions, and within neurons themselves. In the past two decades, direct study of the intact, functioning human brain in healthy and disordered states has been made possible by a variety of neuroimaging modalities. These studies have provided both structural and functional topography at increasing resolution, as well as neurochemical data and, most recently, information regarding neural connectivity ( Behrens et al. 2003 ). The advent of techniques for mapping RNA profiles now permits analysis of several thousand species of RNA even in a single neuron, resulting in exponential increases in information. As these approaches are combined with the experimental behavioral and clinical sciences, opportunities abound for understanding this complex organ and treating its pathologies. The challenge now is to integrate this information into a coherent, accessible form that permits hierarchical analysis from RNA to protein to morphology to connectivity to function in a universal language while preserving fidelity. While earlier comprehensive maps in simpler nervous systems, such as the classic lineage maps of invertebrates ( Stern and Fraser 2001 ), could be completed by single labs, more ambitious projects like a transcriptional map of the mouse brain, the Human Genome Project, and other goal-directed or large-scale research endeavors ( Nass and Stillman 2003 ) will require collaboration of scientists who add value to the enterprise by working in multidisciplinary teams; coordination of efforts to attain a goal; and computation through the use of informatics, models, and simulations. The keystone in this new paradigm is, of course, meaningful data-sharing. Several initiatives serving the brain and behavioral research communities are advancing cooperative research. The Gensat Project ( www.gensat.org ) will soon provide developmental and whole-brain maps of several hundred genes in the mouse nervous system using a bacterial artificial chromosome (BAC) transgenic strategy with fluorescent reporters to provide subcellular resolution. A digital atlas of the mouse brain and associated informatics tools have been developed to organize, visualize, and analyze such gene expression (and other spatial) data generated by researchers ( http://www.loni.ucla.edu/MAP/index.html ). We now have the capability to map the transcriptional expression of virtually the entire mouse genome in the adult and the developing mouse brain, registering these data to a common, digital atlas. Like the galaxy maps generated by the Hubble telescope, this transcriptional atlas will provide important temporal as well as spatial information, revealing genes that may be expressed only at critical stages of brain development. Similarly, the Human Brain Project ( http://www.nimh.nih.gov/neuroinformatics/index.cfm ) is an informatics effort funded through several federal agencies to develop databases, analytical and computational simulations, and other resources to assist human brain-mapping as well as other large-scale coordinated neuroscience programs. While there are several initiatives at NIH aimed at overcoming the informatics barriers to sharing data and facilitating collaboration, coordination, and computation, we recognize that not all of the impediments to data-sharing are technical. The advent of neurobiology as an information science also demonstrates that the academic culture in which our science develops and the publication culture in which our science is communicated will need to change. Promotion decisions at major universities largely depend on the quality and quantity of first-authored or senior-authored papers. Multidisciplinary studies require teams of investigators in which hierarchical schemes for authorship may fail to reflect accurately the magnitude of each individual's contributions. Similarly, contributions to a research database may represent important scientific and scholarly achievements, but generally are underrecognized by promotions committees counting peer-reviewed publications. Indeed, the nature of publication itself needs to change in an era when some of the most important contributions will emerge from comprehensive descriptions of new landscapes (analogous to new genomes and new galaxies) rather than tests of specific hypotheses. These cultural issues are not peculiar to brain and behavioral science, of course, and have recently been considered broadly at the NIH ( http://www.becon.nih.gov/symposium2003.htm ). Scientific publication, as we have known it in print, is slow and expensive, with access limited to those with either the funds to purchase an individual subscription or the proximity to a library with an institutional subscription. Data-sharing also means open-access publishing so that data, whether from mapping efforts or from hypothesis-driven experiments, become available quickly and freely to the scientific community. As we emerge from the “decade of the brain,” we are entering a decade for which data-sharing will be the currency for progress in neuroscience. Efforts driven by collaboration, coordination, and computation should yield the data, tools, and resources that neuroscientists will need in the coming decades. We hope that new electronic publications with open access will accelerate this change and provide the vehicle for disseminating the most exciting discoveries in neuroscience in a rapid, respected, and ready format. A Constellation of Neurons Image courtesy of Miles Herkenham.

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521173

Oxidative Stress Inactivates Cobalamin-Independent Methionine Synthase (MetE) in Escherichia coli

In nature, Escherichia coli are exposed to harsh and non-ideal growth environments—nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper ( Leichert and Jakob 2004 ), which provide evidence that MetE is one of the proteins in E. coli most susceptible to oxidation. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. Our studies suggest a simpler mechanism is employed by E. coli to achieve the same effect.

Introduction As a consequence of living in an aerobic world, organisms face the challenge of maintaining a favorable cellular redox status. Oxidative stress is caused by imbalances between the production and disposal of reactive oxygen species, which can damage proteins, lipids, and nucleic acids. Bacteria encounter reactive oxygen intermediates that are generated as byproducts of aerobic metabolism or during challenge by the immune systems of hosts. Thus, understanding the effects of oxidative stress on the cell, as well as elucidating cellular defense mechanisms, is of considerable interest. Over the past several decades, multiple observations in Escherichia coli have been reported that collectively suggest a link between oxidative stress, methionine limitation, and the enzyme that catalyzes the final step in methionine biosynthesis, cobalamin-independent methionine synthase (MetE). In E. coli, the sulfur of methionine comes from cysteine, which in turn obtains the sulfur group from sulfate via the sulfate assimilation pathway ( Figure 1 ). In catalyzing the formation of methionine, MetE lies at the intersection between the methyl cycle and the one-carbon pathway. Several studies involving cellular adaptations to stress conditions have reported unusual expression of MetE under stress conditions. Following the transition from anaerobic to aerobic growth in minimal medium, wild-type E. coli cells were found to rapidly induce MetE to a level comprising almost 5% of the total cellular protein ( Smith and Neidhardt 1983 ). Similarly, MetE was massively overexpressed in a temperature-sensitive mutant of groEL . GroEL is a heat-shock protein that is instrumental in helping to correctly fold cellular proteins. When the groEL strain growing in rich medium was shifted to the nonpermissive temperature, MetE was the major soluble protein synthesized ( Horwich et al. 1993 ). The additional inability of this strain to grow on minimal medium suggests that MetE may not be correctly functioning in these cells. Furthermore, Candida albicans MetE has been found to be heat and estrogen inducible, and is believed to play an important role in the yeast stress response and virulence ( Burt et al. 1999 ). The basis for the overproduction of MetE in all of these experiments has been unclear. Figure 1 Pathways of Methionine Biosynthesis and One-Carbon Metabolism in E. coli In addition, several independent researchers have reported an association between stress conditions and methionine limitation in E. coli . Early studies found that strains lacking the genes coding for the manganese and iron superoxide dismutases ( sodA and sodB, respectively) were sensitive to oxidative challenge and had an oxygen-dependent growth requirement for the sulfur-containing amino acids cysteine and methionine ( Carlioz and Touati 1986 ; Benov et al. 1996 ), which was attributed to leakage of sulfite from the cell. In trxA grxA (thioredoxin 1 and glutaredoxin 1) and grxA grxB grxC gor (glutaredoxins 1, 2, and 3 and glutathione reductase) strains of E. coli, cells required cysteine or methionine for growth, presumably because of oxidative inactivation of 3′-phosphoadenylylsulfate (PAPS) reductase ( Russel et al. 1990 ; Vlamis-Gardikas et al. 2002 ; Lillig et al. 2003 ). Similarly, the growth of E. coli was dramatically slowed following a shift to higher temperature, unless the medium was supplemented with methionine ( Ron and Davis 1971 ). This heat-induced methionine auxotrophy was traced to temperature sensitivity of homoserine transsuccinylase (MetA), the enzyme that catalyzes the first step in methionine biosynthesis ( Ron and Davis 1971 ; Ron and Shani 1971 ; Gur et al. 2002 ); however, MetE has also been subsequently identified as a thermolabile protein in heat-treated E. coli ( Mogk et al. 1999 ). It is intriguing that MetE was a major aggregation-prone protein at a temperature (45 °C) well below the melting temperature of the purified protein (55 °C) (E. Hondorp and R. Matthews, unpublished data). Aerobic heat shock appears to be associated with oxidative stress ( Benov and Fridovich 1995 ), suggesting that the aggregation of MetE might relate to oxidative stress, rather than heat per se. Though oxidative stress undoubtedly has multiple effects, taken together, these observations suggest that MetE may be inactivated by biological oxidants, thereby decreasing the production of methionine within the cell. Methionine biosynthesis involves several enzymes ( Figure 1 ); the final step is catalyzed by either cobalamin (B 12 )-independent methionine synthase (the metE gene product) or B 12 -dependent methionine synthase (the metH gene product). Both enzymes employ a catalytic zinc to transfer a methyl group from methyltetrahydrofolate to L -homocysteine to form methionine ( equation 1 ). While mammals have only the MetH homolog, organisms that do not synthesize or transport vitamin B 12 (e.g., yeast, fungi, and plants) use only the B 12 -independent isoform ( Matthews 1984 ). The E. coli genome contains both metE and metH but they are differentially expressed: functional MetH is only expressed in the presence of B 12 , which also serves to repress MetE expression. In the absence of exogenously supplied B 12 , MetE is the sole source of de novo methionine synthesis ( Greene 1996 ). In this study, we investigated the effects of oxidative stress on MetE and methionine availability within the cell. Transient oxidative stress conditions induced by diamide were found to elicit a methionine auxotrophy in E. coli growing in minimal medium, which was traced to an effect on MetE. In vitro experiments demonstrated that oxidized glutathione (GSSG) reversibly oxidizes MetE at a rate that correlates with enzyme inactivation. GSSG was found to specifically glutathionylate MetE and induce a conformational change. In vivo thiol-trapping experiments provide direct evidence that MetE is readily oxidized in cells experiencing a variety of oxidative stress conditions. Moreover, oxidation of MetE is associated with a cellular dependence upon methionine for growth, suggesting that oxidant-mediated inactivation of MetE may have a profound impact on the stressed cell. Results Methionine Becomes Limiting for Growth in Cells Subjected to Oxidative Stress Previous observations have suggested a link between oxidative stress and a growth requirement for methionine in E. coli . We investigated the duration of growth lags induced by oxidative stress in cells growing in glucose-minimal medium containing or lacking methionine. In the experiment shown in Figure 2 A and 2 B, oxidative stress was induced by addition of the thiol oxidant diamide, which rapidly penetrates cells and oxidizes intracellular thiols. Diamide treatment does not kill cells, but results in an abrupt arrest of growth for a length of time that is dependent on the initial concentration of diamide. Cells resume normal growth once the thiol status is restored ( Kosower and Kosower 1995 ). E. coli cultures logarithmically growing in glucose-minimal medium with and without methionine experienced a lag in growth upon challenge with diamide ( Figure 2 A). However, cells grown in medium containing methionine resumed growth significantly faster than those needing to synthesize methionine. Higher diamide concentrations increased the duration of the growth lag and augmented the effect of methionine on resumption of growth. Figure 2 Methionine Limitation during Oxidative Stress Induced by Diamide and H 2 O 2 (A) Cells of wild-type E. coli strain W3110 growing exponentially in glucose-minimal medium were diluted into the same medium with (filled circles) or without (open circles) 0.2 mM L -methionine. Cultures were allowed to grow to an OD 600 of approximately 0.5, and then diamide was added to a final concentration of 2 mM (red) or 3 mM (blue), and growth was monitored. (B) Cultures were grown as in (A), except that they were diluted into medium with (filled circles) or without (open circles) 0.2 mM L -homocysteine. At an OD 600 of approximately 0.5, diamide (final concentration, 2 mM) was added to one set of cultures (red). (C) Cells of wild-type E. coli strain W3110 were grown in the presence or absence of L -methionine as described in (A). At an OD 600 of approximately 0.2, H 2 O 2 was added to one set of cultures (green) to a final concentration of 3 mM (a concentration found to be high enough to induce a significant slowing of growth). (D) Cultures were grown as in (C) except that they were diluted into medium with (filled circles) or without (open circles) 0.2 mM L -homocysteine. Note the different time scales between (A,B) and (C,D). The biosynthesis of methionine involves several reactions (see Figure 1 ). If a step upstream of the reaction catalyzed by MetE were responsible for methionine limitation during diamide treatment, addition of homocysteine to the medium should decrease the growth lag imposed by diamide. No effect of homocysteine addition was seen on the duration of the growth lag in diamide-treated cells ( Figure 2 B), even though homocysteine can support the growth of cells that lack enzymes upstream of MetE in the biosynthetic pathway ( Urbanowski and Stauffer 1989 ). Similar results were obtained when hydrogen peroxide (H 2 O 2 ) was used to induce oxidative stress. Addition of methionine to the medium significantly decreased the growth lag produced by H 2 O 2 treatment ( Figure 2 C), while homocysteine was ineffective and even amplified the lag ( Figure 2 D). The mechanism by which homocysteine appears to have potentiated the H 2 O 2 stress is not known; however, it was clearly not protective. Taken together, the growth experiments suggest that methionine becomes limiting under oxidative stress, and that the bottleneck to de novo biosynthesis of methionine lies in the terminal step catalyzed by MetE. Inactivation of MetE In Vitro Is Correlated with Cysteine Oxidation The methionine auxotrophy observed under oxidative stress conditions could reflect an increased demand for methionine or a decreased rate of de novo synthesis due to inactivation of MetE. To distinguish between these possibilities, we assayed the activity of the purified enzyme upon challenge with GSSG ( Figure 3 ). Nearly complete loss of methionine synthase activity was observed following addition of GSSG. This process was readily reversible upon incubation with a reductant such as dithiothreitol (DTT) or reduced glutathione (GSH) (data not shown). Activity loss was correlated with thiol oxidation, as assessed by dithio-1,4-nitrobenzoic acid (DTNB) titration of the trichloroacetic acid (TCA)–precipitated protein. GSSG treatment led to thiol oxidation, which proceeded with approximately the same rate constant (0.06 min −1 ) as loss of activity (0.07 min −1 ). Figure 3 In Vitro Treatment of MetE with GSSG Leads to Loss of Activity and Cysteine Oxidation At time zero, GSSG (final concentration, 5 mM) was added to 50 μM MetE in 100 mM Tris chloride (pH 7.2). MetE activity was measured in samples removed at the indicated time points as described in the Materials and Methods . These assays are made over a 30-s time period and initiated by addition of MetE, so that oxidation of MetE by GSSG is minimal during the time required for measurement. Also, at the indicated times, an aliquot of the protein was precipitated with TCA, and the extent of cysteine oxidation was determined by DTNB titration. MetE activity loss (red) occurred with a rate constant of 0.07 min −1 , while cysteine oxidation (blue) occurred with a rate constant of 0.06 min −1 . While the rate of thiol oxidation could be measured with DTNB, the absolute number of cysteines oxidized was ambiguous. MetE contains seven cysteines, yet DTNB titration of the fully reduced enzyme gave values between 7.2 and 9.0 cysteines for different protein preparations, and oxidation of MetE led to an average decrease of 1.8 cysteines per mol. Thus it was important to use independent means to determine the number of cysteines oxidized by GSSG. Oxidation of MetE by GSSG would be expected to proceed by initial formation of a mixed disulfide between MetE and glutathione ( equation 2 ). An inter- or intramolecular disulfide bond could then be generated by attack on the mixed disulfide by a second cysteine residue. These two possibilities can be distinguished by using mass spectrometry to determine the molecular mass of the oxidized protein. The reduced protein was found to be 84,530 ± 6 Da (expected mass, 84,542, −0.01% deviation), whereas the mass of the protein oxidized by GSSG was 84,835 ± 8 Da. The mass difference of 305 ± 10 Da between oxidized and reduced MetE is consistent with the addition of a single glutathione adduct (expected mass, 306 Da). Formation of an intramolecular disulfide bond would only decrease the mass by 2 Da, which would not be detected by mass spectrometry in such a large protein. To obtain evidence for stoichiometric glutathionylation of MetE, the oxidized protein was reduced with DTT to release the glutathione adduct. The DTT and GSH were separated from the protein using a Microcon concentrator. The filtrate was assayed for GSH using fluorescamine, which reacts with primary amines to form a fluorescent product. The reduced protein did not release GSH (0.0 mol GSH per mol protein) while oxidized protein released 0.9 mol GSH per mol of protein. Thus both mass spectrometry and the fluorescamine assay were consistent with glutathionylation of MetE by GSSG. MetE Is Glutathionylated at Cysteine 645 In order to determine the site of oxidation and verify that an intramolecular disulfide was not being formed concomitant with oxidation, we performed disulfide mapping experiments based on the method of Wu and Watson (1997) . MetE was cyanylated with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under denaturing conditions at pH 3, and then cleaved immediately N-terminally to the cyanylated cysteines in 1 N ammonia, resulting in the formation of 2-iminothiazolidine-4-carboxyl (itz) peptides ( Figure 4 ). Cleavage will not occur at cysteines that have been protected from cyanylation by oxidation. Following cleavage, any disulfide bonds were reduced with DTT and the resulting peptide fingerprint analyzed by liquid chromatography mass spectrometry (LC-MS). Figure 4 Strategy for Mapping of Disulfide Bonds Formed during Oxidative Inactivation of MetE In Vitro Protein sulfhydryls are cyanylated with CDAP under denaturing, acidic conditions, and then cleaved N-terminally to each cyanylated cysteine with 1 N ammonia. This treatment results in an N-terminal fragment and a series of itz peptides. The mixture is then completely reduced with DTT and analyzed by LC-MS. Cleavage does not occur adjacent to cysteines that are oxidized. Thus sites of oxidation can be deduced from the mass fingerprint. This approach allows for relatively straightforward mapping of disulfide bonds. The acidic pH employed minimizes the possibility of disulfide bond scrambling. Furthermore, the ability to cleave N-terminally to each cyanylated cysteine allows cleavage between closely spaced, even adjacent, cysteines. These factors provide a significant advantage over traditional methods of peptide mapping, particularly for MetE, which contains cysteines spaced only two residues apart at the active site (cysteines 643 and 645). Moreover, for large proteins, most proteolytic and chemical cleavage methods have the potential to generate numerous peptides, whereas this approach produces a more limited number. LC-MS analysis of the cyanylated and cleaved reduced MetE allowed for identification of all but one of the expected peptides ( Figure 5 A; Table 1 ). High performance liquid chromatography (HPLC) peaks with masses corresponding to each of the expected peptides were observed, except for the dipeptide itz643–644, which is too small to be detected. Cyanylated cysteines can also undergo a β-elimination side reaction instead of cleavage. Susceptibility of cyanylated cysteines to β-elimination depends on the neighboring residues ( Wu and Watson 1998 ). In MetE, cysteine 726 readily formed the β-elimination product I, and β-elimination at cysteine 560 produced a small amount of product H . Identification of these side products provides additional evidence that cysteines 560 and 726 were reduced and available for cyanylation. Disulfide mapping of a mutant MetE, MetEC>A1–4, in which cysteines 323, 353, 516, and 560 were changed to alanines, further confirmed the mass assignments. Only peaks corresponding to cleavage next to the three remaining cysteines (643, 645, and 726) were observed ( Figure 5 B; Table 1 ). Figure 5 Disulfide Mapping of Reduced MetE Reduced MetE should be cleaved N-terminally to each of its seven cysteines to generate eight protein fragments as shown in the diagram, where peptide A comprises residues 2–322, B is itz323–352, C is itz353–515, D is itz516–559, E is itz560–642, F is itz645–725, and G is itz726–753. It is assumed that itz643–644 is too small to be resolved. β-elimination of cyanylated cysteines can also occur; H is itz516–642 with β-elimination at cysteine 560, and I is itz645–753 with β-elimination at cysteine 726. (A) The HPLC trace at 280 nm for disulfide mapping of fragments derived from cyanylation and cleavage of reduced MetE. Following derivitization, the samples were chromatographed on a C4 reversed-phase column as described in the Materials and Methods . Peaks were assigned from comparison of masses determined by mass spectrometry and predicted masses (see Table 1 ). (B) The HPLC trace for disulfide mapping of MetEC>A1–4 (green), which only contains cysteines 643, 645, and 726, overlaid with that of the wild-type reduced protein (blue). Peaks corresponding to ABCDE , G , and I were identified from the mass data (see Table 1 ). The mass for one peak could not be determined, but was assigned as F by comparison to the wild-type trace. Table 1 Mass Spectrometry Data for Disulfide Mapping Experiments The average mass obtained for each peak described in Figures 5 and 6 is listed. The β-elimination products were found to react with the low levels of acetic acid present in the LC-MS solvent to generate an acetate (Ac) adduct. Masses for both the β-elimination product and the Ac adduct were observed as indicated. Peak G consistently contains two unidentified fragments in addition to itz726–752 Disulfide mapping data for the oxidized protein following cyanylation, cleavage, and reduction with DTT ( Figure 6 A; Table 1 ), were similar to results for the reduced protein, suggesting that most of the cysteines in MetE remained reduced. Peptides A, B, C, D, E, and H provide evidence that cysteines 323, 353, 516, 560, and 643 were reduced. Peptides G and J indicate that cysteine 726 was reduced. The pattern is consistent with the initial oxidation occurring at cysteine 645 alone. Peptide J is the expected product obtained when MetE is initially oxidized at cysteine 645, with β-elimination at cysteine 726, and is then reduced. Figure 6 Disulfide Mapping of Oxidized MetE Reveals That Cysteine 645 Is Glutathionylated (A) LC-MS analysis of fragments generated by cyanylation, cleavage, and total reduction (see Figure 4 ) of GSSG-oxidized (red) and reduced (blue) MetE. For oxidized MetE, peaks corresponding to F and I were not found. Instead, a peak was observed with a mass corresponding to J, itz643–753, with one reduced uncyanylated cysteine and β-elimination at the other cysteine. As discussed in the text, β-elimination occurs preferentially at cysteine 726 in the reduced enzyme, and we infer that the reduced uncyanylated cysteine is cysteine 645. (B) LC-MS analysis of the cleavage products in (A) prior to DTT reduction (black) or following total reduction (red). Prior to DTT reduction, a peak is observed that has a mass corresponding to that expected for K, itz643–753, with a glutathione adduct at one cysteine and β-elimination at the other cysteine. As discussed in the text, β-elimination occurs preferentially at cysteine 726 in the reduced enzyme, and we infer that the glutathionylated cysteine is cysteine 645. The peak containing fragment J, the product of DTT reduction, is no longer apparent. The broad peak, indicated by the asterisk, appeared to contain a mixture of fragments with masses that could not be deconvoluted. These were attributed to disulfide-linked byproducts formed under the basic cleavage conditions prior to reduction with DTT. LC-MS analysis of the oxidized protein fragments prior to DTT reduction yielded an almost identical pattern to that obtained after reduction ( Figure 6 B; Table 1 ). However, a peptide, K, corresponding to itz643–753 with a glutathione adduct and β-elimination, was observed instead of J . It is not possible to distinguish which cysteine, 645 or 726, is glutathionylated and which has undergone β-elimination based simply on the masses of peptides J and K . However, the presence of peptide G in all traces at a level similar to that of reduced MetE indicates that cysteine 726 must be reduced. Moreover, the high susceptibility of cysteine 726 to undergo β-elimination (as evidenced by peak I in Figure 5 A)—contrasted with the complete lack of a β-elimination product at cysteine 645—argues that β-elimination occurs at cysteine 726 in the oxidized enzyme and glutathionylation occurs at cysteine 645. In order to confirm that cysteine 645 is the only site of oxidation, the inactivation of various MetE mutants was analyzed. MetEC>A1–4 contains only cysteine 645 and the zinc-binding ligands, cysteines 643 and 726; the latter ligands are required for catalytic activity. This mutant has approximately 90% of the activity of the native protein, and the kinetics of inactivation by GSSG are nearly identical to that of the wild-type protein ( Figure 7 A). However, mutation of cysteine 645 to alanine (MetEC>A5) dramatically altered the course of inactivation by GSSG. The slow partial loss of activity seen with MetEC>A5 may reflect weakening of zinc binding for the mutant enzyme, allowing glutathione to compete with MetE for zinc. Figure 7 Cysteine 645 Is Critical for Sensitivity of MetE to Oxidation (A) The activity of 50 μM wild-type MetE (black), MetEC>A1–4 (blue), and MetEC>A5 (red) was assayed following addition of GSSG to a final concentration of 5 mM. 100% activity refers to the specific activity of the fully reduced enzyme prior to GSSG addition (27 min −1 for wild-type, 24 min −1 for mutant). (B) The change in absorbance at 325 nm was used to monitor diamide reduction after addition of 500 μM diamide to buffer (light blue), 50 μM wild-type MetE (black), and 50 μM MetEC>A5 (red). (C) Scheme for the reaction of MetE with diamide. In the first step, attack of cysteine 645 on diamide forms an inactive but stable complex. In vivo , where GSH is present in high concentration, attack of GSH leads to the formation of MetE-S-SG and reduced diamide. Further evidence implicating cysteine 645 as the site of sensitivity to oxidation was obtained by monitoring the oxidation of MetE by diamide. The reaction of diamide with thiols can be followed at 325 nm, where diamide absorbs strongly, but the reduced product does not ( Kosower and Kosower 1995 ). Addition of MetE to diamide results in a rapid decrease in absorbance at 325 nm ( Figure 7 B). However, MetEC>A5 does not appear to be oxidized by diamide. This observation suggests that diamide oxidation of MetE in vitro involves an initial attack of cysteine 645 on diamide to form an inactive covalent complex ( Figure 7 C). In the presence of accessible thiols, such as the millimolar concentrations of GSH present in vivo, this complex can then readily react to form glutathionylated MetE and reduced diamide. Thus, although diamide oxidation does not result in glutathionylation of MetE in the absence of glutathione, this experiment demonstrates the extreme reactivity of cysteine 645 to oxidizing agents. Glutathionylation of MetE Induces a Conformational Change In order to further characterize the effects of GSSG oxidation on MetE, the reduced and oxidized forms of the protein were subjected to gel filtration. The elution of glutathionylated MetE was markedly shifted in comparison to that for the reduced protein ( Figure 8 A). The change in hydrodynamic radius seen upon oxidation suggested that GSSG oxidation had altered the conformation of MetE. Therefore, to confirm a conformational difference between reduced and oxidized MetE, we analyzed the two forms of the protein by circular dichroism (CD) spectroscopy and limited tryptic digestion. The similarity between the CD spectra of oxidized and reduced MetE suggests that the oxidized protein has not undergone a gross structural rearrangement and is still largely intact ( Figure 8 B). However, the deviation in the two spectra between 210 and 230 nm is consistent with a conformational change upon glutathionylation. Tryptic digestion of oxidized MetE appeared to occur with different kinetics compared to that of the reduced protein ( Figure 8 C). The oxidized protein was more stable against tryptic digestion than the reduced form, again consistent with a conformational change upon MetE oxidation. Figure 8 Glutathionylation of MetE Induces a Conformational Change (A) Gel filtration analysis of reduced (blue), GSSG-oxidized (red), or a mixture of reduced and GSSG-oxidized (black) MetE. (B) Far UV CD spectra of reduced (blue) and GSSG-oxidized (red) MetE. (C) SDS-PAGE analysis of a digestion of native MetE with 0.08% trypsin. The band corresponding to the molecular weight of the holo-protein is marked with an asterisk. (D) GroEL (2 μM) was incubated with 1 μM reduced (blue) or GSSG-oxidized (red) MetE and the proteins were then subjected to gel filtration. MetE has previously been shown to be an in vivo substrate of the chaperone GroEL ( Houry et al. 1999 ). Since oxidation of MetE was found to induce a conformational change, we tested whether GroEL bound preferentially to either form of MetE. GroEL and reduced MetE were incubated together and then injected onto a gel filtration column ( Figure 8 D). Separate peaks corresponding to GroEL and reduced MetE were observed, indicating that reduced MetE did not associate with GroEL. However, when the glutathionylated protein was incubated with GroEL, only the GroEL peak was observed. The GroEL complex is large enough (approximately 800 kDa) that binding of MetE would not be expected to shift the elution time significantly. Thus, the disappearance of the peak corresponding to oxidized MetE strongly suggests that GroEL specifically binds glutathionylated MetE, leading to coelution. The Equilibrium Constant for Glutathionylation of MetE Is Consistent with MetE Oxidation In Vivo To gain a better understanding of the thermodynamic sensitivity of MetE to oxidation, we measured the equilibrium constant for formation of the inactive mixed disulfide. MetE was incubated with varied ratios of GSH and GSSG until equilibrium was reached, and then activity was assayed ( Figure 9 A). The equilibrium constant K mix ( equation 2 ) was determined to be 1.4 and found to be independent of GSH concentration. If formation of an intramolecular disulfide bond were responsible for MetE inactivation, the equilibrium constant should be dependent on the GSH concentration ( Gilbert 1995 ). The independence of the equilibrium constant from GSH concentration provides further support that glutathionylation is the mechanism of inactivation. Figure 9 The Equilibrium Constant for MetE Oxidation Is Consistent with MetE Inactivation In Vivo (A) MetE was incubated with the indicated [GSH]/[GSSG] ratios at 1.1 mM (yellow), 2.3 mM (red), 4.6 mM (green), or 9.1 mM (blue) GSH until equilibrium was reached, as judged by a constant level of activity with time. MetE activity was then assayed to determine the relative amount of active reduced enzyme. The equilibrium constant, K mix (see equation 2 ) was determined to be 1.4 from the plot of relative MetE activity versus [GSH]/[GSSG]. This value was independent of the GSH concentration and dependent only on the [GSH]/[GSSG] ratio, consistent with glutathionylation as the mechanism of inactivation. (B) Cellular redox potentials are often referenced in terms of the GSH–GSSG couple, which depends upon [GSH] 2 /[GSSG]. Since inactivation of MetE occurs via glutathionylation (which is dependent on [GSH]/[GSSG]), a redox potential (which is dependent on [GSH] 2 /[GSSG]) for inactivation of MetE (by equilibrium titration with GSH/GSSG) cannot be determined. In order to provide a physiological context for the K mix determined in vitro, the equilibrium fraction of MetE expected to be active at different glutathione concentrations, 1 mM (red), 2 mM (green), and 5 mM (blue), was determined as a function of the cellular potential as described in the text. The regions designated “normal growth” and “oxidative stress” were inferred from published estimates of typical redox potentials in cells experiencing these conditions ( Gilbert 1990 ). In order to estimate the extent that MetE inactivation may be expected in vivo, both the redox potential, which is determined by the [GSH] 2 /[GSSG] ratio, and the total glutathione concentration in the cell ([GSH] + 2[GSSG]) need to be taken into account. Based on the equilibrium constant for GSSG oxidation, MetE activity at equilibrium was calculated for typical cellular glutathione concentrations (1–5 mM [ Kosower and Kosower 1978 ; Åslund et al. 1999 ]) at various potentials as determined by the GSH–GSSG redox couple ( Figure 9 B). (At each potential, E h , the ratio of [GSH] 2 /[GSSG] was calculated from the Nernst equation: where E o is –0.252 V at pH 7.2 (using a value of –0.24 V at pH 7.0 with an adjustment of –6.2 mV per 0.1 increase in pH [ Schafer and Buettner 2001 ]), R is 8.314 V·C/mol·K, T is 310 K, n is 2, and F is 96,485 C/mol. Based on a total glutathione concentration ([GSH] + 2[GSSG]) of 1, 2, or 5 mM, and the calculated [GSH] 2 /[GSSG], [GSH] and [GSSG] can be determined at each cellular potential. The ratio of [GSH]/[GSSG] may then be used to calculate the relative amount of MetE activity expected at equilibrium by where K mix is 1.4.) For E. coli growing under normal conditions (–250 mV to –280 mV [ Hwang et al. 1992 ]), more than 97% of the cellular MetE would be expected to be reduced and active. However, in a severely oxidizing environment (greater than –150 mV), inactive MetE may accumulate to an appreciable level. In comparison, Hsp33, which is a redox-regulated chaperone, has a midpoint potential of –170 mV ( Jakob et al. 1999 ). Therefore, thermodynamically, under conditions where Hsp33 is 50% active, MetE would be expected to be 29%–54% oxidized for glutathione concentrations of 1–5 mM. Although GSSG activates Hsp33 via formation of an intramolecular disulfide bond and inactivates MetE by glutathionylation, this suggests that both proteins may have similar sensitivities to oxidation. However, these calculations assume that the oxidation state of MetE is in equilibrium with GSH and GSSG. Deviations from theoretical values could occur if MetE were oxidized or reduced in vivo by a mechanism other than simple thiol-disulfide exchange. Comparison of the actual amount of oxidized MetE to measurements of the physiological concentrations of GSH and GSSG could therefore provide an indication of the in vivo mechanism of oxidation. Nevertheless, while MetE inactivation by GSSG may be energetically favorable under oxidizing conditions, the apparently slow kinetics make it difficult to conclude whether or not oxidation actually occurs in vivo. MetE Is Oxidized In Vivo during Oxidative Stress Thus, to determine whether our observations are physiologically relevant, we needed to develop a method whereby the oxidation of MetE could be directly observed in vivo. To this end, thiol-trapping experiments were performed as outlined in Figure 10 A. Cells growing under normal or oxidative conditions were briefly incubated with iodoacetamide, which is able to quickly penetrate the cell membrane and efficiently alkylate all available reactive thiols. The proteins were precipitated with TCA, and oxidized cysteines were reduced with DTT. The sulfhydryls exposed under these conditions were trapped with iodoacetic acid, which adds a negative charge to the protein for each cysteine trapped. Differently charged forms of the protein were separated by vertical slab isoelectric focusing, and MetE was visualized by immunoblotting. This method of analysis should readily distinguish between glutathionylated MetE (one additional negative charge as compared to reduced MetE) and MetE with an intramolecular disulfide bond (two additional negative charges as compared to reduced MetE). Glutathionylated MetE prepared in vitro is more acidic than fully reduced MetE, as indicated by comparing lanes 4 and 3 in Figure 10 B. Figure 10 In Vivo Thiol-Trapping Experiments (A) The strategy for in vivo thiol trapping involves brief incubation with iodoacetamide, which is able to efficiently alkylate all available thiols. Oxidized cysteines in the TCA-precipitated proteins are reduced, and the sulfhydryls exposed are then trapped with iodoacetic acid, which adds a negative charge. The two forms of the protein are separated by isoelectric focusing, and MetE is visualized by immunoblotting. Oxidized MetE results in a more acidic band on the gel than reduced MetE. (B) Thiol-trapping experiments were performed on a trxA gor strain (WP843) of E. coli (lane 2) as well as the isogenic wild-type strain (DHB4) (lane 1). The position of MetE on the gel may be compared with that of the reduced (lane 3) and GSSG-oxidized (lane 4) purified protein. In vivo thiol-trapping experiments were performed with logarithmically growing wild-type and trxA gor strains of E. coli . Strains mutant in genes for one of the thioredoxins (trxA) and glutathione reductase (gor) are impaired in their ability to reduce disulfide bonds, resulting in a highly oxidizing cytoplasmic environment ( Prinz et al. 1997 ). Comparison of the thiol status of MetE in a trxA gor mutant strain (WP843) with that in the isogenic wild-type strain (DHB4) revealed that approximately 50%–60% of the MetE in the trxA gor strain was oxidized in vivo ( Figure 10 B). Moreover, the shift in the isoelectric point for MetE that was oxidized in vivo is identical to the mobility of glutathionylated MetE prepared in vitro. E. coli strains containing mutations in trxB (which specifies thioredoxin reductase) and gor experience more severe disulfide stress than trxA gor strains ( Prinz et al. 1997 ). Cells require the presence of DTT in the medium to grow at rates comparable to wild-type strains; growth rapidly halts when DTT is removed. This strain cannot be grown in minimal medium, where wild-type MetE is expressed, so a trxB gor strain containing a plasmid specifying MetE (which expresses MetE independently of the cellular methionine concentration) was grown in Luria-Bertani (LB) medium containing DTT. Oxidized MetE was observed within 90 min of DTT removal, while MetE was fully reduced in the presence of DTT ( Figure 11 A and 11 B). Again, the shift in the isoelectric point of MetE oxidized in vivo was indistinguishable from that of glutathionylated MetE prepared in vitro. Together, these experiments provide evidence that MetE is glutathionylated during steady-state growth in two different strains of E. coli that are intrinsically oxidatively stressed. Figure 11 MetE Is Oxidized When E. coli Experience Oxidative Stress (A) Growth of a trxB gor strain (WP778) containing a plasmid expressing MetE was monitored by measurement of the OD 600 during growth in LB medium supplemented with 4 mM DTT (filled symbols). At an OD 600 of approximately 0.4, cells were filtered to remove the DTT, resuspended in fresh LB medium, and growth was monitored (open circles). (B) In vivo thiol-trapping experiments were performed before and after removal of DTT from the medium. Lanes 1 and 7 contain thiol-trapped samples of purified reduced MetE, and lanes 2 and 8 contain thiol-trapped samples of glutathionylated MetE prepared in vitro by treatment of MetE with GSSG. (C) Growth of wild-type E. coli (strain W3110) in glucose-minimal medium was monitored by the OD 600 with (filled circles) and without (open circles) the addition of diamide (at OD 600 approximately 0.5) to a final concentration of 0.9 mM. (D) In vivo thiol-trapping experiments were carried out to assess the oxidation state of MetE at various times following diamide addition. Lanes 1, 2, and 8 contain thiol-trapped samples of purified reduced and/or diamide-oxidized MetE. In order to establish a direct link between oxidative inactivation of MetE and the methionine auxotrophy observed during transient oxidative stresses, we performed thiol-trapping experiments on cells challenged with diamide. When wild-type E. coli cells growing exponentially in glucose-minimal medium were treated with 0.9 mM diamide, MetE was found to be completely oxidized during the lag in growth ( Figure 11 C and 11 D). Identical levels of reduced MetE reappeared within 60 min of the challenge, just prior to the resumption of normal cellular growth. Thus oxidation of MetE is associated with the cellular methionine limitation (described earlier) that is imposed by addition of diamide. Furthermore, these results are consistent with those of Leichert and Jakob (2004) , which independently show that MetE is rapidly oxidized in E. coli (wild-type strain DHB4) within 2 min of diamide treatment. Discussion Protein glutathionylation has been increasingly recognized as an important mode of regulation in eukaryotes; however, in E. coli, only the activities of OxyR and PAPS reductase have been reported to be modulated by formation of mixed disulfides with glutathione ( Kim et al. 2002 ; Lillig et al. 2003 ; Potamitou Fernandes and Holmgren 2004 ). In this study, we have expanded this list by clearly demonstrating that E. coli MetE is also inactivated by glutathionylation in a manner that is stoichiometric and specific for cysteine 645. Moreover, oxidation of MetE was directly observed in vivo concomitant with a methionine auxotrophy induced by oxidative stress. In general, protein glutathionylation may occur by two different mechanisms. Proteins can undergo thiol-disulfide exchange with GSSG to generate the glutathionylated product (K mix equilibrium in equation 2 ). Alternatively, either cysteine residues on the protein or the GSH sulfhydryl may be oxidized to a more reactive intermediate, such as a sulfenic acid, followed by formation of the mixed disulfide. In vivo glutathionylation of proteins via thiol-disulfide exchange is dependent on changes in the [GSH]/[GSSG] ratio, while formation of a reactive intermediate would result in glutathionylation independent of the [GSH]/[GSSG] ratio. Alterations in [GSH]/[GSSG] ratios have been associated with increased protein-glutathione mixed disulfides ( Chai et al. 1994a ); however, there are also reports of extensive glutathionylation occurring in vivo without a measurable change in GSSG concentration or total glutathione levels ( Chai et al. 1994a , 1994b ). This has been taken as evidence to support a mechanism involving a reactive sulfhydryl intermediate ( Thomas et al. 1995 ). The ratios of [GSH] to [GSSG] in wild-type (DHB4) and trxA gor strains (FÅ378) growing in LB medium were previously reported to be 223 ± 35 and 18 ± 7, respectively ( Åslund et al. 1999 ). Though our experiments were performed with E. coli growing in minimal medium, assuming that the [GSH]/[GSSG] ratios are comparable, more than 99% of the MetE in the wild-type strain (DHB4) should be reduced. Similarly, based on the equilibrium constant for thiol-disulfide exchange with MetE, only approximately 7% of the MetE is expected to be oxidized in the trxA gor strain (WP843). Yet 50%–60% of the MetE was found to be oxidized in this strain by thiol-trapping experiments. A [GSH]/[GSSG] ratio of less than 1.4 would be required to accumulate that much oxidized MetE (which corresponds to a redox potential above –174 for 5 mM glutathione and above –152 mV for 1 mM glutathione). Moreover, in vitro oxidation of MetE by GSSG is rather slow, further suggesting that glutathionylation of MetE via thiol-disulfide exchange is not the primary mechanism functioning in vivo. Rather, the in vitro and in vivo data for MetE are consistent with initial formation of a reactive intermediate followed by glutathionylation of cysteine 645, resulting in enzyme inactivation. Our results are complementary to the observations of Leichert and Jakob (2004) that MetE is one of the proteins most susceptible to oxidation in a trxA strain. Since E. coli lacking trxA do not experience a substantial amount of cytosolic disulfide bond formation ( Prinz et al. 1997 ), the appearance of oxidized MetE within this environment delineates the extreme sensitivity of the protein to oxidation. The concurrent presence of oxidized peroxide-detoxifying enzymes (alkylhydroperoxide reductase [AhpC] and thioredoxin-linked thiolperoxidase [Tpx]) suggests that endogenously produced reactive oxygen species (e.g., H 2 O 2 ) accumulate in the trxA strain, since thioredoxin appears to be required to regenerate the reduced thiol status of these proteins. Our preliminary in vitro experiments indicate that glutathionylated MetE is inefficiently reduced by thioredoxin (data not shown), which is not unexpected since glutaredoxins are generally more specific for glutathione-protein adducts ( Potamitou Fernandes and Holmgren 2004 ). Thus, taken together, our study and that of Leichert and Jakob (2004) suggest that the oxidation of MetE observed in trxA strains is mediated by elevated reactive oxygen species, perhaps due to an overwhelmed glutaredoxin system. Leichert and Jakob also observed significant oxidation of MetE in wild-type cells treated with H 2 O 2 , further supporting the premise that MetE is acutely vulnerable to reactive oxygen species. Protein glutathionylation has been suggested to play a protective role in oxidative stress. Studies with purified eukaryotic proteins have shown that incubation with oxidants (e.g., H 2 O 2 ) results in irreversible modification, whereas oxidation in the presence of GSH (as would be expected in vivo) yielded reversible glutathionylation ( Klatt and Lamas 2000 ; Hamann et al. 2002 ; Mallis et al. 2002 ). Similarly, we found that addition of an excess of H 2 O 2 to purified MetE appeared to cause irreversible oxidation, presumably because of the formation of sulfinic (–SO 2 ) or sulfonic (–SO 3 ) acid adducts (data not shown). Thus, glutathionylation of MetE following the formation of a reactive intermediate may serve the dual purpose of modulating enzyme activity while protecting the active site from more extensive damage. Reduction by cellular thioreductases could then readily reactivate the enzyme. Our data strongly suggest that glutathionylation is reversible in vivo as well as in vitro. Upon the addition of diamide to cells growing in medium lacking methionine, growth is abruptly halted and cells become limited for methionine. In the experiments shown in Figure 11 C and 11 D, all of the MetE in the cell is oxidized at the 15- and 30-min time points, whereas it is completely reduced after 60 min, just prior to the resumption of growth. Within this time period, the concentration of MetE within the cell does not appear to change, based on the intensity of the band on the immunoblot. In particular, there is no decrease in intensity of the band corresponding to oxidized enzyme between 15 and 30 min, suggesting that MetE is not being degraded. While a lack of change could indicate a balance between new protein synthesis and degradation, we argue that cells that are not growing and are limited for methionine should have a decreased rate of protein synthesis. If new synthesis of MetE were responsible for the reappearance of reduced MetE, all of the oxidized MetE (estimated at 1% of the total protein or 60 μM MetE) must be degraded and new protein synthesized to the same level between 30 and 60 min. Thus, although we cannot definitively rule out the possibility that the reappearance of reduced MetE is due to new protein synthesis, we feel that it is far more likely that MetE is re-reduced once the diamide in the cell is dissipated. Proteins containing mixed disulfides with glutathione are typically reduced by one of the three glutaredoxin isoforms. The glutaredoxins Grx1 (grxA) and Grx3 (grxC) are structurally distinct from Grx2 (grxB), yet all three proteins are capable of reducing glutathionylated PAPS reductase ( Lillig et al. 2003 ). The levels of Grx2 and Grx3 in E. coli are considerably higher than that of Grx1; however, the three proteins employ different modes of regulation. Grx3 concentrations are relatively constant, whereas OxyR induces Grx1, and levels of Grx2 are growth phase dependent. Grx2 has the highest catalytic activity of the isoforms and has been estimated to contribute up to 80% of the total glutaredoxin activity ( Potamitou Fernandes and Holmgren 2004 ). Since glutaredoxins catalyze the reduction of proteins with high efficiency and specificity, it will be of interest to determine the precise protein or proteins that are responsible for reduction and reactivation of oxidized MetE in vivo. Based on a recent crystal structure of the Thermatoga maritima MetE homolog, cysteine 645 is located at the entrance to the active site (R. Pejchal and M. Ludwig, personal communication). This places the sulfhydryl in direct contact with the solvent, where it is accessible to oxidation and reduction. Glutathionylation of cysteine 645 at the entrance to the active site may sterically prevent substrate binding, leading to the observed inactivation of the enzyme. Addition of the glutathione adduct also appears to correlate with a weakened binding of the catalytic zinc to the enzyme (data not shown). The active site of MetE is located in a cleft formed between two domains (R. Pejchal and M. Ludwig, personal communication). Addition of the glutathione tripeptide to cysteine 645 may provide enough bulk to push the two domains apart, giving rise to the observed conformational change and increased hydrodynamic radius. CD spectroscopy indicates that oxidation does not result in gross unfolding of MetE (see Figure 8 B), yet GroEL was found to bind only to the glutathionylated protein, and not to reduced MetE. Separation of the domains upon addition of glutathione could expose hydrophobic surfaces that allow GroEL to recognize and bind to one of the domains, since the holoenzyme (84.5 kDa) is too large to be accommodated within the cavity formed by the GroEL–GroES complex ( Xu et al. 1997 ). Earlier observations identified MetE as an in vivo substrate for GroEL ( Houry et al. 1999 ); here, we showed that GroEL specifically binds to the glutathionylated protein. GroEL binding may be required to present the enzyme for reduction, as well as to prevent oxidized enzyme from aggregating. Alternatively, binding to GroEL could target the inactive enzyme for eventual degradation. Oxidant-mediated inactivation of MetE may have broad implications for the cell. Initiation of translation in E. coli requires formylated methionine, and it has been suggested that depletion of methionine and one-carbon pools could block protein translation ( Gold 1988 ). We postulate that the development of methionine auxotrophy via inactivation of MetE may therefore protect stressed cells by slowing the initiation of protein translation. This could safeguard cells from rapid synthesis of peptides under adverse conditions, allowing cellular processes to attend to managing and detoxifying the stress. Interestingly, the conditions that induce a heat shock response in E. coli also affect the translational capacity of the cell, implicating the rate of protein synthesis in triggering of this stress response ( VanBogelen and Neidhardt 1990 ). Studies with rat hepatocytes provide support for a role of protein glutathionylation in the control of protein synthesis under oxidative stress conditions. Upon the addition of t -butyl hydroperoxide, rapid inhibition of protein translation was observed, accompanied by an increase in the levels of protein mixed disulfides. It was hypothesized that glutathionylation of key proteins involved in protein synthesis was responsible for the inhibition ( Latour et al. 1999 ). Furthermore, peroxide stress has been found to result in reversible inhibition of protein synthesis in Saccharomyces cerevisiae ( Shenton and Grant 2003 ). This effect was irreversible in cells that lack glutathione, suggesting that protein glutathionylation may protect the cellular translational capacity from irreversible damage. Thus, mechanisms for downregulating the rate of protein translation may be employed by a wide spectrum of organisms experiencing oxidative stress. The methionine limitation imposed by oxidative stresses could also play a significant role in bacterial quorum sensing. S -adenosylmethionine, which is directly formed from methionine, is the precursor to autoinducer AI-2 (see Figure 1 ) ( Chen et al. 2002 ). A growing body of evidence suggests that AI-2 communicates the metabolic state and growth potential of the cell rather than simply providing a density-dependent signal ( DeLisa and Bentley 2002 ; Xavier and Bassler 2003 ). Moreover, a shift in the metabolic activity of the cell is postulated to be responsible for modulation of AI-2 signaling in response to a host of environmental stresses ( DeLisa et al. 2001a , 2001b ). Aerobiosis, H 2 O 2 treatment, and heat shock were all found to result in decreased AI-2 levels, while addition of DTT or glucose served to increase the accumulation of AI-2 ( DeLisa et al. 2001b ). Oxidant-mediated inactivation of MetE imposes a methionine limitation, which would then lower the availability of substrate for AI-2 production. Decreased levels of AI-2 in response to methionine-limiting conditions could provide a simple means of communicating the cellular metabolic potential under stress conditions, thereby helping cells to adapt to a non-ideal or even hostile environment. In conclusion, we have established that MetE, a highly expressed protein in E. coli, is inactivated by glutathionylation of cysteine 645 in vitro. Our results, coupled with the findings of Leichert and Jakob (2004) , demonstrate oxidation of MetE in E. coli undergoing oxidative stress, as evidenced by high levels of the oxidized protein in three intrinsically stressed strains (trxA, trxA gor, and trxB gor) as well as wild-type cells challenged with diamide or H 2 O 2 . Our results further show that oxidation of MetE is associated with a methionine limitation imposed by oxidative stress. Hence this study provides insight into a previously unknown, but important aspect of the E. coli cellular response to oxidative stress. Materials and Methods Materials. Electrophoresis-grade urea, Chaps, ampholytes (pH 4–8), ammonium persulfate, TEMED, 10 N sodium hydroxide, and 85% phosophoric acid were obtained from Genomic Solutions (Ann Arbor, Michigan, United States). Iodoacetate and iodoacetamide were purchased from Fluka (Sigma-Aldrich, St. Louis, Missouri, United States). ( 6S )-5-methyltetrahydropteroyltriglutamate was synthesized from pteroyltriglutamate (Schircks Laboratories, Jona, Switzerland), as described previously ( Matthews 1986 ). L -Homocysteine was prepared by hydrolysis of L -homocysteine thiolactone ( Drummond et al. 1995 ). Polyclonal rabbit antibodies to purified wild-type MetE were generated by Lampire Biological Laboratories (Pipersville, Pennsylvania, United States). All other chemicals were obtained from Sigma (St. Louis, Missouri, United States). GroEL was generously provided by Z. Xu (University of Michigan, Ann Arbor, Michigan, United States). Bacterial strains and plasmids. Wild-type K-12 strain W3110 was obtained from F. C. Neidhardt (University of Michigan, Ann Arbor, Michigan, United States). Wild-type E. coli K-12 strain DHB4 and isogenic E. coli K-12 strains WP843 (trxA gor) and WP778 (trxB gor) ( Prinz et al. 1997 ) were obtained from U. Jakob (University of Michigan). Overexpression of mutant MetE proteins was performed in E. coli K-12 strain GW2531 (metE) ( Mulligan et al. 1982 ). The MetE mutants MetEC>A5 and MetEC>A1–4 were constructed by overlap extension PCR ( Ge and Rudolph 1997 ) using Pfu Turbo (Strategene, La Jolla, California, United States). Plasmid pJG816 ( González et al. 1992 ), specifying wild-type MetE, was used as the template, and the primers used for overlap extension are shown in Table 2 . The products were purified by gel electrophoresis and ligated into alkaline phosphatase–treated pJG816. Plasmids containing the mutant genes were isolated after transformation into competent XL1-blue cells by electroporation. Single Cys→Ala mutations of Cys323 (pEM1), Cys353 (pEM2), Cys560 (pEM4), and Cys645 (pEM5) were constructed by overlap extension using pJG816 as the template. For the construction of pEM3 (Cys353Ala, Cys516Ala), pEM2 was used as the template instead of pJG816. For construction of pEM1, pEM2, pEM3, and pEM4, the purified products and pJG816 were digested with BssHII and StuI, while for pEM5, AatII and BssHII were used. MetEC>A1–4 was constructed by subcloning of fragments containing the individual mutations from the appropriate plasmids. Plasmids pEM1 and pEM3 were digested with MluI; the fragment containing the Cys323Ala mutation from pEM1 was ligated into the alkaline phosphatase–treated pEM3 vector to generate pEM6. Similarly, pEM6 was digested with AgeI and BssHII and then ligated into pEM4 that had been digested with the same enzymes to generate pEM7. Plasmids containing pEM5 (MetEC>A5) and pEM7 (MetEC>A1–4) were transformed into E. coli strain GW2531. Mutations were confirmed by DNA sequencing of the entire metE gene. Table 2 Sequences of Primers Used in the Construction of MetE Mutants a A pair of complementary primers was used for each mutant. Only the sequences of the A primers are shown. Primers nB are the reverse complement of primers nA for each mutation, n = 1 to 5. Changes in the sequence are underlined b Nucleotide number is assigned according to the GenBank sequence M87625 of metE . Translation begins at base pair 1 in the sequence Expression and purification of wild-type and mutant MetE proteins. Wild-type MetE was expressed using strain GW2531/pJG816, MetEC>A5 was expressed using strain GW2531/pEM5, and MetEC>A1–4 was expressed from strain GW2531/pEM7. Six 1-l portions of LB medium supplemented with 100 μg/ml ampicillin and 0.5 mM zinc sulfate were inoculated with 3 ml of an exponentially growing culture in the same medium (OD 600 ∼1). Cultures were incubated at 37 °C with shaking at 250 rpm for approximately 30 h before harvesting. The proteins were purified as described previously ( González et al. 1992 ), except that an 800-ml gradient from 100 to 400 mM potassium phosphate (pH 7.2) was used. Purified proteins were dialyzed into 50 mM potassium phosphate buffer (pH 7.2) containing 500 μM DTT, concentrated, and stored at –80 °C. Culture conditions For examining the effect of oxidative stress in minimal medium, cultures were grown aerobically at 37 °C in a rotary water bath shaker in glucose-minimal morpholinopropane sulfonate (MOPS) medium ( Neidhardt et al. 1974 ) supplemented with 10 μM thiamine. Exponentially growing cultures in the same medium were diluted to an OD 600 of approximately 0.02–0.04. Where diamide was added to cultures, the concentration of the diamide stock was determined spectrophotometrically (ɛ 296 = 3,000 M −1 cm −1 ) ( Kosower and Kosower 1995 ). H 2 O 2 concentrations were determined similarly (ɛ 240 = 43.6 M −1 cm −1 ) ( Hildebrandt and Roots 1975 ; Poosch and Yamazaki 1986 ). Protein concentration determination and activity assay. The molar absorption coefficient for MetE at 280 nm was determined to be 157,000 ± 1000 M −1 cm −1 using the Endelhoch method ( Pace et al. 1995 ). The specific activity of MetE was measured using the modified enzyme assay previously described ( González et al. 1996 ), but omitting DTT from the assay mix. Exclusion of DTT did not affect the measured activity. For experiments monitoring enzyme inactivation, aliquots containing 650 pmol of MetE were withdrawn from the oxidation reaction and mixed with activity assay reagents which had been pre-equilibrated to 37 °C. The final assay mixture contained 10 mM potassium phosphate buffer (pH 7.2), 50 mM Tris chloride (pH 7.2), 100 μM magnesium sulfate, 2 mM L -homocysteine, 66 μM (6S) -5-methyltetrahydropteroyltriglutamate, and enzyme in a total volume of 400 μl. After a 30-s incubation at 37 °C, the reaction was quenched by addition of 100 μl of 5 N hydrochloric acid/60% formic acid, followed by heating at 88 °C for 10 min. After cooling on ice, the reaction was centrifuged at 14,000 g for 5 min at 4 °C to remove precipitated protein. The amount of tetrahydrofolate product (converted to methenyltetrahydrofolate by formic acid) was quantitated by its absorbance at 350 nm. Excess GSSG used to oxidize the enzyme did not significantly alter activity during the 30-s assay. Oxidant effects on cysteine oxidation and enzyme activity. MetE (50 μM) in 100 mM Tris chloride (pH 7.2) was equilibrated to 37 °C, and GSSG was added (final concentration, 5 mM) to initiate the oxidation reaction. The GSSG concentration was established by assay with glutathione reductase ( Zander et al. 1998 ). At time points, aliquots were removed for measurement of cysteine oxidation or enzyme activity (described above). To determine the extent of oxidation, aliquots (100 μl) of the reaction mixture were removed and quenched with 100 μl of ice-cold 20% TCA (w/v). Samples were incubated on ice at least 30 min and then pelleted by centrifugation at 14,000 g for 30 min at 4 °C. The supernatant was removed, and the pellet was dissolved in 100 mM potassium phosphate buffer (pH 7.3) containing 0.25 mM DTNB, 6 M guanidinium chloride, and 1 mM EDTA. Complete dissolution of the pellet required vortexing for 15–20 min. Reduced cysteines were quantitated by measuring the absorbance at 412 nm after subtraction of a blank containing DTNB and buffer but no protein. The extent of oxidation was determined using the fraction of reduced cysteines at the indicated time compared to the value at time zero (taken as 100%). Preparation of oxidized MetE. Purified MetE was desalted into 100 mM Tris chloride (pH 7.2) to remove DTT from the storage buffer. The reduced protein (50 μM) was then incubated with 10 mM GSSG for 90 min at 37 °C. Samples were concentrated using Microcon concentrators (Millipore, Billerica, Massachusetts, United States), and GSSG was removed using Bio-Gel P-6 columns (BioRad, Hercules, California, United States) equilibrated with 100 mM Tris chloride (pH 7.2). Reduction and reactivation of GSSG-oxidized MetE was accomplished by incubation of the oxidized enzyme with 2 mM reductant at 37 °C. For preparation of samples of diamide-oxidized MetE to be used as standards for in vivo thiol-trapping assays, diamide (1 mM) was incubated with MetE (10 μM) in 100 mM Tris chloride (pH 7.2) at 37 °C. After 45 min, diamide was removed by gel filtration using Bio-Gel P-6 columns. Fluorescamine assay for glutathionylation of MetE. Reduced and GSSG-oxidized MetE preparations were exchanged into 50 mM potassium phosphate (pH 7.2) using Bio-Gel P-6 columns. Proteins were then washed three times with the same buffer using Centricon concentrators. MetE (50 μM) was incubated with 2 mM DTT in 50 mM potassium phosphate (pH 7.2) in a volume of 200 μl for 1 h at 37 °C. Each solution was then loaded into a Microcon concentrator and centrifuged at 12,000 g for 5 min at room temperature. Aliquots (100 μl) of the filtrate were added to 1.7 ml of 200 mM borate buffer (pH 9.0) containing 1 mM p -(hydroxymercuri)benzoic acid. While vortexing, 0.6 ml of 1 mg/ml fluorescamine (in acetone) was added. The fluorescence emission of the samples was measured at 485 nm with excitation at 390 nm. A range of GSH concentrations (0–125 mM) was assayed to generate a standard curve. GSH concentrations were determined by DTNB titration ( Riddles et al. 1983 ). The stoichiometry of glutathionylation was calculated by subtracting the results obtained in the absence of DTT from the amount of glutathione released by incubation with DTT and dividing by the enzyme concentration (50 μM). Each form of MetE was assayed in duplicate from two separate preparations. Preparation of disulfide mapping samples. CDAP dissolved in 200 μl of 100 mM citrate buffer (pH 3) containing 6 M guanidine hydrochloride was added to 100 μl of MetE in 100 mM Tris chloride (pH 7.2) and incubated for 15–20 min at room temperature. The stock concentrations were adjusted so that CDAP was approximately 25-fold in excess over the total thiol concentration. CDAP was removed from the solution by gel filtration, using Bio-Gel P-6 columns equilibrated with 6 M guanidine hydrochloride solution (brought to pH ∼7–8 with potassium hydroxide). Concentrated ammonium hydroxide was added (final concentration 1 M [pH ∼12]), and the reaction was incubated at room temperature for approximately 75 min. Samples were then lyophilized to remove the ammonia and stored at –80 °C. Prior to LC-MS analysis, DTT was added (final concentration was approximately 200 times the concentration of MetE) and allowed to incubate at room temperature for 30 min. Solutions were then diluted into 6 M guanidine hydrochloride solution (adjusted to pH 3 with acetic acid) and characterized by LC-MS. LC-MS analysis. Samples (250–500 pmol) were injected onto a Vydac (Hesperia, California, United States) 250 mm × 2.1 mm C4 column (214MS52) using an analytical HPLC Surveyor from ThermoFinnigan (San Jose, California, United States). The column was equilibrated with 97% solvent A (water with 0.1% v/v acetic acid and 0.02% v/v TFA) and 3% solvent B (acetonitrile with 0.1% v/v acetic acid and 0.02% v/v TFA), and samples were eluted at a flow rate of 0.25 ml/min with a three-step linear gradient: 3% B for 15 min, 3% to 30% B in 9 min, and 30% to 65% B in 35 min. For LC-MS of the holo-protein, samples were desalted using a two-step linear gradient: 20% B for 10 min, and 20% to 70% B in 25 min. Fractionation was monitored by the UV chromatogram recorded at 280 nm. The eluent was diverted to waste for the first 15 min, and then directly infused into the electrospray ionization source of a ThermoFinnigan LCQ mass spectrometer. Mass spectral data were obtained in the positive mode, and ESI spectra were deconvoluted using the BioMass software provided by ThermoFinnigan. Diamide oxidation of purified MetE. Oxidation of MetE by diamide was monitored at 37 °C using a Hi-Tech Scientific (Salisbury, United Kingdom) SF-61DX2 stopped-flow spectrophotometer equipped with a xenon light source for single wavelength detection. Diamide (1 mM) and MetE (100 μM) in 100 mM Tris chloride (pH 7.2) were mixed, and reduction of diamide was monitored at 325 nm. Size exclusion chromatography of oxidized and reduced MetE and interaction with GroEL. MetE (20 pmol) was injected onto an Alltech (Lexington, Kentucky, United States) macrosphere size exclusion HPLC column (250 mm × 4.6 mm) with a 300-Å pore size (7-μm particle size) and run at 0.5 ml/min with 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride. For GroEL interaction experiments, GroEL (2 μM) was incubated with MetE (1 μM) in 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride for 30 min at 37 °C before analysis by size exclusion chromatography. CD measurements. Reduced and oxidized MetE were buffer exchanged into 10 mM potassium phosphate buffer (plus 500 μM DTT for the reduced protein) (pH 7.2). The final filtrates were collected and used as a blank. Samples were centrifuged for 1 h at 14,000 g at 4 °C and then diluted to 0.4 mg/ml. Far UV CD spectra were recorded using a JASCO (Easton, Maryland, United States) J-810 spectropolarimeter and 0.1 cm cuvettes. Absorbance spectra of the same sample were recorded on a JASCO V-550 UV/Vis spectrophotometer to determine the precise protein concentration. The data were then normalized to a protein concentration of 4.5 μM MetE. Limited tryptic digestion. Aliquots containing 2.5 mg/ml reduced or oxidized MetE in 50 mM potassium phosphate (pH 7.2) were digested with 0.02% (w/v) trypsin at room temperature. At indicated times, 5-μl samples were quenched with 10 μg of 1-tosylamido-1-lysyl chloromethylketone, and then analyzed by SDS-PAGE (10% gel). Determination of the equilibrium constant for oxidation of MetE by GSSG. MetE (10 μM) was incubated with varying concentrations of GSH and GSSG in 100 mM Tris chloride (pH 7.2) at 37 °C. GSH ( Riddles et al. 1983 ) and GSSG ( Zander et al. 1998 ) stock concentrations were measured as described. After 5.5 h, aliquots were removed and MetE activity was assayed. Since A 350 directly correlates to the amount of product in the activity assay, relative MetE activity was calculated by: where A 350 (x) is the absorbance at 350 nm for sample x, A 350 (oxid) is for completely oxidized MetE (2 mM GSSG, no GSH), and A 350 (red) is for completely reduced MetE (2 mM GSH, 4 μM [trace] GSSG). The equilibrium constant, K mix , was then determined by plotting relative MetE activity versus the [GSH]/[GSSG] ratio. In vivo thiol-trapping experiments. All E. coli cultures used for in vivo thiol-trapping experiments were grown aerobically at 37 °C. E. coli wild-type (DHB4) and trxA gor (WP843) strains were grown in glucose-minimal MOPS medium supplemented with 10 μM thiamine and all amino acids except methionine ( Neidhardt et al. 1974 ), with 0.4 mM arginine used instead of 5.2 mM. Tetracyclin (20 μg/ml) was included in the medium for the trxA gor strain. Overnight cultures of exponentially growing cells were diluted to an OD 600 of approximately 0.02 into the same medium. Cultures were allowed to grow to an OD 600 of approximately 0.4, and samples were removed for thiol trapping as described below. For in vivo thiol-trapping experiments performed on the trxB gor strain, a plasmid expressing MetE (pJG816) was transformed into electrocompetent trxB gor (strain WP778) cells by electroporation. An overnight culture of WP778/pJG816 in LB medium containing 20 μg/ml kanamycin, 50 μg/ml ampicillin, and 4 mM DTT was diluted to an OD 600 of approximately 0.02 into the same medium (without kanamycin). Care was taken to ensure that an overnight culture from the same inoculum failed to grow when DTT was not included in the medium. The 50-ml culture was grown to an OD 600 of approximately 0.4, and then filtered into a prewarmed sterile filter flask. The cells were washed twice with prewarmed sterile LB medium to remove residual DTT, and resuspended in 50 ml of prewarmed LB medium. At the indicated time points following DTT removal, samples were taken for alkylation as described. To assay the oxidation status of MetE in diamide-treated cells, wild-type E. coli were grown in glucose-minimal MOPS medium without methionine as in the growth experiments described above, except that diamide (0.9 mM, final concentration) was added at an OD 600 of approximately 0.4. Aliquots were removed at intervals following diamide addition for treatment with alkylating agents. In order to trap oxidized thiols in vivo, 1 ml of culture was added to 250 μl of 0.5 M iodoacetamide in 100 mM Tris chloride (pH 7.2) that had been pre-equilibrated at 37 °C. Samples were incubated 2 min at 37 °C, quenched by the addition of 140 μl of 100% (w/v) ice-cold TCA, and incubated on ice for at least 30 min. Precipitated proteins were pelleted by centrifugation (14,000 g ) for 30 min at 4 °C, washed with cold 10% TCA (once) followed by ethanol (twice), and then dried by incubating approximately 20 min in a 37-°C incubator. The pellet could then be stored at –80 °C prior to secondary alkylation with iodoacetic acid. Pellets were dissolved in 50 μl of alkylation buffer containing DTT (0.1 M Tris chloride [pH 8.7] containing 0.2 M potassium chloride, 1 mM EDTA, 8 M urea, and 10 mM DTT), and incubated 15 min at room temperature. Newly exposed cysteines were trapped by adding 12.5 μl of 0.5 M iodoacetate (104 mg dissolved in 0.5 ml of 1 M potassium hydroxide, followed by addition of 0.5 ml of 1 M Tris chloride [pH 7.3]) and incubating 2 min at room temperature. For some samples, excess DTT was added to quench the reaction; however, this was found to not be necessary. Alkylated proteins were then frozen in liquid nitrogen and stored at –80 °C prior to analysis by isoelectric focusing. For the reduced and oxidized MetE standards, 25 μl of 0.5 M iodoacetamide was added to 100 μl of 10 μM MetE in 100 mM Tris chloride (pH 7.2) and incubated for 1 min at 37 °C. The reaction was quenched by addition of 125 μl of 20% (w/v) ice-cold TCA and incubation on ice for at least 30 min. The proteins were pelleted by centrifugation (14,000 g ) at 4 °C for 30 min and then washed with cold 10% TCA (once) and either ether (3 times) or 1 M Tris chloride (pH 7.2) followed by water. Pellets were then alkylated with iodoacetate as described for the in vivo culture samples. Vertical slab isoelectric focusing. The vertical slab isoelectric focusing protocol developed was based on that of Savinova and Jagus (1997) and Robertson et al. (1987) . Gels contained 8 M urea, 9.6% glycerol, 3.75% acrylamide, 5% ampholytes, and 1.25% Chaps. Briefly, 6.0 g of urea was dissolved in 2.4 ml of 18 MΩ water, 2.4 ml of 50% glycerol, and 1.56 ml of acrylamide solution (28.36% acrylamide/1.62% bis-acrylamide). 600 μl of ampholytes (pH 4–8) and 780 μl of 20% Chaps were added, and the resulting solution was degassed for approximately 10 min. 25 μl of 10% APS and 20 μl of TEMED were added, and 8 cm × 8 cm × 1.0 cm gels were poured. Gels were allowed to polymerize approximately 30 min and then used immediately. Lanes were carefully washed with cathode buffer. The sample buffer solution contained 9.5 M urea, 5% chaps, 4.5% glycerol, 0.5 mM lysine–hydrochloride, 2% ampholytes (pH 4–8), 50 mM DTT, and 0.15% SDS. Care was taken not to increase the solution temperature above 30 °C to avoid breaking down urea, which could then carbamylate proteins. Aliquots of the sample buffer were snap-frozen in liquid nitrogen and stored at –20 °C. An Xcell Surelock Mini-cell gel apparatus from Invitrogen was assembled with the freshly prepared gel, and degassed cathode (upper, 0.1 M sodium hydroxide) and anode (lower, 10 mM phosphoric acid) solutions were added. Samples were diluted at least 10-fold into sample buffer and loaded. Power was applied for 1 h at 100 V followed by 1 h at 200 V and finally 30 min at 500 V. Gels were then stained as described previously ( Garfin 1990 ) or blotted. Immunoblotting. Gels were electroblotted onto PVDF membrane (Amersham Pharmacia, Piscataway, New Jersey, United States) according to the manufacturer's (Invitrogen) protocol using 1/2X Towbin buffer. Proteins were immunoblotted with a polyclonal rabbit antibody to MetE (1:3,000) and immune complexes were revealed using a fluorescein-linked anti-rabbit antibody (ECF western blotting kit, Amersham Pharmacia) followed by direct detection using a Molecular Dynamics (Sunnyvale, California, United States) Storm phosphorimager. Supporting Information Accession Numbers The Swiss-Prot ( http://www.ebi.ac.uk/swissprot/ ) accession numbers for the gene products discussed in this paper are AhpC (P26427), glutaredoxin 1 (P00277), glutaredoxin 2 (P39811), glutaredoxin 3 (P37687), glutathione reductase (P06715), GroEL (P06139), iron superoxide dismutases (P09157), manganese superoxide dismutase (P00448), MetA (P07623), MetE (C. albicans) (P82610), MetE (E. coli) (P25665), MetE (T. maritima) (Q9X112), MetH (P13009), OxyR (P11721), PAPS reductase (P17854), thioredoxin 1 (P00274), thioredoxin reductase (P09625), and Tpx (P37901).

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555751

Antioxidant effects of insulin-like growth factor-I (IGF-I) in rats with advanced liver cirrhosis

Background The exogenous administration of Insulin-like Growth Factor-I (IGF-I) induces hepatoprotective and antifibrogenic actions in experimental liver cirrhosis. To better understand the possible pathways behind the beneficial effect of IGF-I, the aim of this work was to investigate severe parameters involved in oxidative damage in hepatic tissue from cirrhotic animals treated with IGF-I (2 μg. 100 g -1 . day -1 ). Iron and copper play an important role in oxidative mechanisms, producing the deleterious hydroxyl radical (*OH) that peroxides lipid membranes and damages DNA. Myeloperoxidase (MPO) and nitric oxide (NO) are known sources of free radicals and induce reduction of ferritin-Fe 3+ into free Fe 2+ , contributing to oxidative damage. Methods Liver cirrhosis was induced by CCl 4 inhalation in Wistar male rats for 30 weeks. Healthy controls were studied in parallel (n = 10). Fe and Cu were assessed by atomic absoption spectrometry and iron content was also evaluated by Perls' staining. MPO was measured by ELISA and transferrin and ferritin by immunoturbidimetry. iNOS expression was studied by immuno-histochemistry. Results Liver cirrhosis was histologically proven and ascites was observed in all cirrhotic rats. Compared to controls untreated cirrhotic rats showed increased hepatic levels of iron, ferritin, transferrin (p < 0.01), copper, MPO and iNOS expression (p < 0.01). However, IGF-treatment induced a significant reduction of all these parameters (p < 0.05). Conclusion the hepatoprotective and antifibrogenic effects of IGF-I in cirrhosis are associated with a diminution of the hepatic contents of several factors all of them involved in oxidative damage.

Background Insulin-like growth factor-I (IGF-I) is an anabolic hormone produced in different tissues in response to growth hormone (GH) stimulation [ 1 ]. Liver synthesis of IGF-I accounts for 90% of the circulating peptide. In cirrhosis the reduction of receptors for GH in hepatocytes and the diminished synthesis ability of the hepatic parenchyma cause a progressive fall in serum IGF-I levels. The clinical impact of the decreased in IGF-I production in advanced cirrhosis is largely unknown [ 2 - 5 ]. Recent studies from our laboratory in rats with carbon tetrachloride-induced cirrhosis have demonstrated that short courses of treatment with low doses of IGF-I are able to produce systemic beneficial effects [ 6 - 13 ] and are associated to hepatoprotective [ 14 , 15 ] and antifibrogenic [ 16 ] effects. In order to give a better insight into the pathways by which IGF-I seems to exert its the hepatoprotective and antifibrogenic actions, this study was aimed at analyze several parameters involved in oxidative stress or inflammation in the liver, such as metals ions (iron and copper), iron transport and store proteins (transferrin and ferritin) and enzymes (myeloperoxidase -MPO- and inducible nitric oxide synthase -iNOS-) both in IGF-I treated and untreated cirrhotic rats. Metal ions, such as iron and copper, exhibit the ability to produce reactive oxygen species, resulting in lipid peroxidation, DNA damage, depletion of sulfhydryls and altered calcium homeostasis [ 17 - 19 ]. Iron-dependent processes play a pivotal role in the development of oxidative-induced cell injury. Specifically, the generation of hydroxyl radicals from hydroperoxide and the formation of aldehydes and lipid peroxy radicals from lipid hydroperoxides are catalyzed by redox-active metals, including iron and copper [ 17 , 20 , 21 ]. MPO and NO are known sources of free radicals and induce reduction of ferritin-Fe 3+ into free Fe 2+ contributing to oxidative damage [ 22 , 23 ]. Methods Induction of liver cirrhosis Cirrhosis was induced as previously described [ 9 , 12 ]. Briefly, male Wistar rats (3 weeks old, 130–150 g) were subjected to CCl 4 inhalation (Merck, Darmstadt, Germany) twice a week for 11 weeks with a progressively increasing exposure time from 1 to 5 minutes. From that time until the 30 th week rats were exposed to CCl 4 once a week for 3 min. During the whole period of cirrhosis induction animals received Phenobarbital (Luminal, Bayer, Leverkusen, Germany) in the drinking water (400 mg/L). Rats were housed in cages placed in a room with 12-hour light-dark cycle and constant humidity and temperature (20°C). Both food (standard semipurified diet for rodents; B.K. Universal, Sant Vicent del Horts, Spain) and water were given ad libitum . Healthy, age and sex-matched control rats were maintained under the same conditions but receiving neither CCl 4 nor Phenobarbital. All procedures were performed in conformity with The Guiding Principles for Research Involving Animals [ 24 ]. Study design The treatment was administrated the last three weeks (27 th -30 th ) of CCl 4 exposure (from day 0 to day 22 nd ). In the morning of day 0, animals were weight and blood samples were drawn from the retroocular venous plexus from all rats with capillary tubes (Marienfeld, Germany) and stored at -20°C until used for analytical purposes. Cirrhotic rats were randomly assigned to receive either vehicle (saline) (CI, n = 10) or recombinant human IGF-I (Pharmacia-Upshon, Sweden) (2 μg × 100 g bw -1 × day -1 in two divided doses, subcutaneous) (CI+IGF, n = 10) for three weeks. Control rats (CO, n = 10) received saline during the same period. The last dose of IGF-I was administrated the day 21 st at 6 p.m. In the morning of the 22 nd day, animals were weight and killed by decapitation. After the abdominal cavity was opened, the liver was dissected and weight. A sample from the left major liver lobe was processed for histological examination (fixed in Bouin's solution). The rest of liver samples were stored at -80°C. Liver histopathology, Perls'stain and immunohistochemistry Bouin-fixed tissues were processed and sections (4-μm.) were stained with Haematoxylin and Eosin and Masson's trichrome. Liver cirrhosis was diagnosed according to the criteria previously described [ 14 , 16 ]. Liver sections were stained for iron detection with Perls' Prussian Blue [ 25 , 26 ]. A semiquantitative score was given since 0 to 6 points: 0 when no staining was observed, as it was observed in controls; 6 points were assigned to sections with the maximal staining (full staining), that it was observed in liver macrophages and fibrous septa from cirrhotic rats; 2–5 points when the staining were less extent. Four fields from each preparation (×100 magnification) were evaluated twice by two different observers. The arithmetical mean of the two punctuations was taken as the final score. Immunohistochemical staining of iNOS in paraffin sections (4 μm) was performed using an avidin-biotin peroxidase technique as described by Shu el al. [ 27 ], with some modifications. The primary antibody anti-iNOS (1:500) was obtained from Oxford Biomedical Research, INC, NS 01. The procedure for negative controls was performed by omission of antigen retrieval part of the protocol. The positive staining was estimated blindly in the entire preparation by using a numerical scored from 1 to 8 points attending to the staining area and the intensity of the color. The arithmetical mean of the two evaluations was taken as the final score. Analytical methods Sample processing Hepatic samples were homogenized in a Potter homogenizer in 7 volumes of cold buffer (0.1 M Tris-HCl, 0.25 M sucrose, pH= 7.4) containing 5 mM 2-mercaptoethanol, 0.5 μg/mL Leupeptin, 0.7 μg/mL pepstatin A and 100 μg/mL PMFS. Fibrous parts and unbroken cells debris were eliminated by centrifugion at 500 g for 5 min. Supernatans were used as the whole homogenate. Analytical determinations on hepatic homogenates MPO was measured by ELISA, using a commercial kit from BIOXYTECH ® (OXIS Int. Portland, OR, USA). Transferrin and ferritin were determined by immunoturbidimetry, using a Hitachi 710 autoanalyzer (Roche Diagnostic, Basilea, Switzerland) and kits for clinical human, from the same laboratory. MDA was assessed after heating samples at 45°C for 60 minutes in acid medium. It was quantitated by a colorimetric assay using LPO-586 (Bioxytech; OXIS International Inc., Portland, OR, USA), which after reacting with MDA, generating a stable chromophore that can be measured at 586 nm (Hitachi U2000 Spectro; Roche). Total proteins were assessed by Bradford's method [ 28 ]. Determinations of iron and copper by Atomic Absorption Spectrophotometry Representative samples (approximately 1 g. of each rat liver) were collected, weighed and later dried in stove (70°C) to constant weight. Iron and copper concentrations were determined by flame atomic absorption spectrophotometry (Perkin Elmer 460, Uberlingen, Germany) [ 25 ]. Statistical Analysis Data were expressed as mean ± SEM. To analyse the homogeneity among groups, Kruskall-Wallis test was used, followed by multiple post-hoc comparisons using Mann-Whitney U tests with Bonferroni adjustment. Any P value < 0.05 was considered to be statistically significant. Calculations were performed with SPSS program version 6.0 (SPSS Inc., Chicago, IL). Results Liver cirrhosis was histologically proven and ascites was observed in all rats treated with CCl 4 . Table 1 shows the values of parameters involved in oxidative damage in hepatic homogenates. Compared with healthy controls, untreated cirrhotic rats (CI group) showed increased hepatic levels of the following variables: Fe (p < 0.01); transferrin and ferritin (p < 0.01); Cu (p < 0.001); MPO and iNOS expression (p < 0.001). However, cirrhotic animals treated with IGF-I (CI+IGF group) showed significant reductions in hepatic Fe and Cu contents, ferritin, transferrin and MPO levels and iNOS expression (p < 0.05 for all the parameters). As shown in Figure 1 , untreated cirrhotic rats (CI) have significantly greater scores of iron (ferric iron) in the liver using Perls' Prussian blue staining as compared with controls (CO = 0.68 ± 0.11) and cirrhotic rats treated with IGF-I (CI = 5.50 ± 0.22; CI+IGF = 1.70 ± 0.40; AU, p < 0.01). As mentioned before, hepatic levels of iron, assessed by atomic absorption spectrophotometry, were also significantly higher in CI group compared to controls and CI+IGF group (see Table 1 ). On the other hand, hepatic levels of copper were also increased in untreated cirrhotic rats and returned to normal in CI+IGF group. Figure 2 shows the immunohistochemical expression of iNOS that was increased in CI group compared both to control and CI+IGF groups. In order to find a relationship between the studied parameters and oxidative liver damage, MDA levels, an index of lipid peroxidation, were evaluated [ 29 ]. Hepatic levels of MDA (nmol/mg protein) were increased in untreated cirrhotic rats compared with control group (CI = 1.741 ± 366; CO = 0.565 ± 0.030; p < 0.05) as it was previously reported in similar protocols [ 14 , 16 ]. This marker of lipid peroxidation was again reduced in CI+IGF (0.99 ± 0.11 nmol/mg protein, p = ns vs controls). A significant direct correlation was found between hepatic iron and hepatic MDA levels (see Figure 3 , r = 0.857 p < 0.001). In addition, MPO correlated with hepatic levels of iron (r = 0.719, p < 0.001), iron content with hepatic ferritin (r = 0.656, p < 0.001) and hepatic levels of Cu with MDA (0.649 p < 0.01). Discussion These results show that the treatment with low doses of IGF-I induces a reduction of all studied parameters involved in oxidative damage mechanisms in this model of cirrhosis. These findings support the hepatoprotective and antifibrogenic effects previously reported [ 14 , 16 ]. This study also provides evidence for the involvement of oxidative stress in the cell injury occurring in CCl 4 -induced cirrhosis associated with iron and copper overload and an increase of myeloperoxidase and iNOS expression. It is well known that iron and copper promote oxidant forces [ 17 , 18 , 21 , 30 ]. Oxidant stress is considered present when there is either an overproduction of free radicals or a significant diminution in antioxidant defenses, the result of either being excessive levels of free radicals [ 29 , 31 ]. In both iron and copper storage disorders, generation of free radicals and depletion of antioxidants may be critical factors determining the intensity of liver injury [ 18 , 19 , 30 , 31 ]. In a previous work we showed that antioxidant enzymes (superoxide dismutase, SOD, Glutathione peroxidase, GSHPx, and catalase) were reduced in the liver of cirrhotic animals and improved by low doses of IGF-I administration [ 14 ]. Of interest, in the present study we demonstrate that hepatic levels of iron and copper metals (both involved in oxidative damage), increased in untreated cirrhotic rats, reverted to normal levels after IGF-I treatment. Free iron (or low molecular iron or chelatable iron pool) facilitates the decomposition of lipid hydroperoxides resulting in lipid peroxidation and induces the generation of OH radicals and also accelerates the nonenzymatic oxidation of glutathione to form O 2 *- radicals [ 18 , 19 , 30 , 32 ]. The direct and significant correlation between lipid peroxidation and hepatic iron content presented here provides new evidence of the relationship between these parameters. Most of the body's iron is tightly bound to transferrin, entering cells via receptor-mediated endocytosis. Transferrin avidly binds 2 moles of Fe 3+ per mole of protein [ 32 ]. Normally the average of transferrin iron saturation is about one third of the full capacity, thereby ensuring that there is virtually no free iron circulating in the extracellular fluids. At pH 7.4, the iron-transferrin complex does not participate in the Fenton reaction. Under more acidic conditions, the complex breaks down with release of iron. This is of important physiological relevance, since the iron-transferrin complex, within endocytotic vesicles, is subjected to an acidic environment (pH 5–6). Intracellular iron released from transferrin is rapidly incorporated into ferritin, minimizing its inherent toxicity [ 17 , 30 , 31 ]. Iron can be released from the ferritin within the cell by a number of factors that occur in inflammation: acidic pH, proteolysis, myeloperoxidase, NO, O 2 *-, etc. [ 33 ]. Enhanced degradative proteolysis, which also occurs in oxidative stress, may lead to proteolytic modification of ferritin, causing an increase in cellular iron. Although in this study free iron could not be quantified, all of the factors certainly involved in inducing an increase of free iron pool appeared elevated in untreated cirrhotic rats (MPO, iNOS, Cu,...) and returned to normal levels after IGF-I-treatment. In the present study, we have found that hepatic transferrin and ferritin levels increased in cirrhotic rats with a parallel rise in iron deposition, whereas in cirrhotic rats treated with IGF-I all the above-mentioned parameters appeared diminished (see Table 1 and Figure 1 ). High serum ferritin levels and hepatic iron storage have also been reported in hepatitis B virus and hepatitis C virus-related chronic hepatitis and alcoholic liver disease [ 26 , 34 , 35 ]. It has also been shown that iron induces ferritin biosynthesis [ 21 , 22 , 35 - 39 ]. A result here presented shows a direct correlation between hepatic iron and ferritin levels which is consistent with the over-mentioned Authors statements. In liver cirrhosis the increase in iron content is not a real iron overload as in hemochromatosis, because iron is stored mainly inside the macrophages [ 40 ]. In agreement with this data, the present work shows that the iron scores detected in this experimental model of cirrhosis were found in Kupffer cells, as it is shown in Fig. 1 . Transferrin is mainly produced by the liver when hepatic regeneration takes place, as occurs in cirrhosis [ 37 , 41 , 42 ]. Thus, the reported increase of transferrin in untreated cirrhotic animals could be due to regeneration. However, cellular proliferation does not explain our findings, because in a parallel study in this series we showed that cellular proliferation (assessed by PCNA expression) was higher in IGF-treated cirrhotic animals [ 15 ] than those which showed lower hepatic levels of transferrin. Therefore, the hepatoprotective effect of IGF-I in cirrhotic animals could be mediated partly by enhancing the endogenous regenerative response, aimed al the restoration of functional liver mass [ 14 ]. In the present work, the described increase of transferrin in untreated cirrhotic animals seems to be a defensive response to the enhanced iron content [ 17 , 18 , 21 , 22 , 32 ]. On the other hand, the mechanisms responsible for the effects of IGF-I described in this article are not fully understood. The beneficial effects of IGF-I could be a result of many properties of this hormone that require further investigation. The well known erythropoietic activity of IGF-I [ 43 , 44 ] could even contribute to an extrahepatic utilization of iron, decreasing its storage in the liver. Hepatic copper overload leads to progressive liver injury and eventually cirrhosis in Wilson disease and Indian childhood cirrhosis [ 45 ]. Copper is absorbed into the intestine and transported by albumin to the liver. Any excess in copper levels is excreted into the bile mainly through a lysosome-to-bile pathway. Hepatic copper accumulation results from a reduction in the bile excretion of copper, as occurs in patients with Wilson disease, biliary obstruction, or other types of cholestasis [ 45 ]. Cirrhotic animals included in this protocol showed severe cholestasis after receiving CCl 4 for 30 weeks. As previously reported [ 14 ] IGF-I-treatment induced a reduction in cholestasis parameters (serum levels of bilirubin, alkaline phosphatase and cholesterol). This may account for explanation of the decreased copper hepatic content revealed in the present work. After hepatic injury, several kinds of cells (endothelial cells, Kuppfer cells, and circulating platelets, neutrophils and monocytes) are activated in the subsequent inflammatory response [ 23 ]. Free radicals produced mainly by macrophages cause local tissue damage in inflammatory conditions [ 23 , 31 ]. Neutrophil and monocyte activation is a critical step in both the host defense system against microorganisms and the inflammatory response. When neutrophils are activated, they begin to produce superoxide radicals (O 2 *-) and secrete myeloperoxidase (MPO) [ 23 ]. The majority of the O 2 *- formed during this respiratory burst is converted to the bactericidal oxidant hypochlorous acid (HOCl) via a series of reactions catalyzed by superoxide dismutase and MPO [ 23 , 29 ]. Numerous MPO-expressing cells have been detected in fibrous septa of human cirrhotic livers [ 46 ]. MPO has been identified as a component of human Kupffer cells [ 46 ]. The same authors suggest that the oxidative damage resulting from the action of MPO may contribute to acute liver injury and hepatic fibrogenesis [ 46 ]. In our study, the increase of MPO in cirrhotic animals and its decrease in those treated with IGF-I suggests an anti-inflammatory effect of this hormone. Another result which deserves particular mention is that iNOS expression was significantly lower in cirrhotic rats treated with IGF-I compared to untreated cirrhotic animals. This finding is in accordance with those reported by other groups [ 47 - 53 ]. However the versatility of this molecule, small changes in the experimental conditions or the studied cell line can show results that seem to be an apparently contradiction [ 54 - 58 ]. For example, in our experience, we did not find a similar response in early stage of cirrhosis animals (data not shown). Probably, in early stages of cirrhosis NO induces an improvement in parenchyma irrigation by vasodilatation, but in advanced liver cirrhosis, where exist thick collagen septa, the increase of NO results to lead enhancing oxidative damage by N-derived radicals. Conclusion In conclusion, these results show that the hepatoprotective and antifibrogenic effect of IGF-I in rats with liver cirrhosis is associated with a significant reduction of the hepatic levels of several parameters such as Fe, Cu, MPO, iNOS, ferritin and transferring, all of them involved in oxidative damage. In this work, iron and copper overload have been demonstrated in the liver from rats with CCl 4 -induced cirrhosis. The hepatic levels of both metals diminished in cirrhotic animals treated with IGF-I. MPO content, iNOS immunohistological expression and hepatic ferritin and transferrin levels were increased in untreated animals and returned to normal in cirrhotic animals treated with IGF-I. The IGF-I effects described in the present study suggest that a therapeutical approach targeted at lowering oxidative stress marker levels could be effective in the chronic liver disease. Abbreviations IGF-I, insulin-like growth factor-I; Fe, iron; Cu, cooper; MDA, malondialdehyde; CO, control healthy group; CI, untreated cirrhotic rats; CI + IGF, IGF-treated cirrhotic rats; O 2 *-, superoxide radicals; MPO, myeloperoxidase; iNOS, inducible nitric oxide synthase; AU, arbitrary units. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MG : Analytical studies, hypothesis and paper elaboration. ICC : Experimental design and treatment (induction of liver cirrhosis and IGF-I administration), hypothesis, histopathological study and scores. MDS : Analytical studies and in vivo assay. IN : Atomic absorption spectrometry assay. JEP : In vivo assay. AC : Hypothesis and experimental design and revision. ADC : Experimental treatment and documentation. EC : Histopathological study and measurements. SGB : Revision. Pre-publication history The pre-publication history for this paper can be accessed here:

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Mothers' education but not fathers' education, household assets or land ownership is the best predictor of child health inequalities in rural Uganda

Background Health and nutrition inequality is a result of a complex web of factors that include socio-economic inequalities. Various socio-economic indicators exist however some do not accurately predict inequalities in children. Others are not intervention feasible. Objective To examine the association of four socio-economic indicators namely: mothers' education, fathers' education, household asset index, and land ownership with growth stunting, which is used as a proxy for health and nutrition inequalities among infants and young children. Methods This was a cross-sectional survey conducted in the rural district of Hoima, Uganda. Two-stage cluster sampling design was used to obtain 720 child/mother pairs. Information on indicators of household socio-economic status and child anthropometry was gathered by administering a structured questionnaire to mothers in their home settings. Regression modelling was used to determine the association of socio-economic indicators with stunting. Results One hundred seventy two (25%) of the studied children were stunted, of which 105 (61%) were boys (p < 0.001). Bivariate analysis indicated a higher prevalence of stunting among children of: non-educated mothers compared to mothers educated above primary school (odds ratio (OR) 2.5, 95% confidence interval (CI) 1.4–4.4); non-educated fathers compared to fathers educated above secondary school (OR 1.7, 95% CI 0.8–3.5); households belonging in the "poorest" quintile for the asset index compared to the "least poor" quintile (OR 2.1, 95% CI 1.2–3.7); Land ownership exhibited no differentials with stunting. Simultaneously adjusting all socio-economic indicators in conditional regression analysis left mothers' education as the only independent predictor of stunting with children of non-educated mothers significantly more likely to be stunted compared to those of mothers educated above primary school (OR 2.1, 95% CI 1.1–3.9). More boys than girls were significantly stunted in poorer than wealthier socio-economic strata. Conclusions Of four socio-economic indicators, mothers' education is the best predictor for health and nutrition inequalities among infants and young children in rural Uganda. This suggests a need for appropriate formal education of the girl child aimed at promoting child health and nutrition. The finding that boys are adversely affected by poverty more than their female counterparts corroborates evidence from previous studies.

Background Health outcomes are a result of a multi-layered conceptual framework with proximal and distal determinants [ 1 ]. Proximal (nearer in time and place to the terminal event) determinants such as feeding practices, injury and disease especially the infectious diseases are mediated through factors prevailing within households or the institutions serving children directly. At the distant level are factors operating through the socio-political and economic dimensions of the environment. In recent decades, development agencies and governments have emphasized efficacious interventions to improve child survival and healthy development. However, most of these interventions have traditionally addressed proximal determinants with limited effort on distal determinants since many of them lie outside the health sector. Recent calls were made for interventions to address the more distal determinants of health outcomes [ 2 , 3 ]. To minimize inequities in child health and nutrition emphasis is made on correct identification and service delivery to the poor and most vulnerable in society. To assess inequalities of child health and nutrition, the World Health Organisation recommends use of linear growth retardation (stunting) [ 4 - 6 ]. In the biomedical arena stunting is attributable to a wide range of prenatal and postnatal factors. These include: low birth weight [ 7 - 9 ], inadequate care and stimulation [ 10 ], and insufficient nutrition and recurrent infections [ 11 ]. Use of stunting avoids the measurement pitfalls like improper case definitions, time, and others usually associated with more traditional measures such as morbidity, mortality, and life expectancy. It also eludes problems associated with using monitory variables such as income or expenditure to compare health inequalities [ 12 , 13 ]. Stunting varies systematically with socio-economic status with the poorest most affected by it. This relationship is observed in a number of studies in the African region [ 14 - 18 ]. Unfortunately, proxies used to construct socio-economic indicator(s) vary. The relative household socio-economic position is sometimes developed by lumping indicators of socio-economic status into one global indicator, or derived from non-interventional feasible indicators. Additionally control for unmeasured effects of each indicator on others is often never done. This is detrimental to development of effective policy since choice of an indicator has interventional implications. This is especially critical in sub-Saharan Africa where evidence suggests that macro-economic growth alone is unlikely to significantly reduce stunting in medium terms [ 19 ]. Therefore efforts to address distal determinants of child health inequalities especially through the socio-economic pathway, requires that great care is taken in selecting indicators that are amenable to easy monitoring and feasible interventions [ 20 ]. The present study was conducted in a rural setting, in Uganda. It examines the independent influence of four different components of socio-economic status: mothers' education, fathers' education, household asset index, and land ownership on growth stunting, a proxy for health inequalities. Results of this study should provide policy makers and programme managers with useful information in child health and nutrition planning for rural areas. Methods Study area The study was carried out in Hoima district, which is located in western Uganda and shares a border with the Democratic Republic of Congo along Lake Albert. The district has a population density of 80 per square kilometre with a total population of approximately 370,000, comprised of mainly peasant farmers of the Banyoro tribe [ 21 ]. The district has fertile soils and enjoys a bimodal rainfall pattern ranging from 800 – 1500 mm per annum enabling two harvests a year. Farming activities include cattle rearing, growing tobacco and coffee for cash; and maize, cassava, potatoes, beans and groundnuts for consumption. Design and sampling The present survey utilised a two stage probability proportional to size cluster design [ 22 ]. Data was collected with respect to infant- and young child feeding knowledge and practices, anthropometry and indicators for household socio-economic status. The sample was made large enough to estimate a prevalence rate of wasting similar to that assessed at the national level (4.0%), at 95% level of confidence [ 23 ], with 2% precision error and an assumed intra-cluster correlation coefficient of 0.11. The national census data [ 21 ] was used to estimate the population under-2 years of age in Hoima district (24,000). A sample size of 720 children was calculated based on the formula by Cochran [ 24 ], using the SampleXS software. At the first stage of sampling, a total of 72 villages were selected on probability proportional to population size basis. At the second stage, a total of 10 households were systematically sampled from each village selected at the first stage, providing a self-weighting sample with each child/mother pair in the district having an equal probability to be selected into the sample. A household was defined as a group of people living, cooking and eating together. One child under 2 years was enrolled per household after an informed verbal consent of the mother/caregiver. In case a household had two children in the target age group or twins one was selected randomly. Children with major handicap, disability or malformation were excluded from the sample. Problems encountered with sample selection included the existence of newly formed villages hitherto unrecorded in the census register. In one case an old village had been split; one of the "offspring" villages was selected randomly. Data Collection A structured questionnaire constructed in English and translated into Runyoro, the local language, was pilot tested and updated in survey similar settings and administered face-to-face to mothers/caregivers in their home settings. Trained non-medical university students gathered data in September and October 2002. To minimise bias, training of interviewers emphasized proper and random identification of respondents, eligible children, and questionnaire administration. A village leader followed data collectors through the village, and traditional village protocol was observed. Information about the educational level of parents, household durable assets, materials of the dwelling structure and land ownership was collected as proxy makers of household socio-economic status. Child's age was obtained through birth certificates, health cards, or recall using a calendar of local events while taking precautions to minimize field errors [ 25 ]. Recumbent length measurements were taken with specially designed length boards that measured to the nearest 1 mm following standard recommendations [ 26 ]. The questionnaire was based on the demographic and health survey (DHS) questions [ 27 ] to avoid separate validity and reliability checks of the data collection instrument. Measurements The dependent variable (stunting) was constructed using child length, age and sex, and was defined as length less than minus 2 standard deviations (<-2 SD) from the median of the NCHS/WHO reference population [ 5 ]. The education level of fathers and mothers was assessed on scales ranging from (1) never went to school, to (7) college/university. Two categorical variables were constructed yielding four categories for mothers: (1) no formal education, (2) stopped in primary, (3) completed primary (minimum 7 years of school) and (4) stopped above primary. Five categories were yielded for fathers because they were more educated: (1) no formal education, (2) stopped in primary, (3) completed primary, (4) stopped in secondary, and (5) completed secondary 4 or above (minimum 11 years of school). Household durable assets (cupboard, hurricane lamp, radio, bicycle, fuel, boat, telephone, refrigerator, motorcycle and car) were assessed as (1) available/ in working condition and (2) not available/ not in working condition. Four components of the dwelling structure were assessed including number of rooms, roof – (1) thatched, (2) corrugated iron sheets or tiles; floor – (1) mud, (2) cement; wall – (1) thatched, (2) mud and pole, (3) unburned bricks, (4) burnt bricks built with mud, (5) burnt bricks built with cement and (6) cement/concrete blocks. A household wealth (asset) index was constructed from 15 variables (household durable assets and dwelling structure) using principal components analysis [ 28 ]. The index scores were divided into quintiles (1) poorest, to (5) least poor. Land is usually given special socio-economic status in many subsistence agricultural communities in Uganda [ 29 ]. In order to assess for its independent contribution to child well being land ownership was analysed independent of other household assets. It was assessed as a continuous variable using an open question "How much land does your family have for agriculture or any other activity?" A football pitch was used to estimate the size equivalent to one acre. A categorical variable was constructed in terms of; (1) no land at all, (2) one acre, (3) two acres, (4) three acres and (5) four or more acres. Child age in months was assed as a continuous variable and a categorical variable was constructed yielding four categories: (1) 0–5 months, (2) 6–11 months, (3) 12–17 months, and (4) 18–23 months. Child sex was ordinal (1) male, (2) female. Analysis Data was entered in Epidata software [ 30 ], and was analysed in epi info (version 6.04d) and SPSS (version 12.0). Of 720 children recruited, 23 (3%) had missing data on anthropometric measurements or were extreme outliers and were dropped from the analysis of stunting. First, socio-economic indicators (mothers' and fathers' education, household wealth index and land ownership) were evaluated for the extent of shared variation by obtaining bivariate correlation coefficients (Spearman's rho). Second, unadjusted associations of stunting with socio-economic and demographic variables were examined by use of odds ratios. Third, backward conditional logistic regression was done simultaneously controlling for socio-economic and demographic factors. The pattern of stunting with sex across different socio-economic strata was evaluated with chi-square tests and bivariate logistic regressions. Mean age differences between boys and girls were assessed by the student t-test. Tests for trend across categories were performed by treating the categories as continuous variables in the logistic regression analyses. The clustering effect was not controlled for as the large number of primary sampling units (72) in the study minimizes it. The goodness-of-fit for regression models was assessed with Hosmer and Lemeshow test; with the null hypothesis that the model adequately fits the data (significant chi-square p-value implies that the model does not fit). Ethics Approval of the study was granted by Makerere University Faculty of Medicine Ethics and Research committee, the Uganda National Council for Science and Technology and the Regional Committee for Medical Research Ethics, West Norway (REK vest). Results Of the children studied 366 (51%) were females. The median age (inter-quartile range) was 10 (5–16) months for females and 11 (5–16) months for males respectively. The mean age difference between boys and girls was not statistically significant (p = 0.34). A total of 148 (21%) mothers and 59 (9%) fathers never attained any formal education (Table 1 ), while only 52 (7%) of mothers were educated up to secondary 4 or above. Ninety percent of mothers were married. One hundred seventy two (25%) of the children in the study were stunted out of which 105 (61%) were boys. The status of stunting ranged from 12% among children aged 0–5 months to 41% in children 18–23 months (Figure 1 ). There were 21 twins included in the study and 6 of them were stunted. Ninety percent of households owned any land. Median acreage was 2.0 and the mean was 3.6 acres. Table 1 Frequency distribution, unadjusted and conditional multiple logistic regressions of stunting with socio-economic predictors (coefficients are expressed as odds ratios and p-values for the test of trend are indicated) Variables Total Stunted b Unadjusted Adjusted n a (%) n (%) OR 95%CI OR 95%CI Child age in months 18–23 150 (22) 61 (41) 5.2 2.9–9.3*** 5.1 2.6–9.8*** 12–17 189 (27) 56 (30) 3.2 1.8–5.7*** 3.0 1.6–5.8** 6–11 195 (28) 36 (19) 1.7 0.9–3.1 1.8 0.9–3.5 0–5 164 (23) 19 (12) 1.0 1.0 p-value for trend test < 0.001 < 0.001 Child sex is male 354 (49) 105 (31) 1.9 1.3–2.7*** 2.0 1.3–3.0** Mother's education None 148 (21) 45 (31) 2.5 1.4–4.4** 2.1 1.1–3.9* Stopped in primary 282 (40) 80 (30) 2.3 1.4–3.9** 2.1 1.2–3.8* Completed primary 131 (18) 22 (18) 1.2 0.6–2.2 1.0 0.5–2.0 Above primary 152 (21) 23 (15) 1.0 1.0 p-value for trend test < 0.001 0.03 Father's education None 59 (9) 17 (30) 1.7 0.8–3.5 Stopped in primary 179 (29) 47 (27) 1.5 0.9–2.6 Completed primary 151 (24) 36 (24) 1.3 0.8–2.3 Stopped in secondary 95 (15) 17 (18) 0.9 0.5–1.8 Secondary 4 or above 142 (23) 27 (20) 1.0 p-value for trend test 0.03 Household wealth index 1 st quintile (Poorest) 139 (20) 38 (29) 2.1 1.2–3.7** 2 nd quintile 140 (20) 36 (26) 1.7 0.9–2.9 3 rd quintile 150 (21) 33 (24) 1.3 0.8–2.5 4 th quintile 140 (20) 36 (26) 1.1 0.6–2.0 5 th quintile (Least poor) 140 (20) 23 (17) 1.0 p-value for trend test 0.01 Land ownership None 73 (10) 16 (22) 0.9 0.5 – 1.8 1 acre or less 179 (25) 39 (23) 0.9 0.6 – 1.6 2 acres 167 (24) 44 (28) 1.2 0.7 – 2.1 3 or 4 acres 152 (22) 37 (25) 1.0 0.6 – 1.8 5 acres or more 135 (19) 32 (24) 1.0 p-value for trend test 0.78 Hosmer-Lemeshow goodness-of-fit chi-square p-value 0.90 *p < 0.05; **p < 0.01; ***p < 0.001 a Totals for some categories do not add up to 720 because of missing values b Below -2 SD height-for-age of the NCHS/WHO reference Figure 1 Proportion of stunted children (<-2 SD of NCHS/WHO reference) among 720 children by sex and age group in western Uganda Stunting distribution by socio-economic status Unadjusted logistic regression analysis indicated that 3 socio-economic indicators (fathers' education, mothers' education and household wealth index) had a graded pattern with stunting across strata with the least educated or most poor being more likely to have stunted children. The corresponding p-values for test of trend was <0.001, 0.03 and 0.01 for mothers' education, fathers' education and household wealth index, respectively (Table 1 ). Land ownership however, showed no differentials with stunting across strata. The extent of association between socio-economic indicators was examined. The correlations (Spearman's rho) between mothers' and fathers' education was 0.50; mothers' education and household wealth index = 0.37; mothers' education and land ownership = 0.12; fathers' education and household wealth index = 0.39; fathers' education and land ownership = 0.14; and household wealth index and land ownership = 0.18. Their colinearity was sufficiently low to allow them into the same model. The four socio-economic indicators (mothers' education, fathers' education, household wealth index and land ownership), child age and sex were simultaneously entered in a backward conditional logistic regression. Only mothers' education, child age and sex appeared in the last step model. Children belonging to mother with no formal education or to mothers who stopped in primary school were significantly more likely to be stunted compared to their counterparts with mothers who were educated beyond primary school (OR = 2.1, Table 1 ). Being a boy (OR = 2.0) or older in age was also significantly associated with stunting. Stunting distribution by sex and socio-economic status Stunting differentials for both sexes within and across socio-economic indicators did not favour male children. There was a graded relationship of stunting among males unlike females with almost all socio-economic groups except land ownership (Table 2 ). More males were stunted in poorer socio-economic strata than their peers in better off strata. The trend was more marked with mothers' education (p < 0.001) and household wealth index (p = 0.01), less with fathers' education (p = 0.06) and none with land ownership (p = 0.64). The magnitude of the difference in stunting between males and females diminishes with improvement in socio-economic status. The socio-economically advantaged groups show no differences in stunting status between the two sexes. Compared to the proportion of stunting between boys and girls in the strata of mothers educated above primary, mothers with no formal education were significantly more likely to have stunted boys than girls (OR = 3.0). Table 2 Comparison of stunting status by sex and socio-economic position with unadjusted odds ratios and 95% confidence intervals (CI) derived from bivariate logistic regression with girls in the comparison group (coefficients are expressed as odds ratios, p-values for test of trend and Pearson chi-square are also indicated) Stunted children p-value Unadjusted odds ratio 95%CI Males n(%) Females n(%) Mother's education None 33 (43) 12 (17) 0.01 f 3.00 1.05–8.59* Stopped in primary 44 (34) 36 (26) 0.18 1.33 0.53–3.38 Completed primary 15 (23) 7 (12) 0.16 2.34 0.69–7.87 Above primary 11 (17) 12 (14) 0.65 1.0 p-value for test of trend < 0.001 0.14 0.11 Father's education None 12 (40) 5 (19) 0.09 f 1.65 0.45–6.03 Stopped in primary 30 (35) 17 (20) 0.04 f 1.21 0.46–3.20 Completed primary 24 (31) 12 (17) 0.05 f 1.37 0.49–3.87 Stopped in secondary 9 (21) 8 (16) 0.59 0.77 0.23–2.63 Ordinary level or above 16 (25) 11 (15) 0.14 1.0 p-value for test of trend 0.06 0.39 0.37 Index of household wealth 1 st quintile (Poorest) 27 (40) 17 (25) 0.05 f 2.17 0.81–5.81 2 nd quintile 25 (34) 14 (21) 0.05 f 2.44 0.88–6.73 3 rd quintile 21 (35) 11 (15) 0.01 f 2.60 0.90–7.56 4 th quintile 20 (28) 10 (13) 0.02 f 2.73 0.92–8.09 5 th quintile (Least poor) 11 (15) 15 (22) 0.34 1.0 p-value for test of trend 0.01 0.34 0.28 Land ownership None 9 (27) 7 (18) 0.42 0.77 0.23–2.61 1 acre 19 (25) 20 (22) 0.59 0.57 0.22–1.48 2 acres 28 (41) 16 (18) 0.01 f 1.05 0.41–2.70 3 or 4 acres 26 (30) 11 (18) 0.07 f 1.42 0.52–3.87 5 acres or more 20 (30) 12 (18) 0.08 f 1.0 p-value for trend test 0.64 0.66 0.17 f Fishers exact test; *p < 0.05 Discussion This study examined how socio-economic indicators namely mothers' education, fathers' education, household asset index and land ownership relate with inequalities in child health and nutrition using growth stunting as the proxy for the inequalities. Findings showed that mothers' education was a robust predictor for inequalities of child health and nutrition. Secondly, boys were more affected by low socio-economic status than girls. Indirectly the study also indicates that it may be unsuitable to bunch together socio-economic indicators as they could have low covariance or could conceptually not be interchangeable and thus unable to serve as adequate proxies for one another. While interpreting findings of this study one should consider the fact that some of the fixed factors known to influence linear growth such as birth weight [ 7 , 8 ] and maternal stature [ 9 , 31 ] were not controlled for in the analysis. Birth weight was dropped because of many missing data attributable to the fact that majority of mothers in Uganda give birth outside the health units, consequently missing information on birth weight. Unfortunately the study design excluded assessment of maternal stature. Additionally, in a cross-sectional study such as the present one, it is impossible to say anything about cause and effect relationships. Nonetheless, considering that our agenda was to examine socio-economic predictors for inequalities in health and nutrition, these limitations do not greatly trivialise the importance of the study. The socio-economic predictor variables used in the study were assumed to represent distinct dimensions reflecting different domains of influences from the society. The relatively low colinearity between the indicators used supports this assumption. This favours discussions on choice of socio-economic indicators where researchers are urged to use a number of socio-economic indicators rather bunching them together as each could have its unique contribution [ 20 , 32 ]. Our results regarding the relationship of socio-economic status with stunting are similar to findings in other studies [ 14 - 18 ]. As one climbs up the socio-economic ladder, there is a remarkable drop in the rate of stunting observed. Interestingly despite taking care of some covariance that existed between different socio-economic indicators during the analysis, mothers' education emerged as the only independent socio-economic predictor for inequalities of child health and nutrition. This indicates that the association of fathers' education and household wealth index with stunting could be confounded by mothers' education. This emphasizes the need for promoting the education of the girl child with a principal aim of improving child health, which is in line with recommendations by UNICEF [ 33 ]. However, for this to be achieved there is need for a rational approach that involves a greater strategic and collaborative relationship between different sectors of government and agencies. Additionally, mothers' education could be used as a suitable indicator to guide effective targeting especially with regard to identifying the most vulnerable families in rural subsistence agricultural communities. This is in appreciation of the fact that achieving universal coverage for child-health interventions presently lies far beyond the capacity of many health systems in low-income countries [ 34 , 35 ]. It is also consistent with the recommendation that modern programming has to go beyond equitable targeting [ 2 ] in order hasten improvements in health outcomes. It was surprising that land ownership and access, perceived by many in Uganda as the ultimate form of security or socio-economic status [ 29 ], showed no differentials with stunting. Obviously, this may be different in more densely populated parts of the globe. The fact that majority (90%) of the households had a piece of land could also reduce differences in nutritional status among children. However, assuming no extraneous confounding or misclassification biases these findings could imply that land ownership in rural subsistence agricultural set-ups is not critical for child health and nutrition or is at least not as vital as parents' education and household wealth index in child well being. Concordant with findings in other studies [ 31 , 36 ], age correlated positively with stunting. However, disturbing results were observed with child sex. Among well-nourished children, sex differences are attributed to a normal pattern of dimorphism, with males tending to be taller and heavier than females. Finding of this study indicate that male children were more likely to be stunted than females. Surprisingly several studies report a similar pattern in Africa [ 8 , 16 , 23 , 37 ]. What was more disturbing however was that larger proportions of male children were stunted as one descends the socio-economic profile as compared to females. Stunting differentials with socio-economic status observed in this study could be solely attributed to boys. Unfortunately none of the literature cited disaggregate stunting with sex across socio-economic groups. There are also no documented beliefs, attitudes or practices that segregate against the boy child in Uganda. However, in evolutionary biology there is evidence of male vulnerability in response to environmental stress in early life [ 38 ]. Conclusions This study highlights the importance of maternal education in child well being. From our data any increment in maternal education is likely to have a positive influence on child growth. Governments of low-income countries need to ensure that the girl child receives appropriate formal education. For effective targeting of families with children in greatest need or at highest risk of health and nutrition hazards, policy makers and programme managers could be guided by mothers' education. Findings that males appear to be more adversely affected by poverty than their female counterparts corroborate evidence from previous research. Authors' contributions All authors participated in the design of the study, the interpretation of findings and write-up of the manuscript. HW coordinated and supervised field data collection and performed the statistical analysis. All authors read and approved the manuscript. Competing interests The authors declare that they have no competing interests.

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529315

A Chemoattractant Role for NT-3 in Proprioceptive Axon Guidance

Neurotrophin-3 (NT-3) is required for proprioceptive neuron survival. Deletion of the proapoptotic gene Bax in NT-3 knockout mice rescues these neurons and allows for examination of their axon growth in the absence of NT-3 signaling. TrkC-positive peripheral and central axons from dorsal root ganglia follow proper trajectories and arrive in close proximity to their targets but fail to innervate them. Peripherally, muscle spindles are absent and TrkC-positive axons do not enter their target muscles. Centrally, proprioceptive axons branch in ectopic regions of the spinal cord, even crossing the midline. In vitro assays reveal chemoattractant effects of NT-3 on dorsal root ganglion axons. Our results show that survival factor NT-3 acts as a short-distance axon guidance molecule for muscle sensory afferents as they approach their proper targets.

Introduction Neurotrophin-3 (NT-3) is a key requirement for the development of proprioceptive inputs to motor neurons ( Chen and Frank 1999 ; Chen et al. 2003 ). Mice deficient in NT-3, its tyrosine kinase receptor, TrkC, or in TrkC-positive neuron-specific transcription factor Runx3 display severe ataxia associated with the absence of muscle spindles, and loss of proprioceptive neurons in dorsal root ganglia (DRGs) or their axons ( Ernfors et al. 1994 ; Klein et al. 1994 ; Tessarollo et al. 1994 ; Fariñas et al. 1996 ; Liebl et al. 1997 ; Inoue et al. 2002 ; Levanon et al. 2002 ). NT-3 is expressed in the ventral spinal cord, in the developing limb buds, and in intrafusal bag fibers of muscle spindles later in development ( Copray and Brouwer 1994 ; Fariñas et al. 1996 ; Tojo et al. 1996 ). When sensory axons contact developing myotubes, they induce muscle spindle differentiation, forming ring-like spiral nerve endings around them. In the chicken embryo, limb ablations or anti-NT-3 antibody injections into limb buds lead to elimination of TrkC-positive neurons and decreased innervation of motor neurons ( Oakley et al. 1995 , 1997 ). Is NT-3 only a chemotrophic survival factor for muscle sensory afferents, or does it have additional roles in the development of the proprioceptors and the establishment of the monosynaptic reflex arc? Here we provide evidence that NT-3 acts as a chemoattractant for sensory axons during the final phase of their target-directed pathfinding. Results TrkC-Positive DRG Neurons Are Rescued in Bax/NT-3 Double Knockout Mice Mice lacking proapoptotic protein Bax allow for distinguishing survival effects of neurotrophins from other effects. Bax-deficient sensory neurons no longer require neurotrophins for survival ( White et al. 1998 ; Patel et al. 2000 ), thus they can be used to examine axonal effects. We bred NT-3 heterozygote and Bax knockout (KO) mice to obtain mice with double KO of both NT-3 and Bax genes, and examined proprioceptive axonal projections. All NT-3 and double KOs died within 48 h after birth ( Tessarollo et al. 1994 ). We performed TrkA/TrkC double immunohistochemistry ( Huang et al. 1999 ), enabling detection of both proteins in the same sample. TrkC-positive cells ( Figure 1 A) and fibers ( Figure 1 E) were absent in NT-3 KOs at embryonic day (E) 15. Two subsets of DRG cells expressing either TrkA or TrkC were detected in double KOs, similar to wild-type (WT) or Bax KO animals. Surprisingly, at postnatal day (P) 0, a few cells expressed TrkC even in NT-3 KO animals in the Bax +/+ genetic background, and some cells co-expressed TrkA and TrkC, regardless of the genotype ( Figure 1 B). In order to quantify our results, we analyzed the ratio of TrkA and TrkC immunopositive cells from four different DRGs of animals of different genotypes. At all ages studied, Bax/NT-3 double null DRGs had TrkA/TrkC ratios similar to those of Bax null DRGs, and higher than those of NT-3 null DRGs ( Figure 1 D). TrkC-positive neurons rescued by Bax deletion, however, failed to differentiate properly, as evidenced by the lack of expression of the proprioceptive molecular marker Parvalbumin (PV) ( Figure 1 C). Figure 1 TrkA/TrkC and PV Immunohistochemistry in DRG and Spinal Cord Red represents TrkA, green represents TrkC (and PV in [C]), and yellow represents co-expression. (A) TrkA/TrkC immunostaining in E15 DRG. TrkC-positive neurons normally eliminated in NT-3 KOs are rescued in double KOs. (B) TrkA/TrkC immunostaining at P0 in DRG. (C) PV immunostaining in P0 DRG. Rescued TrkC-positive cells fail to express PV. (D) Ratio of TrkA-immunopositive cells to TrkC-immunopositive cells in E15 and P0 DRGs. Data are presented as percentage of cells with standard deviation. Double KOs always had similar ratios to Bax KOs, and NT-3 KOs had the least amount of TrkC-positive cells, if any. (E) TrkA/TrkC immunostaining in E15 spinal cord. Arrow points to group Ia fibers. Dorsal is up. Scale bar: 50 μm (A–C), 1 mm (E). NT-3 Is Necessary for Proper Innervation of Motor Neurons TrkA/TrkC-positive fibers in the spinal cord could be detected at E15 ( Figure 1 E). TrkA-positive fibers were restricted to and terminated in the dorsolateral spinal cord, whereas TrkC-positive fibers entered the cord dorsomedially, and descended into the ventral horns in WT ( Ozaki and Snider 1997 ) and Bax KO embryos. There was no detectable TrkC expression in NT-3 KO spinal cord, indicating complete absence of proprioceptive fibers. In double KO spinal cord, TrkC-positive fibers entered the dorsal spinal cord and descended medially in a manner similar to that seen in WT or Bax KO cases. However, it was not possible to follow TrkC immunolabeled fibers all the way to their terminal zones in any of the cases. Next we examined the central projections of DRG axons with the lipophilic tracer DiI at P0. In WT and Bax KO pups, proprioceptive afferents entered the dorsal spinal cord and followed a medial course towards the ventral horn. They then turned laterally towards motor neurons in the lateral motor column, where they branched and terminated ( Figure 2 A). DiI labeling was confined to dorsal spinal cord in NT-3 KOs ( Figure 2 A), as reported earlier, consistent with a complete absence of proprioceptive innervation ( Ernfors et al. 1994 ; Tessarollo et al. 1994 ). In double KOs, proprioceptive afferents initially followed a trajectory similar to that of WT counterparts, but most of them failed to project all the way to the ventral cord and into the lateral motor column. Instead, they arborized near the ventral midline; some crossed the midline and extended into the contralateral ventral cord ( Figure 2 A and 2 C). In order to distinguish between a role for NT-3 in initiation of motor neuron innervation and a role for maintenance, we repeated the DiI labeling at E17. Innervation patterns of E17 spinal cords ( Figure 3 ) were similar to those at P0 ( Figure 2 ). Dorsal horns of all genotypes were filled with DiI-labeled fibers corresponding to nerve growth factor–dependent nociceptive axons. In WT and Bax KO embryos, proprioceptive fibers extended towards ventral horn motor neurons ( Figure 3 A and 3 B), whereas the ventral horns of the NT-3 KO embryos were devoid of innervation ( Figure 3 C). In the Bax/NT-3 double KOs, DiI-labeled fibers entered the ventral spinal cord, but extended towards the midline instead of the ventral horn ( Figure 3 D), in a pattern similar to that observed at P0. Our data point to a complete absence of proprioceptive innervation of the ventral horn of the Bax/NT-3 null spinal cord throughout the developmental stages investigated. As the sensory axons never reach motor neuron dendrites in the ventral horn ( Figure S1 ), the stretch reflex arc circuit is not established. The failure to initiate contact between sensory axons and motor neurons in the absence of NT-3 suggests a requirement for NT-3 for proper axon targeting in addition to a role in sensory axon maintenance. Figure 2 Axonal Projections in the Spinal Cord after DiI Labeling of DRG at P0 (A) Rescued DRG proprioceptive neurons fail to properly innervate motor neurons in double KOs. Instead, some axons are directed towards the ventral midline; they cross the midline and branch. (B) Schematic drawing of the monosynaptic reflex arc as it normally develops. Small black dots represent NT-3 released centrally by the motor neurons and peripherally by the muscle spindles. (C) High-power magnification of the inset in (A). Arrow points to the midline, and arrowheads point to synaptic bouton-like structures. Scale bar: 1 mm (A), 400 μm (C). Figure 3 Sensory Axons Labeled with DiI through the DRG at E17 (A) DiI-labeled fibers in WT spinal cord. Notice proprioceptive axons extending towards the motor neurons located in the ventral horn of the spinal cord in cross section. (B) Bax null spinal cord. (C) NT-3 null spinal cord. Stained fibers are restricted to the nociceptive axons in the dorsal horn, as evidenced by the complete absence of labeling in the ventral spinal cord. (D) Bax/NT-3 double null spinal cord. Although fibers extend into the ventral spinal cord, they never grow towards the motor neurons, but are directed towards the midline instead. Scale bar: 100 μm. NT-3 Is Required for Proper Peripheral Innervation In order to study peripheral innervation in double KO animals, we investigated spindle development in the gastrocnemius muscle. Muscle spindles could be identified easily in P0 WT and Bax KO animals by their characteristic morphology ( Figure 4 A and 4 B). Proprioceptive fibers labeled with neurofilament-M (NF-M) antibody formed ring-like spiral endings wrapped around intrafusal bag fibers labeled with S46 antibody, specific for slow developmental myosin heavy chain protein ( Miller et al. 1985 ). As reported earlier, there were no muscle spindles in NT-3 KO animals ( Ernfors et al. 1994 ). On the other hand, Bax KO animals had more spindles than WT, and spindles were in clusters similar to animals over-expressing NT-3 in the muscle ( Wright et al. 1997 ). Although NT-3-dependent cells were rescued, no muscle spindles were detected in the limbs of double KOs ( Figure 4 A and 4 B). Muscle spindles could be observed with TrkC antibody in WT and Bax KO animals, but not in NT-3 or double KOs ( Figure 4 E). In both NT-3 and double KO animals, NF-M-labeled fibers could be detected in muscles, thus the muscles of these animals were not completely devoid of nerve fibers. Since there were no TrkC-positive fibers in these muscles, we think that these NF-M-labeled fibers correspond to motor axons. Absence of muscle spindles might be due to a failure in initiation of differentiation by proprioceptive axons, or a failure of maturation and maintenance in the absence of NT-3. To distinguish between these two possibilities, we investigated muscle spindle development at E15 and E17 with S46/NF-M immunostaining as well as DiI labeling. No structures with the characteristic muscle spindle shape could be detected in any of the genotypes at E15 ( Figure S2 ). However, numerous developing spindles could be identified at E17 in WT and Bax KOs, but not in NT-3 KO embryos ( Figure 4 C). Bax/NT-3 double null muscles were devoid of spindles ( Figure 4 C), except for one spindle-like structure observed in one leg of an embryo ( Figure 4 C, inset, denoted by the asterisk). DiI labeling through the DRGs at E17 yielded similar results, with muscle spindles identified in parallel sections in WT and Bax null muscles ( Figure 4 D), but not in NT-3 or double KO animals. Although DiI-labeled fibers could be detected in double null muscles, they never formed ring-like structures characteristic of muscle spindles. We also examined TrkA/TrkC expression at P0 in the tibial nerve, which carries sensory fibers to the gastrocnemius muscle as well as the skin of the lower leg. In NT-3 KOs, TrkA-positive axons could be seen in the tibial nerve but there were no TrkC-positive axons; in contrast, TrkA- and TrkC-labeled axons are both present in WT, Bax KO, and double KO animals ( Figure 4 F). These results suggest that although proprioceptive axons follow proper trajectories in distal peripheral nerves, they fail to innervate their target muscles in the absence of NT-3. Figure 4 Muscle Spindles in Gastrocnemius Muscle and TrkA/TrkC Staining in the Tibial Nerve at P0 (A) NF-M (red) and S46 (green) immunostaining in cross section of gastrocnemius muscle at P0. There are no muscle spindles detected in double KOs. (B) NF-M (red) and S46 (green) immunostaining in parallel sections of gastrocnemius muscle at P0. (C) NF-M (red) and S46 (green) immunostaining in parallel sections of gastrocnemius muscle at E17. Double null muscles are mostly devoid of muscle spindles, except for one spindle-like structure detected (shown in inset, denoted by the asterisk). (D) Muscle spindles detected by DiI labeling through the DRG. Gastrocnemius muscle is sectioned at 40 μm thickness in parallel plane to the muscle fibers. (E) Muscle spindles detected by TrkC staining in cross section of gastrocnemius muscle at P0. (F) TrkA (red) and TrkC (green) immunostaining in the tibial nerve cross section at P0. TrkC-positive fibers are missing in NT-3 KOs. Red-green overlap (yellow) is due to the thickness of the section and overlapping of red- and green-labeled (TrkA and TrkC) fibers present at different focal depths, rather than co-localization. Arrows indicate muscle spindles. Scale bar: 50 μm (A, B, D), 25 μm (C, E), 75 μm (F). NT-3 Is a Chemoattractant for DRG Axons In Vitro To test the hypothesis that NT-3 acts as a chemoattractant for sensory axons, we performed a series of in vitro assays. Proprioceptive axons in mice enter the gray matter in the spinal cord and advance ventrally parallel to the midline by E13 and reach the motor neurons by E15 (Ozaki and Snider 1994). We co-cultured collagen-embedded E13 WT DRG explants with NT-3-soaked sepharose beads ( n = 26). Control cultures were set up using bovine serum albumin (BSA)– or phosphate buffer saline (PBS)–soaked beads ( n = 12). DRG axons began extending towards the localized NT-3 source by the end of the first day and consistently displayed a strong chemoattraction by 3 d in vitro, whereas they did not show such preference for BSA-loaded control beads ( Figure 5 A and 5 B). This attraction was not due to survival support of NT-3 because Bax null ganglia displayed the same chemoattraction ( Figure 5 C; n = 16). NT-3 may act through either TrkC, or the p75 NTR . We repeated the co-culture experiments with DRG explants derived from p75 NTR KO mice ( n = 18). Axons of these ganglia also showed strong chemoattraction towards the NT-3 beads ( Figure 5 D). Finally, we used diffusible TrkC receptors conjugated to IgG constant regions (TrkC-Fc) added to the medium ( n = 6) to deplete soluble NT-3 from the collagen gels ( Figure 5 E). In the presence of TrkC-Fc the chemoattraction was completely blocked, demonstrating that the effect we see is specific for NT-3. In order to investigate the active range of our beads, we have repeated the cultures with WT E13 DRGs by placing the beads at increasing distances from the ganglia ( n = 4 each) ( Figure 5 F). There was still preferred growth towards the bead at 1,200μm, the longest distance studied, although the number of axons and the extent of growth were not as robust. Next, we set up E13 DRG spinal cord explant cultures using NT-3-loaded beads placed at the midline at mid-spinal cord level as an ectopic NT-3 source ( n = 15). Control cultures were set using PBS-loaded beads ( n = 6). DiI labeling through the DRGs revealed numerous fibers entering the spinal cord at ectopic regions and growing towards the NT-3 beads ( Figure 6 A), surrounding the beads and forming bundles around them ( Figure 6 C– 6 E). In control cultures, all labeled fibers were directed towards the dorsal spinal cord and terminated there, where they normally enter the gray matter at E13 ( Figure 6 B). No axons were observed around the PBS-loaded control beads ( Figure 6 F). Axons that normally enter the gray matter through dorsal spinal cord grow towards the midline when presented with a localized NT-3 source, and new axon growth towards the NT-3 bead is initiated from DRGs, entering the spinal cord at ectopic loci at lateral mid-spinal cord ( Figure 6 G). NT-3 is therefore capable of acting as a chemoattractant for DRG axons. Figure 5 Chemoattraction of E13 DRG Axons to Local NT-3 Observed by In Vitro Co-Culture Assays (A) WT DRG with NT-3-loaded bead. (B) WT DRG with BSA-loaded bead. (C) Bax null DRG with NT-3-loaded bead. (D) p75 null DRG with NT-3-loaded bead. (E) WT DRG with NT-3-loaded bead and TrkC-Fc in the medium. (F) WT DRG with NT-3 loaded beads placed at increasing distances away from the ganglia (range, 500–1,200 μm). Scale bar: 150 μm (A–E), 350 μm (F). Figure 6 Chemoattraction Towards NT-3 Beads Placed in E13 Spinal Cord DRG Explant Co-Cultures (A) NT-3 bead placed in the midline of E13 WT spinal cord. Notice axons labeled through the DRGs (circled with black dashed lines) growing towards the bead (circled with white dashed lines) enter the spinal cord at ectopic loci instead of dorsal spinal cord. (B) PBS-loaded bead in E13 spinal cord. All labeled axons extend along the dorsal spinal cord, where they terminate. (C) High-power image of the bead in (A). Notice labeled axons surrounding the bead. (D) High-power image of an NT-3-loaded bead. Notice axons bundled around the bead. (E) High-power image of an NT-3-loaded bead. Notice the axons approaching the bead via the dorsal spinal cord. (F) High-power image of a PBS-loaded bead. No labeled fibers were observed around control beads. (G) Summary of our observations from E13 spinal cord DRG organotypic cultures. In control cultures fibers extend along the dorsal spinal cord, where they normally enter the gray matter at E13. In the presence of an ectopic NT-3 source localized at the midline, these axons grow towards the NT-3 bead. NT-3 also initiates axon growth from the DRGs, entering the spinal cord at ectopic lateral loci, growing towards the bead, surrounding the bead, forming nerve bundles, and branching around it. Scale bar, 175 μm (A and B), 100 μm (C–F). Discussion Relatively few studies have implicated NT-3 as a chemoattractant agent for sensory and motor axons. Previously noted chemoattractant action of the embryonic mouse maxillary process on trigeminal ganglion neurons ( Lumsden and Davies 1986 ) is now attributed to NT-3 and brain-derived neurotrophic factor (BDNF) ( O'Connor et al. 1999 ). Recently, Tucker et al. (2001) showed that developing sensory and motor axons in limb slice cultures preferentially grow towards neurotrophin-soaked beads instead of following their normal trajectories. Conversely, beads soaked with neurotrophin function-blocking antibodies led to reduction of sensory and motor axon growth towards the limb. In transgenic mice, which over-express NT-3 under the nestin promoter in the central nervous system, the course of the proprioceptive afferents are altered and directed towards the regions with high levels of ectopic NT-3 expression in the spinal cord ( Ringstedt et al. 1997 ). Ringstedt et al. considered the possibility that NT-3 may play a chemoattractant role during the innervation of ventral horns by proprioceptive afferents. However, earlier findings of normal proprioceptive afferent trajectories in chicken embryos despite injection of function-blocking NT-3 antibody into the spinal cord ( Oakley et al. 1995 ) led them to discount this possibility. In that study, though, assays on the effectiveness of antibody perturbation on NT-3-dependent cell survival showed that effectiveness was significant (approximately 90%) but not complete ( Oakley et al. 1995 ). Ringstedt et al. (1997) argue that while central sensory axons may still navigate properly in the absence of NT-3, ectopic expression of NT-3 disrupts their targeting. Recently, another member of the neurotrophin family, BDNF, has been suggested to act as a chemoattractant for sensory axons innervating ear ( Tessarollo et al. 2004 ). In a gene replacement strategy in which BDNF expression was driven by the NT-3 promoter, vestibular axons rerouted towards ectopic sources of BDNF in the cochlea that normally expressed NT-3 and were not innervated by these axons. Ectopic NT-3 supplied using osmotic pumps and adenovirus-mediated expression induce sensory axon growth during regeneration ( Zhang et al. 1998 , Bradbury et al. 1999 , Oudega et al. 1999 , Ramer et al. 2002 ), and induce axonal plasticity of corticospinal axons in injured adult spinal cord ( Zhou et al. 2003 ), where the sprouting axons from the intact site cross the midline towards the NT-3 source on the lesion side of the spinal cord. Our present results are in agreement with these observations, and provide further evidence that NT-3 acts as a chemoattractant for sensory afferents. Previously, normal motor neuron innervation was rescued in NT-3 KO animals by over-expressing NT-3 selectively in their muscles ( Wright et al. 1997 ). It appears that peripheral NT-3 alone is sufficient for rescuing proprioceptive neurons in NT-3 KO animals and proper axonal pathfinding in the spinal cord. However, it is unclear whether NT-3 is absent from the ventral spinal cords of these animals. The possibility has been raised that motor neurons may retrogradely transport NT-3 in muscle to the spinal cord ( Chen and Frank 1999 ). Assays to determine whether any NT-3 is present in the ventral horns of these mice would be informative. Recently, Patel et al. (2003) reported their observations on Bax/NT-3 double KO mice they bred. Their results are significantly different from ours. They see no proprioceptive afferents in the periphery of the double knockouts and note that central proprioceptive afferents terminate in the intermediate spinal cord without extending ventrally. Their observations are based on PV immunostaining and DiI labeling of the peripheral nerves. We noted that PV immunolabeling is diminished in our Bax/NT-3 double KOs. In various manipulations of the neurotrophins it has been noted that molecular markers for proprioceptive axons such as PV or calcitonin gene-related peptide for nerve growth factor–responsive axons are compromised ( Ringstedt et al. 1997 ; Patel et al. 2000 ), thus PV immunostaining in Bax/NT-3 double KOs cannot reveal the extent of proprioceptive axons in the periphery. Seventy-three percent of retrogradely labeled gastrocnemius muscle afferents were reported to be expressing TrkC RNA in the adult rat DRG, although some of these (about 10%) may represent cutaneous innervation ( McMahon et al. 1994 ). Presently, it is not clear whether TrkC protein is also made by cutaneous afferents and if so at what stage in development this receptor is expressed by cutaneous axons. Based on the available evidence showing that all proprioceptive neurons are eliminated in NT-3 or TrkC null mice, we think that TrkC staining in the tibial nerve mostly represents proprioceptive axons in the vicinity of their peripheral target. Altogether, our findings suggest a role for NT-3 in initiation of muscle innervation and spindle differentiation by the proprioceptive axons. Patel et al. (2003) examined DiI-labeled central DRG axons in the spinal cord of three E17 and two P0 cases, and report that central proprioceptive axons stop in the intermediate laminae, never entering the ventral cord. We have examined nine Bax/NT-3 double KO cases, and often incomplete DiI labeling gives the impression that there are no axons reaching the ventral spinal cord. We have also seen cases similar to theirs ( n = 4), but at higher magnification these axons did not have terminal boutons and were not completely labeled. However, with complete fills ( n = 5), it was possible to trace these axons into the ventral midline and across to the contralateral side, and visualize terminal boutons at their tips (see Figure 3 C). Previously, Arber et al. (2000) reported that members of the Ets family of transcription factors, Er81 and Pea3, are expressed by DRG neurons as well as motor neurons and their target muscle fibers. They found that in the spinal cord of Er81 KO mice, ventral projections of proprioceptive axons were mostly absent, and very few axons made it to the ventral cord. Patel et al. (2003) note that the phenotype they observed in their Bax/NT-3 double KO mice is quite similar to that of Er81 KO mice. They provide evidence that NT-3 induces Er81 expression in DRG explants in vitro. Patel et al. (2003) report that Er81 mRNA expression is diminished (but not abolished) in both NT-3 KO and Bax/NT-3 double KO mice, while their immunohistochemistry shows much less protein expression in the double KO mice. It is highly possible that a small but considerable number of DRG cells express the transcription factor Er81 and that their axons grow beyond the intermediate levels of the spinal cord in Bax/NT-3 double KO mice. While the phenotype of Er81 KO mice is quite dramatic, and most proprioceptive axons stop within the intermediate spinal cord, it is important to note that a few axons still find their way to the ventral spinal cord and target properly to the motor neurons ( Arber et al. 2000 ). Patel et al. (2003) also present observations from islet2 DTA mice, which lack a significant portion of the motor neurons in the ventral cord. In these mice PV immunostaining shows axons in the ventral horns. Patel et al. argue that since motor neurons are absent in these mice, NT-3 secreted by them could not be a signal for proprioceptive axons to enter the lateral motor columns. However, there is no evidence showing that NT-3 mRNA or protein expressed in the ventral spinal cord is exclusively from motor neurons, and there are no available data indicating that in islet2 DTA mice, NT-3 expression in the ventral spinal cord is abolished ( Yang et al. 2001 ; Pun et al. 2002 ). Studies in embryonic mice reported NT-3 mRNA in the ventral horns of the spinal cord, but it is not definitive that both mRNA and protein are expressed solely by motor neurons. In the adult spinal cord, while motor neurons express high levels of NT-3, other cells, including glia, also express it ( Zhou and Rush 1994 ; Dreyfus et al. 1999 ; Buck et al. 2000 ). Our present results, along with those from transgenic mice with NT-3 over-expression in ectopic regions of the spinal cord ( Ringstedt et al. 1997 ), argue for a role of NT-3 in chemoattractant axon guidance of proprioceptive axons in the spinal cord. Finally, in culture assays, we see a strong chemoattraction of DRG neurons to localized sources of NT-3. This response is seen in WT, Bax null and in p75 null DRG explants, and in the absence of any other neurotrophins or target-derived axon guidance molecules. Furthermore, in vitro, sensory axon response to NT-3 does not appear to be dose-dependent ( Ringstedt et al. 1997 ; Tucker et al. 2001 ). NT-3 is capable of attracting axons along distances of up to 1 mm in collagen gel matrix, covering the physiological range it needs to attract axons during development. Previous studies with exogenous or local applications of NT-3 to developing primary sensory axons have indicated that this neurotrophin can attract and induce axonal branching ( Ulupınar et al. 2000 ; Özdinler and Erzurumlu 2001 , Özdinler et al. 2004 ). Along the monosynaptic stretch reflex pathway, only Wnt-3 has been implicated as an axon arborization factor in the spinal cord ( Krylova et al. 2002 ). Another molecule, Slit2, expressed in the midline and by motor neurons ( Wang et al. 1999 ) is capable of inducing axonal branching ( Nguyen Ba-Charvet et al. 2001 ; Özdinler and Erzurumlu 2002 ). DRG neurons express Robo receptors, which bind to Slits, and proprioceptive axons are therefore capable of responding to Slit signals ( Wang et al. 1999 ). Slit2 does not cause repulsion of NT-3-responsive DRG axons in vitro ( Nguyen Ba-Charvet et al. 2001 ), but causes ectopic branching and arborization of trigeminal axons in the brainstem ( Özdinler and Erzurumlu 2002 ). Thus, Slit2 might also be involved in terminal branching of propioceptive axons in the ventral cord and in the midline branching observed in our double KOs. Lack of PV expression in Bax/NT-3 KO mice suggests that PV expression might be responsible for proper axon targeting and muscle spindle differentiation. Presently we cannot completely rule out this possibility. However, no defects in axon pathfinding along the monosynaptic reflex arc or in muscle spindle differentiation have been noted in PV KO mice, which develop normally and show no apparent changes in their behavior or physical activity ( Schwaller et al. 1999 ). These observations suggest that axonal targeting defects in Bax/NT-3 double KO mice cannot be simply due to lack of PV expression in proprioceptive cells. Our present results suggest that NT-3 acts as a short-range axon guidance cue for proprioceptive axons centrally and peripherally, as they navigate to their targets using other axon guidance cues. In its absence, these axons terminate in inappropriate loci. However, NT-3 may not be the only molecule that plays a role in targeting and terminal branching of sensory axons in the ventral spinal cord. NT-3 most likely acts cooperatively with other axon guidance molecules or by regulating expression of yet to be identified proprioceptive neuron-specific receptors/ligands for numerous axon guidance cues. Materials and Methods Generation of double KOs. We crossed Bax KO females on a C57BL/6 background (Jackson Laboratory, Bar Harbor, Maine, United States) to NT-3 heterozygote males on a 129 Sv background to generate double heterozygote animals. Progeny was genotyped with PCR, and animals heterozygous for both genes were bred to obtain the double KOs. Primers used for the Bax locus were R661, GTT GAC CAG AGT GGC GTA GG; R662, CCG CTT CCA TTG CTC AGC GG; and R663, GAG CTG ATC AGA ACC ATC ATG. Primers used for the NT-3 locus were NT3A, CGT GGT GAG GTT CTA TTG GCT AC; NT3B, CAG AGC ACC CTG CCC AAA GCA GAG; NT3R, CCT TGA CAA TAC TGA ATG CC; and NEOF, GGG AAC TTC CTG ACT AGG. WT, Bax KO, and NT-3 KO littermates were used as controls. A total of 11 Bax/NT-3 double KO mice were analyzed, of these nine were P0 pups. The p75 colony on a 129 S1 background was received from Jackson Laboratory. We used tail DNA to genotype animals using the primers IMR0013, CTT GGG TGG AGA GGC TAT TC; IMR0014, AGG TGA GAT GAC AGG AGA TC (generic neo primers); IMR0710, TGT TAC GTT CTC TGA CGT GGT GAG; and IMR0711, TCA GCC CAG GGT GTG CAC TC ( p75 locus). For embryonic experiments, day of plug positivity was considered E0. All of the protocols used in this study were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guidelines for use of experimental animals. TrkA/TrkC immunohistochemistry. Frozen spinal cord sections (10 μm thick) were blocked and incubated in a cocktail of rabbit anti-TrkA and goat anti-TrkC antibodies (gift of Dr. Reichardt; Huang et al. 1999 ), followed by a cocktail of CY3 conjugated donkey anti-rabbit and FITC conjugated donkey anti-goat antibodies (Chemicon, Temecula, California, United States) in the presence of 0.3% TritonX-100 and 10% normal donkey serum. For PV immunohistochemistry, sections were reacted with monoclonal mouse anti-PV antibody (Sigma, St. Louis, Missouri, United States), and developed by Vector fluorescein mouse on mouse kit (Vector Laboratories, Burlingame, California, United States). For quantification purposes, TrkA- and TrkC-labeled cells from four different DRGs of each genotype and age studied were counted, and ratios plotted. Muscle spindle detection. Gastrocnemius muscle was dissected out in P0 pups and sectioned longitudinally, or, in some cases, the whole leg at the level of gastrocnemius muscle with tibial nerve was sectioned in cross section. Sections were incubated with monoclonal S46 antibody (gift of Dr. Stockdale) reactive to the spindle-specific slow-tonic myosin heavy chain isoform and developed by Vector mouse on mouse kit as described above, followed by rabbit anti-NF-M antibody (Chemicon) and CY3 conjugated goat anti-rabbit antibody (Chemicon) in a sequential double-labeling protocol. In another method, DiI crystals were placed in DRGs of E17 embryos from different genotypes ( n = 2), and the gastrocnemius muscle was isolated and sectioned into 40-μm-thick slices on a vibratome. DiI labeling. Spinal cords were dissected out with the DRGs attached, motor root was cut to prevent backfilling of motor neurons, and crystals of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes, Eugene, Oregon, United States) were inserted in DRGs. Labeled spinal cords were incubated at 37 °C for 8 d and sectioned on a vibrotome at 100 μm. Sections were observed under the fluorescent microscope and photographed, and images were transferred to Adobe Photoshop, inverted, and adjusted for brightness and contrast. Photoconverted in 0.15% DAB (3,3′-diaminobenzidine; Sigma) in 0.1M Tris buffer (pH 8.2). In vitro co-culture assay. DRG explants were derived from Swiss Webster, Bax null, and p75 null mouse embryos (E13). For collagen matrix assays, DRGs were dissected out under a stereomicroscope using tungsten needles. Collagen matrix was prepared with 430 μl of collagen (3 mg/ml dissolved in 0.1M acetic acid, Sigma), 50 μl of 10X DMEM medium, and 2.5 μl of 0.8 M NaCa 2 , and the pH was adjusted to 7.5. Individual ganglion explants were placed in 24-well plates and covered with freshly prepared collagen. Sepharose beads with an average diameter of 150 μm were used. Beads were washed twice with PBS, air dried, and loaded with 10, 20, 50, or 100 ng/μl NT-3 (Collaborative Research, Chemicon) at 4 °C overnight with constant shaking. For negative control, beads were loaded with BSA (10–100 ng/μl) or PBS. Either a single neurotrophin-loaded bead or a single BSA (or PBS)–loaded bead was implanted about 200–1,200 μm away from the ganglion explant. Collagen-embedded cultures were then placed at 33 °C for 15 min for the matrix to harden. Serum-free culture medium was then added to each well. In cultures with WT DRG and control beads, 10% serum was added to ensure viability of the explants. TrkC-Fc (Regeneron Pharmaceuticals, Tarrytown, New York, United States) was added (20 μg/ml) into the culture medium. Explant co-cultures Spinal cords with attached DRGs were dissected out from Swiss Webster mouse embryos at E13, and sectioned into 300-μm-thick slices. Tissue slices were placed on Millicell Tissue Culture Inserts (Millipore, Billrica, Massachusetts, United States). NT-3-loaded ( n = 15) or PBS-loaded ( n = 6) sepharose beads were prepared as described above, and placed in the midline at mid-spinal cord level. Inserts were placed in six-well plates with serum-free medium at the bottom of the wells, and kept at 33 °C for 3 d in the presence of 5% CO 2 . Cultures were then fixed with 4% paraformaldehyde in PBS, and small crystals of DiI were placed into the DRGs. Samples were incubated in a 37-°C incubator, allowing the dye to diffuse, and photographed under a Nikon (Tokyo, Japan) inverted epifluoresence microscope. Supporting Information Figure S1 Motor Neuron Innervation Initiated in WT E17 Embryos Cannot Be Detected in Bax/NT-3 Double KOs In some of the samples, motor neurons were labeled by backfilling through the ventral root in addition to the sensory axons labeled through the DRGs. (A) WT embryo showing proprioceptive axons contacting motor neuron dendrites in the ventral horn, forming synapses. (B) Bax/NT-3 double null spinal cord. Although labeled fibers enter the ventral spinal cord, they extend towards the midline instead of the ventral horn and never contact the motor neuron dendrites. (C) High-power image of the inset in (A). Arrows point to the proprioceptive fibers contacting motor neurons (asterisk). (D) High-power image of the inset in (B). Notice that there are no sensory axons contacting labeled motor neurons (asterisk). Scale bar: 100 μm (A and B), 50 μm (C and D). (24 MB TIF). Click here for additional data file. Figure S2 S46/NF-M Immunohistochemistry at E15 Gastrocnemius Muscle Although numerous muscle and nerve fibers were labeled, no muscle spindles could be identified because the characteristic morphology of sensory nerve ending wrapped around muscle bag fiber had not begun to develop in any of the genotypes yet. Scale bar: 25 μm. (13 MB TIF). Click here for additional data file.

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519003

A Molecular Model of Blood Cell Renewal

A developing organism captured on time-lapse video is a wonder to behold. If you're watching a chick embryo, by day 3, you'll see millions of cells engaged in a frenzy of activity, as rapidly dividing cells migrate to new positions, acquire the characteristics of specialized cells, and craft well-defined tissues, organs, and limbs in just under two weeks. In addition to the cells destined for specialization is another important group, stem cells, whose progeny have two very different fates. They can either “self renew”—that is, make identical copies of themselves—or generate intermediate progenitor cells that give rise to mature, differentiated cells. Both differentiation and self renewal are guided by an elaborately regulated genetic program, which transforms embryonic stem cells into the many different cell types that make up the body. Adult stem cells share the hallmark trait of self renewal, but are relatively rare: in bone marrow, the source of hematopoietic, or blood-forming, only an estimated one in 10,000–15,000 cells is an adult hematopoietic stem cell (HSC). Studies that have compared the gene expression profiles of different types of stem cells to identify genetic signatures of “stemness” have found only a limited number of signature genes. And the molecular mechanisms that regulate this so-called potency and the self renewal process have remained obscure. Now, focusing on HSCs, Margaret Goodell and colleagues have undertaken a systematic evaluation of HSC renewal. The study identifies molecular signatures associated with discrete stages of the HSC self renewal cycle and proposes a molecular model of the process. HSC renewal passes through three stages: quiescence, activation and proliferation, and a return to the dormant state. HSCs give rise to both red blood cells, which carry oxygen and carbon dioxide, and white blood cells, which fight infection. Certain stressors—including blood-cell-inhibiting chemotherapy and bone marrow transplants—trigger HSC activation, which induces rapid proliferation, generating both progenitors to deal with the threat and new stem cells that return to quiescence. Once activated by a trigger, dormant HSCs engage a regulatory program that rapidly churns out billions of cells, then puts the brakes on cell division, prompting the return to a nondividing, quiescent state. To understand the genetic programs underlying this process, Goodell and colleagues induced proliferation in HSCs (with the chemotherapeutic drug, 5-fluorouracil, or 5FU), then allowed the cells to return to quiescence, so they could characterize the changes in gene expression that occurred during each stage. They compared these time-specific patterns to the gene expression profiles of naturally proliferating fetal mouse HSCs (which undergo massive proliferation) and quiescent adult mouse HSCs (which hardly divide at all) to find genes associated with the two different states. Genes were grouped into proliferating or quiescent groups based on when they were expressed after 5FU treatment, and these groupings were refined based on comparisons to previously published HSC gene expression data. Functional analysis of these genes found a bias toward genes involved in cell division processes in the proliferation stage and toward cell division inhibitors in the quiescent stage, supporting the logic of the groupings. To understand the activation process at a global level, the authors employed some novel analysis strategies, including the “Gene Ontology” (GO) system for classifying genes. With these results, Goodell and colleagues constructed a model of the HSC self renewal cycle: quiescent HSCs maintain a “state of readiness,” molecularly speaking, that allows a quick response to environmental triggers. A stressor (like the chemotherapy mentioned above) triggers a “prepare to proliferate” state—a kind of pregnant pause—and then the proliferation machinery kicks in, going through an early and late phase before quiescence returns. By shedding light on the molecular mechanisms of stem cell renewal, this study will aid efforts to develop stem-cell-based clinical therapies, which depend on replicating the HSC self renewal cycle to replenish diseased or damaged tissue, and will ultimately guide efforts to grow stem cell colonies outside the body, a long-standing goal that would have many clinical applications. The authors suggest their findings may also be relevant to studies of cancer stem cells, tumor cells with self renewal properties.

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551537

Identification of ciliated sensory neuron-expressed genes in Caenorhabditis elegans using targeted pull-down of poly(A) tails

An mRNA-tagging method was used to selectively isolate mRNA from a small number of cells for subsequent cDNA microarray analysis. The approach was used to identify genes specifically expressed in ciliated sensory neurons of Caenorhabditis elegans .

Background Recent advances in technologies for analyzing whole-genome gene-expression patterns have provided a wealth of information on the complex transcriptional regulatory networks and changes in gene-expression patterns that are related to phenotypic changes caused by environmental stimuli or genetic alterations. Changes in gene expression are also fundamental during development and cellular differentiation, and differences in gene expression lead to different cell fates and eventually determine the structural and functional characteristics of each cell type. Comparative analyses of gene-expression patterns in various cell types will therefore provide a framework for understanding the molecular architecture of these cells as cellular systems. Caenorhabditis elegans is an ideal model organism for investigating development and differentiation at high resolution, because adult hermaphrodites only have 959 somatic nuclei, whose cell lineages are all known. About 19,000 genes were identified by determination of the C. elegans genome sequence [ 1 ]. Functional genomic approaches, including systematic inhibition of gene functions by RNA interference [ 2 - 5 ], large-scale identification of interacting proteins [ 6 ], systematic generation of deletion mutants [ 7 - 9 ], and determination of the time and place of transcription [ 10 - 12 ], are currently in progress to accumulate information on all genes in the genome. Genome-wide gene-expression profiling using DNA or oligonucleotide microarray technology has also been applied to this organism. Microarrays containing more than 90% of C. elegans genes have been constructed and used in global gene-expression analyses under a wide variety of developmental, environmental and genetic conditions [ 13 - 15 ]. Genome-wide gene expression analyses of the germline have also been carried out [ 16 , 17 ]. Mutants lacking functional gonads and those with masculinized or feminized gonads were used in these studies to identify germline-expressed genes and genes correlated with the germline sexes. To analyze gene-expression patterns in various cells, particularly those forming small tissues, selective isolation of mRNA from these cells is necessary. As an example of this approach, mRNA was prepared from mechanosensory neurons after cell culture of their embryonic precursors followed by selection of the cells by flow cytometry [ 18 ]. Although embryonic cell cultures allow the collection of cells at early stages of development, methods for the separation, culture and collection of fully developed tissues have not been established and might be technically difficult. C. elegans modifies its behavior by sensing environmental cues such as food, chemicals, temperature or pheromones. These cues are recognized by approximately 50 sensory neurons positioned in the head and tail. Although the overall functions of the chemosensory or thermosensory neurons have been examined by laser-killing experiments, the molecular mechanisms that underlie the functions of each sensory neuron have not yet been fully explored. Profiling of genes that are expressed in sensory neurons might therefore provide insights into the genes required for the specific functions of neurons. To identify sensory neuron-expressed genes, we adopted the mRNA-tagging method [ 19 ]. In this method, poly(A)-binding protein (PABP), which binds the poly(A) tails of mRNA, is utilized to specifically pull-down poly(A) RNA from the target tissues. By employing this method, we successfully identified novel genes that are expressed in the ciliated sensory neurons of C. elegans . Results Preparation of mRNA from particular types of neurons using mRNA tagging To isolate sensory neuron-expressed transcripts, we devised a method that utilizes PABP. This approach involves the generation of transgenic animals that express an epitope-tagged PABP using cell-specific promoters. Since PABP binds the poly(A) tails of mRNA [ 20 ], in situ crosslinking of RNA and proteins, followed by affinity purification of the tagged PABP from lysates of these animals, is expected to co-precipitate all the poly(A) + RNA from cells expressing the tagged PABP (Figure 1 ). This method was independently devised by Roy et al . and used to identify muscle-expressed genes [ 19 ], but whether the procedure was applicable to smaller tissues, such as neurons, was unknown. We applied this technology, mRNA tagging [ 19 ], to the ciliated sensory neurons of C. elegans ; these comprise approximately 50 cells whose cell bodies are typically 2 μm in diameter compared to the approximate animal body length of 1 mm. PABP is encoded by the pab-1 gene in C. elegans . Nematode strains expressing FLAG-tagged PAB-1 from transgenes were generated using tissue-specific promoters. To prepare mRNA from sensory neurons, we generated the JN501 strain (hereafter called che-2 ::PABP) in which the transgene was expressed in most of the ciliated sensory neurons using a che-2 gene promoter [ 21 ]. A second strain, JN502 ( acr-5 ::PABP), was generated to prepare mRNA from another subset of neurons using an acr-5 promoter, which is active in B-type motor neurons, as well as unidentified head and tail neurons [ 22 ]. A third strain, JN503 ( myo-3 ::PABP), which expressed the transgene in non-pharyngeal muscles using the myo-3 promoter [ 23 ], was generated to serve as a non-neuronal control. Expression of FLAG-PAB-1 was confirmed by western blotting analyses, and immunohistochemistry using an anti-FLAG antibody (data not shown). Expression patterns were essentially the same as those reported for the promoters used, but we note that expression of FLAG-PAB-1 in ventral cord motor neurons was weak in the acr-5 ::PABP strain compared to that in sensory neurons in the che-2 ::PABP strain. As a measure of the functional integrity of FLAG-PAB-1-expressing cells, responses of the che-2 ::PABP strain to the volatile repellent 1-octanol, which is sensed by ASH amphid sensory neurons was tested. The sensitivity of the che-2 ::PABP animals was indistinguishable from the wild type (data not shown). The ability of the exposed sensory neurons to absorb the lipophilic dye diQ was also tested. Amphid sensory neurons in the head stained normally, whereas phasmid neurons, PHA and PHB, in the tail showed weak defects in dye-filling (90% staining of PHA and 91% staining of PHB, compared to 100% in wild type for both neurons). The acr-5 ::PABP and myo-3 ::PABP strains appeared to move normally, suggesting overall functional integrity of motor neurons and body-wall muscles, respectively. Poly(A) RNA/FLAG-PAB-1 complexes were pulled-down from whole lysates of these transgenic worms using anti-FLAG monoclonal antibodies. Poly(A) RNA was then extracted and concentrated. The amounts of known tissue-specific transcripts were examined by reverse transcription PCR (RT-PCR) (Figure 2 ). The mRNA for tax-2 , which is expressed in a subset of sensory neurons [ 24 ], was enriched in RNA from che-2 ::PABP. The mRNA for odr-10 , which is expressed in only one pair of sensory neurons [ 25 ], was also highly enriched in che-2 ::PABP. On the other hand, mRNA for acr-5 and del-1 , both of which are expressed in B-type motor neurons [ 22 ], was enriched in RNA from acr-5 ::PABP. The mRNA for unc-8 , which is expressed in motor neurons and ASH and FLP sensory neurons in the head [ 26 ], was contained in RNA from both che- 2::PABP and acr-5 ::PABP. The mRNA for unc-54 , which is expressed in muscles [ 23 ], was enriched in RNA from myo-3 ::PABP. Representatives of housekeeping genes, eft-3 [ 27 ] and lmn-1 [ 28 ], were detected in RNA from all transgenic strains. Quantitative RT-PCR was performed to estimate the relative amounts of neuron type-specific transcripts. The amount of the odr-10 transcript in RNA from che-2 ::PABP was 39-fold higher than that from acr-5 ::PABP, and mRNA for gcy-6 , which is expressed in only a single sensory neuron [ 29 ], was enriched 10-fold. On the other hand, the mRNA for acr-5 was enriched eightfold in RNA from acr-5 ::PABP compared with that from che-2 ::PABP. mRNA for the pan-neuronally expressed gene snt-1 [ 30 ] was equally represented in RNA from both acr-5 ::PABP and che-2 ::PABP. Therefore, selective enrichment of sensory neuron-, motor neuron- and muscle-expressed genes in RNA from che- 2::PABP, acr-5 ::PABP and myo-3 ::PABP strains, respectively, have been achieved as intended. Of these, the enrichment of motor neuron-expressed genes appeared less efficient, because weak bands were sometimes seen for these genes in RT-PCR from che- 2::PABP or myo-3 ::PABP RNA. cDNA microarray experiments We used a cDNA microarray to compare the properties of mRNA prepared from che- 2-expressing ciliated sensory neurons with that from acr-5 -expressing cells. RNA purified from che-2 ::PABP was labeled with Cy5 and that from acr-5 ::PABP was labeled with Cy3. The two types of labeled RNA were mixed and hybridized to the cDNA microarray and the che-2 ::PABP/ acr-5 ::PABP (Cy5/Cy3) ratio was calculated for each cDNA spot. The cDNA microarray contained 8,348 cDNA spots corresponding to 7,088 C. elegans genes. Two sets of independently prepared RNA samples were hybridized to two separate arrays. The logarithm of the hybridization intensity ratio for each spot, log 2 ( che-2 ::PABP/ acr-5 ::PABP), was calculated and values from the two experiments were averaged. This calculation allowed us to order the genes represented on the microarrays according to the log 2 ( che-2 ::PABP/ acr-5 ::PABP) value (see Additional data file 1). Genes specifically expressed in che-2 -expressing cells should have higher rank orders in this list, whereas those expressed in acr-5 -expressing cells should have lower rank orders. To evaluate the results of the microarray experiments, we searched for genes that are known to be expressed in amphid sensory neurons, but not in ventral cord motor neurons, or vice versa, using the WormBase database (WS94). Of these, 20 sensory neuron-specific genes and five motor neuron-specific genes were present on the arrays (see Additional data files 1 and 2). These genes showed a highly uneven distribution, with sensory neuron-specific genes concentrated in the highest rank orders and motor neuron-specific genes distributed in lower rank orders (Figure 3a ). Muscle-expressed genes (also found using WormBase) were almost evenly distributed. However, intestine-expressed genes were concentrated in the lower rank orders. These results demonstrate that our mRNA isolation procedure specifically enriched ciliated sensory neuron- and motor neuron-expressed genes as intended. The unexpected distribution of the intestine-expressed genes will be discussed later. daf-19 encodes a transcription factor similar to mammalian RFX2. Several genes expressed in ciliated sensory neurons and essential for ciliary morphogenesis, such as che-2 and osm-6 , are under the control of daf-19 and have one or more copies of the cis -regulatory element X-box in their promoter regions [ 31 ]. We therefore examined the distribution of genes that harbor X-boxes in their promoter regions. Again, the distribution of X-box-containing genes was highly uneven (Figure 3b , see also Additional data files 1 and 2), further demonstrating the successful enrichment of ciliated neuron-expressed genes. Expression analysis of candidate sensory neuron-expressed genes by reporter fusions The above analyses showed that sensory neuron-expressed genes were enriched in the mRNA population purified from che-2 ::PABP. However, only a few genes were previously known to be expressed in these tissues. In fact, the expression patterns for most top-ranked genes in our list were not known. To determine which of these genes were actually expressed in sensory neurons, we examined the expression patterns of 17 genes with the highest rank orders using translational green fluorescent protein (GFP) fusions. The expression patterns for these genes had not been reported previously. We did not observe any GFP fluorescence for two clones, K07B1.8 and C13B9.1, probably because the promoter region we selected did not contain all the functional units or expression was below the level of detection. GFP-expressing cells were identified for all the remaining 15 genes (Figure 4 , Table 1 ). For 13 of these GFP fusions, expression was observed in ciliated sensory neurons, namely amphid, labial and/or phasmid sensory neurons. Of these, expression in the intestine, in addition to the sensory neurons, was observed for Y55D5A.1a and T07C5.1c, whereas expression of K10D6.2a was also observed in seam cells and the main body hypodermis (hyp7). Expression of K10G6.4 was observed in many other neurons in addition to sensory neurons. Expression in the intestine and coelomocytes, but not in sensory neurons, was observed for two other clones, C35E7.11 and F10G2.1, respectively. In summary, of the 15 genes whose expression patterns could be determined, 13 (87%) were expressed in sensory neurons. These results showed that most of the genes with the highest rank orders were expressed in ciliated sensory neurons. We also examined the expression patterns of two genes with the lowest rank orders (Y44A6D.2 and T08A9.9/ spp-5 ). Expression in the ventral nerve cord was observed for Y44A6D.2, while only weak expression in the intestine was observed for T08A9.9 (data not shown). These results also suggested that our procedure was somewhat less effective in enriching motor neuron-expressed genes than sensory neuron-expressed genes (Figure 3a ). Categorization of che-2 ::PABP-enriched genes reveals specific features In an attempt to characterize ciliated sensory neuron-expressed genes as a set, we first referred to functional annotations of each gene generated by the WormBase. It was noted that the fraction of genes with functional annotations was smaller for the highest ranked genes (Figure 5a ). BLASTP searches of the nonredundant (nr) protein sequence database and proteome datasets for several representative animal and yeast species showed that nematode-specific genes were enriched, while those with homologs in yeast and other animals tended to be under-represented in the top-ranked genes (Figure 5b,c ). Among the genes with Gene Ontology (GO) annotations, top-ranked genes showed a significantly larger fraction with a 'nucleic acid binding' functional capacity ( P = 0.004, Figure 6 ). Protein motifs found to be enriched among the che-2 ::PABP-enriched genes included 'cuticle collagen', 'chromo domain', 'linker histone' and 'laminin G domain'. Another prominent characteristic of the che-2 ::PABP-derived mRNA fraction was enrichment of genes homologous to nephrocystins. Nephrocystins are responsible for a hereditary cystic kidney disease, nephronophthisis, and to date, nephrocystin 1 ( NPHP1 ) through nephrocystin 4 ( NPHP4 ) have been identified [ 32 - 35 ]. C. elegans homologs of NPHP1 and NPHP4 were ranked at positions 15 and 25 in our list, suggesting a link between these disease genes and the functions of worm sensory neurons. Discussion Preparation of mRNA from a subset of neurons in C. elegans We prepared poly(A) RNA from a subset of neurons using the mRNA-tagging technique. The genome-wide identification of muscle-expressed genes demonstrated that mRNA tagging is a powerful technique for collecting tissue-specific transcripts in C. elegans [ 19 ]. The method is especially useful in this organism because dissection and separation of the tissues are difficult because of the worm's small size and the presence of cuticles. However, it was not known whether this method was applicable to smaller tissues, such as subsets of neurons. In this study, we attempted to isolate mRNA from ciliated sensory neurons using mRNA tagging. Although the volume of target neurons was much smaller than that of muscles, transcripts of various sensory neuron-expressed genes, ranging from those expressed in many sensory neurons to those expressed in only one or two sensory neurons, were successfully enriched. The procedure of mRNA tagging is based on immunoprecipitation of poly(A)-RNA/FLAG-PAB-1 complexes. A potential problem with this technique is that once the cells are broken, poly(A) RNA released from non-target cells might bind unoccupied FLAG-PAB-1. To reduce this possibility, we adopted stringent washing conditions in addition to in situ formaldehyde crosslinking. Although this procedure reduced the recovery of immunocomplexes, it ensured minimal contamination by mRNA from non-target cells. As there are many characterized promoters that can deliver FLAG-PAB-1 to small numbers of neurons in C. elegans , profiling of the gene-expression pattern of each type of neuron should be possible with this technique. Another potential problem with this method is that PABP might have different binding affinities for different transcript species, rendering some tissue-specific transcripts difficult to recover. Although PABP binds tightly to the poly(A) tails of most mRNA [ 36 ], RNA species co-immunoprecipitated with PABP from cultured cells do not represent the total RNA of the cells [ 37 ]. This might also cause another problem in that transcripts with strong PABP affinity might be undesirably enriched in the precipitates and cause unexpected biases. Analysis of purified mRNA using a cDNA microarray Preparation and characterization of EST clones led to the identification of more than 10,000 cDNA groups corresponding to different genes of C. elegans ([ 12 , 38 ] and Y.K., unpublished results). We used a cDNA microarray on which such cDNA clones were spotted to identify the genes expressed in ciliated sensory neurons. Using a cDNA microarray rather than a genome DNA microarray has the advantage that genes on the array have guaranteed expression, and hybridization to the corresponding mRNA species is efficient. The microarray we used contained 7,088 genes of C. elegans , representing 40% of the predicted genes on the genome [ 1 ]. On the other hand, there are also genes that were not represented in our cDNA collection, including characterized sensory neuron-specific genes such as osm-6 [ 39 ] and most seven-transmembrane receptor genes including odr-10 ; this might be a disadvantage of using a cDNA microarray. Acquisition of cDNA clones for rare mRNA species and use of whole-genome microarrays are complementary approaches for improving the applicability of the method described here. Evaluation of microarray experiments Previously known sensory neuron- and motor neuron-expressed genes were used to evaluate the results of our microarray analyses. Most genes were enriched in our che-2 ::PABP-derived mRNA preparations or in the acr-5 ::PABP-derived mRNA preparations depending on their expression patterns. However, several genes were not enriched as expected. Furthermore, enrichment of motor neuron-expressed genes in the acr-5 ::PABP-derived mRNA preparations appeared less efficient. The reasons for these occurrences are unknown, but the expression of FLAG-PAB-1 in motor neurons were low in the acr-5 ::PABP strain, which could account for the low efficiency of enrichment for this tissue. Another potential problem is that the expression pattern of the acr-5 promoter has not been fully characterized [ 22 ], and both the che-2 and acr-5 promoters are active in labial neurons, where expression of the acr-5 promoter was relatively strong compared to motor neurons (data not shown). Genes expressed in the intestine were enriched in the acr-5 ::PABP-derived mRNA preparations. FLAG-PAB-1 was weakly expressed in both the che-2 ::PABP and acr-5 ::PABP strains in intestine, with the latter showing higher level of expression (data not shown). Low-level expression of artificially manufactured genes in the intestine seems to be quite common, either due to readthrough transcription from the vector or the 3' regulatory sequences. Our results may suggest that in future applications one must be very careful about this type of low-level expression of FLAG-PAB-1. We determined the expression patterns of genes highly enriched in the che-2 ::PABP-derived mRNA preparations. Thirteen of 15 genes that showed clear expression patterns of GFP reporters were expressed in multiple sensory neurons. None of these genes has previously been characterized. In addition, quantitative PCR analysis shows that genes expressed in only one or two neurons, gcy-6 and odr-10 , respectively, can be enriched. Therefore, our procedure is effective for identifying genes that are preferentially expressed in a particular subset of cells. On the other hand, the presence of small fractions of genes that are predominantly expressed in tissues other than sensory neurons was also evident. Therefore, mRNA-tagging technology should be regarded as enrichment of candidate cell-specific genes and the real expression pattern of each gene should be verified independently. Characterization of the sensory neuron-expressed gene set Since most of the genes enriched in the che-2 ::PABP-derived mRNA preparations proved to be sensory neuron-expressed, characterization of the enriched genes as a set should lead to molecular characterization of the sensory neurons of C. elegans . A prominent feature of the genes enriched in the che-2 ::PABP-derived mRNA preparations is that they include nematode-specific genes more often than the rest of the genes, as judged from inter-species BLASTP comparisons, suggesting that many of the genes identified have functions unique to nematode sensory neurons. The existence of many nematode-specific gene families has previously been noted, and was proposed to be related to the nematode-specific body plan [ 40 ]. Since we were obviously counter-selecting for ubiquitously expressed genes that serve common cellular functions, a lower representation of highly conserved genes is expected. In addition, these observations indicate that our approach is effective for identifying hitherto uncharacterized genes that might be important for specific functions of differentiated cell types. Identification of panels of genes expressed in particular cells will also be useful for understanding the regulatory network of gene expression. In this context, it is of interest to examine whether we can identify cis -acting elements commonly found in the promoter regions of the sensory neuron-expressed genes. The enrichment of X-boxes in the che-2 ::PABP fraction suggests that this might be plausible. In addition, other reports have identified cis -acting elements in genes expressed during particular developmental stages or in particular neurons (see, for example [ 41 , 42 ]). However, searches for common sequences using the MEME program did not reveal any motifs that were enriched in the che-2 ::PABP fraction. This is likely to be due to the heterogeneity of our sensory neuron-expressed gene collection (see the expression patterns in Table 1 ). Further refinement of our gene sets by expression analysis of each gene will be required to identify cis -acting elements that regulate cell-specific gene expression. By surveying GO annotations and protein motifs, genes whose predicted functions are related to nucleic acids and/or chromatin were found to be enriched in the che-2 ::PABP gene set. This might indicate that C. elegans sensory neurons have specialized regulatory mechanisms for gene expression, although it remains to be seen which of these 'chromatin' genes are actually expressed in a sensory neuron-specific manner. It was also apparent from visual inspection or computer searches that two homologs of nephrocystins are included in the highest rank orders. It has recently been shown that nephrocystin 1 and nephrocystin 4 interact with each other and are both components of cilia. These studies have led to the hypothesis that the kidney disease nephronophthisis is caused by malfunctions of cilia on the tubular epithelium [ 33 - 35 , 43 ]. C. elegans ciliated sensory neurons also have prominent ciliary structures [ 44 ], but none of the other cell types in this organism has any cilia. It has also been found that all C. elegans homologs ( bbs-1, 2, 7 and 8 ) of the human genes responsible for Bardet-Biedl syndrome, which is also thought to be a ciliary disease, are specifically expressed in ciliated sensory neurons [ 45 ]. It is therefore likely that the gene set revealed by our analysis includes C. elegans homologs of as yet unidentified ciliary disease genes. Conclusions The present study demonstrates that a combination of mRNA tagging and microarray analysis is an effective strategy for identifying genes expressed in subsets of neurons. Systematic reporter expression analyses following this approach will facilitate the accumulation of information regarding gene expression patterns. In particular, profiling of the gene expression patterns of subsets of neurons, in combination with analyses of neural functions, might provide insights into understanding the distinct roles of cells within the neural network. Materials and methods Generation of strains expressing FLAG-PAB-1 in a tissue-specific manner The initiation codon of a cDNA for pab-1 , yk28d10, was replaced with a linker composed of two complementary oligonucleotides, 5'-AATTGCTAGC ATGGATTACAAGGATGATGACGATAAG T-3' and 5'-CTAGACTTATCGTCATCATCCTTGTAATCCATGCTAGC-3', in which the underlined sequence encodes an initiation codon followed by a FLAG peptide. The resulting epitope-tagged gene was cloned into the pPD49.26 vector (donated by Andy Fire, Stanford University). The promoter of che-2 [ 21 ], acr-5 [ 22 ] or myo-3 [ 23 ] was inserted 5' upstream to the fusion gene to generate the FLAG-PAB-1 expression plasmids pche2-FLAG-PABP(FL), pacr5-FLAG-PABP(FL) and pmyo3-FLAB-PABP(FL), respectively. Wild-type animals were transformed with each expression construct, along with the pRF4 plasmid, which carries a dominant rol-6 allele, as a marker [ 46 ]. Stable integrated transgenic strains were generated from unstable transgenic lines as described [ 47 ]. Each integrated strain was outcrossed twice with wild-type N2. The genotypes of these strains were: JN501: Is [ che-2 p:: flag - pab-1 pRF4]; JN502: Is [ acr-5 p:: flag - pab-1 pRF4]; and JN503: Is [ myo-3 p:: flag - pab-1 pRF4]. mRNA tagging To purify poly(A)-RNA/FLAG-PAB-1 complexes from subsets of neurons, we modified a protocol for chromosome immunoprecipitation [ 48 ]. Transgenic animals were grown in liquid as described previously [ 49 ]. The worms were then harvested and washed twice with M9 [ 50 ]. To crosslink poly(A) RNA with FLAG-PAB-1 in vivo , worms were treated with 1% formaldehyde in M9 for 15 min at 20°C with gentle agitation. The formaldehyde was then inactivated by 125 mM glycine for 5 min at 20°C and washed out by replacing the buffer with four changes of TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl). At this point, worms were dispensed into 0.4 g aliquots, placed in 2-ml microtubes and stored frozen until lysate preparation. Worms were resuspended in 0.45 ml lysis buffer (50 mM HEPES-KOH pH 7.3, 1 mM EDTA, 140 mM KCl, 10% glycerol, 0.5% Igepal CA-630 (Sigma), 1 mM DTT, 0.2 mM PMSF, protease inhibitor cocktail (Complete-EDTA, Roche) at the recommended concentration) supplemented with 20 mM ribonucleoside vanadyl complexes (RVC, Sigma) and 1000 U/ml of human placental ribonuclease inhibitor (Takara). Animals were disrupted by vigorous shaking with 2 g acid-washed glass beads (Sigma), and worm debris was removed by centrifugation at 18,000 g for 20 min. Five hundred microliters of supernatant, with the protein concentration roughly adjusted to 20 mg/ml, was incubated with 50 μl of anti-FLAG M2 affinity gel beads (Sigma) for 2 h. The affinity beads were sequentially washed three times with lysis buffer supplemented with PMSF, twice with wash buffer (50 mM HEPES-KOH pH 7.3, 1 mM EDTA, 1 M KCl, 10% glycerol, 0.5% Igepal CA-630, 1 mM DTT) and once with TE (10 mM Tris-HCl pH 7.5, 0.5 mM EDTA). Lysate preparation and purification of RNA-protein complexes were performed at 4°C. Precipitated materials were eluted with 100 μl elution buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS, 20 mM RVC) by incubation for 5 min at 65°C. Elution was repeated and the two supernatant fractions were combined. The eluted RNA/FLAG-PAB-1 complexes were incubated for 6 h at 65°C to reverse the formaldehyde crosslinks. Proteins were digested with proteinase K and removed by phenol-chloroform extraction. Nucleic acid was recovered by ethanol precipitation. Typically, 100 ng nucleic acid was obtained from 0.5 ml cleared lysate of myo-3 ::PABP. Under the above washing conditions, binding of free poly(A) RNA to PABP was severely impaired (data not shown). Examination of the functional integrity of FLAG-PAB-1-expressing cells in the che-2 ::PABP strain For staining of living animals with lipophilic dye, we followed the procedure described before [ 51 ] except that diQ (Molecular Probes) was used instead of FITC. Forty-six wild type and 56 che-2 ::PABP worms at L4 to young adult were observed. Cells were identified by their positions and the percentage of stained cells was scored. Responses of the che-2 ::PABP strain to 1-octanol was assessed as described [ 52 ] except that Eppendorf Microloader (Eppendorf) was used to deliver 1-octanol to animals' noses. RT-PCR Fifty nanograms of RNA was converted to cDNA using an RNA PCR Kit (AMV) Ver. 2.1 (Takara) according to the manufacturer's protocol. One-tenth of the cDNA from each sample was subjected to a gene-specific PCR reaction in a total volume of 20 μl. Quantification of the PCR products was performed using a FastStart DNA Master SYBR Green I Kit (Roche) with the Light Cycler system (Roche). Serial dilutions of cDNA prepared from poly(A) RNA of wild-type worms were used to generate a standard curve. The ratio of expression levels for each gene was calculated using the amount of eft-3 as a reference, and the results of three independent experiments were averaged. The primers used for the amplification of each gene were: lmn1-52: 5'-CGTTCACCACCCACCAGAA-3' and lmn1-32: 5'-CAAGACGAGCTGATGGGTTATCT-3' for lmn-1 ; eft3-52: 5'-ATTGCCACACCGCTCACA-3' and eft3-32: 5'-CCGGTACGACGGTCAACCT-3' for eft-3 ; tax2-54: 5'-GATTAATCCAAGACAAGTTCCTAAATTGAT-3' and tax2-34: 5'-TTCAATTCTTGAACTCCTTTGTTTTC-3' for tax-2 ; unc8-52: 5'-TCTCAGATTTTGGAGGTAATATTGGA-3', and unc8-32: 5'-GATCTCGCAGAAAAGTTCTGCAA-3' for unc-8 ; unc54-52: 5'-AACAGAAGTTGAAGACCCAGAAGAA-3', and unc54-32: 5'-TGGTGGGTGAGTTGCTTGTACT-3' for unc-54 ; snt1-51: 5'-GAGCTGAGGCATTGGATGGA-3' and snt1-31: 5'-CCAAGTGTATGCCATTGAGCAA-3' for snt-1 ; acr5-52: 5'-AATCGATTTATGGACAGAATTTGGA-3' and acr5-32: 5'-ATGTTGCAAAAGAAGTGGGTCTAGA-3' for acr-5 ; odr10-51: 5'-TCATTGTGTTTTGCTCATTTCTGTAC-3' and odr10-31: 5'-ATATTGTTCTTCGGAAATCACGAAT-3' for odr-10 ; del1-51: 5'-TAAACTGCCTCACGACAGAAG-3' and del1-31: 5'-GCCATCAAGTTGAACCAAGAAT-3' for del-1 . All primers were designed to include one intron in the PCR product amplified from the genomic DNA for each gene, such that the length and melting point were different from the product amplified from the cDNA. In Figure 2 , eft-3 was amplified for 25 cycles, lmn-1 , snt-1 and unc-54 for 30 cycles and tax-2 , unc-8 , odr-10, del-1 and acr-5 for 35 cycles. Amplified DNA was visualized by electrophoresis followed by staining with ethidium bromide. cDNA microarray analysis Microarrays were prepared using a 16-pin arrayer constructed according to the format of Patrick Brown (Stanford University [ 53 ]) on CMT-GAPS-coated glass slides. Two micrograms of RNA prepared from JN501 was reverse-transcribed using oligo(dT) primers and SuperScript II reverse transcriptase (Lifetech) with the addition of Cy5-dCTP to generate Cy5-labeled probes. RNA prepared from JN502 was similarly used for the generation of Cy3-labeled probes. Equal amounts of the two probes were mixed and hybridized to a single array overnight at 42°C in Gene TAC Hyb Buffer (Genomic Solutions). Each array was then washed in 1× SSC/0.03% SDS at 42°C, followed by successive washes in 0.2× SSC and 0.05× SSC at room temperature. The fluorescence intensity of each spot was scanned using a ScanArray Lite (Perkin Elmer) and analyzed by QuantArray (GSI Lumonics). Reporter constructs for determination of expression patterns A genomic DNA fragment for each gene was amplified by PCR such that it contained an upstream promoter region followed by a partial or full-length predicted coding region. The 3' PCR primers were designed to introduce a restriction site in-frame with GFP in the vectors. The 5' PCR primers were designed to anneal to a sequence 0.6-5.0 kilobases (kb) upstream of the predicted coding region of each gene. The upstream-predicted gene was essentially not included in the promoter fragment. The amplified PCR fragments were cloned into pPD95.70, pPD95.75 (donated by A. Fire) or a Gateway vector (Invitrogen) to create GFP fusions (see Additional data file 3 for details). The resulting reporter plasmid for each gene was introduced into wild-type animals. Transgenic worms at all developmental stages were observed under a differential interference contrast (DIC)-fluorescence microscope. Cells were identified according to their positions [ 54 ] by comparing the fluorescence images of GFP and DiQ staining with Nomarski images of the same animal. At least two independent transgenic lines were observed to confirm the expression patterns. Images were obtained as described previously [ 17 ]. Bioinformatics cDNA clones were mapped to the C. elegans genome using BLAT [ 55 ] and BLASTN programs, corresponding gene models were identified in the WormBase annotations [ 56 ] and protein sequences were obtained from WormPep122. The gene ontology annotation dataset for C. elegans was obtained from the Gene Ontology Consortium [ 57 ]. For Figure 3 , genes known to be expressed in specific tissues were searched for using the expression pattern search interface of WormBase [ 58 ] or using the AcePerl AceDB server [ 59 ]. The gene set 'Sensory neurons' was defined as genes expressed in all or some ciliated sensory neurons (including amphid neurons), but not in motor neurons or the ventral nerve cord, according to WormBase. The gene set 'Motor neurons' was genes expressed in VB or DB ventral cord motor neurons and no more than one type of ciliated sensory neuron, or those expressed in cholinergic neurons. The gene set 'Muscles' was genes expressed in some muscles, but not in neurons or the intestine, while 'Intestine' was genes expressed in the intestine, but not in neurons or muscles. X-boxes were searched for using MEME and MAST [ 60 ] based on the definition matrix deduced from Swoboda et al . [ 31 ]. Only between 60 and 160 base-pairs (bp) upstream of the initiation codon of each gene were considered, since Swoboda et al . observed that X -boxes were present about 100 bp upstream of the initiation codons [ 31 ]. The proteome data set for Caenorhabditis briggsae , whose draft genome sequence has recently been released, was downloaded from WormBase [ 61 ]. Proteome data sets for humans, mice, Drosophila melanogaster , Schizosaccharomyces pombe and Saccharomyces cerevisiae were obtained from NCBI [ 62 ], and BLASTP searches were performed using the WormPep protein sequence as a query for each C. elegans gene. Protein motifs that preferentially appeared in genes at the higher rank orders were searched for as follows. For all genes represented in our microarrays, the protein motifs contained in each gene product were obtained from the AcePerl server. For each motif, deviation of the average log 2 ( che-2 ::PABP/ acr-5 ::PABP) value for all genes that carried the motif was calculated. To avoid artifactual results due to gene families with close sequence similarities (such as major sperm proteins), groups of genes whose mRNA are expected to cross-hybridize were treated as a single imaginary gene with the average log 2 ( che-2 ::PABP/ acr-5 ::PABP) value. Additional data files The following additional data are available with the online version of this article. Additional data file 1 is a table listing the results of the microarray experiments. Additional data file 2 lists genes expressed in sensory neurons, motor neurons, muscles and the intestine, and those with X-boxes shown in Figure 3 . Additional data file 3 lists the primers and vectors used for reporter constructions. Additional data file 4 contains the legends to the above three tables. Supplementary Material Additional data file 1 a table listing the results of the microarray experiments Click here for additional data file Additional data file 2 Genes expressed in sensory neurons, motor neurons, muscles and the intestine, and those with X-boxes shown in Figure 3 Click here for additional data file Additional data file 3 The primers and vectors used for reporter constructions Click here for additional data file Additional data file 4 The legends to the above three tables Click here for additional data file

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Failed attempts at experimental transplantation and transmission of nocturnally-periodic simian Loa from monkey to man

This paper describes unsuccessful attempts to induce a nocturnally-periodic infection with simian Loa in a human volunteer (the author of this paper) by means of 1. Transplanting adult simian Loa worms from a wild drill ( Mandrillus leucophaeus ) to man; and 2. Infecting the same volunteer by sub-cutaneous inoculation with infective larvae of simian Loa from a laboratory-bred, experimentally infected Chrysops silacea .

Findings In the mid 1950s, one of the main lines of research followed by the Helminthiasis Research Unit (HRU), at Kumba in the then British Cameroons was to establish the relationship between human infections with Loa loa and the highly similar Loa parasites which were found in several species of local monkey, especially in the drill ( Mandrillus leucophaeus ) but also, less frequently, in several long-tailed monkey species, Cercopithecus nictitans martini , C. mona mona and C. preussi . Observations on 109 young drills (found to be free from natural Loa infection after a quarantine period of 6–8 months in screened cages), which were experimentally infected by transplantation of live adult simian Loa worms extracted from wild drills shot in the local forests, revealed that the parasites exhibited a nocturnal microfilarial periodicity with microfilariae (mf) that were significantly longer than those of human L. loa . By contrast, drills which had been infected by inoculation of infective larvae from the day-biting Chrysops silacea or C. dimidiata , that had been experimentally infected 10 days previously by feeding on the blood of Loa -infected human volunteers, demonstrated diurnally-periodic microfilarial infections, whose parasites (both adult worms and mf) were of shorter length than the normal wild monkey parasites [ 1 ]. Further studies on the biting habits of local Chrysops species revealed that in nature the diurnally periodic human Loa loa was transmitted among humans by the day-biting species C. silacea and C. dimidiata , whereas the nocturnally periodic simian parasite was being transmitted among monkeys by two forest-canopy-dwelling species, C. langi and C. centurionis , both of which bite in the forest canopy at night and are presumed to feed mainly on sleeping monkeys [ 2 ]. No naturally-acquired diurnally periodic microfilarial infection was seen in 21 wild drills, the periodicities of whose adult worms were examined by transplantation into uninfected animals; despite the fact that male and female worms of human and simian Loa were quite capable, when transplanted together under experimental conditions into uninfected drills, of inter-breeding and producing microfilariae of intermediate dimensions and periodicity [ 3 ]. (This is not to say that under natural conditions diurnally-periodic worms of human Loa are never transmitted to monkeys, but such events, if they do occur, appear to be rare). Later, Belgian workers in the Mayumbe District in the south-western part of the Democratic Republic of the Congo (DRC), commented on the local human Loa infections in that area being particularly liable to give rise to cases of Loa -encephalopathy after treatment with diethylcarbamazine citrate (DEC). They also noted that out of 547 patients from this area, whose blood films were examined both by day and night, 197 showed Loa mf only by day, 322 showed them by day and by night; and 16 showed mf only at night. Although the latter cases, with a complete reversal of the normal periodicity, showed only light microfilarial loads (12 cases with 1–5 mf per examination, and 4 cases with 6–9 mf per examination), among those persons who showed Loa mf by day and by night, 62 showed microfilarial concentrations that were nearly as high by night as by day and 10 showed more mf by night than by day (two of them showing 500 – 1,000 mf by night as compared with 300 – 500 mf by day) [ 4 - 6 ]. As some of the cases of loiasis from Mayumbe were abnormal in displaying a primarily nocturnal periodicity of the microfilariae, it is possible that the local strain of Loa responsible for them may be closely related to the simian parasite. Recently in the Republic of Cameroon, cases of Loa -encephalopathy have been reported following mass treatment with ivermectin by the African Programme for Onchocerciasis Control (APOC) in areas where loiasis is co-endemic with onchocerciasis [ 7 - 11 ]. A remarkable clustering of many of these cases was found in the Lékié Division, a forest and forest/savannah mosaic area some 80 km from the capital, Yaoundé. So far, the reason for this clustering has not become apparent but the occurrence of these cases of Loa -encephalopathy has had a deleterious effect on the popularity of the APOC campaign in that area [ 12 ]. Furthermore, at the end of 2003 in the Mayumbe area of the DRC, some 100,000 persons were treated with a standard single dose of ivermectin distributed as part of the activities of APOC, and 41 cases of serious adverse reactions (SAEs) were reported, of which 14 were fatal despite appropriate management of the patients. This is an incidence rate even higher than that reported in Lékié Division of Cameroon and has led to the establishment of a commission to examine the matter (Dr B. Thylefors, personal communication ). The patho-biological reasons for the occurrence of Loa -encephalopathy following treatment with DEC or with ivermectin, mainly seen in patients heavily infected with Loa microfilariae, are not well understood, and co-factors may exist that account for the fact that some patients do not develop SAEs despite having high Loa microfilaraemia. Experimental work by Dr Samuel Wanji using an animal model is currently trying to reproduce heavy microfilaraemic infections of human L. loa in experimentally infected monkeys (mainly Mandrillus spp) and to investigate the biochemical and pathological changes that accompany the development of any Loa -encephalopathy following ivermectin treatment. In the original work at the HRU, Kumba, where it was relatively easy to infect young drills experimentally (either by inoculation of infective larvae or by transplantation of adult worms) with either the nocturnally-periodic simian Loa parasite or with the diurnally-periodic human parasite, it was obviously far more difficult to determine whether the nocturnally-periodic simian parasite could be transferred to man. Nevertheless, at that time, before the discovery of the potentially deadly viruses such as Ebola, Marburg and HIV that are believed to originate from monkeys, attempts to infect a human (the author) experimentally with a simian strain of the Loa parasite were undertaken. Today, such experiments would not only be viewed as unethical but also as potentially life-threatening. In 1954 and 1955, the author (who at that time had no signs or symptoms of loiasis and who was not taking any medication, apart from 200 mg proguanil (Paludrine) daily as a prophylactic for malaria), took part in two such experiments, which have not been previously published but are relevant in the light of the localised occurrence of Loa -encephalopathy in some individuals following treatment with ivermectin. These attempts at experimental infection of a human being with simian Loa are as follows: In July 1954, a large male drill, which had been shot in the forest near Kumba some 3–4 hours previously, was brought into the laboratory by the Unit's hunter. It was immediately dissected and a total of seven male and fifteen female mature simian Loa worms, all alive, undamaged and motile, were collected from the subcutaneous and intermuscular tissues. The worms were placed in sterile normal saline solution, along with a small quantity of merthiolate, in the same manner that had been used previously to transplant adult simian Loa worms successfully into other monkeys. Two and a half hours later, 12 of these live, motile, adult female worms and five males were inserted, under local anaesthesia, into the upper, anterior part of the right thigh of the author by the Medical Officer-in-Charge of the Kumba Medical Field Unit. After making a 3-inch, longitudinal incision through the skin and the superficial fascia, the simian Loa worms were inserted, some into the sub-cutaneous tissue and others under the deep fascia of the rectus femoris muscle. The fascial layers were then sewn up, the skin closed with nylon sutures, and intramuscular penicillin was given to counteract infection. Over the ensuing week the transplant area became considerably swollen and painful over an area of approximately 6–8 × 5–6 inches and over the following month it itched frequently. Day and night blood films (50 cu. mm) were taken once a week over a period of six months, and thereafter fortnightly for the next six months, but none of them detected any microfilariae. No Loa worm(s) appeared under the skin or crossing the eye, nor did any Loa mf appear in the peripheral blood over the next 46 years. (It is possible that all the worms died fairly soon after being inserted, or it may be that they remained alive but failed to produce a detectable microfilarial infection). Eighteen months after the transplant, the author also injected himself subcutaneously in the left thigh with 35 live, motile, infective larvae of simian Loa , which had been dissected out in normal saline from a laboratory-bred female Chrysops silacea that had taken a blood-meal 10 days previously from a captive drill infected with the nocturnally-periodic simian strain of Loa . Previous experimental work with monkeys had shown that this method of infection was more effective than trying to induce a Chrysops containing infective larvae of Loa to feed on, and thus transmit infective Loa larvae to, an uninfected person. There was a slight reddening and itching of the skin in the area around the injection site over the following week but otherwise no papular eruption or other reaction developed. No Loa microfilariae were detected in the peripheral blood by day or by night over the ensuing eight years; nor, over the same period was any skin reaction seen that could have been attributed to the death of L3 or subsequent stages of L. loa dying in or under the skin, as were reported subsequently when infective larvae of L. loa from the bite of an experimentally-infected Chrysops silacea were subsequently killed by dosage with diethylcarbamazine citrate (DEC) used as a chemoprophylactic [ 13 ]. Both these attempts failed to infect a healthy human volunteer with the nocturnally-periodic simian strain of Loa . Obviously the failure to infect a single individual must be interpreted with caution. Nevertheless these observations are worth recording especially now that research is in progress to try and ascertain: 1. The factors leading to the localised occurrence of Loa -encephalopathy in certain areas of central Africa, where ivermectin is now being used currently for the control of onchocerciasis, or where in the past it has occurred following treatment with diethylcarbamazine; and 2. The biochemical and histological changes that may be associated with a Loa -encephalopathy if it can be induced in drills heavily infected with Loa mf when treated with ivermectin. Competing Interests None declared. Authors' contributions Brian Duke was sole author

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Dual practice in the health sector: review of the evidence

This paper reports on income generation practices among civil servants in the health sector, with a particular emphasis on dual practice. It first approaches the subject of public–private overlap. Thereafter it focuses on coping strategies in general and then on dual practice in particular. To compensate for unrealistically low salaries, health workers rely on individual coping strategies. Many clinicians combine salaried, public-sector clinical work with a fee-for-service private clientele. This dual practice is often a means by which health workers try to meet their survival needs, reflecting the inability of health ministries to ensure adequate salaries and working conditions. Dual practice may be considered present in most countries, if not all. Nevertheless, there is surprisingly little hard evidence about the extent to which health workers resort to dual practice, about the balance of economic and other motives for doing so, or about the consequences for the proper use of the scarce public resources dedicated to health. In this paper dual practice is approached from six different perspectives: (1) conceptual, regarding what is meant by dual practice; (2) descriptive, trying to develop a typology of dual practices; (3) quantitative, trying to determine its prevalence; (4) impact on personal income, the health care system and health status; (5) qualitative, looking at the reasons why practitioners so frequently remain in public practice while also working in the private sector and at contextual, personal life, institutional and professional factors that make it easier or more difficult to have dual practices; and (6) possible interventions to deal with dual practice.

Introduction Doctors and nurses in government employment are labelled "unproductive", "poorly motivated", "inefficient", "client-unfriendly", "absent" or even "corrupt". These labels are often associated with coping strategies associated with widespread "demotivation", due partly to "unfair public salaries". These are presented as the de facto justification of "inevitable" predatory behaviour and public-to-private brain drain [ 1 - 6 ]. In many countries, developed and developing alike, this has eroded the implicit psychological and social contracts that underlie the civil service values of well-functioning public organizations [ 7 ]. As a result, public servants often resort to dual or multiple employment. This paper reports on income generation practices among civil servants in the health sector, with a particular emphasis on dual practice. It first approaches the subject of public–private overlap. Thereafter it focuses on coping strategies in general and on dual practice in particular. Public–private overlap Private providers capture a significant and growing share of the service delivery market for health care, and ensure an important part of the uptake of services. For a sample of 40 developing countries, an average of 55% of physicians worked in the private sector and an average of 28% of health care beds were private beds (21% private, for profit) [ 8 ]. Asia has more than 60% of private sector contributions to health care financing (excluding China and India) and is the part of the world where the private sector normally plays the dominant role. In Malaysia, for example, the proportion of physicians in private practice increased from 43% in 1975 to 70% in 1990. In Indonesia half of the hospitals are privately run. In Thailand the share of beds in private hospitals grew from 5% in 1970 to 14% in 1989. The contribution of the private sector to health care financing in Africa is 50%, with a significant preponderance of private, not-for-profit, nongovernmental (PNFP-NGO) entities, particularly church organizations. In Zimbabwe church missions provide nearly 70% of all beds in rural hospitals and in Tanzania they run 40% of the hospitals. In Kenya, about one third of the total health services and 40%–50% of the family planning services are provided by PNFP-NGOs. In Latin America the private sector finances 40% to 60% of the health sector [ 9 ]. For many populations, especially in rural areas, these PNFP-NGOs are the main – if not the only – providers of health care. For example, in a mail survey of 88 nongovernmental hospitals in sub-Saharan Africa at the end of the 1980s, 39 were the only service provider in their district [ 10 , 11 ]. It is also the case, for example, of rural Congo in the 1990s, where only NGO-supported districts continued working [ 12 ]. Following the reforms of the early 1990s in Mali, most ambulatory health care – nearly all in rural areas – is now provided by community-owned PNFP health centres organized in a nongovernmental federation [ 13 ]. But these are mainly rural situations. In urban areas PNFP-NGOs usually share the work with private, for-profit (PFP) providers and government services. In Alexandra, South Africa – a poor periurban township – for example, a PNFP university clinic, a municipal clinic and PFP "general dispensing cash practices" worked side by side [ 14 , 15 ]. But in general, the market share of private providers is more limited. PFP health providers are no doubt an important source of ambulatory care throughout the developing world, but tend to concentrate on the more profitable niches of the market. The development of private practice in most developing countries is notoriously unregulated. Private practices are not easily forthcoming with information, at times for fear of tax implications, at times because existing regulations are not respected, often because of a lack of respect for discredited ministries of public health and not infrequently because of the non-existence of information systems. In transitional countries, such as Tunisia, the growth of the entrepreneurial sector is well documented. Although hard data are hard to come by for the poorer countries, it is sufficient to walk around Luanda or Dar es Salaam or to look at the advertising sections of any newspaper in Maputo to see that private health care provision is a thriving growth industry. If there are ever more private providers on the market, not all provide the whole range of health services. They tend to select niches according to demand and competition rather than providing comprehensive care. There are huge differences between and within countries. This (patchy and scarce) evidence confirms that both PFP and PNFP providers have a significant and growing share of the market of health care, but this statement needs qualification. There are wide differences between and within countries. Overall, the market share of the private sector is smaller for inpatient than for ambulatory care, and limited for preventive and public health services [ 8 ]. Coping strategies Individual coping strategies represent the health professionals' ways of dealing with unsatisfactory living and working conditions. In many countries their prevalence has increased over recent years. The notion of the full-time civil servant exclusively dedicated to his/her public sector job is disappearing. Were this without consequences for the performance of the public health sector, it would not be much of a problem. Not all coping strategies can be classified as predatory behaviour or corruption, and their effects on the health care system can be positive as well as negative. They cannot, however, be ignored as, in many countries – particularly the poorest – the situation has gotten out of hand. Most would agree that public sector salaries are most often "unfair". For example, in 1999 a Mozambican nurse's salary was only 10% to 15% of what it had been 15 years before [ 16 ]. In Sierra Leone most health professionals, physicians included, are employed by the public sector at salaries under USD 100 per month [ 17 ]. In many other countries health staff is going through similar experiences. In Russia, state doctors earn between USD 15 and USD 50 per month [ 18 ]. In the Dominican Republic, in 1996, physicians with 20 years of experience earned the same as new medical graduates, rewards for good performance were impossible and personnel were paid regardless of whether they performed their duties [ 19 ]. In such a context, demotivation, overall lack of commitment and low productivity are to be expected. To compensate for unrealistically low salaries, health workers rely on individual coping strategies [ 1 - 6 , 18 , 20 - 54 ]. Many clinicians combine salaried public sector clinical work with a fee-for-service private clientele [ 3 - 6 , 25 - 29 , 32 , 38 - 41 , 44 , 45 , 53 ]. Others resort to absenteeism [ 24 , 44 , 45 ], or predatory behaviour, asking under-the-counter payments for access to services intended to be free of charge [ 33 , 34 , 46 ] or goods and/or misappropriating drugs or other supplies [ 20 , 21 , 27 , 43 , 49 ] and referral of public sector patients to private practices [ 23 ]. In Thailand 37% of patients pay their public sector obstetricians "gratitude money" [ 55 ]. In France, mainly for surgical interventions, under-the-table remuneration is very common in hospital or ambulatory settings [ 56 ]. In Greece, doctors and nurses have been criticized for receiving gifts and bribes [ 57 ]. Another example is fee splitting, whereby a specialist shares a fee with the referring physician [ 23 ]. In 1998, for example, a group of Italian general practitioners was suspended for accepting payments to send their patients to a particular private centre for radiology examinations [ 58 ]. It is common practice in the United Kingdom for consultants to spend time in private clinics when they should be attending to their public duties [ 23 ]. Patients and practitioners may collude to deceive a third agent: in Kazakhstan, for example, it is reported that doctors regularly provide false health reports in return for a fee, so that patients can obtain driving licenses [ 23 ]. A further form of fraud through misinformation is exemplified by the case of the English GP who forged consent forms for patient participation in medical trials in order to boost income [ 53 ]. The problems these coping strategies create are increasingly recognized [ 6 , 38 - 40 ]., although the subject remains taboo for many ministries of health and development agencies. Drugs are – in the current context of scarce resources, health care reform, promotion of generics, the HIV epidemic and growing demand for health care – a sensitive issue, as in many low-income countries, pharmaceuticals make up 50% or more of health care costs [ 59 ]. With health sector reforms, private sector pharmacies are increasingly becoming the first and sometimes the only outlet for the delivery of health services [ 60 - 63 ]. In this environment, and for several reasons – including the "business profession dilemma" in private pharmacy practice – irrational prescription can become a major problem [ 64 - 67 ]. Antibiotics are often sold without a prescription [ 68 , 69 ]. In other settings, such as hospitals and health centres, misappropriation is a widespread practice by all categories of professionals. This is infrequently acknowledged explicitly or documented, even in studies that have looked into the coping strategies of health professionals [ 3 - 5 , 28 , 29 , 41 , 44 , 45 ]. It has nevertheless been documented in a number of African [ 20 , 21 , 27 , 43 , 49 ] and Latin American countries [ 22 ]. Where documented it is perceived as common practice. In Uganda, for example, misuse of pharmaceuticals was reported by facility health workers as well as by the District Health Teams and the Health Unit Management Committees; resale of drugs represented the greatest single source of income for health workers in most units [ 27 , 43 ]. In many other developing countries the situation is supposed to be similar if not worse. Dual practice We have stated that many clinicians combine salaried public-sector clinical work with a fee-for-service private clientele [ 3 - 6 , 25 - 29 , 32 , 38 - 41 , 44 , 45 , 53 ]. Dual practice may be considered present in most countries, if not all. Nevertheless, there is surprisingly little hard evidence about the extent to which health care workers resort to dual practice, about the balance of economic and other motives for doing so, or about the consequences for the proper use of the scarce public resources dedicated to health. Dual practice is often a means by which health workers try to meet their survival needs, reflecting the inability of health ministries to ensure adequate salaries and working conditions. In this paper dual practice is approached from six different perspectives: (1) conceptual, regarding what is meant by dual practice; (2) descriptive, trying to develop a typology of dual practices; (3) quantitative, trying to determine its prevalence; (4) impact on personal income, the health care system and health status; (5) qualitative, looking at reasons for its prevalence and for staying in public practice while working in the private sector and at contextual, personal life, institutional and professional factors that make it easier or more difficult to have dual practices; and (6) possible interventions to deal with dual practice. What is meant by dual practice Dual practice is approached in the literature with great diversity. It can mean health professionals with multiple specializations – for example, among Egyptian physicians the most popular areas of multiple specializations are "cardiology and internal medicine, internal medicine and fever and cardiology and chest" [ 70 ]. It can also mean health professionals working within different paradigms of health (allopathic medicine combined with traditional medicine – Chinese, African or otherwise – or with other paradigms such as osteopathy, homeopathy and others). It may involve combining different forms of health-related practice – clinical with research, with teaching or with management. It can also mean health professionals combining their professional health practice with an economic activity not related to health (such as agriculture). In this paper, dual practice will be used to describe multiple health-related practices in the same or different sites. An operational typology of dual practices Therefore, in terms of sector location, dual practice may be public on public, public on private or private on private (Table 1 ). Table 1 Typologies of dual practice Public Private, for-profit Private, not-for-profit Public + + + Private, for-profit + + Private, not-for-profit + Overtime may be considered a form of dual employment. It is increasing because of cost-containment measures or shortages of staff. In 1992, the German Union of Salaried Employees estimated that the overtime worked by health workers was equivalent to 20 000 extra full-time staff posts. Today, trade unions in Canada and the United Kingdom express particular concern about the use of overtime to substitute for recruitment and about the increase in unpaid overtime [ 71 ]. The extent of dual practice As mentioned and supported by the limited literature available (Table 2 ), dual practice is probably present in all countries regardless of income, even in settings – such as China – where there are major regulatory restrictions [ 29 , 55 , 70 , 72 - 82 ]. In Latin America physicians usually hold jobs in both public/social security and private systems [ 83 ]. Table 2 The extent of dual practice in several countries Country Type and frequency of dual practice Angola Dual (public and private) practice is ubiquitous and unregulated [72]. Cambodia Dual (public and private) practice is ubiquitous [89]. Egypt Rural-based Egyptian physicians in private practice are more likely to have a second job (85%) than urban-based physicians (71%). However, there is not much difference between urban and rural physicians in the likelihood of having a third or fourth job: 15% of urban and 11% of rural physicians have a third job and 2% of urban and 1% of rural physicians have a fourth job. Twenty percent of 113 single private practice dentists work only in their private clinic; 73% have 2 jobs, 6% have 3 jobs and 1% have 4 jobs. Among 261 pharmacists 91% have only one job, 8% have two jobs and 1% have 3 jobs. Among 80 other health service providers, mainly unlicensed, who are officially not allowed to operate, but yet provide a significant amount of health care, 66% of the sample have a second job and 1% has a third job [70]. Indonesia Most doctors have dual practices in the public and private sectors [77]. Malawi The government allows serving medical personnel in its facilities to set up private surgeries where they can practice after official duty hours; it further allows those without professional qualifications (e.g. "paramedics") to set up a health care business for minor health complaints [78]. Mozambique Common among urban, but not rural, health professionals [29]. Papua New Guinea Semi-private wards in public facilities are well patronized in the larger hospitals but tend to be underutilised in the smaller provincial centres [79]. Peru Almost all physicians have both public and private practices [77]. Portugal 23% of public sector health centre workers have a second job, the highest rate being for doctors – 43%; 58% of public sector hospital workers have a second job, the highest rate being for doctors – 50% [75, 76,75, 76]. South Africa Half of general practitioners in private practice have other employment. While 36% worked in the public sector, this was more common in rural (62%) compared with urban (21%) areas [80]. Syria Most physicians have dual practice [77]. Thailand An estimate suggested that in Bangkok alone there were over 2000 private clinics, many of these run by government doctors [81]. Private practice by public sector obstetricians is very frequent [55]. Viet Nam Most doctors complement public sector work with private practice [77]. Full-time government employees are supplementing their incomes through part-time private practice. One village-based health survey found that 70% of the drug sellers were moonlighting government workers [82]. The impact of dual practice Predatory behaviour Dual practice may lead to predatory behaviour (behaviour in which self-gain is pursued to the detriment of the legitimate interests of colleagues, services and/or patients)[ 84 ] by health workers. This is particularly strong in situations where market conditions – usually high physician supply, as is the case of capital cities in Africa and other urban more than rural areas – would otherwise reduce their incomes. In these situations clinicians use their authority to prescribe treatment for their patients to generate additional demand for their own services. This hypothesis, controversial for some practices such as caesarean sections, seems consensual regarding other surgical practices [ 85 ]. In Thailand, for example, it is well demonstrated that antenatal care is sought in private ambulatory facilities, while caesarean sections are offered in public hospital facilities. Caesarean-section rates among private patients (46%) are three times higher than among non-private patients (16%), which indicates that private practice by public obstetricians is a strong determinant of caesarean sections [ 55 ]. The predatory behaviour of individual clinicians constitutes, in many cases, a de facto financial barrier to access to health care [ 21 ]. More important, in the long run, is that it delegitimizes public sector health service delivery and jeopardizes the necessary relation of trust between user and provider [ 20 ]. Conflicts of interest A more insidious problem is that of conflicts of interest. Effects on the system can best be looked at separately for each type of side activity. When health officials set up in dual practice to improve their living conditions – or merely to make ends meet – this may not interfere with their work as civil servants (although it is likely to compete for time and to reinforce rural-to-urban migration). When they take up teaching as an extra job, usually in public sector institutions, that may actually be beneficial to the public agenda, as it reinforces the contact of trainees with the realities of the health services. For doctors who are basically managers, moonlighting in private practice presents less of a conflict of interest than for clinicians. The latter must compete for patients with themselves, and thus they have an incentive (and the opportunities) to lower the quality of the care they provide in the public services. This is not the case for managers: involvement in NGO projects or work for donors can foster better coordination in the provision of services, but may constitute a conflict of interest when NGO or project policies are not necessarily congruent with national health policies or the agenda of the public service [ 4 , 44 , 52 , 55 ]. Other business activities, such as agriculture, are neutral towards health services, although they may constitute a de facto internal brain drain [ 3 ]. Brain drain Dual practice is to a large extent a question of creating and making use of opportunities. The pursuit of such opportunities contributes also to brain drain. Brain drain of health professionals is often thought of only in terms of intercountry migration [ 86 ]. But failure to post and retain the right person at the right place is not merely a question of a Congolese doctor's deciding to move to South Africa or a Philippine nurse's moving to the United States. It is also a question of internal – including public to private sector – "migration". This public-to-private migration compounds the rural-to-urban migration because cities also offer more opportunities to diversify income generation [ 28 , 29 ]. The need to make up for inadequate salaries – and to be in a setting where there are opportunities to do so – thus fuels rural-to-urban migration and resistance against redeployment to rural areas [ 4 , 28 , 29 ]. Professionals who have successfully taken advantage of these urban opportunities increase their market value over time, until they are ready to leave public service. Rural-to-urban brain drain thus is later compounded by public-to-private brain drain. Competition for time and limits to access Coping strategies, including dual practice, also affect access, but through competition for time. In many countries, civil service medical staff is available only nominally full-time to fulfil its assigned tasks. This has been well described regarding Colombia, Costa Rica and Venezuela. In Venezuela doctors and head nurses missed about one third of their contracted service hours, 37% and 30% respectively, while resident doctors and nurses were absent about 7% and 13% of the time, respectively [ 47 ]. In Peru 32% of doctors and nurses considered absenteeism common or very common [ 24 ]. In Costa Rica 65% of doctors and 87% of nurses felt that physicians were unjustifiably absent from work or, even if present at work, often saw private patients on public time in public facilities [ 22 ]. Absenteeism in the hospitals of Bogotá, Colombia, may cost over USD 1 million a year [ 35 ]. If public sector medical staff is moonlighting in private practice, this evidently limits access by public patients. This corresponds to a net flow of resources out of the public sector. Competition for time is also something that concerns managers, whose coping strategies are often more oriented towards collaboration with development agencies [ 44 , 45 ]. Competition for time is a nagging problem for many development agencies and ministries of health. At times it is blatant. In Mali, for example, regional health staff was found to spend 34% of its total working time in (income-generating) workshops and supervision missions supported by international agencies; for chief medical officers it was 48% (El Abassi & Van Lerberghe, unpublished data, 1995). The 73% of working time spent on official duties that was self-reported by the respondents to one of the surveys reviewed may well be an overly optimistic estimate [ 44 , 45 ]. In Egypt the number of hours worked in the private clinic falls as the number of a physician's jobs increases, indicating that the physician replaces hours away from the private clinic with other employment. Secondly, as the number of jobs increases, the amount of time spent in the second job, usually a government job, decreases. This reduces the access of low-income people to medical care, as they cannot afford to seek care in the private sector. Econometric analysis of the data also found that physicians replace hours they should be working at the government job by hours in the private clinic. This has important policy implications, as multiple employment is not increasing the access of the population to medical care. On average, physicians see one patient per hour in their private clinic. This increases to 3.2 patients per hour in the second job, 4.4 in the third job and 2.9 in the fourth job. An extreme example is that of general practitioners who see one patient per hour in their private clinic, approximately four in the second job and 12 in their third job. In general, the number of patients seen per hour increases with the job number. This is true for physicians as well as dentists [ 70 ]. Competition for time automatically results in a transfer of salary resources out of the public sector through reduced availability – at least the equivalent of 27% of the salary mass [ 44 , 45 ]. Outflow of resources Besides competition for time, in many cases the use of the public sector's means of transportation, office infrastructure and personnel represent additional hidden outflows of resources, often in association with dual practice. The overall impact of this outflow of resources is hard to quantify in any country. Reports from Moscow suggest that up to 30% of the federal budget is not accounted for [ 18 , 34 ]; in the United Kingdom, estimates of GBP 115 million are given for prescription fraud alone [ 23 ]. The loss to the public sector associated with redirection of diagnostic and therapeutic resources, such as pharmaceuticals, to private practice or into the black market is obviously difficult to assess. In Uganda, for example, it results in a significant loss to the public health facilities: the median drug leakage in health facilities was estimated at 78% [ 27 , 43 ]. In the Dominican Republic, almost one third of total hospital expenditure remains unaccounted for, representing some combination of theft of materials and supplies, diversion of funds and gross mismanagement [ 37 ]. In Panama, high-value medications were stolen on a daily basis, with significant losses to the hospital [ 87 ]. In Venezuela, between 10% and 13% of all medical supplies and medications were stolen [ 47 ]. In Costa Rica, 71% of doctors and 83% of the nurses reported that equipment and materials had been stolen in their hospital [ 22 ]. In the United Kingdom it is estimated that pilfering – of bandages, medications and stationery, for example – adds up to more than GBP 15 million annually. In an Andalusian hospital in Spain, it was estimated that pilfering of food supplies led to per capita catering costs that were higher than those of a good restaurant [ 23 ]. The ultimate purpose of this stealing is not studied, but it is possible to speculate that many stolen resources find their way into the dual practices of public servants. Competition for time and transfer of resources are compounded by the fact that the best-trained and most competent officials are also the most likely to divert their time to other activities outside the health sector (a de facto brain drain). This in turn reinforces the attraction of what starts out as a job-on-the-side, and quickly becomes not only more rewarding financially but also professionally and in terms of social prestige. The impact of these coping strategies is seen as negative. These strategies weaken the public sector health structure and damage people's health [ 20 , 88 ]. Impact on income Individual income topping-up strategies allow professionals a standard of living that is closer to what they expect. In one study, these strategies more than doubled the median income of public sector health managers, and brought it from 20% to 42% of that of a full-time private practice [ 44 , 45 ] (Tables 3 and 4 . Fig. 1 : The box-plot chart represents for each variable the maximum, 75th percentile, 25th percentile and the minimum [ 44 , 45 ].) Table 3 Median and interquartile range of take-home salaries of civil servant health service managers Low-income countries (61 respondents) Middle-income countries (39 respondents) In USD at official exchange rate 3802 (2137–5249) 11 253 (6704–18 900) In USD corrected for purchasing power parity 13 890 (9411–20 956) 26 376 (18 416–38 931) As % of the income of a private practice serving 15 patients per day 14% (10%–33%) 29% (22%–41%) As % of the income of full-time consultancy work (250 days / year) 31% (23%–44%) 81% (45%–108%) Source: [44, 45] Table 4 Median and interquartile range of total income (salary plus extra activities) of civil servant health service managers Low-income countries (61 respondents) Middle-income countries (39 respondents) In USD at official exchange rate 5899 (2712–8137) 11 372 (6000–23 040) In USD corrected for purchasing power parity 21 438 (4081–84 640) 39 377 (26 149–64 338) As % of the income of a private practice serving 15 patients a day 26% (17%–52%) 42% (29%–64%) As % of the income of full-time consultancy work (250 days/year) 49% (30%–96%) 115% (74%–172%) Source: [44, 45] Figure 1 Distribution of income in USD purchasing power parity, with the increase from extra jobs, compared to distribution of potential income through consultancies or private practice In Phnom Penh, Cambodia, 90% of a physician's income in dual practice is derived from the private sector activity [ 89 ]. In Thailand earnings from private practice among physicians constitute 55% of their total income [ 55 ]. The upside is that income topping-up helps to retain valuable expertise in public service [ 90 - 96 ]. Corruption in the health sector Corruption has been a long-standing concern in development circles. The literature is rich on theories ranging from macrosociological analyses of sociocultural processes to dyadic game theory modelling. Although impressive, this theorizing has not resulted in useful, empirically validated tools to redress this problem [ 97 ]. For these reasons, we prefer to understand this issue from the perspective of the context that expects health professionals to have a standard of living that cannot be met by existing social systems, sometimes to a level where they cannot even satisfy their basic needs through their public sector salary. In Mozambique, for example, medical students seem to know they will be needed in the public sector, and that this would represent an opportunity to contribute to the public's welfare. Nevertheless, in order to improve their earnings their expectations are to combine their public sector practice with private medical work. One third of the medical students expect an income of between USD 715 and USD 1071 a month, and another third expects over USD 1429, at a time when the salary of a newly graduated doctor is about USD 357 a month. This sets the scene for the reality, often unregulated, of dual practice that plagues many countries [ 98 ]. It is not surprising that once graduated they resort to dual practice (often already initiated as medical students). One must distinguish between individual coping strategies and orchestrated activities, acknowledging, nevertheless, that they may be closely interrelated. For example, hospitals with limited budgets in Mozambique, Portugal, Russia and other countries see dual practice as a means of retaining the most senior personnel. There is a fine line separating coping strategies, including dual practice, from corruption. The difficulty in differentiating between the two starts with the definition of corruption. Widely accepted definitions often have ideological connotations. The definition of corruption as the "private use of public goods" is frequently associated with authors who ultimately defend a greater role for the private sector in the provision of public services such as education and health (as proposed by Van der Geest) [ 21 ], but without sufficient evidence of its effectiveness. They fail to acknowledge that corruption in the private sector may be a significant problem [ 99 ] and that liberalization and transition from state-controlled systems to systems in which the market plays a greater role have often resulted in more corruption, not less [ 100 ]. Another problem with current research is its treatment of corruption as something that would be the same everywhere, with essentially the same causes and implications wherever it occurs [ 101 ]. A third problem is that of the moralistic and criminal connotations of the word "corruption". It should be kept in mind that not all that is illegal is corrupt and not all that is corrupt is illegal, and that also a distinction should be maintained between corrupt transactions and those that are immoral [ 99 ]. The literature tends to focus on the "corrupt", failing to acknowledge that contexts that generate so-called corrupt behaviours generate them across the whole spectrum of society, to the extent that they become an ingrained and acceptable part of society, a necessary evil to survive in a very harsh environment. This shift from focusing on the persons involved to the system in which the professionals are integrated has taken place in other areas, such as in quality management [ 102 ] and in the prevention and control of industrial accidents [ 103 ] or medical errors [ 104 ]. Sooner or later this is likely to happen with the corruption literature as well. Corruption has a negative impact on development in general. It hits the poor the hardest, directly and indirectly by, inter alia, reducing their access to public services such as education and health care [ 105 , 106 ]. This negative impact is also felt in indicators of health status. There is an inverse relationship between indices of corruption and ratio of public health spending to GDP and child mortality rates [ 107 ]. As such, it cannot be ignored by health sector managers, but labelling is not only misleading and counterproductive: it also does not help mobilize the coalitions necessary to address the problem. When compared with other sectors, health is frequently classified as being of median to high levels of corruption, with sectors such as public works contracts and construction, arms and defence, energy and industry appearing as more corrupt [ 108 ]. The professional literature hardly touches on corruption in the health sector. It is anecdotal (Yudkin reports, from two Kenyan newspaper articles, that two ministry of health officials had been bribed to purchase sufficient quantities of two medicines made by one company to last the nation for more than 10 years, whereas at least one of the medicines would expire in two to three years) [ 109 ] (see also Baxter, 1998) [ 110 ], biased, peripheral to the core issues [ 22 ] or lacking in empirical data. Empirical data are, nevertheless, available from a number of studies and reports [ 16 , 18 , 21 - 23 , 105 , 110 - 113 ], most of which were reviewed in previous sections. One of the earlier papers on this was by Van Der Geest [ 21 ]. It attempted to explain why health services in southern Cameroon functioned so inefficiently, with special attention to the distribution of medicines. It calculated that the "elementary health centres received on the average about 65% of the medicines they should have received", this proportion increasing for the "more developed health centres" and even more for the hospitals. Once in the institutions, "many of the medicines which finally arrive...and which should be distributed freely among patients is taken by health personnel for private use or distribution among friends and relatives. Medicines are also sold to petty traders or directly to patients visiting health workers in their private homes", resulting in a further loss of medicines of 30% for health centres and of 40% for hospitals. The main consequences of these practices were: underutilization of health services known to be without medicines; a very limited stock of medicines, which forced health professionals to treat patients with inadequate doses of medicines; referral of patients with prescriptions to expensive private pharmacies; and an increase in inequity, as rural populations were clearly at a disadvantage compared to urban populations. The author came to the reluctant conclusion that the root cause of the observed inefficiency was corruption, deeply embedded in socially accepted practices of "gift-giving, with the preponderance of traditional loyalties over obligations to the state, and with a proprietary view of public offices". The most important single factor encouraging corruption, was, however, "the position of the state as the main source of goods, services and employment and the relative underdevelopment of the private commercial sector". In the end Van Der Geest reluctantly acknowledged that "suggestions to ameliorate the situation are hard to make". Reasons for dual practice With current salary levels in many countries, it is surprising that many people remain in public service, when they could earn much more in private practice. Dual practice allows a standard of living that is closer to what clinical doctors – still a rare resource in many situations – expect, and thus helps retain valuable elements in public service. Many spend comparatively little time, or none at all, on private practice. It is unlikely that this is only for lack of opportunities, such as a saturated private health care market, or too much competition from the "real" clinicians. There must be other sources of motivation to keep working in public services. The involvement in (relatively unrewarding) teaching, or in unpaid NGO work shows that other factors – social responsibility, self-realization, professional satisfaction, working conditions and prestige – still play a significant role [ 3 , 4 , 44 , 45 ]. A study from Phnom Penh, Cambodia, suggests that the links with the public sector are highly valued, as they give physicians access to information, opinions of influential doctors, recruitment of patients, privileges for treating and referring patients and an opportunity to make a contribution to the community [ 89 ]. The gap between income and expectations makes it unavoidable that managers, like other health care workers, will seize opportunities that are rewarding, professionally and financially. Some are of the opinion that dual practice can at times be justified. Health workers in Mozambique and Cape Verde rationalize it: "... to help a sick neighbour" or to help patients "because there are patients that, on account of the long waiting times, do not go to the hospital, they will rather go to these persons in order to avoid wasting their time in the hospitals" [ 20 ]. Most, however, implicitly or explicitly condemn such practices while still attempting to explain and/or justify them in various ways. An obvious explanation is that of "serious lack of motivation" and insufficient salaries: "economic reasons, and low salaries ... those are the reasons ... it is a means of surviving" [ 20 ]. The reasons for dual practice are not well studied. A number of Portuguese case studies [ 72 , 74 - 76 ] suggest that these reasons are contextual and vary between professional groups and site of employment (hospital versus health centres). The extent of dual practice seems to vary according to urban or rural residence, according to professional group (it is more common among health workers with university degrees and, for these, more common for doctors than for other professional groups) and within a professional group, according to specialty or occupation. For example, health system managers have fewer opportunities for dual practice than clinicians. This limited evidence suggests that being a migrant worker, being on temporary contracts and doing shift work are important determinants. This evidence is not conclusive or generalizable, but is welcome as it suggests that dual practice depends not so much on the personal (age and sex), social (marital status) and professional characteristics of health workers – although these are not insignificant – but on factors that are manageable. Sometimes dual practice may be the unexpected result of health care reform. In Canada, within the public system, designating sites for different levels of surgical acuity during the early stages of regionalization has resulted in a 3.5-fold increase in the number of surgeons working in more than one setting after this restructuring compared to before, as most surgeons do both high- and low-acuity surgeries. This resulted in interference with continuity of care, increased commuting time for both surgeons and medical residents and increased reliance on house staff (with whom surgeons spent less time and are thus less familiar with the limits of their skills) [ 114 ]. Another area where provider strain was reported as high was among paid home care staff: low wages, irregular hours, inadequate training and high turnover resulted in lack of continuity of care, staff shortages, waiting lists, health risks to both workers and recipients and impoverishment. Some home workers reported working several jobs to make ends meet [ 114 ]. Reforms frequently result in the increase of staff employed on fixed term and temporary contracts [ 71 ]. This trend seems to induce dual employment [ 72 , 74 , 75 ]. Interventions to deal with dual practice At the core of the reliance on individual coping strategies, including dual practice, is a very strong motor: the gap between the professional's financial (but also social and professional) expectations and what public service can offer. Most public responses to individual coping strategies, including dual practice, fail to acknowledge the obvious: that individual employees are reacting individually to the failures of the organizations in which they work, and that these de facto choices and decisions become part of what the organization is. Adequate responses also imply that the main underlying reason for the observed dual practice can be identified. They call for an understanding of how endemic are the practices observed: Are they isolated, individual cases? Are they specific to the health sector? Or are they widespread in other sectors of society? It is equally important to identify the impact of these practices, particularly the impact in terms of reduced access, inequity and other dangers for the health of the public. Dual practices have, in some countries, become so prevalent that it has been widely assumed that the very notion of a civil service ethos has completely – and possibly irreversibly – disappeared. But some of the literature reviewed reflects, from the health workers themselves, a conflict between what it means to be an honest civil servant who wants to do a decent job, and the brute facts of life that make them betray that image. The manifest unease that this provokes is an important observation as such. It suggests that even in the difficult circumstances observed in many countries, behaviours that depart from traditional civil servant duty and ethics have not been interiorized as a norm. This ambiguity suggests that interventions to mitigate the erosion of proper conduct would be welcome. The most relevant conclusion is that there is no single recipe to address the reality of dual practice. Its cause and logic vary, and the resulting differences among situations need to be taken into account in the design of corrective measures [ 115 ]. What does not work Pretending that the problem does not exist or that it is a question merely of individual ethics, or approaching it as a problem merely of corruption, does not do justice to the complex nature of the problem and will not make it go away. Prohibition is equally unlikely to meet with success, certainly if the salary scales remain blatantly insufficient. In situations where it is difficult to keep staff performing adequately for want of decent salaries and working conditions, those who are supposed to enforce such a prohibition are usually in the same situation as those who have to be disciplined. As an isolated measure, restrictive legislation, when not blatantly ignored, only drives dual practice underground and makes it difficult to avoid or correct negative effects [ 6 ]. Despite this, governments still resort to prohibition as the main means of controlling dual practice [ 116 ]. Closing the salary gap by raising public sector salaries to "fair" levels may not be enough to break the vicious circle. This was attempted by Louro, in Greece, in his restructuring of the health sector in 1945. When public-sector doctors were prohibited by law from pursuing private practice, their average remuneration was raised to take account of their lost income. But there was great resistance by doctors not prepared to give up private practice and professional autonomy [ 57 ]. The 1983 Greek NHS Act again required doctors to have a heavily disputed exclusive full-time status in their public sector employment; correspondingly the hospital doctors' salaries were raised so that seniority was favoured – at an average of 112%, or at a range of 11% for junior doctors to almost 211% for directors. Categories such as university and military doctors escaped from the restrictions introduced. The 1990–1994 reform again allowed doctors to become part-timers if they wished to practice simultaneously in private surgeries or to treat on a per-case basis, but in this case their salaries would be reduced. Notably, though the Ministry of Health expected that around 2500 doctors would become part-timers, only 150 opted for it. In 1994 an international committee chaired by Abel-Smith reopened this issue, recommending that either rights to private practice would be denied to all doctors working for the NHS or they would be confined to senior doctors for a limited number of sessions. Higher salaries were to be paid to professors to compensate them for their loss of unlimited private practice rights. In 1997 this still had not been implemented [ 57 ]. And it is not a realistic option in many poor countries. In the average low-income country, salaries would have to be multiplied by at least a factor of five to bring them to the level of the income from a small private practice [ 44 , 45 ]. Doing this for all civil servants is unimaginable; doing it only for selected groups would be politically difficult. Downsizing central bureaucracies and delinking health service delivery from civil service [ 117 ] would make it possible to divide the salary mass among a smaller workforce, leaving a better individual income for those who remain. However, experience shows that such initiatives often generate so much resistance among civil servants that they never reach implementation [ 118 ]. Where retrenchment becomes a reality it is rarely followed by substantial salary increases, so that the problem remains and the public sector is even less capable of assuming its mission. Lastly, a mere increase in salary would not automatically reinstate the sense of purpose that is required to make public services function: as such it would not be enough to make moonlighting disappear spontaneously [ 119 , 120 ]. That does not mean that nothing can be done. Improvement is likely to come from a combination of small piecemeal measures that rebuild a proper working environment. Addressing the problem of dual practice openly A prerequisite is to address the problem of dual practice openly. Where it is not realistic to expect health care workers to dedicate 100% of their time to their public service job, this should be acknowledged. That is the only way to create the possibility of containing and discouraging income-generating activities that present conflicts of interest, in favour of safety valves with less potential for negative impact on the functioning of the health services. Besides minimizing conflicts of interest, open discussion can diminish the feeling of unfairness among colleagues. It then becomes possible to organize things in a more transparent and predictable way. There are indications that the newer generations of professionals have more modest expectations and are realistic enough to see that the market for dual practice is finite and to a large extent occupied by their elders. This gives scope for the introduction of systems of incentives that are coherent with the organization's social goals [ 121 ]. Incentives Where, for example, financial compensation for work in deprived areas is introduced in a context that provides a clear sense of purpose and the necessary recognition, this may help to reinstate lost civil service values [ 122 ]. The same goes for the introduction of performance-linked financial incentives [ 121 ]. These can, in principle, address the problem of competition for working time, one of the major drawbacks of dual practice. However, such approaches require well-functioning and transparent bureaucracies, making the countries most in need also those where they are a priori most difficult to implement on a large scale [ 117 , 123 ]. Improving working conditions It makes no sense to expect health workers to perform well in circumstances where the equipment and resources are patently deficient. But improving working conditions involves more than providing an adequate salary and the right equipment. It also means developing career prospects and providing perspectives for training [ 119 , 120 ]. Perhaps most important, it requires a social environment that reinforces professional behaviour free from the favouritism and arbitrariness prevalent in the public sector of many countries. Professional value systems However ill-defined they may be, the value systems of the professionals are a major determinant in making the difference between good service to the public and bad. It would be naïve to think this could be achieved through mere bureaucratic regulation by governments or donor agencies. With the building up of pressure from donors and from peers as well as from users, civil servant health professionals will be more likely to invest in patterns of behaviours and practices that visibly uphold the professional value system [ 119 , 120 ]. Peer pressure The social and professional culture within a profession may have a major impact on the practice [ 64 , 120 , 121 ]. Peer influence, building on the concept of group responsibility for self-education and monitoring, as well as multi-component interventions, have been shown to be effective in improving professional practice in the public sector of high-income countries [ 124 , 125 ]. The effect of peer pressure may be positive or negative. Pressure from local practice styles is particularly relevant in situations where there is the most uncertainty concerning the most appropriate treatment protocol [ 85 ]. This reflects the practice-styles hypothesis of Wenberg and colleagues in 1982 [ 126 ]. Practice styles can be changed through "peer influence meetings", particularly if the change is seen as building up public reputation and status, once more showing that simple income topping-up is not the principal driving force of professional behaviour [ 127 ]. This points to the importance, in the absence of effective regulatory mechanisms, of the role of professional societies in ensuring peer-pressure mechanisms to reduce undesirable coping strategies associated with dual practice. A further possibility is workers' forming peer pressure groups to reduce undesirable coping strategies associated with dual practice. These groups could function to support members to maintain their personal stance as well as to inform the public of their rights. Making public the membership of such a group could be a way of identifying the non-members, an indirect way of increasing pressure [ 23 ]. A significant problem with individual coping strategies associated with dual practice is the difficulty of assigning individual responsibilities in situations where these are endemic. In these circumstances it might be relevant to introduce legislation that makes the head of an organization or department legally responsible for the actions of that body. This would be a further means of increasing peer pressure and accountability [ 23 ]. Pressure from users Civil society has a particularly important role, specifically in linking reform measures to the experiences and expectations of real people. But civil society must not be seen as a neutral body, particularly in developing countries where patron–client networks or kinship networks have a strong influence on the state and on the patterns of corruption and/or of coping strategies observed. In these situations the reform of civil society itself should be an objective of the interventions to correct such strategies. In many countries, users/clients/patients are not protected against the consequences of the asymmetry of information they face – with health and financial consequences. From the history of the workers' movement in Europe and as the recent evolution in a number of middle-income countries – such as Thailand's National Forum on Health Care Reform [ 128 ] – points out, perhaps the most effective way to help the State regulate professional practice is to increase pressure from civil society. (Fear of malpractice may have a paradoxical effect in that may result in excessive and inappropriate recourse to caesarean sections, for example [ 85 , 129 ].) Creating opportunities for users to voice their discontent effectively implies that patient's rights must be clear, channels for complaints must be simple, regulatory agencies must be strong and trusted by the public, processes must be explicit and transparent and the judiciary system must be strengthened [ 23 ]. Recruitment practices International development agencies, even when they do not have formal, explicit policies regarding dual practice, have become more sensitized to the problem over recent years. This has resulted in a number of recommendations to help minimize the problem. To limit the brain drain due to their own employment policies, organizations such as the World Bank, Norwegian Agency for Development Cooperation (NORAD), German Technical Cooperation (GTZ) or the World Health Organization in principle implement human resources recruitment policies that emphasize the employment of task-specific and short-term consultants, with a commitment of national institutions to retain such staff [ 90 - 92 , 130 ]. Regulating the private sector The anti-corruption literature, without the necessary empirical evidence to support such claims, actually blames government monopoly of service provision as one of the key determinants of the emergence of some of the coping strategies reviewed above [ 105 ]. It has also been argued that the presence of a significant quasi-private system operating within the public sector, i.e. the form of dual practice most common in transitional economies and in developing countries, is detrimental to the development of a strong private sector [ 23 ]. The claims for a greater role for the private sector in the provision of health care are based on a number of assumptions that are not all based on empirical evidence and ignore that private practice, in most developing countries, is notoriously unregulated. The fragmentary evidence shows that blanket recommendations regarding the role of the private sector are inappropriate [ 131 ]. There is a case for public sector support of the private sector where this serves the public's interest and allows redirection of scarce resources. If that is not the case, support has no rationale. Support, but also mere control, carries costs for the public sector administrative machinery. The costs of the "new" state responsibilities must be compensated for by savings resulting from gains in efficiency and from complementarity [ 124 , 131 , 132 ]. A key policy question is whether doctors should be allowed to work in both the public and private sectors. As discussed before, prohibition is unlikely to be effective. The real issue is what types of private practice should be allowed in order to minimize conflicts of interest, and what forms of regulatory mechanisms can be introduced to isolate coping strategies that are associated mostly with lack of regulation rather than just with low income [ 23 ]. It seems that efforts should be undertaken to ensure multiple and independent channels of accountability, by means of penalties for not satisfying contractual obligations, through channels of accountability to professional councils and associations and to the public. Regulation is one important factor influencing the coping strategies that result from the interface with the private sector [ 62 , 124 ]. Even when regulations exist, effective enforcement mechanisms are often absent in low- and middle-income countries [ 125 , 133 ]. Therefore, good legislation is not enough. The state must have the means to enforce it. In India, for example, private clinics and mobile teams promote prenatal sex determination by advertising in local newspapers, in spite of government prohibition of the practice [ 134 ]. Pressure on donors International collaboration is seen as particularly important regarding the support of international development agencies for actions such as: good-governance interventions in specific domains; supporting methods to curb corruption, including policy dialogue, capacity building, documentation and analysis of best practices and support to national programmes; and making reformers aware of the importance of country conditions in programme development [ 115 ]. Anti-corruption strategies have also been approached by donors with different objectives: to reduce poverty, to improve the functioning of democratic institutions, to sustain economic development, political stability and social justice. The lesson for the management of coping strategies and dual practice is that international collaboration cannot be neglected, as donors may be important inducers of coping strategies and dual practice as well as essential partners in the search for solutions. One way to increase donors' and governments' commitment to deal with the causes of individual coping strategies as well as dual practice might be to include a formal "human resources impact assessment" as a condition for the approval of health projects or components of sector-wide approaches. This could force governments and their partners to face the problems caused by dual practice before it becomes part of the public organization's culture. This would not be a guarantee that it would be effectively dealt with, but might limit the damage [ 135 ]. Conclusions In terms of sector location, dual practice may be public-on-public, public-on-private or private-on-private. Dual practice is probably present in all countries regardless of income, even in settings where there are major regulatory restrictions, such as China. Dual practice may lead to predatory behaviour by health workers. This constitutes, in many cases, a de facto financial barrier to access to health care. It delegitimizes public sector health service delivery and jeopardizes the necessary relation of trust between user and provider. Clinicians in dual practice have to compete for patients with themselves, which is an incentive to lower the quality of the care they provide in the public services. Dual practice contributes also to brain drain, specifically public-to-private brain drain. If public sector medical staff is moonlighting in private practice, this limits access. Besides competition for time, in many cases, the use of the public sector's means of transportation, office infrastructure and personnel represent additional hidden outflows of resources, often in association with dual practice. This is not the case for managers: involvement in NGO projects or work for donors can foster better coordination in the provision of services, but may constitute a conflict of interest when NGO or project policies are not necessarily congruent with national health policies or the agenda of the public service. On the other hand, dual practice allows professionals a standard of living that is closer to what they expect, as well as a standard of practice closer to their own perceptions of good professional practice, resulting in higher professional satisfaction. There is no evidence that dual practice by public sector health professionals complements public practice or promotes greater equity of health care distribution. The reasons for dual practice are contextual. The extent of dual practice seems to vary according to urban or rural residence, according to professional group (more common among health workers with university degrees and, for these, more common for doctors than for other professional groups) and even within a professional group, according to specialty or occupation. The limited evidence suggests that being a migrant worker, being on temporary contracts and doing shift work are important determinants. This evidence suggests that dual practice depends not so much on the personal (age and sex), social (marital status) and professional characteristics of health workers, although these are not insignificant, but on factors that are manageable. Sometimes dual practice may be the unexpected result of health care reform. Reforms frequently result in the increase of staff employed on fixed-term and temporary contracts. This trend seems to encourage dual employment. Therefore, at the core of the reliance on dual practice is the gap between the professional's expectations and what public service can offer. Adequate responses imply the identification of the main underlying reason for the observed dual practice. The most relevant conclusion is that there is no single recipe to address the reality of dual practice. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AB, IF, FH were responsible for the Portuguese case studies. PF and WVLconceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529467.xml

544847

Directed evolution of single-chain Fv for cytoplasmic expression using the β-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation

Background Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli . To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli . Results We used the β-galactosidase α-complementation system to monitor and evolve two antibody fragments for high expression levels in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>10 7 clones), clones exhibiting high levels of β-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the β-galactosidase activity present in the cells. Conclusion This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free α-peptide released from the protein fusion by the host proteases. This means that the α-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released α-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein.

Background Because of their high affinity and specificity against their antigen, antibody molecules and their fragments have many applications in research, diagnosis and therapy [ 1 ]. E. coli is a widely used organism for the production of proteins, including antibody fragments such as Fab or single chain variable domains (scFv). Active scFv can be obtained by targeting the protein to E. coli periplasm, where the two disulfide bonds needed for protein folding and stability [ 2 - 4 ] can form. The amount of scFv produced is however usually low, in the range of 0.1–1 mg l -1 of culture at an optical density (OD 600 ) of 1, even if expression levels of 10 mg l -1 have sometimes been reported [ 5 ]. An alternative strategy to produce antibody fragments in E. coli has been to maintain the scFv in the cytoplasm by removing its signal sequence. Under those conditions, scFv might be expressed at very high levels, albeit in an insoluble and inactive conformation. Even if highly efficient in vitro refolding procedures have been developed for scFv and Fab [ 6 , 7 ], it would be more suitable to directly recover soluble active protein from the cytoplasm of the cell. This has been partially accomplished by modifying the cytoplasmic oxido-redox environment by mutating components of the thioredoxin and glutaredoxin pathways [ 8 ], resulting in the accumulation of soluble intra-cytoplasmic oxidized antibody fragments [ 9 - 11 ]. The soluble expression levels reported, however, are not higher than those obtained in the periplasm of the cell. Another interest in expressing soluble active scFv in the reducing environment of the cytoplasm is that the expression of scFv molecules inside the cell can be used to block viral replication and to inhibit oncogene products [ 12 - 15 ]. The use of so-called intrabodies opens many interesting possibilities in gene therapy [ 16 ] and in the in vivo study of protein function [ 17 ]. Several methods have been proposed in recent years to increase the soluble expression levels of foreign proteins expressed in E. coli cytoplasm [ 18 ]. Most of these methods rely on fusion between the protein of interest and a reporter enzyme. If the protein folds into a soluble conformation, the fused enzyme will be active; but if the protein ends up as an inclusion body, the fused enzyme will be inactive, resulting in a null phenotype. Three reporter enzymes have been used to date, the green fluorescent protein (GFP [ 19 ]), the chloramphenicol acetyl transferase (CAT [ 20 ]) and the β-galactosidase (βgal [ 21 ]). In the case of βgal, the whole enzyme was not fused to the protein of interest but only a small N-terminal fragment of about 50 aminoacids called the α-fragment [ 22 ]. The inactive enzyme remainder was expressed in trans by the bacteria (β-galactosidase ΔM15 protein), resulting in in vivo complementation and a lactose+ (lac+) phenotype [ 23 ]. The role of the α-fragment is to promote the tetramerization of the inactive dimeric ΔM15 mutant [ 24 ], resulting in βgal activation. Using the GFP fusion method, Waldo and collaborators [ 18 , 25 , 26 ] isolated several soluble variants of aggregating proteins. However, since the method is based on a phenotypic screen, it is limited to the exploration of libraries of about 10 5 clones and does not allow the isolation of mutants from very large libraries. This is not the case for the two other methods which should allow the selection of very rare events by selecting for chloramphenicol resistance or growth on lactose as the carbon source. To date, there is, however, no report of protein evolution and selection using these latter two methods even if the CAT system has been used to pre-select libraries of hybrid proteins to avoid stop codons and enrich in properly folded molecules [ 27 ]. We previously showed that it is possible to evolve an scFv molecule for very high expression levels in E. coli cytoplasm [ 4 , 28 , 29 ]. This evolved scFv is active in the cell and its expression level is as high as 100 mg l -1 at an OD 600 of 1. This was, however, a very rare event, and the selection had to be conducted using large libraries of 10 7 –10 8 mutants. The selection procedure used was restricted to the properties of a particular antibody molecule able to activate βgal mutants in vitro and in vivo . To extend this result to other scFv molecules, we constructed fusions between two scFv and the α-fragment of βgal to monitor the soluble expression level in vivo . The lactose phenotype of the strain was then used to select mutants with improved lactose utilization and thus presumably expressing scFv at a higher level in the bacterial cytoplasm. Results System design In order to easily fuse scFv to the α-fragment of βgal, we constructed the plasmid pPM170, derived from pUC119 and which contains a lac promoter followed by a Nco I and a Not I site in which an scFv can be easily cloned in frame with the α-fragment present in the pUC119 plasmid. The α-fragment is separated from the protein by a linker consisting of two tags (Fig. 1 ). To verify that the system was indeed able to discriminate soluble scFv expression levels in the cytoplasm, we used a set of mutant scFv derived from the human scFv13 and presenting a gradual increase in soluble expression [ 28 ]. The expression levels of these scFv have been previously studied in E. coli cytoplasm using the tryptophan promoter: The best mutant, 13R4, is expressed at about a 50 times higher level than the wild type scFv13 and the expression levels increased gradually from scFv13 to scFv13R4 (in the rank order 13 < 13R1 < 13R2 < 13R3 < 13R4). These scFv were cloned in pPM170 (plasmids pPM173 to pPM173R4). Fig. 2A shows the phenotype on MacConkey lactose plates of strain TG1 containing the plasmids. As expected, the phenotype gradually increased from lac- (white colonies) for pPM173 expressing the wild type scFv13 to a strong lac+ phenotype when the plasmid contained the best expressed mutant 173R4. The phenotype of this latter strain is comparable to the phenotype of the strain containing an unmodified pUC119 plasmid giving normal α-complementation (T+). The level of α-complementation was further characterized by measuring the βgal activity present in the cell using the whole cell assay developed by Miller [ 30 ]. The result shown in Fig. 2B demonstrated that the βgal activity in TG1(pPM173) was close to background level and that the activity increased gradually to 100% of the activity of the positive α-complementation control. This clearly demonstrated the correlation between the soluble cytoplasmic expression levels of the scFv and the lactose phenotype of the strain, opening the way to direct selection of mutant proteins using this assay. Spontaneous lactose plus mutants When TG1(pPM173) was incubated 2 to 3 days at 37°C, red papillae appeared on the MacConkey plates. These clones were isolated and showed a stable lac+ phenotype, comparable to a wild type lac+ strain. One of the clones, 173S1, had even a higher βgal activity in its cytoplasm than the pUC119 rescued ΔM15 strain (Fig. 2B ). The increase in βgal activity was shown to be linked to the plasmid by transforming TG1 with the plasmid extracted from TG1(pPM173S1). To determine if the mutation was located in the scFv, we excised the gene from the plasmid using the Nco I and Not I sites and cloned it back in pPM170. The resulting clone was lac- (data not shown), demonstrating that the mutation responsible for the lac+ phenotype was associated with the plasmid but not with the scFv. To understand the origin of this phenotypic change, we sequenced the α-fragment of two of the clones, 173S1 and 173S2. Both contained a mutation in the α-fragment of the βgal, resulting in a stop codon (Fig. 1 ) at positions 49 and 50. It should be noted that the stop codon of 173S1 is an amber codon which is partially suppressed in TG1 and replaced by a Gln, resulting in this case in two different fusions, one stopping at position 49 of the βgal and the second with a complete α-fragment, showing that the mutant is dominant over the wild type. Analysis of the sequence of the α-fragment present in pUC119 (and pPM170), showed that a 30 aminoacid long peptide of unknown origin was fused to the C-terminal extremity of the α-fragment (Fig. 1 ). This peptide (or part of it) was present in all the pUC derived plasmids. Sequence analysis showed that this peptide originated from a fusion between a DNA sequence present in pBR322 plasmid between the Nde I and Ear I sites (nucleotides 2297 to 2351 of pBR322; LMR...HRI translated sequence) and the 3' end of M13 gene IV (nucleotides 5470 to 5511 of M13; RQS...AAH translated sequence). The pBR322 sequence, located between the rop gene and the origin of replication, is not normally translated, and the M13 gene IV sequence is read out of frame. Two explanations could account for the stronger lactose phenotype of the 173S1 clone when the extra peptide was removed from the fusion. Either this peptide impaired the α-complementation, or the presence of this peptide changed the expression level of the scFv. To test these hypotheses, we expressed the scFv13 and scFv13R4 clones as fusion proteins either to the α-peptide followed by the 30 aminoacid long peptide (173 and 173R4) or to the shorter α-peptide of clone 173S1 (173S1 and 173S1-R4). As shown in Figure 3B , the expression level of the fusions was much higher with the shorter α-peptide. This demonstrated that the additional C-terminal peptide lowered the protein expression level, presumably by directing the scFv to the cell degradation machinery since both the insoluble and the soluble expression levels where decreased. In all cases, the fusion proteins were degraded, and a band migrating to about 30 kDa was visible. This band may correspond to an unfused scFv liberated by host proteases. However, despite a strong increase in the expression level of 173S1-R4 versus 173S1, the lactose phenotypes of both clones expressed in strain TG1 were comparable (Fig. 3A ). This showed that the link between the lactose phenotype and the intracellular expression levels of scFv13 shown in Figure 2 was due to the presence of the 30 aminoacid long peptide. Indeed, in the case of 173S1, the α-complementation detection system is saturated and the strong lactose phenotype of strain TG1(173S1) would not allow the selection of evolved mutants with a stronger phenotype. This prompted us to retain this foreign 30 aminoacid long peptide for selection and to adapt the procedure to avoid the selection of spontaneous stop codons. Molecular evolution The outline of the selection procedure is shown in Figure 4A . We first introduced random mutations at a low rate (0.2%) in the scFv gene using error-prone PCR. The mutagenized gene was cloned in the pPM170 vector and transformed in TG1 bacteria to give a library of at least 10 7 clones. Transformed bacteria were plated on selective medium containing lactose as the unique carbon source, allowing the selection of lac+ clones. After 1 to 3 days at 37°C, about 10 to 100 clones able to grow under these conditions were cultured in 96-well microtiter plates, then pooled before plasmid extraction. To avoid the isolation of spontaneous lac+ mutations in the α-fragment (see above), the Nco I- Not I fragment containing the scFv gene was excised from the pooled plasmids and cloned back in pPM170. After transformation in TG1, a small number of clones (typically 100 to 1000) was screened on MacConkey lactose agar plates and the deepest red colonies further analyzed. This last screen was done after a 24-h incubation at 37°C in order to avoid the selection of spontaneous red colonies that appeared after about 2 to 3 days. Two scFv were chosen as models. We first used a humanized D1.3 anti-lysozyme antibody, HuLys11 [ 31 ]. This antibody has been extensively studied and its structure solved by X-ray crystallography. The second scFv was derived from the mouse monoclonal antibody 225.28S and recognizes the high molecular weight melanoma-associated antigen [ 32 ]. As such, this antibody might have potential applications in the treatment of that type of cancer. Four rounds of mutagenesis and selection were done for both scFv. The size of the libraries and the number of clones isolated after each round are shown in Figure 4B . The phenotype of the clones increased during the selection from an almost lac- to a clearly lac+ phenotype. This is shown in Figure 5A for 225.28S scFv, where the best five isolated clones (R4.1 to R4.5) exhibited a deeper red color than the original clone (175), demonstrating a greater ability to use lactose as a carbon source. In addition, the phenotype was close to the phenotype obtained with the pUC119 plasmid, that is, normal α-complementation. The results obtained with HuLys11 were comparable, but the colonies showed a weaker lactose phenotype (data not shown). All the isolated clones were different at the DNA and protein level. The mutants of scFv225.28S contained 8–14 nucleotide substitutions and 3–9 mutations in the protein (see additional files 1 and 2 , 22528Sdna.txt and 22528Saa.txt). In the case of HuLys11, the three isolated mutants contained 4–5 nucleotide substitutions and 2–3 aminoacid mutations (see additional files 3 and 4 , HuLys11dna.txt and HuLys11aa.txt). The mutations did not cluster together either in the DNA sequence nor in the protein or in the structure of the scFv. In the case of HuLys11, for which a high resolution structure is available, three of the four isolated mutations are located in β-strands and are solvent exposed (see additional file 4 , HuLys11aa.txt and Fig. 6 ). To confirm the phenotype shown on the plates, βgal activity was measured using the assay described by Miller [ 30 ]. The assay relies on cell lysis by chloroform/SDS and as such measures the amount of βgal activity present in the whole cell. As shown in Figure 5B , the βgal activity present in the mutants was about five to seven times higher than in the strain expressing the wild type 225.28S scFv fused to the α-fragment and five times lower than in the control cells expressing the unfused α-fragment from the pUC119 plasmid. In oder to confirm that this increase in βgal activity was localized in the soluble protein fraction, we measured the activity in soluble extracts of the cells prepared by lysozyme treatment and centrifugation. The results were comparable to those obtained with the whole cell assay (Fig. 5B ), demonstrating that indeed the increase in βgal activity was due to an increase in α-complementation in the soluble protein fraction. Fused scFv-α characterization To determine whether the expression levels of the mutant scFv were higher than those of the wild type protein, soluble and insoluble extracts were prepared and analyzed (Fig. 7 ). No protein was detectable by western blot in the case of the wild type scFv225.28S or mutants R4.2 and R4.4, and only a faint band was visible in the soluble extracts of the other clones (R4.1, R4.3 and R4.5. Fig. 7C & 7D ). This showed that indeed, as expected from the βgal levels measured in Figure 5 , some of the mutants were expressed at a higher level in the cytoplasm than the wild type but that most of the scFv was degraded by the host proteases. To test this hypothesis, we expressed the clones in a TG1 strain deficient in the cytoplasmic Lon protease. This protease has been shown to be involved in the degradation of many recombinant proteins in E. coli [ 33 ]. When expressed in such a strain, the wild type scFv225.28S was detected in the soluble fraction, albeit at a very low level (Fig. 7C ), and no scFv was present in the insoluble fraction. All the evolved scFv were expressed at a much higher level than the wild type, mainly as soluble proteins (Fig. 7C & 7D ), showing that the scFv225.28S proteins were predominantly degraded by the Lon protease in vivo . The same type of result was obtained with scFv HuLys11 since the wild type scFv was not detected in any of the extracts, and the evolved scFv were detected in the soluble fraction of the Lon deficient strain (Fig. 7F & 7G ). The Lon protease is not the only protease present in the E. coli cytoplasm [ 34 ] and in its absence some degradation of the fusion is still present as shown by the additional band at about 30 kDa in Fig. 7C & 7D . This molecular weight is consistent with a degradation site located between the scFv and the α-peptide and could be one of the sources of the free α-peptide liberated in the cell cytoplasm (see below). It should be noted that in the reported cases of the successful isolation of soluble variants of aggregating proteins using the GFP system [ 25 , 26 ], the authors used an E. coli B strain, naturally deficient in the Lon protease [ 33 , 35 ]. The soluble extracts of scFv225.28S and its mutants were tested for the presence of βgal activity (Fig. 7E ). The results obtained in strain TG1 were comparable, as expected, to those obtained previously (Fig. 5 ). There is however no clear correlation between the soluble expression level and the βgal activity since clone R4.1, which is expressed at the highest level, did not give a higher signal than the non-detected clones, R4.2 and R4.4. In the Lon-deficient strain, all the clones, including the wild type scFv225.28S, gave the same signal despite the fact that the wild type scFv was only barely detectable in the extract. These results showed that there was no correlation between the soluble expression level of the fusion protein and the βgal signal. The most likely explanation of this phenomenon is that the βgal signal is not due to the fusion protein detected by western blot but to some α-peptide released from the fusion by the host proteases. This free α-peptide is presumably also much more efficient for βgal complementation than the fusion protein since it may penetrate the tunnel present in M15 protein [ 36 ] at a much faster rate. Unfused scFv characterization The previous results were obtained with the scFv fused to the α-peptide. Since it has been shown in several systems that fusion to a partner may influence the fate of a protein [ 37 - 40 ], we next examined the expression level of scFv225.28S and its mutants as unfused proteins. After cloning in plasmid pPM210, the scFv were expressed in the Lon-deficient TG1 strain. The wild type scFv225.28S was only detected in the soluble fraction (Fig. 8 ) and the mutants were present in both the soluble and insoluble fractions. All the mutant were expressed at a higher level than the wild type scFv, but the increase in expression was less pronounced than when the proteins were fused to the α-peptide (Fig. 7 ). This was however mainly due to the fact that the wild type scFv225.28S was expressed at a higher level when not fused to the α-peptide because the fusion was degraded by the host degradation machinery as previously described for other βgal fusions [ 37 ]. We next compared the expression level of the best isolated clone (scFv225.28S R4.1) in the periplasm and in the cytoplasm. It must be noted that the two vectors used, pPM210 and pAB1, are derived from the same pUC119 plasmid and that in both cases the scFv gene is under the control of the lac promoter. The scFv expressed by the two plasmids are exactly the same except for the pel B leader peptide present at the N-terminal extremity of the scFv expressed in the periplasm. After cleavage of this signal sequence, the two scFv only differ by an additional N-terminal Met-Ala dipeptide present in the scFv produced in the cytoplasm. As shown in Figure 9 , the soluble expression level of mutant R4.1 is higher both in the periplasm and in the cytoplasm than that of the wild type scFv225.28S. In addition, the soluble cytoplasmic expression level of the evolved R4.1 clone is higher than the wild type periplasmic expression level. This suggests that evolving scFv for cytoplasmic expression is a valuable approach for increasing the production of scFv in E. coli even if in this case it is only a twofold increase much lower than in previously reported cases [ 28 ]. In an attempt to increase further the cytoplasmic expression, the genes were cloned under the control of the strong T7 promoter and expressed in strain BL21(DE3)pLysS [ 41 ], which is naturally deficient in the Lon protease [ 33 , 35 ]. As seen in Figure 10 , the mutant proteins were expressed at much higher levels than the wild type. This is particularly the case for scFv225.28S (Fig. 10B ). To further characterize the mutant proteins, soluble and insoluble protein fractions were prepared. As shown in Figure 10 , the increase in expression was due to an increase in the insoluble fraction. In all cases, the amount of soluble protein produced by the mutants was comparable or even lower than that produced by the wild type. This was true both for HuLys11 and 225.28S. Discussion In this report, we used the α-complementation assay as a probe to detect soluble scFv expression in E. coli cytoplasm [ 21 ]. The system was used to evolve two scFv to increase their expression levels. After four rounds of mutation and selection, we were able to select for scFv fusions giving high βgal activity in vivo and in vitro . Characterization of these mutant proteins showed that they were expressed at higher levels in the cytoplasm than the wild type scFv. The proteins were however quickly degraded in the cell cytoplasm, and only a faint band was detected in the soluble fraction and no protein at all in the insoluble fraction (Fig. 7 ). This is not surprising for scFv expressed in the reducing environment of the cytoplasm since the lack of the two disulfide bonds results in only marginally stable proteins quickly degraded by the host proteases [ 42 ]. Since it has been shown that the Lon protease is involved in the degradation of many recombinant proteins in E. coli [ 33 ], we expressed the scFv in an isogenic strain deficient in this protease. Under those conditions, the mutant scFv were expressed at a much higher level than the wild type scFv. This was true for both scFv, HuLys11 and 225.28S. This increase in soluble cytoplamic production of the scFv-α fusion was however not correlated with the βgal activity (Fig. 7 ). For example, clone scFv225.28S R4.1 and R4.2, which were expressed at a very different soluble level (Fig. 7C ), gave the same βgal signal (Fig. 5 & 7E ). This indicates that most, if not all, of the βgal activity present in the cell was not due to the scFv-α-peptide fusion but to some free α-peptide released from the fusion by the host proteases. This means that the α-complementation assay does not sense the scFv-α expression level, as hypothesized, but rather the amount of free α-peptide in the cell cytoplasm. This could also explain why the system was not able to detect differences between the clones in a lon background (Fig. 7E ). Recently, Betton and collaborators [ 43 ] presented a model of the possible in vivo competition between folding, aggregation and degradation. Although this model was presented in the case of periplasmic proteins it might also apply in the cytoplasm. The possible model and the results for in vivo βgal complementation are shown in Figure 11 . The "classical" competition between aggregation and folding is represented by the green arrows at the top of Figure 11 . The scFv, emerging from the ribosome, will fold into folding intermediates. A folding intermediate may fold into a soluble native conformation, or may misfold, leading to aggregation. Such a model has been extensively studied in vivo and in vitro [ 44 - 47 ]. In this model, an increase in α-complementation would result in an increase in soluble protein expression and is the basis of the tag-based systems to detect and evolve soluble proteins [ 18 ]. As proposed by Betton and collaborators [ 43 ], there is however a third pathway, leading to protein degradation, in kinetic competition with aggregation. When the proteins are expressed at a low level under the lac promoter, most of the protein ends up as degraded peptide fragments. However, since aggregation is a high order kinetics, this is favored over degradation under the high transcription rate due to the T7 promoter. This would not be the case if degradation had not originated from the same misfolded conformation as the aggregated protein. However, since the soluble expression levels of the scFv were higher in a lon strain, we must also admit that there is a supplementary pathway involving the Lon protease and leading from soluble fusion to degraded protein as suggested by Parsell and Sauer [ 42 ]. There is a striking difference between the properties of the fused and the unfused scFv. Indeed, in the structural context of the selection (as fusion to the α-peptide), all the scFv, albeit highly sensitive to degradation, were mainly expressed as soluble proteins. This is particularly the case of HuLys11 for which no insoluble protein was detected in Fig. 7G . As unfused scFv, all the clones were, however, mainly found in the insoluble fraction, particularly when expressed using the strong T7 promoter (Fig. 10 ) but also when we used the same promoter than during the selection process (Fig. 8 ). This shows that interactions could take place between the scFv and the fused α-peptide either during or after folding of the fusion protein. This may explain why all the mutations where localized on the surface of HuLys11 (Fig. 6 ) since these residues are more likely to take part in such an interaction. This is particularly the case of the H46 mutation, which is present in all three isolated mutants. In this case a hydrophilic and charged residue (Glu) is replaced by an hydrophobic Gly. As the α-peptide and the 30 aminoacid peptide fused to the scFv contain some highly hydrophobic patches of residues (data not shown), this increase in the hydrophobicity of the scFv surface may favor interaction between the two partners that could enhance the solubility of the fusion by a chaperone-like effect, as proposed for other fusions [ 48 ]. When the scFv is expressed alone, without the C-terminal α peptide, increasing the hydrophobicity of the surface residues could result in an increase in aggregation [ 49 ], as noted in our case (Fig. 8 & 10 ). Finally, another problem during the selection originated in the presence of the 30 aminoacid peptide at the end of the α-peptide. As we showed in the case of the model antibody scFv13, this peptide is needed to obtain good correlation between protein soluble expression levels and in vivo α-complementation since the introduction of a stop codon at the end of the α-peptide resulted in a very strong complementation with all the scFv, even the wild type (clone 173S1 in Fig. 2 & 3 ). This may explain why the selection was biased towards degraded C-terminal α-peptide. Indeed, Figure 3 shows that the role of the additional 30 aminoacid long C-terminal peptide is to decrease the expression level of the fusion in order not to saturate the detection system with the wild type scFv. This decrease in the expression level is presumably accomplished through targeting of the fusion to the cell degradation systems, resulting in a rapid conversion of the fusion to free α-peptide and in a bias in the selection procedure towards degradation and thus aggregation. It must be noted that Schwalbach and collaborators [ 50 ] recently selected a mutant scFv using the GFP system, and that despite an increase in GFP activity, they did not notice any increase in soluble protein expression but rather an increase in protein aggregation. They also noticed that protease degradation of the fusion released free GFP in the cytoplasm, particularly when cells were induced for long periods, that is, the conditions used during the selection process. It should be noted that the authors used a lon + strain for the selection instead of the E. coli B lon - strain used by other authors [ 25 , 26 , 35 ]. These failures may be due, in the specific case of cytoplasmic scFv expression, to the lack of disulfide bond formation, leading to marginally stable reduced scFv, quickly degraded by the cell proteases [ 42 ]. Despite the difficulties associated with cytoplasmic scFv expression, some scFv have been previously evolved for folding under reducing conditions [ 28 , 51 ]. The systems used relied on the binding activity of the scFv molecule, avoiding the problem of protein degradation since degraded antibody cannot bind to its antigen anymore. It would thus be more appropriate to use yeast or bacterial two-hybrid systems to evolve scFv [ 52 , 53 ]. The use of such systems could also avoid the selection of mutations that modify or abolish the antigen-binding properties of the scFv as is presumably the case in our selection since several of the mutations are located in the CDR loops of the antibody fragments (see additional files 4 and 2 , HuLys11aa.txt and 22528Saa.txt). Another possibility could be to use E. coli protease-deficient strains to limit protein degradation during the selection, but the results shown in Fig. 7E demonstrate that even in such a strain there is no correlation between the soluble expression level and the βgal activity present in the cell. Conclusions In this report, we used the α-complementation assay as a probe to detect soluble scFv expression in E. coli cytoplasm. The system was used to evolve two scFv in order to increase their expression levels. After four rounds of mutation and selection, we were able to select for scFv fusion giving a high βgal activity in vivo and in vitro . Characterization of these mutant proteins showed that their expression levels were much higher in the cytoplasm than those of the wild type scFv, particularly in a Lon-deficient strain. There was however no correlation between the βgal activity present in the cell and the soluble expression level of the scFv, showing that the βgal signal is presumably due to some free α-peptide released from the fusion protein by the host proteases and not to the non-degraded soluble scFv-α fusion. Methods Media, plasmids and bacterial strains MacConkey agar, M9 and LB media were previously described [ 30 ]. Strain TG1 is E. coli K-12, [F' traD 36 lac I q Δ( lac Z)M15 pro A + B + ] sup E44 Δ( hsd M- mcr B)5 thi Δ( lac - pro AB). CAG626F' is E. coli K-12, [F' lac I q Δ( lac Z) M 15 zzf ::mini- Tn 10(Kan R ) pro A + B + ] lac Z(am) pho (am) lon trp (am) tyrT [ supC (ts)] rpsL mal (am) [ 54 ]. TG1 lon strain was constructed by P1 transduction. First, zaj - 3054::Tn 10, located at 9.95 minutes, was inserted near to the lon gene in strain CAG626F' by P1 transduction from CAG12017 [ 55 ]. In a second step, lon and zaj - 3054::Tn 10 were co-transduced in TG1 using P1 phage. The introduction of the lon mutation was verified by comparing the resistance to UV irradiation and to nitrofurantoin of TG1 and TG1 lon . Plasmid pPM170 for the cytoplasmic expression of scFv fused to the α-fragment of βgal was constructed as follows (Fig. 1 ). A fragment containing a Shine Dalgarno sequence followed by a Nco I site containing the ATG initiator was obtained by PCR amplification of pPM160 [ 28 ] with pTrpFOR (CGGGAATAAGCTTCAACGCCAG) and EcoAlpha.for (GTGAAT GAATTC GAATGGTGATGATGG) primers. The underlined sequence corresponds to an Eco RI site designed in order to get in frame fusion of the scFv with the α-fragment of βgal present in pUC119 when the fragment was cloned in the Eco RI site of pUC119. The amplified fragment also contained an Hpa I site located 34 bp upstream from the ATG initiator of the scFv gene. This PCR band was digested with Hpa I and EcoR I enzymes and ligated with Hinc II- Eco RI-digested pUC119 plasmid, leading to the pPM170 plasmid. Plasmid pAB1 for the periplasmic expression of scFv under the lac promoter has been described previously [ 28 ]. Plasmid pPM210 is identical to pPM170 except for the presence of a stop codon between the scFv and the α-fragment of βgal. It was obtained by cloning the Nco I- Eco RI fragment of pAB1 in the Nco I and Eco RI sites of pPM170. scFv cloned in pPM210 were transcribed from a lac promoter and tagged at their C-terminal extremity by both a c-myc and a polyhistidine flags. Plasmid pET23NN was designed to easily clone Nco I- Not I fragment containing scFv from pPM170-derived plasmids under the T7 promoter with C-terminal c-myc and His6 tags. pET23d(+) obtained from Novagen was first digested with Xho I enzyme, filled-in with T4 DNA polymerase then digested with Nco I. This fragment was ligated with a Nco I- Eco RI(filled) fragment excised from the pAB1 plasmid [ 28 ]. The resulting plasmid contained a T7 promoter, followed by a Nco I site containing the ATG initiator, a Not I site followed by a c-myc and a His6 tag [same sequence as pPM170 (Fig. 1 ) but with a stop codon before the Eco RI site]. Due to the removal of a T, presumably during T4 DNA polymerase treatment, ligation between the filled EcoR I and Xho I sites reconstituted the EcoR I site (GAATTCGAG instead of GAATTTCGAG). Error-prone mutagenesis The conditions used are those described to obtain 0.2% mutations with p(AT->NN) = p(GC->NN) and p(AT->GC) = p(AT->TA) for ten duplications [ 28 , 56 ]. The amplified band was digested with Nco I and Not I and cloned in pPM170. The ligation was transformed in TG1 by electroporation. Bacteria were plated on MacConkey lactose and M9 lactose agar plates supplemented with 100 μg ml -1 ampicillin, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 1 μg ml -1 of vitamin B1 in the case of the M9 plates. Preparation of bacterial extracts Expression under the T7 promoter was conducted essentially as described except that LB was used instead of M9ZB [ 41 ]. Freshly transformed cells were grown in LB supplemented with 100 μg ml -1 ampicillin and 20 μg ml -1 chloramphenicol until an OD 600 of about 1 and kept at 4°C overnight. In the morning, the cells were diluted to an OD 600 of 0.1 in the same medium and incubated with shaking for 2 hours at 37°C, then 0.4 mM IPTG was added and the culture continued for 3 hours at 30°C. The cultures were cooled down on ice for 5 to 30 minutes, then centrifuged and resuspended at a concentration of 40 OD 600 in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA. Cells were lysed by freezing/thawing followed by a brief sonication. The insoluble fraction was collected by centrifugation (30 minutes at 17 500 g), washed once, then resuspended in the same volume of buffer. For expression under the lactose promoter (plasmid pPM170), an overnight culture in LB supplemented with 100 μg ml -1 ampicillin and 1% glucose was diluted to a final OD 600 of 0.1 in LB supplemented with 100 μg ml -1 ampicillin and incubated with shaking for 2 hours at 37°C, then 1 mM IPTG was added and the culture continued for 3 hours at 30°C. Cytoplasmic soluble extracts were prepared as follows. 16 OD of culture were cooled down on ice for 5 to 30 minutes, then centrifuged and resuspended in 400 μl of 20 mM Tris-HCl (pH 8.0), 0.7 M sucrose. Hen egg-white lysozyme at a 0.1 mg ml -1 concentration was added and the extract incubated 2 minutes on ice. 800 μl of a cold solution of 1.5 mM EDTA were added slowly within ten minutes. Extract were kept 30 minutes on ice then sonicated 20 s. The insoluble fraction was collected by centrifugation (30 minutes at 17 500 g), washed once, then resuspended in the same volume of buffer than the soluble fraction (1.2 ml). Measurement of βgal activity Cells were grown under the same conditions as those described in "Preparation of bacterial extracts" for the lactose promoter. Five hundred μl of cells were lysed using SDS/chloroform and βgal activity determined and expressed as described in Miller [ 30 ]. For Figure 5 and 7 , we also prepared soluble extracts using the protocol described in "Preparation of bacterial extracts" and βgal activity was determined as above. Authors' contributions PP carried out some of the mutant characterization and participated in manuscript preparation. PM conceived of the study, performed the mutagenesis and selection experiments, and participated in manuscript preparation. All authors read and approved the final manuscript. Supplementary Material Additional File 1 DNA sequences of 225.28S and mutants scFv. The position of the six CDRs are indicated as H1, H2 and H3 for the VH and L1, L2 and L3 for the VL. The scFv 225.28S translated sequence is shown above the DNA. Proteins are numbered according to Kabat [57] Click here for file Additional File 2 Aminoacid sequences of 225.28S and mutants scFv. The position of the six CDRs are indicated as H1, H2 and H3 for the VH and L1, L2 and L3 for the VL. Proteins are numbered according to Kabat [57] Click here for file Additional File 3 DNA sequences of HuLys11 and mutants scFv. The position of the six CDRs are indicated as H1, H2 and H3 for the VH and L1, L2 and L3 for the VL. The scFv HuLys11 translated sequence is shown above the DNA. Proteins are numbered according to Kabat[57] Click here for file Additional File 4 Aminoacid sequences of HuLys11 and mutants scFv. The position of the six CDRs are indicated as H1, H2 and H3 for the VH and L1, L2 and L3 for the VL. Proteins are numbered according to Kabat [57]. The secondary structure of the protein, as indicated in the header of the PDB file 1BVK, is summarized above the sequence (H: helix; E: strand). Click here for file

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The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

Background Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. Results Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. Conclusions Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor. Electronic supplementary material The online version of this article (doi:10.1186/1471-2121-4-13) contains supplementary material, which is available to authorized users.

Background Small GTPases of the ADP-ribosylation factor (ARF) family play a major role in membrane trafficking in eukaryotic cells [ 1 ]. ARF activation is facilitated by specific guanine nucleotide exchange factors (ARF-GEFs). Several ARF-GEFs have been identified, varying in size, structure and subcellular distribution [ 2 – 6 ]. Of particular interest in signaling events are the members of the cytohesin/ARNO family of ARF-GEFs. These proteins have been found to associate with the plasma membrane under certain conditions, and consist of three well-defined motifs: an N-terminal coiled-coil domain (CC domain), a central domain with homology to the yeast protein Sec7 (Sec7 domain), and a C-terminal pleckstrin homology domain (PH domain) (Fig. 1 ). The catalytic activity of ARNO for guanine nucleotide exchange is localized in the Sec7 domain and appears to be regulated through the interaction of the PH domain with phosphatidylinositol (PtdIns) (3,4,5)-P3 [ 7 , 8 ], an intermediate in signaling cascades regulated by insulin and other agonists [ 3 ]. Figure 1 Schematic structure of ARNO constructs. Full length of wild type ARNO and ΔPH-ARNO were subcloned either in pCMV-myc or pEGFP-C1. PH-ARNO and ΔCC-ARNO were subcloned in pEGFP-C1. CC-ARNO was subcloned in pEGFP-N1. Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to produce phosphatidic acid (PA). It is involved in a variety of signaling pathways and membrane traffic processes [ 9 , 10 ]. Many hormones, neurotransmitters, and growth factors, including insulin, have been shown to induce the activation of PLD [ 11 , 12 ]. Several factors are involved in the regulation of cellular PLD activity, such as Ca 2+ , protein kinase C, tyrosine kinases, and G proteins [ 13 – 17 ]. Among these, the members of the ARF and Rho families of GTPases appear to be the most potent physiological activators [ 18 – 24 ]. However, the mechanism of the activation of PLD by ARF and Rho has not yet been fully established. This study was designed to investigate the role of ARNO in the regulation of PLD activity by insulin in HIRcB cells, a Rat-1 fibroblast cell line that overexpresses human insulin receptors. The objectives were: 1) to test if insulin induces the translocation of wild type ARNO to the plasma membrane in transiently transfected HIRcB cells; 2) to determine whether ARNO translocation is accompanied by activation and subcellular translocation of ARF; 3) to explore if overexpression of wild type ARNO in HIRcB cells alters insulin-dependent PLD activity; and 4) to investigate the function of individual domains of ARNO in insulin-dependent PLD and ARF activation. Results Insulin–dependent binding of ARNO to cell membranes The translocation of ARNO and ARNO mutants to the membranes was studied in HIRcB cells using a digitonin permeabilization assay. For these experiments, HIRcB cells were transiently transfected with myc-tagged wild type ARNO and the following mutants: ΔPH-ARNO, PH-ARNO, ΔCC-ARNO, CC-ARNO, E156K-ARNO and R280D-ARNO. This assay is based on the formation of pores in the plasma membrane induced by digitonin to allow cytosolic proteins to leak out of treated cells upon centrifugation. Fig. 2 shows that, after digitonin permeablization, a significant fraction of ARNO proteins leaked out of serum-starved HIRcB cells that transiently overexpressed the wild type ARNO and its mutants. Since these proteins were mostly recovered from the supernatant fractions, suggesting that wild type ARNO and the mutants tested are predominantly cytosolic in non-stimulated cells. In contrast, when digitonin permeablization was performed in the presence of insulin (100 nM), most of wt-ARNO, E156K-ARNO, and ΔCC-ARNO as well as a part of CC-ARNO were recovered from the particulate membrane fraction, suggesting that these ARNO proteins can be recruited to the membrane by insulin to various degrees. However, neither R280D-ARNO nor ΔPH-ARNO was recovered from the particulate fraction after insulin stimulation, suggesting that the translocation of ARNO to the membrane requires an intact PH-domain. It should be noted that, although the CC domain alone binds to the membranes under stimulation conditions, the degree of the binding is much less than that of wild type ARNO (Fig. 2 ). Surprisingly, a construct containing only the PH domain of ARNO could not be recruited to the membranes by insulin, indicating that the PH domain is essential but not sufficient for the translocation of ARNO. Figure 2 Insulin promotes the translocation of ARNO to cell membranes. HIRcB cells were transfected with myc-wt-ARNO, myc-E156K-ARNO, myc-R280D-ARNO, myc-ΔPH-ARNO, EGFP-PH-ARNO, EGFP-ΔCC-ARNO, and CC-ARNO-EGFP. The cells were treated with/without (Control) 10 μM digitonin (Dig). Where indicated, 100 nM insulin, 1 mM ATP, and 100 μM GTPγS were present during permeablization reaction. Pellets and supernatants were separated by centrifugation and the presence of myc-ARNO and its mutants or ARNO-EGFP in each fraction was determined by immunoblotting. ARNO recruits ARF1 to the plasma membrane in an insulin-dependent manner Since ARNO is an activation factor of ARF, we tested the hypothesis that agonist-dependent ARNO translocation facilitates the local binding of ARF proteins to the membrane. An initial set of real-time studies was done using HeLa cells that had been stably transfected with an ARF1-GFP construct [ 25 ]. These cells were transfected with myc-ARNO, serum-starved overnight, and imaged with a confocal microscope equipped with a constant-temperature microperfusion incubator to maintain the temperature at 37°C. Time-lapse images were collected at 30-second intervals. A representative experiment was shown in Fig. 3A . Prior to insulin stimulation, ARF1-GFP protein was mostly cytosolic or bound to the Golgi apparatus, although a small amount of ARF-GFP was localized on the surface of the cells. Ten minutes after the insulin stimulation, most of the ARF1-GFP was found on the plasma membrane. Similar results were obtained with HIRcB cells co-transfected with ARNO-myc and ARF1-GFP (Fig. 3B ). It should be noted that a significant accumulation of ARF1-GFP on the plasma membrane was not observed in the cells that had not been transfected with ARNO (not shown), or that had been transfected with the inactive mutant E156K-ARNO (Fig. 3B ). Since the endogenous levels of ARNO in HeLa cells were so low that the protein could not be detected in Western blots, it is reasonable to assume that under physiological conditions only a very small fraction of ARF1 translocates to the plasma membrane in response to extracellular agonists. Figure 3 A. Real time image of the translocation of ARF1-GFP to the plasma membrane. HeLa cells that had been stably transfected with ARF1-GFP were transiently transfected with myc-ARNO, serum starved overnight, and treated with 100 nM insulin. Images were collected every 30 seconds using a Molecular Dynamics 2001 confocal microscope. The time intervals that were indicated on the upper right hand corner of each panel represent the time after the addition of insulin. B. The translocation of ARF1-GFP to the plasma membrane by the effects of insulin requires ARNO. ARF1-GFP/HeLa cells were transfected with myc-ARNO, treated, fixed, and stained for myc-epitope as described in the Materials and Methods section. Images displaying ARF1-GFP (green) and myc-ARNO (red) were merged us ing Adobe Photoshop software. ARNO interacts directly with the insulin receptor Our previous work has shown that the insulin receptor co-immunoprecipitates with ARF in an agonist-dependent manner [ 23 ]. Furthermore, we have also shown that an ARF-GEF activity is associated with the insulin receptor and that this activity is not a function of the receptor itself [ 23 ]. Given that many receptor tyrosine kinases form complexes with their target proteins, we tested the hypothesis that ARNO binds the insulin receptor. Figure 4 shows that insulin receptors that were immunoprecipitated in the presence of insulin were associated with an ARF-GEF activity (Fig 4 ●), and that the ARF-GEF activity that was co-immunoprecipitated with the insulin receptor was significantly increased in the cells that had been transiently transfected with myc-ARNO (Fig. 4 ■). Insulin receptors that were immunoprecipitated in the absence of insulin did not accelerate the binding of GTPγS to the recombinant ARF1 as much as those obtained in the presence of insulin (Fig. 4 ○), indicating that the association of ARF-GEF activity with the insulin receptor was dependent on the presence of insulin. Figure 4 The ARF-GDP exchange activity of the coimmunoprecipitates with the insulin receptor. The exchange activity was determined as described in Materials and Methods. (○,□) Receptors were immunoprecipitated in the absence of insulin from cells transfected with empty vector (○) or with myc-ARNO (□). (●,■) Receptors were immunoprecipitated in the presence of insulin from cells transfected with empty vector (●) or with myc-ARNO (■). We then transfected HIRcB cells with myc-tagged ARNO constructs. Fig. 5 shows that the wild type ARNO co-immunoprecipitated with the insulin receptor in an insulin-dependent manner. E156K-ARNO was also co-immunoprecipitated with the insulin receptor upon insulin stimulation. However, none of the deletion mutants, including ΔPH-ARNO, PH-ARNO, ΔCC-ARNO, and CC-ARNO, as well as a site-directed mutant R280D-ARNO, was found co-immunoprecipitated with the insulin receptor. These data suggest that ARNO directly interacts with the insulin receptor and that the interaction requires intact PH and CC domains, but the catalytic activity of the Sec7 domain does not alter the interaction. Figure 5 Immunoprecipitation of the insulin receptor with ARNO and its mutants. Immunoprecipitated proteins were resolved by SDS-PAGE and myc-ARNO, myc-E156K-ARNO, myc-R280D-ARNO and myc-ΔPH-ARNO were detected by immunoblotting with a monoclonal anti-myc epitope antibody. PH-ARNO-EGFP, ΔCC-ARNO-EGFP, and CC-ARNO-EGFP were detected by immunoblotting with a polyclonal antibody against EGFP. Effects of the overexpression of ARNO or its mutants on insulin-dependent PLD activity We have shown so far that ARNO mediates the translocation of ARF proteins to the plasma membrane with insulin stimulation. Since ARF proteins mediate the activation of PLD by insulin [ 23 ], we tested the hypothesis that ARNO may play a role in the regulation of PLD activitiy upon insulin stimulation. To prove this point, the PLD activity of HIRcB cells that had been transiently transfected with the wild type ARNO, and mutant ARNO constructs. Fig. 6 shows that the overexpression of the wild type ARNO significantly increased insulin-induced PLD activity when compared with that of non-transfected cells. In contrast, the overexpression of the indicated ARNO mutants significantly decreased the ability of insulin to stimulate PLD. We conclude, therefore, that members of the cytohesin/ARNO family of ARF GEFs play an important role in the regulation of PLD activity by insulin. Figure 6 Effects of overexpression of the wild type and mutant ARNO constructs on the activation of phospholipase D by insulin. HIRcB cells were trans fected with empty vector, myc-wt-ARNO, myc-E156K-ARNO, myc-R280D-ARNO, and myc-ΔPH ARNO, PH-ARNO-EGFP, ΔCC-ARNO-EGFP, and CC-ARNO-EGFP. PLD activity was determined by a transphosphatidylation assay as described in Materials and Methods. Discussion Several studies have demonstrated that ARF proteins may mediate receptor-dependent activation of PLD. Stimulation of cell surface receptors with agonists, such as insulin, promotes the translocation of ARF proteins to the cell membranes and the activation of ARF proteins and the subsequent activation of PLD [ 16 , 18 , 21 , 23 ]. However, the mechanisms by which ARF proteins are activated by cell surface receptors remain obscure. ARF GEFs of the cytohesin/ARNO family have been shown to be recruited to cell membranes by mechanisms that are influenced by extracellular agonists [ 7 , 26 ]. These GEFs have been implicated in the regulation of many cellular processes, ranging from the regulation of cell motility [ 27 ] to cell adhesion [ 28 ] and, more recently, oncogenesis [ 29 ]. It has been speculated that PLD activation may mediate several of the cellular events regulated by cytohesin/ARNO GEFs [ 30 ]. However, a direct proof of a role for these factors in the regulation of the receptor-mediated PLD activation is still lacking. To address these and other related issues, we have studied in detail some of the mechanistic aspects of this pathway using a fibroblast cell line that overexpresses human insulin receptors as a model. This model and other similar ones have been used in our laboratory and others to examine specific aspects of insulin receptor function, such as receptor phosphorylation and traffic [ 23 , 31 – 33 ] and the regulation of the MAPK pathway [ 34 ]. Our studies showed that insulin promoted the translocation of myc-tagged ARNO constructs to the plasma membrane. This result is in agreement with data previously published by Venkateswarlu et al [ 7 ] and Langille et al [ 35 ] who demonstrated the insulin-dependent translocation of ARNO and the related protein GRP-1 to the plasma membrane, respectively. A detailed analysis of ARNO deletion and point mutants demonstrated that: 1) the translocation of ARNO to the membrane is independent of its ARF-GEF activity; 2) ARNO translocation to the plasma membrane requires an intact PH domain; 3) the CC domain of ARNO plays a role in targeting ARNO to the plasma membrane; 4) neither the PH domain of ARNO nor its CC domain alone sufice to target the protein to the plasma membrane; and 5) the plasma membrane translocation of ARNO is strongly regulated by insulin and, perhaps, other extracellular agonists. The linkage between ARNO translocation to specific subcellular fractions and ARF activation was studied using myc-tagged ARNO and ARF-GFP constructs in two different cell types. Our data showed conclusively that insulin promoted the co-localization of wild type myc-ARNO and ARF1-GFP on the surface of HIRcB and HeLa cells. Interestingly, insulin, acting through ARNO, promoted the translocation of ARF1-GFP to the plasma membrane. ARF1, like most members of the ARF family, is primarily a cytosolic protein that exerts its function on specific membranes to which it is recruited by specific activators that promote the binding of GTP. However, ARF1 seems to act primarily at the Golgi, promoting the binding of coatomer proteins to the Golgi membrane [ 36 , 37 ]. Nevertheless, the fact remains that ARF1 is primarily cytosolic, and that only a small fraction of it is bound to the Golgi membrane at any time [ 36 ]. It is not surprising, therefore, that some ARF1 may bind to the plasma membrane after being locally activated by ARNO, which is in turn recruited to the cell surface by the action of insulin. It should be remembered that our cells overexpress ARF1-GFP. Whether ARF1 does in fact work at the plasma membrane under physiological conditions or not remains to be established. Our data simply establish the fact that a receptor-dependent mechanism to recruit ARF1 to the plasma membrane does exist. On the other hand, ARF6 is normally found associated with the plasma membrane [ 36 , 38 ], and there is evidence that ARF6 might be the primary target for ARF-GEFs of the cytohesin/ARNO family [ 27 ]. However, when ARF dominant negative mutants were tested for their ability to inhibit agonist-dependent PLD activation, the data showed that ARF1 dominant negative mutants (T31N-ARF1) were as efficient as ARF6 mutants (T27N-ARF6) [ 23 ]. These observations strongly support the idea that ARF-GEFs of the cytohesin/ARNO family have full access to the cytosolic ARF proteins. Therefore, although ARF6 might be the primary intermediate for ARNO-regulated PLD activation, other ARF proteins may as well play an important role in the pathway. The ability of insulin to promote the translocation of ARNO and ARF to the plasma membrane correlated well with the ability of insulin to promote the activation of PLD. Therefore, our data support the hypothesis that the activation of PLD by insulin is mediated by ARF-GEFs of the cytohesin/ARNO family by a mechanism that involves the interaction of the PH and CC domains of these GEFs with some specific cellular targets. This conclusion is based on the demonstration that ARNO constructs with catalytically inactive domain or the mutants with defective PH and CC domains acted as dominant inhibitors of insulin-dependent PLD activation. The dominant negative effects of E156K-ARNO were not unexpected, since this mutant contains the intact PH and the CC domains and is therefore likely to compete with endogenous ARNO. The dominant negative effect of the PH and the CC domain deletion mutants on PLD activation was of particular interest. These mutants were at best partially translocated to the membrane but blocked the ability of insulin to promote ARF and PLD activation. This result was somewhat surprising since these deletion mutants contain an intact Sec7 domain and, therefore, would have been expected to support ARF and PLD activity. However, this was not the case, suggesting that all regions of ARNO play an important role in the regulation of this protein. Moreover, the failure of the ΔCC mutant to activate ARF and PLD indicates that other cellular targets that bind to the CC doma in of ARNO and regulate the subcellular location or the function of the signaling protein complex may exist. In fact, some proteins that interact strongly with the CC domain of members of the ARNO family, such as CASP and GRASP, have already been identified [ 39 , 40 ]. Consistent with these ideas was the observation that the overexpression of either the PH or the CC domain alone was sufficient to block insulin-dependent PLD activation. Therefore, we propose that cellular targets that recognize both the PH and CC domains of ARNO are important for the regulation of the function of this protein by cell surface receptors. On the other hand, our data also strongly support the hypothesis that the regulation of ARNO activity by insulin involves, at least transiently, a direct interaction of the insulin receptor with ARNO. Consistently, the presence of an ARNO-like activity and ARNO in the immunoprecipitated materials was confirmed by biochemical experiments. Finally, ARNO constructs lacking either the CC or the PH domain, or with a defective PH domain, failed to co-immunoprecipitate with the insulin receptor. These findings suggest a mechanism of the activation in which the binding of ARNO to the membrane is regulated by the insulin receptor at two different levels: 1) ARNO must interact with the receptor; and 2) ARNO must interact with the membrane, either via binding to polyphosphoinositides or through the interaction with specific protein targets. Our data strongly support the idea that both CC and PH domains play a crucial role in this phenomenon. Conclusions This study suggests a general model for the activation of PLD with insulin stimulation. Insulin, upon binding to its receptor, promotes the phosphorylation of IRS-1 and the activation of PI3 kinase. This results in the accumulation of polyphosphoinositides on the plasma membrane. In parallel, the insulin-bound receptor promotes the recruitment of ARNO (and/or other members of the ARNO family, such as GRP-1) to the plasma membrane, either by direct interaction with their CC and PH domains or by promoting the interaction of ARNO with other as yet unidentified targets. The binding of ARF-GEFs to the plasma membrane is stabilized by the interactions of their PH domain with polyphosphoinositides generated by the action of PI3 kinase. Once on the membrane, the ARF-GEFs catalyze the activation of membrane-bound ARF6 or cytosolic ARF proteins that are then recruited to the membrane where they may activate PLD. Cell culture Rat-1 fibroblasts overexpressing the human insulin receptors (HIRcB cells) were cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12, supplemented with 10% fetal bovine serum, antibiotics, and 100 nM methotrexate, as previously described [ 20 ]. Cells were subcultured, transfected as indicated in the figure legends, and serum starved for overnight (approximately 20 hrs) prior to insulin stimulation. HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics. HeLa-ARF1-GFP stable transfectants were obtained by using G418 as a selection agent as described elsewhere [ 25 ]. Clonal populations were obtained and used in the assays described here. Transient Transfection Subconfluent (70–90%) HIRcB cells were transfected with LipofectAMINE (Invitrogen) for biochemical analyses or Superfect (QIAGEN) for imaging analyses. Transfection was performed according to the manufacturer's instructions. Transfection efficiencies were 70–90% for LipofectAMINE and 40–50% for Superfect transfection reagent as previously described [ 41 ]. Generation of fusion proteins It has been reported that the members of the cytohesin/ARNO family of ARF-GEFs each exist in two isoforms in terms of existence of extra G (glycine) in PH domain [ 42 ]. In this study, we used the isoform of ARNO with GGG (tri-glycine), which has similar binding affinities for both PI-(3,4,5)-P 3 and PI-(4,5)-P 3 . The following myc-tagged ARNO constructs were generated: wt-ARNO, ΔPH-ARNO, PH-ARNO, ΔCC-ARNO, E156K-ARNO, and R250D-ARNO. wt-ARNO, ΔPH-ARNO (amino acids 1 to 269), PH-ARNO (amino acids 262–399), and ΔCC-ARNO (amino acids 51–399) (Fig. 1 ) were amplified by PCR and subcloned in the multiple cloning site of the vector pEGFP-C1 (CLONTECH) and fused to green fluorescent protein (GFP) as described by Venkateswarlu and coworkers [ 7 ]. The CC domain of ARNO (amino acids 1 to 55) (Fig. 1 ) was PCR out of wt-ARNO and subcloned into pEGFP-N1 using BglII and EcoRI restriction sites. E156K-ARNO (inactive Sec7 domain) was generated by site-directed mutagenesis as described by Frank and coworkers [ 43 ]. R280D-ARNO was designed on the basis of that a mutation on an analogous arginine impairs the binding of cytohesin-1 to polyphosphoinositides [ 26 ]. The sequences of the constructs were verified by direct sequencing and the expression of appropriate fusion proteins was examined by Western blotting. The level of expression of all constructs was found to be comparable. Immunoprecipitation assay Transfected and serum-starved HIRcB cells were washed with ice-cold PBS, scraped, and collected by centrifugation. The cell pellets were solubilized on ice for 1 hr in a solution of 50 mM Hepes, pH 7.45, containing 100 mM NaCl, 1.5% sodium cholate, 1 mM EDTA, 1 mM EGTA, 5 ug/ml leupeptin, 1 mM PMSF, and 1 mg/ml soybean trypsin inhibitor. Insoluble materials were removed by centrifugation. The cell lysate was immunoprecipitated with anti-mouse IgG agarose that had been equilibrated with a monoclonal antibody 83.7 (which recognizes the α subunit of the human insulin receptor). Immunoprecipitation was carried out overnight (approximately 20 hrs) at 4°C. The immunoprecipitates were washed with lysis buffer, resuspended in SDS-PAGE sample buffer, and subjected to Western blotting analysis. Immunoblotting Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blocked with 5% non-fat milk in PBS containing 0.1% Tween at room temperature for 2 hrs. The membrane was then cut in half horizontally. The upper part was used to detect the β subunit of the insulin receptor with a monoclonal antibody, CT-1, that recognizes the carboxyl terminus of the β subunit of the human insulin receptor. The lower part was used to detect ARNO proteins with a monoclonal antibody anti-myc or a polyclonal antibody anti-GFP. PLD activity assay Serum-starved HIRcB cells were labeled overnight with 3 H-palmitate (5 μCi/ml) in serum-free medium. The cells were stimulated with insulin (100 nM) in the presence of 0.5–1% ethanol for 20 min. The reaction was stopped by addition of chloroform: methanol (1:1). The lipid phase was extracted and developed by thin layer chromatography (TLC) on silica gel 60 plates using ethyl acetate: trimethylpentane: acetic acid (9: 5: 2) as a solvent. The position of major phospholipids was determined using true standards (Avanti Biochemicals) and autoradiography. The TLC plates were scraped and the total amount of radioactivity associated with each lipid species was determined by liquid scintillation counting. The data were expressed as the number of counts associated with the phosphatidylethanol (PtdEtOH) spot normalized by the total number of counts of lipid. Digitonin treatment Serum-starved HIRcB cells were collected, resuspended in PBS, and treated with 10 μM digitonin in the presence or absence of insulin (100 nM), ATP (1 mM), and GTPγS (100 μM) at 37°C for 15 min. To release intracellular proteins, the digitonin-treated cells were centrifuged in a microcentrifuge for 20 min. The supernatants and the cell pellets were collected separately, and subjected to SDS-PAGE. ARNO proteins were detected by immunoblotting as described above. In vitro ARF activation assay ARF activation was determined by the binding of GTPγS to the purified, myristoylated recombinant human ARF1 (mhARF1), as described by Shome and coworkers [ 23 ]. The insulin receptor was immunoprecipitated in the presence or absence of 100 nM insulin as described above. Four to 8 μg mhARF1 and the immunoprecipitated insulin receptors were incubated with 100 nM GTPγ[ 35 S] (1 μCi) in 20 mM Hepes buffer containing 2 mM MgCl 2 / 0.1% Na-cholate / 1 mM ATP. At the indicated time points, the reaction was quenched by addition of 100 μM ice-cold, unlabeled GTPγS and the protein-bound nucleotide was determined by filtration through nitrocellulose filters as described [ 23 ]. Confocal microscopy HIRcB cells were plated on poly-L-lysine coated glass coverslips and transfected with the constructs as indicated above. Cells were serum starved overnight and stimulated with 100 nM insulin. Live cells were imaged in a LSM5 Zeiss laser scanning confocal microscope equipped with a 63X oil immersion objective. For ARF and ARNO colocalization experiments, HIRcB cells were plated on poly-L-lysine coated coverslips as described above and co-transfected with myc-ARNO and ARF-GFP constructs using Superfect transfection reagent according to the manufacturer's instructions. Following insulin stimulation, the cells were fixed with 4% fresh paraformaldehyde in PBS at 4°C for 30 min, and permeabilized in 0.1% Triton X-100 at room temperature for 2 min. After permeabilization, the cells were blocked with 3% bovine serum albumin in PBS at room temperature for 30 min, and immunostained with a monoclonal antibody 9E10 (Upstate Biotechnology) that recognizes the myc epitope. After extensively washing, the cells were incubated with a Cy5-conjugated donkey anti-mouse secondary antibody (Jackson Immunoresearch) and imaged using a Zeiss laser scanning confocal microscope with filters appropriate for the detection of GFP and Cy5.

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Health policy and systems research agendas in developing countries

Background Health policy and systems research (HPSR) is an international public good with potential to orient investments and performance at national level. Identifying research trends and priorities at international level is therefore important. This paper offers a conceptual framework and defines the HPSR portfolio as a set of research projects under implementation. The research portfolio is influenced by factors external to the research system as well as internal to it. These last include the capacity of research institutions, the momentum of research programs, funding opportunities and the influence of stakeholder priorities and public opinion. These dimensions can vary in their degree of coordination, leading to a complementary or a fragmented research portfolio. Objective The main objective is to identify the themes currently being pursued in the research portfolio and agendas within developing countries and to quantify their frequency in an effort to identify current research topics and their underlying influences. Methods HPSR topics being pursued by developing country producer institutions and their perceived priorities were identified through a survey between 2000 and 2002. The response to a call for letters of intent issued by the Alliance in 2000 for a broad range of topics was also analyzed. The institutions that were the universe of this study consisted of the 176 institutional partners of the Alliance for Health Policy and Systems Research producing research in low and middle income countries outside Europe. HPSR topics as well as the beneficiaries or issues and the health problems addressed were content analyzed. Topics were classified into 19 categories and their frequency analyzed across groups of countries with similar per capita income. Agendas were identified by analyzing the source of funding and of project initiation for projects under implementation. Results The highest ranking topic at the aggregate level is "Sector analysis", followed by "Disease burden" and "Management and organization". Categories at the bottom of this ranking are "Equity", "Policy process", "Economic policy and health" and "Information systems". "Disease burden" is more often funded than other topics for which there is more demand or perceived priority. Analysis suggests few although important differences across priorities, demand for funding and actual project funding. The donors' agenda coincides most with the ranking of research topics overall. Ranking across country income groups shows important differences. Topics that gain prominence in low income countries are "Disease burden" and "Accessibility". In lower middle income countries "Insurance" gains prominence. In upper middle income countries "Decentralization/local health systems", "Equity" and "Policy process" are more prominent. "Program evaluation" is the most consistently ranked topic across income regions, showing a neutral influence by donors, governments or researchers. Conclusions The framework proposed offers a basis to identify and contrast research needs, projects and products at the international level and to identify the actor agendas and their influence. Research gaps are suggested when comparing topic ranking against the challenges to health system strengthening and scaling up of disease control programs. Differences across per capita income groups suggests the need for differentiated priority setting mechanisms guiding international support. Data suggests that stakeholders have different agendas, and that donors predominate in determining the research portfolio. High-level consensus building at the national and international levels is necessary to ensure that the diverse agendas play a complementary role in support of health system objectives. The Ministerial Summit for Health Research to be held in Mexico in November 2004 should be an opportunity to analyze further data and to commit funding for priorities identified through sharing and discussion of agendas.

Background Countries and international agencies have made a qualitative leap in the funding of the global disease challenges. The Global Fund for AIDS, TB and Malaria has received pledges totalling over US$ 2 billion. Bilateral donors are also making important funding contributions. In this context, strengthening of health systems has become a critical issue. Research can play a major role to identify the best policies to channel massive efforts, to ensure that vertical approaches do not fragment fragile health systems and to monitor and evaluate progress. How relevant is the research effort being undertaken in developing countries, and how is the agenda being driven? WHO is organizing the Ministerial Summit on Health Research, to be held in Mexico City, 23 to 26 November, 2004. The main theme will be the role of health research in meeting the Millennium Development Goals. Health policy and systems research (HPSR) will have a prominent role in the context of the scaling up of efforts against major diseases and child and maternal mortality. Looking towards the Summit, WHO established a task force to identify HPSR priorities as an effort to advocate for major funding in this area. The Alliance for Health Policy and Systems Research, an initiative of the Global Forum for Health Research in collaboration with WHO, has been promoting since its inception in 2000 the identification of research priorities among producer institutions in developing countries. This paper proposes a conceptual framework and a methodology to think about the HPSR research portfolio, the agendas that influence it and the priority setting process. The paper makes the case for the formulation of HPSR priorities at national, international and global levels. A previous article [ 1 ] looked at research capacity among Alliance partner institutions in the South and identified the need to increase funding to establish long term research projects as a basis for sustainable capacity building. Using data from the same survey, indicators are proposed to assess the priority setting process on the basis of various types of data. The scope and value of the conceptual framework and its indicators are illustrated by presenting the HPSR topic ranking on the basis of Alliance partner contributions and the influence upon it of country income and actor agendas. A forthcoming paper will present findings from a new survey now being completed and covering the universe of research producers in the South. Conceptual framework Priority setting efforts are often bogged down because of inadequate methods of categorization of the research that is undertaken and the influences shaping it. These influences, in turn, are often not adequately understood, including the nature and role of priorities. The conceptual framework proposed here strives to offer some simple definitions of the research portfolio and its various influences, as well as indicators to measure and relate these concepts. The nature of overall health research has been defined in terms of basic, applied and strategic research. These categories are useful to guide investment decisions which might maximize benefit [ 2 , 3 ]. However, within the field of health policy and systems research, little effort has been given to the classification of what is studied. Before priorities can be identified, it is important to be able to agree on what is studied through an analysis of the dimensions that characterize the object of research. Furthermore, it is important to establish the distinction between the object of the research and the factors shaping this choice at various levels. The HPSR portfolio To identify the object of research the concept of the research portfolio can be useful. The HPSR portfolio can be defined as the current set of research projects on health system structures, functions, processes and results at national, international and global levels. Projects, as distinct from plans or priorities, would include the commitment of resources towards a specific, time-bound aim and a set of objectives. It has been proposed that the research portfolio should be analyzed along three dimensions of strategic importance: where to make investments, that is, the identification of the object of research or thematic areas in which investments are made; the type of investment research and development (R & D) instrument used and the resources spent through each area and type of instrument [ 3 ]. While our contribution aims to develop the first dimension, it is useful to expand on the other two to understand their interrelations. The Ad Hoc Committee on Health Research proposed three types of R & D instruments: discovery oriented research to develop new health products and interventions; innovation research to adapt efficacious but unaffordable interventions to make them cost-effective, and implementation R & D to achieve greater efficiency in the use of existing interventions [ 2 ]. Harrison has argued for the need to consider a fourth instrument of equity R & D to ensure that the research portfolio responds to the poor and the underlying health problems in developing countries. With regard to funding, there is a need not only to consider investments, but also funding sources and mechanisms. Four broad types of sources can be recognized: bilateral and multilateral donors, government commissioning, private commissioning, and funding through resources available to research institutions as part of their budgets. Each will have different implications for the kind of knowledge produced and for its possible influence on the health system [ 1 ]. This subject has been explored for Alliance partners in developing countries, identifying the amounts and sources of funding for their research portfolio [ 4 ]. The object of research Previous analyses have revealed a complex heterogeneity along which researchers classify HPSR in developing countries, which is not surprising in an interdisciplinary field [ 5 ]. However, five overarching dimensions can be recognized: • concepts reflecting the health system, such as policy and financial structures, regulatory functions, processes such as technology evaluation and quality monitoring, and results such as satisfaction and health gain • the levels of the health system, such as the households and the community, first level facilities and hospitals • the issues or problems pertaining to the health system such as priorities, equity and the public private mix • the populations addressed by the system, such as children, mothers and the elderly, or rural and urban populations • the health needs addressed, whether in terms of risks or disease. While these dimensions can be useful to characterize the research portfolio, it is clear that there will be overlaps; for example, equity is both an issue and an attribute of the health system, particularly if it has been integrated in monitoring and regulation. In order to make use of these dimensions it is proposed to consider as the project topic the first dimension of concepts pertaining to the health system structures, functions, processes and results. The topic could then be classified following normative or theoretical frameworks or by using the categories researchers apply in their own research. The other four dimensions can be used to qualify the research topic as to provide a more detailed description. These four dimensions could be selectively used or aggregated to facilitate description according to the needs at hand. Analysis of portfolio characteristics Each of the five project dimensions can be analyzed in terms of the range of items considered. The ranking and emphasis of each item can also be revealed by analyzing its frequency. A research portfolio at any level can be very focused and comprise a narrow set of topics. Or it can be wide-ranging across many health system functions, structures, processes and results. It has been argued that research portfolios should be more focused on improving immediate health problems through operations research in low income countries, where funding and human resources are very limited [ 3 ]. However, the challenges of scaling up disease control programs call for research at the health systems level also. The level of the research portfolio The level at which the portfolio is analyzed is important, as it will have different characteristics and uses. At national level, the HPSR portfolio would be the set of projects addressing health and health system problems within the confines of national borders and governmental authority as well as sector-wide and inter-sectoral issues. Examples would be the impact on equity of decentralization policies, or the roles played by conflicting policy actors in scaling up of services. The international research portfolio would be topic areas which are common across a number of countries or regions. Identification of the international portfolio could serve, among other purposes, to fund research at regional or international levels, to strengthen the critical mass of research available to inform country policy making and lesson learning across countries, and to extend the range of methodological approaches through comparative research [ 6 ]. The HPSR portfolio at global level refers to research themes and projects that are, by their very nature, supra-national. This would involve, for example, international financing of immunization efforts, intellectual property rights in health research, and development of international disease control measures. The HPSR agendas The HPSR portfolio at any level is influenced by factors within and outside the research system [ 7 ]. Within the research system the following factors can be identified: research capacity; research trends and preferences expressed by researchers and research institutions; research funding and market opportunities; and research preferences voiced by policy makers, service managers and public opinion. Outside the health system the broad factors shaping the portfolio are the health conditions and health system problems as well as the cultural, economic and political context. As a whole, these factors shape actor-specific research agendas that express ethical, professional and political values and that influence the allocation of scarce resources towards alternative project portfolios. It is clear that if actor agendas have few areas in common there will be no consensus and therefore no overall priorities. Significant overlap of interest, on the other hand, can lead to the identification and formulation of shared research priorities. Research priorities are therefore defined as the explicit areas of agreement on, and ranking of, the object of research across diverse actor agendas. Priorities can then become policy instruments to coordinate diverse agendas towards a common end without forcing a single research agenda. The characteristics of agendas and of priorities can be identified through the same kinds of dimensions and indicators as the research portfolio. That is, preferences can be classified in terms of topics, issues or beneficiaries, and health problems. The characteristics of the HPSR agendas and priorities can also be studied in terms of the range of topics, issues, levels, populations and health problems. They can also be ranked and their emphasis revealed by frequency analysis. In this way, the agendas can be compared across themselves, priorities can be identified as common topics and issues and with similar ranking and emphasis, and the influence of agendas and priorities on the actual research portfolio can be assessed. Priority setting through agenda co-ordination It has been argued that co-ordination of the various influences shaping the HPSR portfolio can increase the impact of research on equity and can contribute to its strategic role for development [ 8 ]. Co-ordination would involve developing a consensus of researcher, policy maker and investor agendas. Such a consensus should ideally result in a highly coherent set of topics across the various actors' research agendas and, eventually, a high degree of correspondence between agendas and the research portfolio. HPSR portfolio change through co-ordination would involve a gradual process of adjustment to new priorities, project completions, maturation of research capacity and funding opportunities. Coordination requires interfaces and mechanisms such as Research Forums and Essential National Health Research Mechanisms [ 9 ] to develop a consensus while allowing, and even encouraging, critical differences. In sum, the HPSR portfolio can encompass a differing range of topics with diverse rankings and emphases; can be more or less coherent with respect to researcher, funding and policy maker agendas; and can be more or less co-ordinated along a set of shared priorities. The interplay of these dimensions can give rise to a number of scenarios, of which three are here illustrated. a) Co-ordination could lead to focusing the HPSR portfolio on a few, highly cost-effective topics in a situation of few resources and well identified, high priority needs. There would be, eventually, a high degree of coherence across the portfolio and agendas held by various actors. The risks here would be lack of diversity to foster innovation and healthy criticism. b) In a situation of plentiful resources the research portfolio and its driving agendas could be wide-ranging and have little overlap. Specific portfolio segments would correspond with particular agendas, thus satisfying multiple interests. However, co-ordination through overarching mechanisms could ensure integration of knowledge around high level priorities. In this manner a unified, although highly diverse, HPSR field of enquiry would be obtained. c) In a situation of lack of coordinating mechanisms, with low resources, the portfolio could focus on a reduced set of topics, each satisfying a particular agenda and thus fragmenting resources and hindering support to health system development. If resources are more plentiful, lack of coordination could lead to a rich but highly dispersed and inefficient research portfolio with little impact on development. Irrespective of the availability of resources, co-ordinating mechanisms are likely to be important to ensure an efficient use of research resources. Methodology and Indicators Two sources of information were used to illustrate the conceptual framework proposed. The first was a survey of Alliance-HPSR partner institutions in developing countries detailing research priorities and project information. Researchers reported here on the priorities they had received from policy makers in the course of diverse consultations in the past year. The second source was a database of letters of intent (LOI) submitted to the Alliance for funding, where projects were justified on the basis of priorities negotiated by researchers and policy makers or service managers. Content analysis This proceeded in several steps. A preliminary list with 24 research topics was identified through an inductive analysis using the research statements expressed in the LOI, which were the most detailed. This list was then used to classify research topics in the projects and priorities expressed in the Alliance partners' profiles. Beneficiaries/issues and diseases/health problems addressed by the LOI, projects and priorities were also categorized and classified at this stage. While projects may have contained more than one topic or beneficiary/issue, the most prominent one was selected. In a few cases where several topics were considered this was indicative of a sector-wide analysis and classified accordingly. The beneficiaries/issues of the research were classified to include any of four alternative dimensions: a) the demographic group: elderly or children/adolescents, b) level of care: community, primary or hospital, c) the geographical focus: urban or rural, and d): gender, equity/poverty, indigenous populations, and public-private mix. Whenever more than one dimension or aspect was applicable (which occurred only in a small number of statements), a decision was made to include the most prominent. The concept "equity" was classified both as a topic and as an issue. It was assigned as a topic whenever equity was the main objective of the research and it was addressed through a number of health system attributes such as financing, access, and service delivery. Equity/poverty was considered as an issue when the poor were identified as the main subjects of research or when the equity implications of research directed mainly to another topic were highlighted as a major concern. The distribution of statements was analyzed according to country income group: low income, lower middle income and upper middle income. These income groups correlate highly with geographical regions, with LI being mostly in Africa and Asia, LMI mostly in Asia and with a particular weight by China, and UMI in Latin America and the Caribbean (table 1 ). Table 1 Glossary of Health Policy and Systems Terms Used for Content Analysis TOPIC TERMS FOUND IN RESPONSES Accessibility Health seeking behaviour, determinants of utilization, coverage, outreach, referral, barriers to care, willingness and capacity to pay, cost-sharing, price regulation, prices, equity in access, demand for health services. Community participation Community-based strategies, community participation in governance, empowerment, school health, family health strategies, social support networks. Costing & cost effectiveness Determination & evaluation of costs, cost-benefit of services, economic evaluation, cost-effectiveness of resource allocation, alternative uses for resources. Decentralisation/local health systems Decentralization policy and process, impact of decentralization on services and health outcomes, district health system development, healthy cities, municipal health services, local government, devolution, community participation in local health services. Disease burden Prevalence and incidence of diseases, mortality and morbidity, disease profiles, health status, health needs, burden of disease studies, risk factors, determinants of health and disease other than economic or social policy. Economic policy and health Free trade agreements and health, TRIPPS and health, economic crises and health, impact of poverty reduction and adjustment policies on health, debt reduction and health, social policy and health, social assistance and health issues, intersectoral co-ordination, labour policies and health. Equity Equity of health system, impact of health reforms on equity, equity and poverty, poverty targeting of services, poverty and health, exclusion. Financing Financial mobilization, financial allocation, financing policies, national & district health accounts, financial equity, community health financing, financing of specific programmes. Human resources Personnel management, deployment, migration, motivation, knowledge, attitudes and practices of health personnel, satisfaction, quality of life, human resource policy, human resource performance, traditional healers, training and education of human resources, medical education curriculum assessment, evaluation of medical and nursing teaching programmes. Information, education and communication (IEC) Information and communication for the general public, health education strategies and impacts, knowledge attitudes and practices (KAP). Information systems Information needs, informatics, surveillance mechanisms and systems, strengthening of information systems, health monitoring systems, establishment of public domain databases, development of indicators for service management and policy. Insurance Risks and benefits covered by insurance schemes, community based health insurance, options for health insurance, insurance reform, impact of insurance on health and service outcomes. Management & organization Health service provider performance, delivery of services, administration, service management strengthening, contracting and provider payment mechanisms, impact of privatization on services, performance agreements, impact of hospital autonomy on service delivery, stakeholders in service management, community participation in management. Pharmaceutical policy & management Rational drug use, procurement, logistics, herbal medicine, dispensing practices, pharmaceutical regulation, national drug policy, essential lists. Policy process Stakeholder analysis, role and relationships of actors in the formulation and implementation of policy, role of government agencies in policy formulation, role of community and NGOs in policy formulation, factors influencing policy process, perceptions of policy, decision-making processes, policy negotiation. Programme evaluation Evaluation and assessment of impact of policies or programmes on specific diseases or services. Quality Clinical practice guidelines, evidence-based medicine, quality assurance, patient satisfaction. Research to evidence Health systems research training, health systems research training, outcomes of research, research impact, policy utilization and impact of research, research methods, creation of national HPSR database, priority setting of health research, research ethics, essential national health research, dissemination of research. Sector Analysis Health sector reforms and implications, health systems development, private health service development, intersectoral collaboration and co-ordination, public/private mix health care, health care organization, regulation, policy formulation on specific diseases, on programmes or on aspects of the health system, sector-wide and system-wide performance. Two researchers classified all statements independently and disagreements were discussed and resolved. The 24 topic categories were reduced to 19 to avoid groups with less than 2% of the total number of statements while maintaining topic coherence. Table 1 presents the glossary of terms included under each topic. The frequency of responses by country for all types of statements is generally proportional to country population, with China, India, Brazil and Bangladesh at the top of the frequency. However, countries with a strong health systems research presence are over-represented, such as Colombia, Argentina, Philippines, Thailand, South Africa, Uganda, Ghana, Cuba, Costa Rica, Benin, Jamaica and Tanzania. Identification of agendas The range and emphases of the HPSR portfolio and agendas were mapped through topic content and frequency analysis (Figure 1 ). Project and agenda data were also aggregated from the two sources to obtain a general mapping of topics. This was used to assess coherence across actor agendas and with the portfolio and to increase the number of observations to enable analysis by income level. Figure 1 Concepts and indicators Two methods were used to assess the coherence between the HPSR portfolio and the agendas held by researchers, policy makers and international donors and partners. The first method used the survey data to infer the agendas by observing the topic frequency of: projects proposed and funded by researchers without external assistance; projects initiated and funded exclusively by government, and projects initiated and funded exclusively by international stakeholders or research partners. The second method compared the research portfolio against the agenda expressed by policy makers. The policy maker agenda was observed through the survey as reported by researchers and through the LOI as negotiated with researchers. A negligible influence by the donor, in this case the Alliance-HPSR, would be expected in the LOI given that the call requested priorities within the generic definition of HPSR presented above. Each of the three modes of identification of agendas could have method-specific biases. In the first method, preferences are derived from the portfolio itself, that is, from research projects in implementation. Furthermore, the method isolates the preferences expressed by each actor. As such, this method could be deemed to reveal most objectively preferences behind each actor funding or initiating a project. However, projects under implementation may hide topic preferences that are not translated into projects or topics that were generated and funded through joint actor participation. The observation of negotiated priorities expressed in LOI captures the mix or balance of researcher-side influences and policy maker needs. It will therefore reflect a consensus position across each actor. However, it will exclude the influence of funding opportunities, will not reveal actor-specific preferences and will be limited by the constraints placed on the LOI (see below). Priorities based on consultations between researchers and policy makers and expressed by researchers through a survey will reveal the understanding and conceptual framework of researchers and may underplay policy-maker needs. Furthermore, these priorities will be influenced by the research projects under execution and reported in the same instrument. Assessment of coordination between portfolio and agendas The analysis of relationships or influences across the portfolio and each of the actor agendas, as well as of similarities or differences between agendas, was undertaken by correlating topic frequencies across lists and by undertaking a qualitative analysis of changes in rank order and emphasis. Analysis of the range and rank of topics across groups of countries by income was undertaken by aggregating project and priority topic data into a reference list representing the combined set of influences on the agenda-setting process, including the portfolio itself. The aggregation of data into a reference list was mainly a strategy to increase the observations and make the analysis more reliable, although it may have validity if it describes the overview of the agenda-setting factors at play. That is, the actual portfolio can be conceived as a force shaping the agendas, together with other factors. Survey and LOI database The survey of HPSR producer institutions in developing countries was described in detail elsewhere [ 1 ] and includes information for 108 of the 176 Alliance-HPSR partners (61% response rate) who produced research in low and middle income countries outside Europe between 2000 and 2001. The database contains information on the current research portfolio (294 projects were declared) as well as research priorities (402 priorities were stated, with a maximum of 5 per survey). Information on project initiation and source of funding is available for 270 projects. A total of 39 developing countries out of a total of 133 were contacted. Respondents are close to one sixth of the close to 650 institutions known to the Alliance to be producing HPSR in developing countries. Biases in the partner database could have occurred as a result of preferences by certain type of institutions in joining the Alliance HPSR and in answering the questionnaire required from partners. Over-representation at both levels could have occurred of more competitive and productive institutions with larger project portfolios and funding, and more interest in international funding. On the other hand, larger institutions may have been discouraged from responding given the larger number of projects to be reported, although they would also have more capacity to respond. Furthermore, the response rate could have been lower among institutions where producing HPSR is not a main function. The LOI database has 403 submissions for research funding in response to a call by the Alliance-HPSR in 2000. Applicants requested funding for one year projects in high priority areas identified jointly by them and national policy makers and stakeholders. A limitation of this database is the exclusion of funding requests for projects over one year as well as topics that would be formulated solely by researchers. Analysis of the partners' database indicates that 24% of projects are of longer duration and that up to 34% of projects undertaken are initiated by the research institution without stakeholder collaboration. Expansion and standardization The frequencies of statements for each income region were expanded proportional to population to make comparisons across regions possible. The frequency of statements across the three types of statements (projects, LOI and priorities) was standardized to give each equal weight when aggregating them to analyze the combined representation of the research portfolio and the agenda-setting process as a whole. Responses show a distribution across income regions proportional to population in some cases and with significant biases in others (table 2 ). The low income region (LI) has 50% of the population and 47% of statements, while the upper middle income (UMI) region has only 12% of the population but twice the number of statements, with 22%. The lower middle income (LMI) region is also somewhat under-represented, with 38% of the population and 31% of statements. Table 2 Distribution of Statements According to Type, Content Category and Geographical Region by Income Group LI LMI UMI TOTAL % Types of statements Total % Total % Total % Priorities 198 49 143 36 61 15 402 100 Letters of intent 193 48 101 25 109 27 403 100 Projects 124 42 97 33 73 25 294 100 TOTAL 515 47 341 31 243 22 1099 100 Content categories Topics 482 46 330 31 237 23 1049 100 Beneficiaries or Issues 217 53 113 28 80 20 410 100 Health Problems 132 58 48 21 48 21 228 100 Total statements by geographical region Africa 247 77 15 5 57 18 319 100 Asia 260 53 214 44 13 3 487 100 Latin America and the Caribbean 8 3 112 38 173 59 293 100 TOTAL 515 47 341 31 243 22 1099 100 Total population in Geographical region 50 38 12 100 The frequency of statements on priorities is as would be expected for the population in each region. However, project statements and demand for funding are biased in favour of UMI, with 25% and 27% of the statements, respectively, against 12% of population share. Ranking This was done for each topic or category within the topic by rounding percentage differences to integers and grouping in the same rank all categories falling within the same percentage. Results This article does not attempt to provide an exhaustive analysis given the fact that data is limited to Alliance partner producer institutions in the South. The purpose here is to illustrate the potential of the proposed methodology and to present the most robust findings. HPSR topics are first presented and analyzed aggregating in a single list the topics in the research portfolio as well as in the policy maker and researcher agendas. This aggregate representation is then analyzed by groups of countries according to their per capita income. The influence exerted on the HPSR portfolio by various actors is then analyzed. Characteristics of HPSR producers in developing countries HPSR producer institutions are generally small with an average of 3 projects, 8 researchers and a project portfolio worth $155,226 [ 1 ]. Only 19% of researchers have a PhD qualification, although researchers in key disciplines are well represented and better qualified. Research capacity and funding are similar across income regions, although inequalities are apparent. Overview of topics A total of 19 research topics were identified when aggregating portfolio (project) and priority (voiced preferences) data into the reference list. Topics ranged in frequency from 2% to 11% and were ranked in 8 classes (Table 3 ). The highest ranking topic is "Sector analysis" with 11% followed by "Disease burden" with 9% and "Management and organization" with 8%. From here three topics rank lower equally at 7%, two rank at 6%, seven rank at 4% and then two each at 3% and 2%. Categories at the bottom of this ranking are "Equity", "Policy process", "Economic policy and health" and "Information systems". The emphasis of topics at the top end is then about five times as greater as those at the bottom end of the range. Table 3 Ranking of Topic at the Aggregate Level Rank Topic % 1 Sector Analysis 11 2 Disease burden 9 3 Management & organization 8 4 Accessibility 7 Programme evaluation 7 Research to evidence 7 5 Financing 6 Human resources 6 6 Community participation 4 Costing & cost effectiveness 4 Decentralisation/local health systems 4 Information, Education and Communication 4 Insurance 4 Pharmaceutical policy & management 4 Quality 4 7 Equity 3 Policy process 3 8 Economic policy and health 2 Information systems 2 Rank Beneficiaries/Issue % 1 Community 15 2 Equity/poverty 14 3 Hospital 12 4 1st level 11 Gender/women 11 Rural areas 11 5 Children/adolescents 10 6 Public private mix 6 7 Urban areas 5 8 Elderly 4 9 Indigenous peoples/traditional medicine 3 Rank Health Problem % 1 Reproductive health 30 2 HIV-AIDS 11 Nutrition 11 TB 11 3 Chronic 7 Environmental health 7 Malaria 7 Mental health 7 4 Other infectious 6 5 Other 3 The fact that "Equity" appears so low in the aggregated ranking could be partly attributable to the fact that this topic was defined to include only projects and priorities having equity as the central topic and measuring it through multi-dimensional approaches such as health conditions, access to services and financing. A subsidiary analysis was thus undertaken to include under "Equity" those projects or priorities addressing equity or poverty as a secondary, qualifying, role of research on other topics. This broadened topic "Equity" climbs to fourth rank, at the same level as "Accessibility", "Program evaluation" and "Research to policy". Public and private institutions show no significant changes in topic ranking (corr = 0.70). "Community participation" and "Accessibility" are the only topics with major differences, ranking higher among private institutions. Topic analysis by beneficiary/issue Out of the total topics classified in the reference list, only 38% (404) were sufficiently focused or detailed to be able to attribute a beneficiary or specific issue (Table 4 ). This was mainly the case with priority statements, which by their very nature were generic. The beneficiary or issue statements were spread across the 11 categories identified through content analysis. The category with least statements had only 2% of the total, and that with most 13%. Table 4 Beneficiaries/Issues According to Topic Beneficiaries or Issues Topic Elderly Children Community Primary Hospital Urban Rural Equity Gender Indigenous peoples/traditional medicine Public private mix TOTAL A B A B A B A B A B A B A B A B A B A B A B n = B Accessibility 2 3 10 7 7 7 10 8 7 12 19 20 17 14 19 17 2 11 7 7 42 10 Community participation 9 20 4 3 43 19 13 7 13 12 4 3 13 7 23 6 costing & cost effectiveness 13 3 13 2 38 6 13 4 13 3 13 2 8 2 Decentralisation/local health systems 13 5 27 7 7 2 13 8 27 10 7 2 7 2 15 4 Disease burden 15 50 21 19 3 2 3 2 6 4 12 15 9 8 6 4 24 17 3 11 34 8 Economic policy and health 14 10 43 8 14 2 29 4 7 2 Equity 6 10 6 2 78 28 11 22 18 4 Financing 39 17 4 4 9 5 39 18 4 2 4 2 23 6 Human resources 29 9 35 15 6 2 6 4 18 8 6 11 17 4 Inform. Educ. & Communication 21 8 7 2 7 2 14 8 14 5 36 11 14 3 Information systems 25 4 38 7 13 2 13 3 13 2 8 2 Insurance 44 13 13 8 19 8 13 4 6 11 6 2 16 4 Management & Organization 2 3 9 7 9 10 49 48 2 4 11 13 2 2 4 4 13 14 47 12 Pharmaceutical policy & Mgmnt. 17 5 33 8 8 3 25 6 8 2 8 11 12 3 Policy process 50 6 17 4 33 4 6 1 Programme evaluation 2 10 22 24 2 2 17 17 5 4 12 19 2 3 2 2 2 2 32 30 41 10 Quality 7 2 20 7 47 15 7 3 13 4 7 2 15 4 Research to evidence 33 2 33 2 33 2 3 1 Sector Analysis 11 11 11 10 8 8 11 8 8 7 3 11 47 40 36 9 None 26 14 5 2 11 5 5 3 47 20 5 11 0 19 5 n 10 37 54 41 48 26 40 50 46 9 43 404 100 % 2 100 9 100 13 100 10 100 12 100 6 100 10 100 12 100 11 100 2 100 11 100 A = % across the row ; B = % down the column The topics with least identification of beneficiary or issue were "Costing and cost effectiveness", "Policy process" and "Research to Evidence", with 79% to 96% in this situation. By contrast, "Community participation" and "Management & Organization" were the topics least frequently unidentified with beneficiary/issue, at between 30% and 41%. The most frequent beneficiary/issues were Community, Equity/poverty Hospital, Gender Primary care and Rural areas. The three beneficiary/issues least identified were Urban areas, elderly and Indigenous peoples/traditional medicine. The analysis of the correlation between beneficiary/issue and topic is tentative at this stage given the low frequency in many of the cells of the 19 by 11 matrix. Two beneficiaries/issues account for a large part of the focus of research topics: Community and Hospital as a focus on levels of care. At the community level the topics of community participation, financing, health insurance, decentralization, policy process, information systems and human resources are all prominent. At the hospital level the topics of costing and cost effectiveness, pharmaceutical policy, quality of care and management and organization are most prominent. By contrast, the topic of program evaluation is fairly widely spread across several issues or beneficiaries. The following topics show also a fairly discreet relationship to beneficiaries/issues: Research on accessibility is mostly focused on rural areas. Research on disease burden is prominent among the elderly and children. Economic policy and health focuses on children. Gender is also an important component of these three topics. Equity is focused on indigenous populations. The topic of information, education and communication is prominent among children. Sector analysis focuses mainly on the public private mix. Topic analysis by income level The differences in ranking of the topics in the reference list across income regions are shown in the first three columns of Table 5 . Larger differences occur in 9 topics, mostly in lower middle income and upper middle income countries. This suggests that the reference list reflects more closely lower income country needs. Largest differences were observed in health insurance, decentralization/local health systems and, equity and policy process, topics that are more highly ranked in upper middle income countries. Table 5 Ranking of Topics in the Reference List and Differences by Income Region and by Project Initiation RANK TOPIC INCOME REGION PROJECT INITIATION Low Lower Middle Upper Middle Donors Govnt. Research Institution 1 Sector Analysis -- - 2 Disease burden -- 3 Management & organization + -- 4 Accessibility + + -- -- Programme evaluation -- Research to evidence - - + 5 Financing - ++ ++ -- Human resources + 6 Community participation - + + ++ Costing & cost effectiveness -- + + +++ Decentralisation/local health systems - +++ + Information, Education and Communication + Insurance -- +++ - + Pharmaceutical policy & management Quality + 7 Equity - +++ + +++ Policy process - +++ 8 Economic policy and health - + + + Information systems + + Cells with a rank difference of 1, 0 or -1 are blank. Changes of 4 or more = +++; 3 = ++; 2 = +; -2 = -; -3 = --; -4 or more = --- Comparing the research portfolio and the agendas The overall ranking of topics in the reference list was compared against the ranking of topics in projects initiated by each actor. The relationship between actor preferences and the reference list was assessed through an analysis of their rank congruence. As described in a previous paper in more detail [ 1 ], the research institution is the initiator in 34% of projects, while 31% are initiated by a donor agency, international research partner or by a private contractor. Governments initiate in 24% of cases. 12% of projects are reported as a mix of the above and are not considered for this analysis. The agendas across actors differ substantially, and none can be said to be close to the other. As a result, the reference list shows marked difference with respect to each actor's agenda. Government initiation shows preference for financing and cost-effectiveness as compared to the overall ranking. Government initiation tends to give lower regard to disease burden studies and for research on accessibility. International donor initiation matches best the reference list but gives somewhat less preference to sector-wide analyses. Research institution initiation is more marked for equity and community participation and less so for management and organization, accessibility and program evaluation. International donor initiation preferences are associated to the top ranking topics in the overall listing, suggesting a predominance of their agenda on the reference list. Conclusions The analysis of the research portfolio and priorities at the international level shows a widely diversified set of topics, ranging from sector wide issues to more focused program evaluation. The emphasis on sector wide issues reflects the challenges to health systems today and suggests that countries consider as important the macro-level analysis as the micro. Micro approaches with a focused attention to beneficiaries or specific issues are well identified, particularly under the topic of program evaluation. However, the evidence also suggests a gap between the research that is actually being undertaken and the challenges for strengthening and scaling up of disease control programs. Such a gap is evident in the low emphasis given to research on human resources, policy process, equity, economic policy and health and information systems. By contrast, the analysis suggests a high degree of attention at the community level, although much attention is also given at the hospital level. Primary care thus seems to be under-emphasized. Considering the disease focus, whenever this was made evident, the data do not suggest a bias towards problems that would not be evidently important at country level. The fact that the public or private character of research institutions is insignificant for the agenda suggests the capacity of diverse institutions to work within a common agenda. There are significant differences in the research portfolio across groups of countries based on per capita income, suggesting the need for priority setting mechanisms at both national and international levels that reflect such diversity. The greater congruence between donor preferences and the international research agenda highlight the importance of consensus building between national and international actors. While it is appropriate for governments and international donors to fund different aspects of the research portfolio, this requires high-level priority setting and consensus mechanisms to ensure they complement each other rather than lead to fragmentation. More research is required to establish the relationships between actors' agendas and the research portfolio at the international level. There is also a need to discuss the most desirable balance of influences and to increase the voice of developing country actors. Evidence-based HPSR priorities emerging through such a process would then be able to support scaling up of research efforts on a par with scaling up of health system strengthening and disease control. Regional and global meetings, such as the Ministerial Summit for Health Research to be held in Mexico in November 2004, are good opportunities to present and discuss the evidence and to commit funding accordingly. Attention must be given to encouraging consensus building on research priorities within regions comprising countries with similar needs. The interests of donors, governments, health workers, the community and researchers must all be taken into consideration so that research funding leads not only to fund relevant research but to build the necessary interfaces for utilization. Competing interests None declared.

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External decontamination of wild leeches with hypochloric acid

Background Medicinal leech, Hirudo medicinalis , has been used in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps. In many countries, wild leeches are still provided from local markets and utilised with antibiotic prophylaxies. In this research, results of identification of bacteria in the transport fluid is reported, oral and intestinal floras and the antibiograms of the identified microorganisms are investigated. Also, to avoid possible infections, the ability of hypochloric acid, a disinfectant, to suppress the relevant microorganisms without changing the life style and behavior of leeches in terms of sucking function, is investigated. Methods Bacterial identifications and antibiograms of oral and intestinal flora and transport medium were performed for 10 leeches. The optimum concentration of hypochloric acid which eliminated microorganisms without affecting the viability and sucking function of the leeches were determined by dilution of hypochloric acid to 100, 50, 25, 12.5, 6.25 ppm concentrations in different groups of 25 leeches. Finally, 20 leeches were applied atraumatically to the bleeding areas of rats, the duration of suction was determined and compared statistically between the leeches treated and not treated with hypochloric acid solution. Results Aeromonas hydrophilia was the most commonly identified microorganism and found to be resistant to first generation cephalosporins, frequently used in prophylaxis at surgical wards. In the next stages of the study, the leeches were subjected to a series of diluted hypochloric acid solutions. Although disinfection of the transport material and suppression of the oral flora of hirudo medicinalis were successful in 100, 50, 25, 12.5, 6.25 ppm concentrations; 12.5 ppm solution was the greatest concentration in which hirudo medicinalis could survive and sucking function was not affected significantly. Conclusions External decontamination of wild leeches with 12.5 ppm hypochloric acid enables bacterial suppression without causing negative effects on leech sucking function and life.

Background The medicinal leech, Hirudo medicinalis , has been used with increasing frequency during the past few decades for salvage of venous compromised pedicled flaps, microvascular free-tissue transfers and replantations. Although the therapeutic use of leeches in medicine dates back 50 BC; for centuries they were collected from various water supplies and utilised under septic conditions with the risk of wound infection and infestation. The supply of leeches was modernized by medicinal leech farm set up in the 1970s [ 1 ]. Today, leech therapy is indicated in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps (Fig 1 , 2 , 3 , 4 ) or replants with an increase in success rate by 60%–83%. However, in the presence of infection as a complication of medicinal use of leeches; the success rate for flap salvage may decrease to ≤ 30% [ 2 ]. Aeromonas is the most common microorganism in leech infections and may cause a wide spectrum of deseases such as cellulitis, ocular infections, arthritis, myocarditis, peritonitis, meningitis, bacteremia and sepsis [ 2 - 5 ]. In many countries, wild leeches are still provided from local markets and utilised in many plastic and reconstructive surgery clinics depending on antibiotic prophylaxies. In the first stage of the study, bacterial content of the transport fluid of leeches that were bought from local markets were studied, also oral and intestinal flora cultures and antibiograms of the identified microorganisms were performed subsequently. In the next stages hypochloric acid, a disinfectant, was tested on animals that were not bred in specific laboratories, to suppress the relevant microorganisms in order to avoid possible infections, without changing the life style and behavior of leeches in terms of sucking function. Methods This study was approved by the Animal Care and Ethics Committee of our institution. Stage 1 Ten leeches were obtained with their 100 cc original water from different stores. Their sizes varied between 6–10 cm in length and 0, 5 – 1, 5 cm in width. They were transported in sterile boxes immersed in the water which the animals were kept at the market. For 2 days they were observed at room temperature. Specimens were taken and smears from transport fluids were prepared. All leeches were held with sterile gloves and prepared in sterile conditions. Specimens were obtained from their mouth (the smaller and more mobile pole) with the help of sterile cotton – swab. After traction was applied between the mouth and posterior muscular organ, the animal was cleaned with alcoholic solution of povidone-iodine, as described by Hokelek et al. [ 6 ]. A longitudinal incision was made on the animal and crop or intestine was exposed (Figure 5 ). Using sterile cotton-swabs, smears were prepared from the intestinal material and they were planted in MacConkey and blood agars in 5 minutes. The growth was observed after 24–48 hours of incubation at 37°C. In addition to classical bacterial identification methods, Analytical Profile Index 32 GN (BioMérieux, France) kit was also used for identification. Further biochemical studies were made on microorganisms that were found to be A. hydro/caviae and A. sobria for definite identification. Since bacterial growth was observed in specimens taken from oral and intestinal flora or transport fluid of all animals except one ; antibiograms for aeromonas isolates were performed. Stage2 30 leeches from different stores were obtained and transported in different sterile boxes with their 100 cc original water as in stage 1. On the following day, samples were taken from transport fluids and oral region and planted in MacConkey and blood agar. Twenty five leeches, which had bacterial growth in either oral flora or transport water were used for the second stage. 100, 50, 25, 12.5, 6.25 ppm solutions were prepared by adding apropriate amounts of hypochloric acid into the transport fluids of 25 leeches seperately with five animals in each concentration group. After 10 minutes of contact with hypochloric acid solutions at 20°C, smears were taken from mouths and transport fluids of each leech under sterile conditions with cotton-swabs. Cotton-swabs were put into a neutralizing media (lecithinized agar media) because they could also contain disinfectant fluid which would prevent microorganism reproduction. Then the neutralized smears were planted into MacConkey and blood agar [ 7 ]. The leeches were then put into boxes containing distilled water and observed. Although the leeches which had been left in the 100, 50, 25 ppm solutions lost their muscular activity and died ;the leeches which were treated with 12.5 and 6.25 ppm hypochloric acid solutions were alive. Bacterial growth was not observed in either concentration group for the specimens taken from oral flora and transport fluid, however bacterial cultures were positive for in specimens taken from the intestine or crop. Of these microorganisms, bacterial colonies for Aeromonas spp were counted. To compare the changes in the number of Aeromonas colonies in leech intestinal flora induced with the application of hypochloric acid, the intestine of five new leeches were exposed as described for Stage 1. Based on the findings of Stage 2, a concentration of 12.5 ppm was chosen for Stage 3 studies. Stage 3 20 leeches were obtained with their 100 cc original water from different stores and equally divided into two groups. One group was treated with hypochloric acid while the others had no treatment at all. After taking specimens from the transport fluids and oral floras, for 10 leeches 12.5 ppm hypochloric acid solution was prepared with the transport fluids. After waiting for 10 min at 20°C temperature, smears were taken from the oral floras and fluids again. All leeches were put into boxes containing distilled water and watched for survival and blood suction function on sedated rats (ketamine 30 mg/kg and xylazine 10 mg/kg). After shaving dorsal region of 20 rats, 2 mm long skin incissions were done in order to produce bleeding. The leeches were applied to the bleeding areas atraumatically and the duration of suction was determined and compared statistically (ANOVA) between the leeches treated and not treated with hypochloric acid solution. Statistical significance was presumed at p < 0.05. Results Stage 1 Bacterial growth was observed in specimens taken from the oral flora in 4 animals (40%), intestinal flora in 9 animals (90%) and transport media in 5 animals (50%). In one leech, no growth was observed in either oral, intestinal and transport fluid cultures. In all culture media, 4 different gram (-) bacterial growth ( aeromonas species , peudomonas species , acinetobacter species , sphingobacterium species ) was observed. In one media, Gram (+) growth was seen : meticilline susceptible coagulase (-) staphylococcus . Dominant microorganisms were aeromonas species , Table 1 . This was an expected result. The biochemical characterization and identification of Aeromonas spp . was investigated by using conventional methods and a commercial kit ID 32 GN ATB System (BioMérieux). In this study, with this system the results were obtained as A. hydro/caviae , A. sobria , A. salmonicidia and A. spp , with the latter nomenclature designating all other Aeromonas species . The most commonly encountered agents, A. hydro/caviae and A. sobria underwent further biochemical studies, as suggested by Abbott, et al.[ 4 ]. As a result, it was seen that A. hydro/caviae was in fact A. hydrophila , and A. sobria was in fact A. veronii biovar sobria (Table 2 ) Antimicrobial susceptibility testing was performed by the disc diffusion method according to NCCLS (National Commity Clinical Laboratory Standart / American). The Aeromonas spp . were found to be susceptible to third generation cephalosporines, aminoglycosides and co-trimoxazole, and all aeromonas spp. were resistant to ampicillin-sulbactam and amoxycillin-clavulanic acid (Table 3 ). Three Aeoromonas spp . were found to have inducable beta lactamase which meant that although they were susceptible, they might develop resistance to beta lactams other than carbapenems during treatment. Stage 2 Fluid and oral flora cultures that were prepared before hypochloric acid application showed multiple microorganism growth, as it was in stage 1. After adding hypochloric acid there was no growth in transport fluid and oral media cultures in either concentration so that no identification and antibiogram study could be performed. After 10 minutes in hypochloric acid solution, leeches were taken to sterile containers which contained distilled water. The muscle activity of all leeches that had been treated in 100 ppm solution ended in 3 min, in 50 ppm ended in 7 min, in 25 ppm ended in 21 min and they died eventually. The ones which had been immersed in 12.5 ppm and 6.25 ppm solutions survived. The Aeromonas colonies in the intestinal flora of these last two groups were compared with the control animals'. With respect to colony suppression, while the difference between the control group, 12.5 ppm group was significant, (p < 0.05, F = 30.1, ANOVA) however the difference between control group and 6.25 ppm group was not significant. Stage 3 All the leeches survived while hyperactivity followed by hypoactivity was seen in the group of leeches treated with hypochloric acid. In this group, two of the leeches never made suction but the other eight leeches sucked the bleeding area for 5–16 min (mean 12.2 ± 3.8). Non hypochloric acid treated group sucked for 9–22 min (mean 15.5 ± 4.4) and the difference between two groups was not significant (p > 0.05, F = 2.6, ANOVA). In 70 % of cultures that were prepared from transport fluids of leeches and in 50% of cultures that were prepared from oral floras of leeches before hypochloric acid application, bacterial growth (mainly Aeromonas spp ) were observed. After hypochloric acid application no growth was observed in any of the specimens taken from animals, therefore no identifications or antibiograms could be performed. Discussion Many reports showed that venous compromise is a more common complication than arterial occlusion in free-tissue transfers. Although surgical correction is the first choice for management of venous compromised flaps ; in cases where surgical correction is not feasible or fails, nonsurgical procedures such as exanguination treatments can be used. Among them, medicinal leech therapy has become widely accepted to promote perfusion of venous compromised flaps and to relieve congestion. However there are reports in the literature concerning about infections specially with aeromonas bacteria after leech therapy calling attention for appropriate antibiotic prophylaxis [ 8 , 9 ]. Many species of Aeromonas survive in water. These also can live in materials embedded in water, drainage tubes, fountains and containers for distilled water. Among other aeromonas species A. hydrophilia is the mostly isolated one from the intestines of leeches. Hemoglobin that is sucked by the leech is denaturated by aeromonas . One of the products, heme, is used by aeromonas and the other, globulin, is used by the leech[ 10 ]. This shows the endosymbiotic relationship of leeches and aeromonas. Many investigators isolated aeromonas spp as the dominant bacteria in infections related with leech usage [ 11 - 13 ]. Aeromonas spp . can cause infections with contamination through the bite point of the leech or on the macerated skin. The infections caused by these microorganisms can be minimized by easy measures. Leeches should not be handled with the forceps from the containers because these traumas can cause regurgitation of the leech into the wound and eventual contamination. Therefore, leeches should be taken with sterilized gloves without traumatisation of the animal. Same care should be taken while carrying the leech from the wound. When the leech is filled up, it puts itself off. If it is taken before this time, the risk of regurgitation is high. Low amounts of cigarette smoke or a heat source can be used in order to cause the leech to put itself off. During leech application, the routine surgical antibiotic prophylaxis must be changed because aeromonas spp. are resistant to first generation cephalosporins which are widely used at surgical wards. These are usually susceptible to third generation cephalosporins, aminoglycosides, tetracyclines and quinolones. Lineaweaver et al. recommended third generation cephalosporins for the prophylaxis of surgical procedures after which leeches were utilised [ 10 , 12 ]. In countries, where special farms for medicinal leeches do not exist, animals obtained from markets should be studied for their floras and antibiotic susceptibilities. Eroğlu et al. investigated the floras and the antibiotic susceptibility of leeches in Black Sea region. Most commonly A. hydrophila , Ochrobacter antropia , nonfermantating gram (-) rods , Acinetobacter Iwofii and A sobria were isolated. All of the isolates were sensitive to ciprofloxacine, cefotaxime, ceftazidime, gentamycine and trimetoprim-sulfamethoxasole [ 14 ]. Likewise, we aimed to investigate the general flora of leeches we use occasionally and antibiotic susceptibility of these bacteria. Our results correlated with the other studies about this subject in the literature. The interesting observation in our study was there were 3 Aeromonas hydrophylia colonies which produced inducable betalactamase. In case of infections with these species, beta lactam antibiotics except carbapenems should not be used. In addition, we aimed to supress possible bacterial contamination of the transport media and oral floras of leeches but not intestinal flora because of the endosymbiotic relationship of the aeromonas and leech, with hypochloric acid solutions at disinfectant concentrations that does not affect the life style of leeches. Investigators have attempted to disinfect the guts of leeches before they are placed on patients by placing the ectoparasites in % 0.02 chlorhexidine for 15 seconds or in antibiotic solutions (tetracycline or cefoperazone solutions) for 12 hours, but these attempts were unsuccessful [ 2 ]. Mackay et al. incubated leeches in solutions of antibiotics to which Aeromonas spp . are sensitive for 12 hours but could not eradicate aeromonas from the intestines [ 11 ]. We preferred hypochloric acid which is an ideal disinfectant and has a wide antibacterial spectrum for desired purpose. Chlorine in the form of hypochloric acid exhibits rapid microbicidal activity by inhibiting of key enzymatic reactions within the cell and eventual protein denaturation. It has a rapid bactericidal effect and can dissolve in water. However, it has some disadvantages as it irritates mucosal membranes, has interactions with some chemicals and metals [ 15 ]. It was showed that bactericidal effect of hypochloric acid of 100 ppm concentrations on pseudomonas was achieved within 10 min exposure [ 7 ]. Aeromonas spp . cause similar nosocomial infections and has similar antibiotic susceptibility with pseudomonas spp . Therefore, in the second stage of our study, beginning with 100 ppm , we applied decreasing concentrations of hypochloric acid in transportation fluids, observed bacterial growth by taking specimens from transport material and oral flora of hirudo medicinalis . We tried to find the dilution ratio that was closest to 100 ppm , suppresing oral flora and transfer liquid and also allowing the leech to survive with normal function. As a result of the second stage of the study we found out that concentration to be 12.5 ppm . In the third stage, we observed that mean sucking duration of the hypochloric acid treated group was shorter than the other group but the difference was not significant (12.2 min & 15.5 min respectively). However hypochloric acid had successful disinfection effect for transport liquid and oral flora. In our opinion, such a decrease in duration of sucking function can be preferable to infection possibility. Since leeches are much cheaper than antibiotics, it is logical to use them once, after treating with hypochloric acid, and never utilise them again. We do not advise to use "full-up" leeches over and over again by putting them in hypertonic solutions to force them vomitting because if they regurgitate, the intestinal flora can readly contaminate the environment where the leeches are kept and cause infection eventually. Although the oral flora and transport enviroment studies do not really address the problem since excretory contamination is probably the major factor in leech infections ; we believe that to take any measure in order to decrease the infection risk during utilisation of ordinary leeches is valuable. Conclusions We can comment that preparation of 12.5 ppm hypochloric acid solution with transport fluids of ordinary leeches obtained from the local market for 10 minutes and then taking the leeches gently from water before application can prevent possible infections caused by contamination from leech oral flora and transport medium. Competing interest None declared. Author's contributions AA and SVK participated in design of the study, involved in the dissections of leeches. EH and ST participated in the design of the study and writing of the manuscript.HN, NG and BO involved in the antibiogram tests. SNK participated in design of the study. Pre-publication history The pre-publication history for this paper can be accessed here:

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Contribution of Noncentrosomal Microtubules to Spindle Assembly in Drosophila Spermatocytes

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules and revealed when asters are kept away. These observations are consistent with a model in which centrosomal and noncentrosomal microtubules contribute to the assembly and are required for the robustness of the cell division spindle in cells that contain centrosomes.

Introduction Two different pathways of spindle assembly are known to operate in the animal kingdom. The first, observed in somatic as well as in male germline cells, requires the microtubule organising activity of centrosomes ( Compton 2000 ; Bornens 2002 ). The second, restricted to female germline and some embryonic cells that lack centrosomes, is thought to depend upon the microtubule stabilisation and organisation activity of the chromosomes themselves ( McKim and Hawley 1995 ; de Saint Phalle and Sullivan 1998 ; reviewed in Karsenti and Vernos 2001 ). Centrosome-independent microtubule growth and sorting into a bipolar spindle have been observed in vitro around chromatin-coated beads in Xenopus egg extracts ( Heald et al. 1996 ). Moreover, some experimental data suggest that a centrosome-independent pathway for spindle assembly also exists in somatic cells ( Bonaccorsi et al. 1998 , 2000 ; Megraw et al. 1999 , 2001 ; Vaizel-Ohayon and Schejter 1999 ; Khodjakov et al. 2000 ; Hinchcliffe et al. 2001 ; reviewed in Raff 2001 ). It is generally assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, so far, the actual process of centrosome-indepen dent microtubule nucleation, polymerisation, and sorting into a bipolar spindle has not been documented in any of the centrosome-containing cell lineages of a living animal. The problem in visualising such microtubules of noncentrosomal origin when centrosomes are present is a technical one. In Drosophila , as in most animal cells, at the onset of cell division the two segregated pairs of centrosomes have a strong microtubule organising activity ( Tates 1971 ; Church and Lin 1982 ; Cenci et al. 1994 ). Consequently, as soon as the nuclear envelope (NE) breaks down, numerous microtubules invade the nuclear region, making it extremely difficult to single out any noncentrosomal microtubules that might be present. To circumvent this limitation, we have taken advantage of two experimental conditions that inhibit the natural process of centriole migration from the plasma membrane to the interior of the cell that takes place at the onset of meiosis in Drosophila spermatocytes. Under such conditions, the centrosomes organise asters, but these are kept at the plasma membrane, away from the nuclear region. In these cells, microtubules can be seen to grow from the remnants of the fenestrated NE and to assemble into anastral bipolar spindles in a centrosome-independent manner. We propose that these spindle-shaped arrays correspond to a subset of microtubules that are normally present in the spindles of wild-type cells. Results Centriole Migration towards the Nucleus in Drosophila Spermatocytes Requires Microtubules and the Function of asp Studies based on electron microscopy ( Tates 1971 ) had shown that soon after the last round of mitotic divisions that precede meiosis in Drosophila spermatocytes, the centrioles migrate towards the periphery of the cell and position themselves underneath the plasma membrane. The same studies revealed that shortly before the onset of prometaphase I, the centrioles are found again close to the nuclear membrane, thus strongly suggesting that they migrate back near the nucleus in preparation for meiosis. Using an endogenously expressed centriolar green fluorescent protein (GFP) marker, we have been able to demonstrate such migration in living spermatocytes ( Figure 1 A; Video 1 ). The entire process takes about 2 h. Initially, the two centriolar pairs move towards the nucleus and start to migrate apart as they approach the nuclear membrane. They finally position themselves at opposite sides of the nucleus, about 30 min before the onset of NE breakdown (NEB). Figure 1 Centriole Migration in Primary Spermatocytes (A) Time-lapse series of confocal images from a wild-type primary spermatocyte expressing GFP-PACT (centrioles) and His2AvD–GFP (chromosomes). The centrioles (arrows) can be seen moving away from the plasma membrane (0) towards the nucleus (N) and then migrating diametrically apart as the chromatin condenses. The chromosomes are fully condensed at timepoint 121 min. (B–D) The two centriole pairs (green) projected over the phase-contrast view (grey) can be seen close to the fenestrated NE and away from the plasma membrane (pm) in control cells (B), while they remain plasma membrane-bound in asp (C) and in colcemid-treated wild-type cells (D). In asp spermatocytes (C), the position of the membrane-bound centrioles correlates tightly with the pointed end of phase-dark protrusions (arrows) that are not present in colcemid-treated cells. These reflect the distribution of phase-contrast membranes known to overlap microtubules in these cells. (E–J) XY projections (E–G) and their corresponding optical sections (H–J) of control (E and H), asp (F and I), colcemid-treated spermatocytes (G and J) expressing an endogenous GFP–α-tubulin confirm that the two major MTOCs in control cells are close to the nucleus, but remain near the plasma membrane in the two experimental conditions. MTOC activity in colcemid-treated spermatocytes was assayed following a 1-s pulse of 350 nm light to inactivate the drug, thus allowing microtubule regrowth. The yellow bar in the XY projections (E–G) marks the position of the corresponding XZ optical sections (H–J). We have identified two experimental conditions that inhibit centriole movement, back from the plasma membrane in Drosophila spermatocytes. The first one is mutation in the gene abnormal spindle, asp ( Ripoll et al. 1985 ; Casal et al. 1990 ; Gonzalez et al. 1990 ; Saunders et al. 1997 ; do Carmo Avides and Glover 1999 ; Wakefield et al. 2001 ; Riparbelli et al. 2002 ). In contrast to wild-type control cells ( Figure 1 B), the two pairs of centrioles in asp E3 /asp L1 spermatocytes at late prophase are still located at the plasma membrane ( Figure 1 C), where they remain throughout meiosis. Centriole migration back towards the NE can also be inhibited by microtubule depolymerisation. Like in asp mutant spermatocytes, the centrioles of wild-type spermatocytes exposed to the microtubule-depolymerising drug colcemid remain close to the plasma membrane throughout meiosis ( Figure 1 D). Top ( Figure 1 F) and lateral ( Figure 1 I) views of living asp mutant spermatocytes expressing a GFP–α-tubulin fusion reveal that the microtubule organising centres (MTOCs) are found at the periphery of these cells. The MTOCs of control cells at this stage can be seen near the nuclear membrane ( Figure 1 E and 1 H). These observations strongly suggest that the membrane-bound centrioles observed in asp mutant spermatocytes are associated to active centrosomes that retain MTOC activity. This conclusion is further substantiated by the localisation of the pericentriolar material (PCM) marker γ-Tub23C ( Zheng et al. 1991 ; Sunkel et al. 1995 ) around the membrane-bound centrioles, coinciding with the position of the MTOCs (data not shown). The same applies to cells in which centriole migration is inhibited by colcemid. Immediately after a short pulse of 350 nm UV light to inactivate the drug (W. E. Theurkauf, personal communication), two asters are organised around the membrane-bound centrioles in these cells ( Figure 1 G and 1 J). Anastral Spindles Are Assembled When the Centrosomes Are Kept Membrane Bound Inhibition of centrosome migration back from the plasma membrane in Drosophila spermatocytes offers an unprecedented opportunity to assay centrosome-independent microtubule polymerisation during spindle assembly in the cells of a living animal that contain centrosomes. Therefore, we decided to follow microtubules by time-lapse confocal microscopy in asp and colcemid-treated cells that expressed a GFP–α-tubulin fusion, as they went through meiosis. At the onset of prometaphase, the NE becomes fenestrated, but does not disappear in Drosophila ( Tates 1971 ; Stafstrom and Staehelin 1984 ; Church and Lin 1985 ). This partial NEB can be readily identified by the sudden entry of GFP–α-tubulin into the nuclear region ( Figure 2 , timepoint 0; Video 2 ). In control cells, microtubule polymerisation and organisation are largely concentrated around the centrosomes ( Church and Lin 1982 ; Cenci et al. 1994 ). Consequently, the abundance of these microtubules makes it extremely difficult to determine the possible contribution of any centrosome-independent microtubule polymerisation activity to spindle assembly ( Figure 2 , control, 10 min to 32 min; Video 2 ). Microtubule organisation is significantly different in the case of asp mutant spermatocytes. At the time of NEB, the membrane-bound centrosomes can be seen organising the two asters at a significant distance from the nucleus, which is kept clear from astral microtubules ( Figure 2 ; Video 3 ). Around 10 min after NEB, a distinct focus of microtubule polymerisation appears within the nuclear region, away from the asters ( Figure 2 , asp , 10 min; Video 3 ). It gives rise to a few bundles ( Figures 2 , 15 min) that grow ( Figure 2 , 22 min) and get organised into a bipolar spindle-shaped microtubule array that in 28% ( n = 43) of the cells is anastral and establishes no contact with the membrane-bound centrosomes ( Figure 2 , 39 min). The remaining 72% was accounted for by cells in which, despite the distance, microtubules from one or both asters reach the spindle so that spindle poles and asters were aligned. Although the acentrosomal origin of the spindle microtubules in these cells is fairly convincing, only those cells that assembled truly anastral spindles that remained so throughout meiosis were considered as cases of noncentrosomal spindle assembly. Figure 2 Time-Lapse Series of Meiosis Progression in Control, asp , and Colcemid-Treated Spermatocytes Timepoint 0 coincides with the time of NEB revealed by the sudden entry of GFP signal into the nucleus. In control cells ( Video 2 ), microtubules are mainly organised around the centrosomes (arrows). However, when centrosomes are kept away from the nuclear region by mutation in asp ( Video 3 ) or colcemid treatment ( Video 4 ), microtubule nucleation and growth are clearly revealed over the nuclear region (N), well away from the centrosomes. Such noncentrosomal microtubules may form bundles that eventually are sorted into spindlelike bipolar microtubule arrays. Microtubules were labelled with an endogenous GFP–α-tubulin fusion. These observations strongly suggested that microtubules can nucleate in a centrosome-independent manner and assemble a spindle-like array in Drosophila spermatocytes. The question remained open, however, as to whether such anastral structures could not simply be a consequence of mutation in asp itself. To rule out such a possibility, we followed spindle assembly in wild-type spermatocytes in which centrosomes had been kept membrane-bound by colcemid treatment. The results were strikingly similar to those observed in asp cells. Seconds after colcemid inactivation, the cortex-bound position of the centrosomes is revealed by the growing asters that were not visible before ( Figure 2 , colcemid inactivation, 0 timepoint; Video 4 ). Like in asp mutant spermatocytes, microtubules can clearly be seen to nucleate over the nuclear region, well away from the asters ( Figure 2 , 11 min), grow ( Figure 2 , 15 min), and get sorted ( Figure 2 , 37 min and 43 min) into anastral bipolar arrays. Of the colcemid-treated cells studied ( n = 32), 22% behaved like the cell shown in Figure 2 . The remaining cells assembled more than one spindle, multipolar spindles, or spindles that were connected to one of the asters. The Nucleation of Noncentrosomal Microtubules in Spermatocytes with Membrane-Bound Centrosomes Has a Late Onset We then decided to time the onset of anastral microtubule nucleation. The timing of the main landmarks of meiosis progression in control, asp , and colcemid-treated spermatocytes is summarised in Figure 3 . In control spermatocytes, chromosome condensation starts within 2 min after NEB, and the first centrosomal microtubules enter the nuclear region shortly afterwards. Chromosome condensation is completed between 10 and 12 min after NEB. In agreement with previous reports, anaphase onset takes place between 32 and 47 min after NEB ( Church and Lin 1985 ; Rebollo and Gonzalez 2000 ; Savoian et al. 2000 ). Remarkably, the timing of meiosis progression from NEB to anaphase onset, both in asp and in colcemid-treated wild-type spermatocytes, seems to be largely unaffected, suggesting that the feeble spindle checkpoint of these cells ( Rebollo and Gonzalez 2000 ; Savoian et al. 2000 ) is not triggered by the membrane-bound centrosomes' condition. The timing of the onset of noncentrosomal microtubule growth within the nuclear region in asp and colcemid-treated cells is tightly controlled. It occurs between 9 and 13 min after NEB, at the same time or marginally later than the end of chromosome condensation. If this process occurs with the same timing in wild-type cells, the noncentrosomal microtubules will intermingle with numerous centrosomal microtubules that are already present at this stage. Figure 3 The Timing of Noncentrosomal Microtubule Nucleation Referred to NEB (timepoint 0), the timing of chromosome condensation and of onset of chromosome segregation is essentially identical in control, asp , and colcemid-treated spermatocytes. In control spermatocytes, aster microtubules can be seen entering the nuclear region 3–6 min after NEB. They do not in asp or following colcemid treatment. In these two cases, however, centrosome-independent microtubule polymerisation can be seen over the nuclear region. It starts between 9 and 13 min after NEB, coinciding with or very shortly after the end of chromosome condensation. Acentrosomal Microtubules Are Nucleated on the Inner Side of the Remnants of the NE and Not around the Chromosomes To determine the nucleation site of the microtubules organised over the nuclear region, we followed the initial stages of microtubule assembly by time-lapse microscopy, acquiring several Z series of XY confocal and phase-contrast sections at different timepoints. From these, we generated a time-lapse series of 3D reconstructions that allowed us to localise the foci of nucleation of anastral microtubules. We were able to draw the following three main conclusions that apply to both asp and colcemid-treated spermatocytes. Firstly, the foci from which microtubules grow may be clustered ( Figure 4 A) or dispersed ( Figure 4 B). Secondly, no significant correlation can be established between the site of microtubule nucleation and the chromosomes ( Figure 4 A and 4 B). Finally, nucleation takes place in close proximity to the remnants of the NE (ten out of ten cells reconstructed; Figure 4 A and 4 B), which in Drosophila ruptures without disassembling completely ( Tates 1971 ; Stafstrom and Staehelin 1984 ). Figure 4 The Place of Noncentrosomal Microtubule Nucleation The initial stages of noncentrosomal microtubule nucleation revealed by an endogenous GFP–α-tubulin fusion (left) and phase contrast (right). Following the corresponding videos, it is possible to unmistakably tell the chromosomes (arrows) apart form the other phase-dark objects that are present over the nuclear region (asterisks). The cell in (A) is shown as a single timeframe and the cell in (B) as a time-lapse series. In both cells, noncentrosomal microtubule nucleation (arrowheads) takes place close to the remains on the NE and does not overlap with the major chromosomes. Nucleation sites can be clustered (A) or dispersed (B). In the time-lapse series (B), only the chromosomes that are in focus are labelled. Timepoint 0 min in these series corresponds to the first sign of noncentrosomal microtubule nucleation, around 11 min after NEB. A white bar marks the growing end of a microtubule bundle that at timepoint 93 min reaches one of the bivalents. The Anastral Spindles Organised in Cells with Membrane-Bound Centrosomes Can Sustain Some Degree of Chromosome Segregation We then decided to study in more detail the extent to which the anastral spindles organised in cells with membrane-bound centrosomes can mediate successful cell division. To this end, we produced transgenic flies carrying a GFP–α-tubulin fusion together with a His2AvD–YFP (yellow fluorescent protein) strain so that both chromosomes and microtubules could be visualised in the same cell ( Figure 5 ; Video 5 ). In asp cells, during prometaphase, the bivalents do not move to the extent that they do in control cells (data not shown). As mentioned before, congression occurs ( Figure 5 ; Video 6 ), but orientation is rarely bipolar. Homologue chromosomes separate at the onset of anaphase, but they barely move, remaining near the center of the spindle. Moreover, they tend to cosegregate ( Video 7 ) and end up included in the same daughter nucleus. All together, these abnormalities result in high levels of aneuploidy in agreement with previous genetic analysis data ( Ripoll et al. 1985 ). In contrast, in half (52%) of the anastral spindles assembled following transient colcemid treatment, homologue chromosomes could be seen to segregate from one another ( Figure 5 ; Video 8 ). Anaphase in these cells is not complete, however, because only the chromosome-to-pole movement (anaphase A) is observed. The further separation achieved in wild-type cells by the extension of the spindle (anaphase B) is very limited in these cells. Figure 5 Chromosome Segregation in Anastral Spindles in Drosophila Spermatocytes (Control [ Video 5 ]) At metaphase I (0), the bivalents (revealed by a His2Avd–YFP fusion, shown by double arrowheads) are aligned in the middle of the spindle (revealed by a GFP–α-tubulin fusion), at the metaphase plate. At the onset of anaphase (3 min), the homologue chromosomes start to migrate towards opposite poles (single arrowheads) and to decondense. During anaphase B (4 min and 6 min), the spindle poles move apart from each other and the two sets of decondensed chromosomes become further separated. ( asp [ Video 6 ]) At timepoint 0, the bivalents align at the metaphase plate. Homologue chromosomes split apart at the onset of anaphase I (4 min). However, anaphase A migration is highly impaired. By the time the chromosomes start to decondense, they have barely moved towards the spindle poles (8 min and 14 min), and often homologue chromosomes end up included in the same daughter nucleus. (Colcemid [ Video 8 ]) As in asp spermatocytes, the asters (arrows) remain at the plasma membrane at metaphase I in colcemid-treated cells, and the bivalents align in a metaphase plate-like within the acentrosomal spindles (0 min). Homologue chromosomes split apart at the onset of anaphase (upper cell, 6 min) and significantly segregate from one another (upper cell, 8 min; lower cell, 3 min). Further separation of the daughter nuclei during anaphase B is very limited in these cells (8 min), and cytokinesis does not occur. The Orientation of the Cytokinesis Furrow Correlates with the Position of the Membrane-Bound Asters, Independently of Spindle Orientation Following colcemid treatment, we have never observed complete cytokinesis. However, as reported before ( Riparbelli et al. 2002 ), cytokinesis does proceed to completion in around half (47%, n = 19) of asp cells. These cells, which contain unconnected centrosomal asters and anastral spindles, provide a valuable experimental system to assess the contribution of asters and spindle to specifying the place of cleavage. To this end, we plotted the angles between the line defined by the two asters, the major spindle axis, and the plane of cleavage in wild-type and asp spermatocytes ( Figure 6 ). Two conclusions can be drawn from these data. Firstly, the anastral spindles assembled in asp cells can be observed at any angle, even up to 90°, with respect to the position of the two asters. Interestingly, in most such cases, furrow progression forces the spindle to rotate and align with the asters so that, at the end, a fairly normal cytokinesis takes place. Secondly, the orientation of the plane of cleavage keeps a tight 90° ± 10° with respect to the axis defined by the asters and does not correlate with the orientation of the anastral spindle. Figure 6 Correlation between the Orientation of the Cytokinesis Furrow, the Asters, and the Spindle in asp Spermatocytes Schematic representation (A–C) of the relative position of the asters (red), the spindle (yellow), and the cytokinesis furrow (blue), corresponding to a control cell (D) and two examples of asp mutant spermatocytes (E and F), respectively. Asters (arrows) and spindles are labelled with a GFP–α-tubulin fusion. The position of the cleavage furrow (double-headed arrow) was determined by time-lapse imaging of these cells (data not shown). In wild-type cells ( n = 10), plotting spindle and furrow orientation relative to the interastral axes shows that asters and spindle are tightly aligned, and cleavage occurs at an angle of 90° ± 10° with respect to them (G). In asp spermatocytes ( n = 10), the plane of cleavage occurs at a 90° ± 10° angle with respect to the asters and does not correlate with the orientation of the anastral spindle. Discussion We have found that when the asters are kept near the plasma membrane during meiosis I in Drosophila spermatocytes, noncentrosomal microtubules appear over the nuclear region and, in a fraction of the cells, are sorted into anastral bipolar spindles (summarised in Figure 7 ). Identical observations are derived whether centrosomes are forced to remain membrane-bound by mutation in asp or by transient colcemid treatment of wild-type cells. The very different nature of these two experimental conditions strongly argues against these spindles being assembled as a consequence of the experimental conditions themselves. It rather suggests that the observation of anastral spindles is due to the impaired ability of the plasma membrane-bound centrosomes to contribute to spindle assembly. Anastral spindles are also assembled following the removal of centrosomes by laser ablation or microdissection in cultured cells ( Khodjakov et al. 2000 ; Hinchcliffe et al. 2001 ) or by inhibiting the formation of centrosomes in mutant Drosophila embryos ( Megraw et al. 1999 ; Vaizel-Ohayon and Schejter 1999 ), thus reinforcing this argument. Figure 7 Noncentrosomal Microtubules and Spindle Assembly (Central column) Spindle assembly in Drosophila spermatocytes with membrane-bound centrosomes. At the time of NEB, the chromatin (pale blue) starts to condense, and the membrane-bound centrosomes (red) organise asters (yellow) at a significant distance from the nuclear region. Around 12 min after NEB, the first noncentrosomal microtubules (green) start to nucleate near the remnants of the NE (grey), as the chromosomes achieve full condensation (dark blue). These microtubules then bundle, associate with the chromosomes, and eventually end up organised into a bipolar anastral array whose shape is reminiscent of the female meiotic spindle. (Left column) Spindle assembly in wild-type Drosophila oocytes ( Theurkauf and Hawley 1992 ; Matthies et al. 1996 ). NEB starts at the beginning of stage 13 of oocyte development. At this stage, the oocyte does not contain centrosomes and the chromosomes (karyosome) are tightly condensed (dark blue). Microtubules (green) appear 11–15 min after NEB within the nuclear region in association with the karyosome. These microtubules form bundles and are sorted around the chromatin into a bipolar spindle. Evidence suggests that ER components may be required for spindle assembly in these cells ( Kramer and Hawley 2003 ). At metaphase I, recombined bivalents are aligned at the spindle equator, while those that have not recombined are found closer to the spindle poles. Meiosis remains arrested at this point (stage 14) until oocyte activation. Despite the obvious morphological similitude, the equivalence between these and the anastral spindles organised in spermatocytes with membrane-bound centrosomes is unclear. (Right column) Hypothesis regarding the contribution of centrosomal and noncentrosomal microtubules to spindle assembly during meiosis I in wild-type Drosophila spermatocytes. Before NEB, the centrosomes are located at opposite positions near the nucleus. Shortly after NEB, astral microtubules enter the nuclear region and make the first contact with the condensing chromatin. No evidence of noncentrosomal microtubule polymerisation near the nuclear region at this stage has been found yet. Once chromosomes are fully condensed, microtubule bundles of centrosomal origin (yellow) connecting centrosomes to chromosomes already exist. At this stage, noncentrosomal microtubules (green) start to polymerise in association with the remnants of the NE. These microtubules form bundles that interact with the chromosomes and intermingle with the microtubules of centrosomal origin. The fully mature spindle in these cells would therefore contain a spindle-shaped structure made of microtubules of noncentrosomal origin (green) embedded in another spindle-shape array made of two overlapping asters (yellow). We propose that each of these subsets may perform to a certain extent some of the functions carried out by normal spindles, but neither of them can on its own mediate robust cell division. The Place of Noncentrosomal Microtubule Nucleation Noncentrosomal microtubule nucleation during cell division is thought to take place over the chromatin ( Nachury et al. 2001 ). This assumption is largely based on the observations carried out in the few acentrosomal systems in which spindle assembly has been followed by time-lapse microscopy (reviewed in Karsenti and Vernos 2001 ). These include wild-type Drosophila female meiocytes ( Theurkauf and Hawley 1992 ; Matthies et al. 1996 ), parthenogenetic Sciara embryos ( de Saint Phalle and Sullivan 1998 ), and Xenopus egg extracts ( Heald et al. 1996 ). It is also consistent with the active role of chromosomes in spindle organisation. For instance, it has been reported that bivalents micromanipulated away from the spindle in Drosophila spermatocytes induce the assembly of anastral minispindles in the cytoplasm ( Church et al. 1986 ). Likewise, the removal of chromosomes before NEB has been shown to inhibit spindle assembly in grasshopper spermatocytes ( Zhang and Nicklas 1995 ), although the phenotype of fusolo mutants, recently described, seems to argue otherwise ( Bucciarelli et al. 2003 ). Moreover, the chromosomal localisation of the RanGEF RCC1 is expected to result in a local enrichment of the GTP-bound form of Ran, known to facilitate spindle assembly ( Nachury et al. 2001 ; Wilde et al. 2001 ; Gruss et al. 2002 ; reviewed in Hetzer et al. 2002 ), thus providing a mechanistic interpretation for the suspected role of chromatin in this process. In contrast, our observations reveal that nucleation of the noncentrosomal microtubules over the nuclear region occurs over the remnants of the NE, which in Drosophila are present throughout cell division despite extensive fenestration at the onset of prometaphase ( Tates 1971 ; Stafstrom and Staehelin 1984 ; Church and Lin 1985 ). However, upon closer examination, our observations may also be consistent with the literature quoted above. The single bivalents that organise minispindles when micromanipulated into the cytoplasm in Drosophila spermatocytes have actually been shown to be surrounded by masses of stacked membranes, whose contribution to microtubule nucleation/stabilisation, according to the authors themselves, cannot be ruled out ( Church et al. 1986 ). In Drosophila oocytes, too, the meiotic spindle is ensheathed in a membrane structure derived from the endoplasmic reticulum (ER). Although the bulk of microtubules has been described by time-lapse confocal microscopy to form over the chromatin ( Theurkauf and Hawley 1992 ; Matthies et al. 1996 ), the contribution of these membranes to the initial stages of microtubule nucleation cannot be discarded either. In this regard, the recent cloning of Axs , which encodes a transmembrane protein associated with the membranes that surround the spindle and is required for the segregation of achiasmate chromosomes, is very tantalising ( Kramer and Hawley 2003 ). Asx is distributed within the ER of the germinal vesicle just before meiotic spindle assembly. Upon germinal vesicle breakdown (GVBD), Axs associates with the developing spindle through all stages of assembly. These observations have been taken as an indication that the ER may be organised into structures that impinge on spindle assembly during meiosis in Drosophila females ( Kramer and Hawley 2003 ), very much in line with our observations in Drosophila spermatocytes. Indeed, our results do not discard the contribution of chromatin to microtubule stabilisation and sorting into a bipolar array, even if chromatin itself is not the place of initial microtubule nucleation, nor do they rule out the possibility of microtubules being polymerised over the chromatin at later stages. They merely show that in these cells, the remaining bits of the fenestrated NE provide a particularly favourable environment to sustain the initial stages of noncentrosomal microtubule nucleation. Moreover, these observations strongly suggest that, unlike centrosomes, the foci of microtubule nucleation over the nuclear region do not behave as stable MTOCs. As soon as the microtubule bundles acquire a certain length, they interact with the condensed chromosomes and are often sorted into a bipolar spindle, regardless of the initial number of nucleation sites. We have not been able to detect γ-Tub23C at these nucleation sites. We still do not know the actual contribution of Ran to spindle assembly in Drosophila spermatocytes, although, given its known conservation across distant species ( Hetzer et al. 2002 ), it is likely to play a major role. Since orthologues of most of the known components of this pathway are known in Drosophila , it is technically possible to address this question both under normal conditions and in cells in which centrosomes cannot contribute to spindle assembly as described in this work. Experiments are underway in our laboratory to address these points. Functional Relevance of the Anastral Spindles Perhaps the most fundamental question regarding the anastral spindles organised in cells that normally contain centrosomes is the extent to which they could provide a back-up, able to mediate successful and robust cell division when the centrosomes cannot contribute to spindle assembly. Our observations suggest that this is an unlikely scenario. Firstly, only a fraction of cells display a single bipolar array, the rest being accounted for by cases in which either the spindle is multipolar or there is more that one per cell or there is no spindle at all. Secondly, chromosome segregation is also significantly less efficient than in control cells. These two points, however, carry a caveat since they could reflect the effect of depleted asp function or residual traces of active colcemid, rather than the anastral nature of the spindle. Finally, cytokinesis is severely disrupted in these cells. Around half of asp spermatocytes containing anastral spindles go through and complete cytokinesis. However, in these cells, the orientation of the cleavage furrow correlates tightly with the position of the two asters and not at all with the orientation of the spindle. This situation gives rise to cases in which the plane of cleavage is nearly parallel to the spindle. In colcemid-treated cells that contain notoriously small asters, cytokinesis does not occur. These observations strongly argue that asters contribute to specify the place of furrow and may be required for cleavage. The contribution of centrosomes to ensure proper cytokinesis has been previously observed in vertebrate cell lines ( Rieder et al. 1997 ; Savoian et al. 1999 ; Hinchcliffe et al. 2001 ; Khodjakov and Rieder 2001 ), human cell lines ( Gromley et al. 2003 ), Dyctiostelium ( Neujahr et al. 1998 ), or Xenopus ( Takayama et al. 2002 ). This conclusion, however, is not consistent with the observation that cytokinesis is not inhibited in asterless Drosophila spermatocytes ( Bonaccorsi et al. 1998 ). Therefore, the anastral spindles organised in spermatocytes with membrane-bound centrosomes seem able to provide only some of the functions required for cell division, with relatively low efficiency. The functionality of the anastral spindles assembled in embryos laid by cnn mutant females, which do not appear to contain centrosomes, is also compromised. These spindles are not always properly shaped, the chromosomes are not tightly aligned at the spindle equator, chromosome movements are nonsynchronous, and their segregation not always faithful ( Megraw et al. 1999 ; Vaizel-Ohayon and Schejter 1999 ). Thus, in this instance, too, when centrosome function is abrogated in a syncytium that normally contains centrosomes and that does not naturally undergo parthenogenesis, anastral spindles can be assembled that are able to perform some of the functions of their wild-type counterparts, but in a rather inefficient manner. Origin of the Anastral Spindles: Neomorphic or Constitutive Two alternative interpretations can account for the origin of the anastral spindles that we have observed ( Khodjakov et al. 2000 ). First, they could be neomorphic structures, assembled through a pathway normally repressed that is only triggered in response to the impaired contribution of centrosomal microtubules. Although we cannot at the moment discard this interpretation, we find it hard to envisage how such an alternative pathway could have evolved, given the extremely low frequency of centrosome loss or inactivation in wild-type populations. Moreover, it is also difficult to imagine what sort of signalling mechanism could trigger the alternative pathway in these cells since centrosomes are still present and active as MTOCs. Alternatively, these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived microtubules, but revealed when asters are kept away. This interpretation is summarised in Figure 7 . In wild-type spermatocytes under normal conditions, the first astral microtubules enter the nuclear area shortly after NEB and start to build a bipolar spindle as chromosome condensation progresses. By the time chromosome condensation is fully achieved, a distinct bipolar spindle can be observed in these cells. However, it is not yet fully mature, as the number of microtubules will still increase until anaphase onset. It is about this time that nucleation of the acentrosomal microtubules occurs in cells with plasma membrane-bound centrosomes. Therefore, if this process occurs at the same time in wild-type cells, the acentrosomal microtubules could significantly contribute to the maturation of the cell division spindle. This interpretation is consistent with the recent proposal put forward by Gruss et al. (2002) to account for their observations regarding spindle assembly in HeLa cells. They found that when the function of the human homologue of TPX2 is inhibited by RNA interference, the centrosomal asters do not interact and do not form a spindle. From these observations, they concluded that, intermingled with microtubules of centrosomal origin, the mitotic spindle may contain noncentrosomal microtubules that are stabilised and organised by the chromatin and are essential for the assembly of functional spindles. In Drosophila , secondary spermatocytes' mutation in fusolo seem to reveal the centrosome-derived component of the spindle ( Bucciarelli et al. 2003 ). Forcing the asters away from the nucleus in Drosophila primary spermatocytes reveals the noncentrosomal component that, indeed, does not require asters to get organised into a spindle-like structure. We propose that both components are required to mediate robust cell division. The very recent finding of peripheral, noncentrosomal microtubules that contribute to spindle assembly in LLCPK1α cells provides additional evidence to substantiate this conclusion ( Tulu et al. 2003 ). Regardless of their neomorphic or constitutive nature, the acentrosomal spindles that we have found in asp and colcemid-treated spermatocytes are, from a morphological point of view, closely reminiscent of the anastral female meiotic spindles found in many animal species, including Drosophila ( Theurkauf and Hawley 1992 ). The same holds true for the anastral spindles assembled when centrosomes are removed from the cell or cannot be organised due to mutation in essential centrosomal components ( Megraw et al. 1999 ; Khodjakov et al. 2000 ; Khodjakov and Rieder 2001 ; Hinchcliffe et al. 2001 ). In the case of the Drosophila female meiotic spindle, the timing of microtubule nucleation is also very similar: between 9 and 12 min after NEB in spermatocytes and 11 to 15 min in oocytes ( Matthies et al. 1996 ). These similarities have led some to propose that experimentally induced anastral spindles could require the same motors and structural components that build the spindles in female meiocytes ( Megraw et al. 1999 ; Khodjakov et al. 2000 ) (summarised in Figure 6 ). In fact, it has been suggested that the absence of some of these components at the time syncytial divisions occur could explain the lack of robustness of the anastral spindles assembled in embryos derived from cnn mothers ( Megraw et al. 1999 ). We still do not know to what extent the anastral spindles of spermatocytes share components with the oocyte spindle. Some essential ones cannot be shared, though, since they are only expressed in the female germline. Given the wealth of probes and mutants available in Drosophila , it should be possible to draw a clear picture of the situation regarding this fundamental question. Materials and Methods Fly stocks Flies from w 1118 ;e 11 asp E3 /TM6C and w 1118 ;red asp L1 /TM6C stocks were crossed to generate w 1118 ;e 11 asp E3 /red asp L1 transheterozygous individuals. The viability of asp E3 /asp L1 males is high, but they are poorly fertile and produce high levels of aneuploid gametes. Transgenes The chromosomes were labeled with transgenes expressing either a His2avD–GFP fusion ( Clarkson and Saint 1999 ) or its derivative, His2avD–EYFP, constructed by us under the control of the polyubiquitin promoter ( Lee et al. 1988 ). To visualise centrioles, we used the transgene expressing GFP-PACT (pericentrin-AKAP450 centrosomal targeting) (kindly provided by J. Raff) that contains the predicted Drosophila homologue of the PACT domain described by Gillingham and Munro (2000) . To visualise microtubules, we constructed a transgene that contained the GFP–α-tub84B fusion as previously described ( Grieder et al. 2000 ) under the control of the polyubiquitin promoter ( Lee et al. 1988 ). Time-lapse recording Live spermatocytes were recorded as previously described ( Rebollo and Gonzalez 2000 , 2003 ). For most applications, we collected a series of timepoints at 15–30 s intervals, each containing four to eight XY sections at different depths along the Z axis and including both the phase-contrast and fluorescence channels. For more-detailed 3D reconstruction, stacks containing 20 sections were obtained. Laser intensity was always kept to a minimum, and only the excitation laser line 488 was utilised. GFP and YFP signals were distinguished by overlying the two recorded channels. Image processing was performed with NIH-Scion Image, Interactive Data Language (IDL), and huygens2. For 3D reconstructions, we wrote macros in NIH-Scion Image to navigate through the three dimensions of the cell stack. Colcemid treatment Newly hatched adult males were fed for 8–12 h with a solution containing 32 μg/ml of colcemid (Sigma, St. Louis, Missouri, United States) in 1 M sucrose. Upon dissection, their testes were prepared for in vivo imaging as described above. Once under the microscope, microtubules were allowed to repolymerise by a 1-s pulse of 350 nm light that inactivates the drug. For simplicity, this entire procedure of exposure to colcemid followed by light inactivation of the drug is referred to through this manuscript as ‘colcemid treatment.’ Supporting Information Accession Numbers The FlyBase accession numbers discussed in this paper are α-tubulin 84B (CG1913), asp (CG6875), Axs (CG9703), cnn (CG4832), γ-Tub23C (CG3157), His2AvD (CG5499). The GeneBank accession numbers discussed in this paper areGFP (U57609.1), Ran (NM_006325), RCC1 (D00679), TPX2 (BC020207), and YFP (U57609.1). Video 1 Centriole Migration in Primary Spermatocytes (849 KB MOV) Video 2 Spindle Assembly in Control Spermatocytes (442 KB MOV) Video 3 Spindle Assembly in asp Spermatocytes (675 KB MOV) Video 4 Spindle Assembly in Colcemid-Treated Spermatocytes (844 KB MOV) Video 5 Chromosome Segregation in Control Spermatocytes (206 KB MOV) Video 6 Chromosome Segregation in asp Spermatocytes I (452 KB MOV) Video 7 Chromosome Segregation in asp Spermatocytes II (377 KB MOV) Video 8 Chromosome Segregation in Colcemid-Treated Spermatocytes (465 KB MOV)

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554103

Negative density-distribution relationship in butterflies

Background Because "laws of nature" do not exist in ecology, much of the foundations of community ecology rely on broad statistical generalisations. One of the strongest generalisations is the positive relationship between density and distribution within a given taxonomic assemblage; that is, locally abundant species are more widespread than locally sparse species. Several mechanisms have been proposed to create this positive relationship, and the testing of these mechanisms is attracting increasing attention. Results We report a strong, but counterintuitive, negative relationship between density and distribution in the butterfly fauna of Finland. With an exceptionally comprehensive data set (data includes all 95 resident species in Finland and over 1.5 million individuals), we have been able to submit several of the mechanisms to powerful direct empirical testing. Without exception, we failed to find evidence for the proposed mechanisms creating a positive density-distribution relationship. On the contrary, we found that many of the mechanisms are equally able to generate a negative relationship. Conclusion We suggest that one important determinant of density-distribution relationships is the geographical location of the study: on the edge of a distribution range, suitable habitat patches are likely to be more isolated than in the core of the range. In such a situation, only the largest and best quality patches are likely to be occupied, and these by definition can support a relatively dense population leading to a negative density-distribution relationship. Finally, we conclude that generalizations about the positive density-distribution relationship should be made more cautiously.

Background Species that are locally abundant tend to be more widespread than species that are locally rare [ 1 , 2 ]. This positive relationship between density and distribution of species has been observed in a variety of species assemblages over a spectrum of spatial scales, and it has been suggested that it may be almost an universal pattern in ecology [ 3 - 6 ]. However, a few studies document a negative relationship between density and distribution [ 7 - 14 ] (but see [ 15 ]). Only recently Gaston et al. [ 5 ] encouraged ecologists to pay more attention to the possibility of a negative relationship. Nine mechanisms have been proposed to explain the positive relationship between density and distribution [ 2 , 3 , 14 , 16 - 25 ]. Of these, two are artefactual (sampling artefact, phylogenetic non-independence) and seven are ecological (pattern of aggregation, range position, niche breadth, resource availability, density dependent habitat selection, dispersal ability and vital rates). Negative relationship between density and distribution can be generated by similar mechanisms that give rise to a positive relationship, but for substantially different circumstances and parameter values [ 5 , 6 ]. Below we discuss briefly all of the mechanisms that can possibly explain the positive relationship between density and distribution, and evaluate whether they could also generate a negative relationship. Sampling artefact Since low density species are less likely to be detected in surveys, a positive density-distribution relationship may result from systematic under-recording of the distribution of species that occur at low density [ 2 , 3 , 20 ]. It follows logically that sampling artefact is not expected to generate a negative density-distribution relationship [ 5 ]. However, there is a possibility that rare species with limited, but known, distribution face proportionally higher sampling effort resulting in inflated estimates of their density. When this is the case, a false negative relationship between density and distribution may be generated. Phylogenetic non-independence Both positive and negative density-distribution relationship may result from related species being considered as independent data points [ 19 , 26 , 27 ]. However, phylogenetic non-independence may be the causative factor of a density-distribution relationship only if species density and distribution are determined by species specific life history characteristics affected by the common ancestry, and if there are differences between taxa in their density and distribution. Phylogenetic non-independence has been rejected as an explanation for the positive density-distribution relationship in all previous studies that have controlled its effects [ 5 , 14 , 25 , 28 , 29 ]. Patterns of aggregation A positive density-distribution relationship may be generated as a result of an underlying theoretical spatial distribution of individuals. For a given level of aggregation, a species with more individuals in a given area is expected to occur in more locations than a species with fewer individuals in the same area [ 20 , 24 ]. However, whether this purely statistical mechanism can actually cause a positive density-distribution relationship rather than serves as a restatement of the relationship in another form, is questionable [ 29 ]. This mechanism is unable to generate a negative density – distribution relationship [ 5 ]. Range position Empirical observations suggest that habitat occupancy and the density of individuals decline when moving along a gradient from the centre of the species geographical distribution range toward its edge [ 2 , 30 - 32 ]. Therefore, a positive density-distribution relationship in any particular region may result when species are at different positions relative to the centre of their geographical range [ 16 ]. Gaston et al. [ 5 ] argued that this mechanism cannot generate negative density-distribution relationship. More recently, Hanski [ 33 ] has pointed out that if patches are more isolated toward range edges, only the largest or best quality patches, i.e. those patches that are able to support the most dense populations, will be occupied. We note that this may lead into a negative density-distribution relationship. Niche breadth Brown [ 2 ] hypothesized that a positive density-distribution relationship arises because species which have an ability to use a broader range of resources are assumed to be widespread and more abundant. However, while some evidence exists that species with greater niche breadth are more widespread [ 3 , 5 , 34 - 40 ], virtually all published studies fail to document a positive interspecific relationship between niche breadth and abundance (see the reviews in [ 5 , 25 ]). On the contrary, many species with wide niche breadth are widely distributed but locally rare [ 5 , 25 ]. However, even if the relationship is commonly and almost predominantly negative, it is not often significantly different from zero. Nevertheless, it seems that the relationship between niche breadth and abundance may in fact generate a negative rather than a positive density-distribution relationship. Resource availability On the assumption that density and distribution of resources determine the density and distribution of the species utilizing them, a positive or a negative density-distribution relationship in the resource will generate the same relationship in the consumer [ 3 , 22 ]. Most of the density-distribution relationships of the resource are reported to be positive [ 2 , 41 ]. Density dependent habitat selection If a species tends to inhabit more habitats when density is high and fewer when density is low, then locally abundant species will tend to occupy more habitats and have wider distributions [ 17 ]. At present, there is little evidence for positive density-dependent habitat selection [ 5 , 25 ]. Instead, some evidence exists for negative density-dependent habitat selection [ 42 - 44 ]. A negative density-distribution relationship arises when a species has a wider distribution, i.e. inhabits more habitats, when the density is low. Dispersal ability Metapopulation theory may explain both positive and negative density-distribution relationships [ 21 ]. A positive relationship may arise where a species that has high density is less likely to go extinct in a given patch than a species that has lower density, or where dispersal increases with density, thereby promoting colonization of empty patches [ 3 , 18 ]. Conversely, a negative relationship may emerge when the species differ in dispersal ability, since dispersal can reduce density but increase distribution [ 21 , 33 ]. Vital rates If a set of species differs only in respect to its mortality rate, then species with higher mortality will have a lower density. Moreover, the species will inhabit fewer patches than a species with low mortality, leading to a positive density-distribution [ 23 ]. Under rather restricted conditions, the vital rates mechanism could also generate a negative density-distribution relationship [ 23 ]; however, to test this mechanism, population level data on density-dependent birth and death rates are necessary [ 5 ]. Here, we will examine the relationship between density and distribution of Finnish butterflies (Hesperioidea and Papilionoidea), and test the possible mechanisms affecting this relationship. The density-distribution patterns of butterflies have been examined in many previous studies, and if a relationship was found, it was generally positive [ 3 , 14 , 25 , 38 , 45 - 47 ]. However, most of the studies were conducted in the British Isles, thus representing a sort of pseudoreplication in terms of range of environments and selection of butterfly species studied. Using density and distribution data from two extensive butterfly censuses, we will assess which of the four mechanisms – phylogenetic non-independence, range position, niche breadth and dispersal ability – are best at explaining the density-distribution relationship of Finnish butterflies. We will also discuss the effects of sampling artefact on the density-distribution relationship. Results Density-distribution relationships A strong negative relationship was detected between the density and distribution of Finnish butterflies (linear regression; F 1,93 = 229.97, P < 0.001, r 2 = 0.71) (Fig. 1 ). Distribution of butterflies To understand the effects of range position and dispersal ability on the distribution of the butterflies, we analysed the data using simple linear regression and with a multiple linear regression to even out correlated effects. In simple linear regressions, both variables had a significant positive effect on butterfly distribution (Table 1 ). The overall multiple linear regression was highly significant (F 2,76 = 121.90, P < 0.001, r 2 = 0.76), and both of the predictor variables had an independent positive effect on the distribution of the butterflies (Table 2 ). The relationship between distribution of the butterflies and both of the predictor variables are shown in figures 2 and 3 . To study the effect of niche breadth on the distribution of the butterflies, we analysed the effects of larval specificity and adult habitat breadth on butterfly distribution using analysis of variance (ANOVA) and Tukey's multiple comparisons test. There was no significant interaction between larval specificity and habitat breadth on butterfly distribution (ANOVA F 4,72 = 1.32, P = 0.270), and thus the interaction term was removed from the analysis. However, both larval specificity and habitat breadth had a significant effect on butterfly distribution (F 2,76 = 8.20, P < 0.001 and F 2,76 = 12.53, P < 0.001, respectively). A significant difference was found in the distribution of butterfly species with different habitat breadths. Specialist butterfly species had smaller distribution than generalist species (Tukey MD, mean difference = -2.03, SE = 0.40, P < 0.001), and intermediate species had smaller distribution than generalist species (Tukey MD = -1.24, SE = 0.44, P = 0.018). Specialist species tended to have smaller distributions than intermediate species, but the difference was not significant (Tukey MD = -0.79, SE = 0.35, P = 0.063; Fig. 4 ). No significant difference was found in the distribution of monophagous and oligophagous butterfly species (MD = -0.441, SE = 0.46, P = 0.611). However, the distribution of the polyphagous butterfly species was greater than mono- or oligo-phagous species (Tukey MD = 1.44, SE = 0.36, P < 0.001 and Tukey MD = 1.00, SE = 0.42, P = 0.051, respectively) (Fig. 5 ). Density of butterflies In simple linear regressions, both range position and dispersal ability had significant negative effects on the density of the butterflies (Table 3 ). The overall multiple regression of the density of the butterflies on their range position and dispersal ability was highly significant (F 2,76 = 38.84, P < 0.001; r 2 = 0.51), and both the predictor variables had independently negative effects on butterfly density (Table 4 ). The relationships between the density and the predictor variables are depicted in Figures 6 and 7 . To study the effect of niche breadth on the density of the butterflies, we analysed the effects of larval specificity and adult habitat breadth by ANOVA and Tukey's multiple comparisons test. No significant interaction was observed between larval specificity and habitat breadth on butterfly density (ANOVA F 4,72 = 0.84, P = 0.505), and thus the interaction term was removed. However, habitat breadth had a significant main effect on butterfly density (F 2,76 = 8.49, P < 0.001). Specialist species had a greater density than intermediate (Tukey MD = 1.53, SE = 0.40, P < 0.001) or generalist species (Tukey MD = 1.62, SE = 0.49, P = 0.003), but there was no significant difference between intermediate and generalist species (Tukey MD = 0.09, SE = 0.52, P = 0.984; Fig. 8 ). Larval specificity also had an overall effect on butterfly density (F 2,76 = 5.63, P = 0.005). Monophagous species had significantly greater density than polyphagous species (Tukey MD = 1.54, SE = 0.40, P < 0.001). However, Tukey test detected no differences between monophagous and oligophagous, or between oligophagous and polyphagous species (Tukey MD = 1.03, SE = 0.52, P = 0.124 and Tukey MD = 0.51, SE = 0.47, P = 0.526, respectively) (Fig. 9 ). Phylogenetic non-independence Controlling for the phylogenetic non-independence by using the method of phylogenetically independent contrasts (CAIC) verified that none of the results reported above were artefacts of treating species as independent data points. The results with the phylogenetically independent contrasts have been tabulated in Table 5 and clearly support the previous studies on distribution, abundance or distribution-abundance relationships in which the phylogenetic non-independence not a causative factor in any of the results [ 5 , 14 , 25 , 28 , 29 , 39 , 40 , 48 ]. Discussion Density and distribution of butterflies A positive relationship between the density and distribution of species is expected to be an almost universal pattern in ecology [ 3 - 6 ]. In contrast to this, we found a strong negative relationship between density and distribution in Finnish butterflies. Here we will discuss three mechanisms-range position, niche breadth and dispersal ability – that could be responsible for generating this negative relationship. Range position According to the range position hypothesis, species are expected to inhabit increasingly fewer localities when moving along a gradient from the centre of the species' geographical distribution range toward the edges of the range [ 2 , 31 , 32 ]. In addition, density should also decline along this gradient [ 2 , 30 ]. If these two assumptions are correct, a positive density-distribution relationship in any particular region will result when species are at different positions relative to the centre of their geographical ranges [ 16 ]. However, there is little evidence for the latter of the two assumptions [ 31 , 32 ]. Moreover, metapopulation theory suggests that if patches are more isolated toward range edges, only the largest or best quality patches will be occupied, i.e. those patches that are able to support the most dense populations [ 33 ]. If this is the case, a negative density-distribution relationship will be observed. In our study, we found a strong negative relationship between range position and density indicating that species on the edge of their geographical distribution are indeed more abundant on the patches they occupy. To illustrate the effect of this relationship on the density-distribution relationship, we divided the range position of the butterflies into three classes (1155 km / 3 = 1 – 385 km, 386 – 770 km and 771 – 1155 km) and plotted these on top of the density-distribution relationship (Fig. 10 ). Species on the edge of their geographical distribution (stars) had the smallest distribution and highest density, whereas the species furthest from the edge of their geographical distribution (filled boxes) had the largest distribution and lowest density. Consequently, being at different positions relative to the centre of the geographical ranges may indeed cause a negative density-distribution relationship. Niche breadth The niche breadth hypothesis predicts that a positive density-distribution relationship arises because species that are able to use a broader range of resources are widespread and also have high density [ 2 ]. There is evidence of a positive relationship between niche breadth and distribution [ 3 , 5 , 34 - 40 ], but it is difficult to see why wider niche breadth should lead to higher density [ 5 ]. Many studies have failed to document a positive interspecific relationship between niche breadth and density (see [ 5 , 25 ]. In fact, most studies summarized in Gaston et al. [ 5 ] gave negative (although not statistically significant) relationships between niche breadth and density (see also [ 25 ]. In our study, we analysed niche breadth with two variables, adult habitat breadth and larval feeding specificity. We found that butterfly distribution was strongly positively related to adult habitat breadth and to larval feeding specificity, but more interestingly, both variables were significantly negatively related to density. To illustrate how these relationships may affect the density-distribution relationship, we plotted both habitat breadth (Fig. 11 ) and larval feeding specificity (Fig. 12 ) on the density-distribution relationship. These figures illustrate that the negative density-distribution relationship may be caused by differences in the niche breadth: habitat specialist species and monophagous species have higher abundance, but simultaneously have smaller distribution, than habitat generalist or polyphagous species. Based on the available empirical evidence, it seems that the relationship between distribution and niche breadth is generally positive [ 3 , 5 , 34 - 40 ]. If we accept this to be the case, and the density-distribution relationship is determined by niche breadth, then the form of the relationship is caused by the relationship between density and niche breadth. Dispersal ability According to the metapopulation theory, differences in dispersal ability of species may generate negative density-distribution relationships [ 3 , 21 , 33 ]. If, for any reason, species differ in their dispersal ability and dispersal ability has a positive effect on distribution, but a negative effect on density, a negative density-distribution relationship will result. We found a strong positive relationship between dispersal ability and distribution and a strong negative relationship between dispersal ability and density, which support earlier studies in butterflies [ 14 , 25 , 40 ]. We divided the dispersal ability of the butterflies into three classes (10/3 = 0 – 3.33, 3.34 – 6.66 and 6.67 – 10) and plotted the classes on the density-distribution relationship. Fig. 13 illustrates that species with the lowest dispersal ability have the highest density but lowest distribution, while species with the highest dispersal ability have the lowest density and highest distribution, leading into the observed negative relationship between density and distribution. Conclusion We conclude that many of the ecological mechanisms proposed to create a positive density-distribution relationship are also able to generate a negative relationship. Indeed, it is intriguing that many studies, which have found a positive density – distribution relationship have failed to find an ecological mechanism that would explain the observed pattern. This may not be surprising if the ecological mechanisms are more likely to generate a negative rather than positive density – distribution relationship. In support of this view we found a strong negative density-distribution relationship but what is more important also empirical support for ecological mechanisms that are able to explain the observed pattern. Our study area (Finland) is a long, northern country extending more than 1100 km from south to north. Many of the studied butterfly species meet the edge of their distributional range in Finland. On the edge of a distribution range suitable habitat patches are likely to be more isolated than in the core of the range. In such a case, only the largest and best quality patches are likely to be occupied, but because these are the best quality patches, they may support a relatively dense population [ 33 ], leading into a negative density – distribution relationship. We suggest that one important determinant of the density-distribution relationships is the geographical position of the study area and, therefore, future studies should place greater emphasis on the comparison of the relationship in different geographical areas. Methods Data set Our data is predominantly based on published literature [ 49 , 50 ] and the results of The National Butterfly Scheme in Finland (NAFI). We included all 95 butterfly species that are classified as resident or fluctuating in Finland [ 50 ] and excluded 21 species classified as migratory, irruptive or extinct [ 50 ]. Distribution of butterflies The distribution of butterfly species is based on the "Atlas of Finnish Macrolepidoptera" [ 50 ]. This atlas contains extensive and detailed distribution data of butterflies in Finland, covering all reliable records and observations of butterflies from 1747 to 1997 [ 50 ]. The data are compiled from many different sources including c. 1500 literature references and information extracted from several large museum and private collections [ 50 ]. The distribution data in the atlas are divided into old observations (before 1988) and new observations (1988 – 1997). We chose to describe the distribution of butterflies by the new observations, which represent the current distribution more accurately and also correspond more favourably with our density data. Moreover, there was a very strong positive correlation between distribution of butterflies based on old and new observations (Pearson correlation: r = 0.966, n = 95, P < 0.001). We describe the distribution for a given species as the number of 10 km × 10 km grid squares on the Finnish national coordinate system from which the species was recorded during the period 1988 – 1997 [ 50 ]. Density of butterflies The National Butterfly Scheme in Finland (NAFI) is a formal study that was established in 1991 for the purpose of monitoring the population trends of the butterflies across the country [ 51 , 52 ]. Monitoring studies may be liable to sampling biases [ 53 ], therefore explicit instructions were drafted in order to avoid any bias in sampling or reporting the abundance of butterflies [ 52 ]. NAFI provides quantitative abundance data including the 10 km × 10 km grid square of the Finnish national coordinate system in which the observation was made, the year, the number of individuals of each species observed and the number of observation days [ 52 ]. During the first ten-year period (1991–2000) of NAFI, 432 lepidopterologists participated in the study, providing data on 1 523 989 individuals, representing a total of 106 butterfly species. To obtain density for each butterfly species per 10 km × 10 km grid square, we divided the total number of individuals of each butterfly species by the number of 10 km × 10 km squares occupied by the species. Some rare butterfly species with known occurrence sites may face proportionally higher sampling effort than common species [ 53 ]. To remove the effect of sampling effort on density, we divided the density by the number of observation days. Note that observation days are the days when the observer was observing at a given square, and thus include also days when a given species was not observed. The number of observation days for each species averaged 20 815, varying between 53 and 50 595 days. It is possible that size confounds the density and distribution data if larger species are more visible and thus reported more often than smaller species. We analysed the possibility that the size of the butterfly species is a confounding factor. There is practically no dimorphism between the sizes of male and female butterflies. Therefore, as a size measure we used only the female wing span reported in Marttila et al. [ 49 ], in which the mean of a sample of 20 females was reported with an exception of some rare species with fewer individuals measured. We found no evidence of any size bias: there was no relationship between the female wing span and the density or distribution of the species (linear regression F 1,93 = 0.04; P = 0.835, r 2 = 0.00 and F 1,93 = 0.99; P = 0.987, r 2 = 0.00, respectively). Range position To determine the range position, we measured the distance (km) between the northernmost distribution record and the southernmost point (Hankoniemi) of Finland from the maps in Huldén et al. (2000). Note that the longest possible range was 1155 km. In analysis including range position, we only included species, the distribution range of which begins from southern Finland [ 50 ]. Thus, we excluded the species which occur only in Northern Finland (n = 14, [ 49 ]), and two species ( Lycaena helle and Clossiana thore ) which do not range to southern Finland. Niche breadth We describe the niche breadth for each butterfly species using two different measures: 1) larval host plant specificity, and 2) the number of habitat types occupied by the adults. We excluded from this analysis the species which occur only in Northern Finland (n = 14, [ 49 ]). This was done because their larval host plants are unknown or are not confirmed and because the habitat types of Northern Finland are unique compared to other Finnish habitat types. The data on larval host plant specificity in Finland are based on Huldén et al. [ 50 ] and Wahlberg [ 54 ]. We classified the larval host plant specificity into three classes: (1) monophages, i.e. species that feed on a single plant species; (2) oligophages, i.e. species that are restricted to one genus of food plants; and (3) polyphages, i.e., species that feed on one or more than one family of food plants. The main habitats that Finnish butterflies occupy have been categorised into four types [ 49 ]: (1) uncultivable lands (e.g. edge zones beside industrial area, harbour and storage areas, loading places, uncropped fields, and other unbuilt areas which have been exposed to human impact), (2) meadows (many kinds of non-cultivated open grasslands), (3) forest edges (e.g. road sides), and (4) bogs. Adult niche breadth is the number of habitat types in which the adults typically are found. As there were only two species ( Pieris napi and Gonepteryx rhamni ) that occupied all four main habitat types, habitat types three and four were combined. Value one represents specialist species that are limited to one habitat type, value two represents intermediate species that occur in two habitat types, and value three represents generalist species that occur in three or four habitat types. Dispersal ability Metapopulation theory predicts that species with greater dispersal ability are likely to occupy more habitat patches than species with lower dispersal ability [ 3 ]. To describe the relative dispersal ability of butterfly species, we adopted the method described in Cowley et al. [ 14 ]. We sent a questionnaire to experienced lepidopterists in Finland and asked them to give a "dispersal ability index" (0–10) for each butterfly species. In the questionnaire, a zero value indicates that a given butterfly species is extremely sedentary and a value of ten means that it is extremely mobile. To obtain a relative dispersal ability for each butterfly species, we calculated the average dispersal ability from returned questionnaires (n = 13). To ensure our measure of dispersal ability is reliable, we compared our measure with the dispersal ability estimated previously by Bink [ 55 ] (nine dispersal ability classes), Pollard and Yates [ 56 ] (three dispersal ability classes based on mark-release-recapture studies), Cowley et al. [ 14 ] (continuous variable based on questionnaires) and Cook, Dennis & Hardy [ 57 ] (continuous variable based on vagrancy in grids). The correlations between our measure and those of Bink [ 55 ], Pollard and Yates [ 56 ], Cowley et al. [ 14 ] and Cook et al. [ 57 ] were all strongly positive and significant (Pearson correlation; r = 0.672, n = 73, P < 0.001, ANOVA; F 3,27 = 8.74, r = 0.567, P < 0.001, Pearson correlation; r = 0.703, n = 31, P < 0.001 and Pearson correlation r = 0.602, n = 11, P = 0.050, respectively), indicating that our dispersal ability is in line with other independent estimates and thus reliable (see also [ 58 ]). Phylogenetic non-independence Lack of statistical independence among species for the traits of interest was tested using the method of phylogenetically independent contrasts [ 19 ] as implemented in the CAIC program [ 59 ]. Statistical control of phylogenetic non-independence requires knowledge of the phylogeny [ 19 , 27 ]. However, knowledge of the general phylogenetic relationships among butterfly species is still in a state of flux [ 60 ], and there are no studies available that look explicitly at the relationships of species in Finland. However, the recent surge of published studies on various groups of butterflies allows us to compile a likely phylogeny for Finnish butterflies (Fig. 14 ). We took the relationships of the butterfly families from [ 60 ], the relationships within Papilionidae from [ 61 ], and the relationships within Nymphalidae from various sources [ 62 - 65 ]. Relationships within Pieridae, Lycaenidae and Hesperiidae are based on current taxonomy, with morphologically well-defined groups shown as monophyletic. In the analysis all branch length were assumed equal because no estimate of evolutionary distance exist for the entire data set. However this option is justified under the assumption of punctuated evolution. Regression analysis was used to investigate the standardized linear contrasts calculated by CAIC [ 19 ]. Note that the regression lines must pass though the origin [ 66 , 67 ]. Authors' contributions JP, VK, AK and JSK compiled and analysed most of the data, and wrote the manuscript. NW constructed the phylogeny and AG conducted the phylogenetic analyses. KS provided the density data.

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555547

On evolutionary origin of cancer

Background The necessary and sufficient capabilities of cancer cell have been identified. Strikingly, this list does not include one that would seem to be a key property, namely the ability of cancer cells to kill their "host". This is believed to be a self-evident consequence of the other capabilities (e.g., metastasis), although the available evidence suggests a distinct killer function. Taking into account this unlisted property can significantly affect the current paradigm of carcinogenesis. Presentation of the hypothesis On the assumption that killer function is a key capability of the cancer cell, it is suggested that cancer has evolved as a mechanism of negative selection of mutant alleles of vitally important genes present in population. Similarly to apoptosis, which is an altruistic suicidal act of a damaged cell, cancer is an altruistic suicidal act of an individual who carries dangerous alleles and presents a hazard for genetic stability of the population. From this point of view, apoptosis is not a protection means against cancer as generally believed, but rather they are the first and second lines of defense against genome instability, respectively. Testing the hypothesis The modern DNA array technology is capable of revealing gene expression profiles responsible for killer function of cancer cell as well as those specific targets in the body that are most strongly affected by the tumor growth. Implications of the hypothesis This hypothesis suggests new avenues of cancer research as well as principally new therapeutic strategies.

Background We are now witnessing a post-genomic era of cancer research. Thousands of papers are devoted to discovering molecular mechanisms of this extremely complicated program, the latter term being understood as a prescribed sequence of events with an inevitable result. In almost all studies cancer is considered as a given entity with no attempts made, to my knowledge, to search into its evolutionary origin. However, cancer almost certainly fulfills some evolutionary tasks. First, although cancer is usually mentioned as a representative of a large group of age-related diseases, it principally differs from all the others. Cardiovascular diseases, diabetes, Alzheimer's disease, and many other diseases are manifestations of the "loss-of-function" phenotype due to degeneration and/or death of corresponding cells. Cancer, on the contrary, is an active "gain-of-function" process. Cancer cells acquire numerous new functions, including the unique abilities to adapt to a changing environment and to dodge the blows of the body's protective means, as well as a striking capacity to recruit surrounding normal tissues. The tumor transforms its natural enemies (normal cells) into unnatural allies, being able to grow only having such a paradoxical support [ 1 - 15 ]. The functional relations between normal and cancer cells are so diverse and the tumor structure is so complex and hierarchical that a growing tumor is sometimes viewed as a special developing "organ" [ 2 , 16 ]. Such an "organ" must have serious evolutionary grounds to have evolved. Second, cancer is an inevitable attribute of the animal world. It hits every species despite the fact that there are huge differences among them in the intracellular machinery, organization of signal pathways and, accordingly, in anticancer defense (or, transformation resistance, see below). In no case, however, is the anticancer defense of a species high enough to exclude completely this illness, despite presumably high cellular plasticity in this respect. This fact would indicate some evolutionary advantages of possessing such a trait. It seems likely that anticancer defense of a species is counterbalanced by opposing forces acting on the population level. In other words, cancer incidence among a species is presumably maintained at certain favorable level, which is coupled in each particular case with an evolutionary adaptation of the intracellular machinery. In conclusion, evolutionary grounds for cancer seem to be evident; it is the explanation of its expediency which presents difficulties. A clue to this problem would be the fact that cancer cell possesses, apart from the well known necessary and sufficient capabilities [ 17 ], a killer function directed against the host. Strikingly, this evident capability that seems to be a key property of the tumor cell does not attract the attention it deserves. It is believed to be a self-evident consequence of the other traits that constitute the malignant phenotype, although the available evidence would rather suggest that killer function is a distinct capability. Taking into account this overlooked property, one can view cancer as a suicidal act of an individual, since the inevitable outcome of this illness is demise (if treatment is absent or delayed). By analogy with apoptosis, which has evolved as altruistic suicidal act of a damaged cell [ 18 ] that otherwise would threaten the genetic stability of the cell population, cancer might be viewed as an individual suicidal act that brings some benefits to the population. Presentation of the hypothesis Killer function is a key capability of the cancer cell Carcinogenesis is a multistage process of accumulation of gene defects that determine the characteristic traits of the cancer cell: self-sufficiency in growth signals, insensitivity to anti-growth and pro-apoptosis signals, limitless replicative potential, sustained angiogenesis, tissue invasion, and metastasis [ 17 ]. These acquired capabilities determine the malignant phenotype of a cell but do not explain the clinical manifestations of cancer. Indeed, it seems astonishing that the human body, which consists of ca. 10 14 cells, can not endure a relatively small fraction of cancer cells (0.01 – 0.1% of the total), this burden often being incompatible with life. Rarely, the immediate cause of death is evident (brain compression, bleeding, perforation of the intestine), but in general it remains obscure. This suggests a deadly influence of cancer upon the body through some unknown mechanisms. Although each of many different forms of cancer has clinical peculiarities, the overall course of the illness and the final result are always the same. So, the notion that the tumor cell, regardless of origin, possesses a special killer function is a statement of an obvious fact. What is surprising in this regard is that no room has been allocated to it in the existing paradigm of carcinogenesis. Paraneoplastic syndromes present evidence that tumors may affect normal tissues remote from the primary site. These syndromes are extremely diverse and affect almost all organs and tissues [ 19 ]. The most frequent clinical manifestations are cachexia, anorexia, nausea, neuropathy, retinopathy, general sickness, and malfunctions of many body systems. A long time (16 to 20 months) before cancer is diagnosed, some patients show body weight loss, which is indicative that even at early stages tumors may have a generalized effect upon the body, which increases progressively with tumor growth [ 20 ]. Since, however, pronounced cachexia (a loss of more than 5% body weight) occurs in about a third of patients and becomes the cause of death in only 20% of the cases [ 21 ], it is clear that tumor cells have other, yet unrecognized means of killing the body. It remains unknown whether the paraneoplastic syndromes are direct manifestations of the killer function itself or they are mere side effects of tumor growth, such as autoimmune diseases. It is noteworthy that cancer is not always accompanied by paraneoplastic syndromes [ 22 ], yet its killer function never fails. On the other hand, the most effective treatment of paraneoplastic syndromes is specific cancer therapy, while the reverse approach, a symptomatic treatment targeted at particular manifestations of tumor growth, rarely gives positive results and never offers a radical cure. This fact suggests that most of the mentioned effects, justifying their name of para neoplastic, take place not within the killer function pathway but somewhere aside. Because of this, the term killer function will be used here, without going into its mechanism, as indicating the obvious capability of cancer cells to kill the body. The killer capability is crucial to the achievement of the final goal, body demise, whereas all the other, promoting proliferation and spread of killer cells are de facto only accessory. It seems to be a distinct capability of cancer cells, rather than a derivative of other capabilities, such as uncontrolled proliferation and metastasizing. It is unlikely that active proliferation can by itself exert such a deleterious effect since dozens of billions of cells divide daily in the human body, which is many-fold greater than the proliferation pool of the biggest tumor. Even the metastases, these relatively small foci of ectopic proliferation, can not account by themselves for the inevitable demise. The killer function is also a universal property of the cancer cell since without treatment the lethal outcome is inevitable no matter what is the type of tumor, its ability to develop metastases, recur, induce cachexia, or affect biochemical indices. Finally, this property is specific to the cancer cell, as in normal cell physiology there are no examples of such activity. The killer function seems radically different from all other acquired capabilities in that it apparently gives no selective advantage to the cancer cell. On the contrary, for the latter, as a part of the body, acquiring such a function is the same as committing suicide. This changes radically the understanding of the role of cancer cells: they can be regarded not as selfish "cheats" [ 23 ], which propagate at the expense of all others, but rather as altruists which sacrifice themselves and the whole body for the sake of some higher (apparently population) benefits, as suggested in the recent hypothesis of phenoptosis [ 24 ]. Mutations as death program trigger As cancer cells do not acquire selective advantages during realization of their killer function, it seems unlikely that the latter is created each time de novo in the same way as the other properties are, namely in the way of numerous step-by-step cycles of mutation-selection. Rather, the cells possess a built-in and ready-for-use program of deadly events, which, just like apoptosis, is launched under certain conditions and then functions automatically. In such a case, mutations of cancer-related genes are not only transformation steps, as generally considered, but also a trigger countdown mechanism that activates the death program directed against the "host". This dual activity leads to appearance of an expanding clone of killer cells progressively strengthening their effect upon the organism (Fig. 1 ). Figure 1 Step-wise accumulation of specific gene defects. Mutations (red triangles) trigger the built-in cell killer function (red asterisks). m(0), normal cells; m(1), m(2), m(3), m(n), mutant cells with 1, 2, 3, and n defects in cancer-related genes, respectively. (a), (b), (c), (n), selection "sieves" which determine the stages of transformation. The trigger "count-down" is shown on the left. Double-headed arrows indicate that transformation and trigger steps are amenable to species- and tissue-specific variations (see text). A specific feature of the genetic defects underlying the tumor phenotype is that they do not decrease cell viability (basic cellular functions, in contrast to specialized, are even enhanced). In a certain sense, these are the defects of not the cell per se but rather of the cell/organism "interface" that mediates subordination of cell functions to the common interests. Under normal conditions, numerous signal pathways tie the cell to the tissue homeostasis mechanism, whereas their defects are nothing but a step-wise liberation of the cell from its "fetters". The number of liberating mutations necessary for cell transformation to occur ranges from 4 to 12 [ 25 , 26 ], this value being a quantitative measure of the cell "transformation resistance". If one can compare the cancer cell with an explosion device aimed at destroying the body, then mutagenesis serves there the function of a trigger countdown clock with 4–12 intermediate positions (Fig. 1 ). Peto paradox Cell transformation is an extremely rare event because it requires the coincidence in a single cell of several very improbable events [ 26 - 28 ]. However, the life-span risk of human cancer is high (ca. 20%) because of a huge number of cells in the body (ca. 10 14 ) and large longevity. Indeed, no matter how unlikely is the event by itself it has a real chance to occur under such conditions. If so, one might suggest that animals with a small body weight and short life-span (e.g., rodents) should not suffer from cancer at all, while big animals (whales) should get cancer in their mothers' wombs. Reality, however, does not follow this theory's predictions (the well known Peto paradox [ 29 ]). All animals regardless of body weight and longevity suffer from cancer, but, on the other hand, cancer incidence does not threaten the species existence. The Peto paradox is explained by various transformation resistance of cells from different species [ 23 , 30 ]. This may be due to (i) different efficiency of DNA repair systems, (ii) difference among species in the degree of selective advantages acquired by the cell from similar mutations [ 30 ], (iii) different setting up of the trigger (e.g., more signal pathways have to be damaged to transform human than mouse fibroblasts [ 31 ]). The trigger is probably most reliable (i.e., has a greatest number of intermediate positions) in whale cells and least reliable in mouse cells. The Peto paradox can apparently be applied to body tissues as well [ 23 ]. Indeed, cancer develops in all human tissues, which differ greatly in the number of cells as well as their proliferation activity. Just as in the case of interspecies variability, it can be assumed that cells of different tissues have different transformation resistance [ 32 , 33 ]. The presented examples suggest that the transformation resistance of cells from different tissues and species underwent evolutionary adaptation to the selective pressure exerted by tumor growth [ 23 ]. Cancer is a local manifestation of generalized mutagenesis Most patients develop only one tumor, which presents carcinogenesis as allegedly a local process. Experiments with exposure of animal skin to chemical carcinogens as well as cases of occupational cancer, which demonstrate clearly the link between site of exposure and tumor localization, support this notion. Without questioning the significance of such observations, they seem to have an exceptional and limited character (see below). In fact, mutagenesis is intimately related to metabolism and is therefore omnipresent: every day in each cell many thousands of DNA lesions occur due to replication and repair errors, spontaneous depurination, methylcytosine deamination, reactive oxygen species attacks, and telomere shortening. This list should perhaps be extended to include the mutagenic effect of apoptosis resulting from uptake by phagocytosis of DNA from dead cells [ 34 ]. Because of imperfect repair of DNA damage, the mutation rate is estimated to vary in the range of 10 -4 –10 -8 per gene per cell division [ 26 , 27 ]; mutations occur in all tissues and increase with age [ 10 , 27 , 35 , 36 ]. Calculations show, for instance, that by the age of 65 over 10 5 mutations accumulate in the normal stem cell of human colonic crypt [ 28 ]. When a tumor nodule appears in the body, it seems to be only the tip of an iceberg, maturing in the body for decades and consisting of a multitude of damaged cells at different stages of transformation. This assumption is supported by the clinical experience showing that overt symptoms of the disease are always preceded by precancerous lesions, such as hyperplasia, metaplasia, and dysplasia. This idea finds further development in the concept of "field cancerization", i.e., large (more than 7 cm in diameter) and surrounding the tumor "patches" of damaged cells, recognized on the basis of mutations in TP53 , but remaining undetectable by routine diagnostic techniques [ 37 ]. Similar conclusions can be drawn from a notion of mutation as a random and rare event and carcinogenesis as accumulation of genetic defects. Mutagenesis can be described as the process both extensive (measured by number of affected cells) and intensive (measured by number of mutations per individual cell). Evidently, these parameters are positively correlated with each other: the wider the damaged zone, the deeper the damage of individual cells. The reverse is also true: the deeper the damage of the individual, most "advanced" cells, the wider the lesion area (this means that the very fact of a tumor appearance is, in general, the sign of a significant mutagenic lesion). In other words, appearance of the cell having a complete set of mutations (i.e., cancer cell) is accompanied by formation of a large pool of precancerous cells. The simple model of accumulation of mutant cells (i.e., cells with 1–4 mutations, the latter being the arbitrary transformation threshold) in an exponentially growing cell population is shown in Fig. 2 . With time mutant cells inevitably appear [ 28 ], since mutagenic load is increasing and the repair systems become less efficient [ 35 ]. The first to appear and start to grow is a layer of cells with one defect, then a layer of cells with two defects, and so on. Each subsequent cell layer grows quicker than the preceding one because each new mutation confers a selective advantage to affected cells [ 38 ]. Additional momentum is conferred to the entire process by acquisition of chromosomal instability [ 39 ] or a mutator phenotype [ 26 ]. At each stage of carcinogenesis, transition from quantity (of damaged cells) to quality (a cell with a new mutation) takes place, the latter having a chance to appear only from a large enough pool of its predecessors. On the whole, maturation of a tumor looks like a "pyramid" growing until a completely transformed cell appears at its top. The latter gives rise to the overt tumor. The overall process may be symbolized by a "mushroom", in which the "stem" and "cap" are the latent and overt stages, respectively (Fig. 2 ). Figure 2 Maturation of cancer "mushroom" in exponentially growing tissue. k , cycles of cell exponential growth; u , 4 × 10 -5 . a – e, stages of carcinogenesis. m(0), normal cells; m(1), m(2), m(3), and m(4), mutant cells with 1, 2, 3, and 4 mutations in cancer-related genes, respectively. Cell numbers in each layer are indicated on the right (see text). It follows from the model of multistage carcinogenesis [ 23 ] that in an exponentially growing tissue the number of mutant cells is approximated by the formula where is the mean number of cells with m mutations after k cell generations and u is the mutation rate. If we assume that the body is composed of 3.5 × 10 13 cells (i.e., 2 45 , k = 45), and u is 4 × 10 -5 (for a single gene u is 4 × 10 -7 , thus if there are one hundred cancer-related genes u becomes 4 × 10 -5 [ 40 ]), then at the moment of appearance in the body of the cell with 4 mutations (a tumor founder), there will be ~3.9 × 10 3 cells with 3 mutations, ~1.4 × 10 7 cells with 2 mutations, and ~3.1 × 10 10 cells with 1 mutation (Fig. 2d ). In other words, in the human body one cell from every thousand (0.1%) will be mutated at least in one of the cancer-related genes. In reality, mutation frequency should be much higher because cell death occurs during developmental growth so that replenishing cell divisions must increase k value significantly. This calculation, though very approximate, agrees with experimental data [ 27 ] and can give an idea of the magnitude of the mutation frequency (see also [ 28 ]). They might indicate that cancer appears as mutation frequency reaches a certain security threshold. On the other hand, this calculation showing that the human body is flooded with mutant cells prior to tumor appearance points to the validity of chemoprevention as an essential approach to controlling cancer [ 41 ]. Cancer as a mechanism of negative selection of mutant alleles As discussed above, the size of the fraction of pre-malignant cells is dependent on trigger tuning – the more mutations needed for a malignant transformation, the bigger the pool of damaged cells. Hence, the first completely transformed cell plays de facto a triple role: (i) a sensor for general mutagenesis (since its "trigger clock" counts down in accordance with accumulation of mutant cells in the body), (ii) an executioner that unleashes a built-in death program after mutagenesis exceeds some threshold level, and (iii) a founder of a clone of killer cells. Any stem cell in an organism can apparently play such a triple role, thus ensuring reliability of the mechanism. Evolutionary conservation of the death program prompts us to look for an explanation for its appearance. It may consist of the counter-selection of genetically defective individuals. As a matter of fact, if an arising tumor manifests significant whole body mutagenesis, then germ cells are most probably not an exception: a similarity of spontaneous germinal and somatic cell mutation rates was shown for human cells in vitro [ 42 ]. High levels of somatic mutation seem to be a direct reflection of the germ line mutation rate selected over evolutionary time [ 35 ]. Due to its prolonged solitary existence and relative lack of protective, repair and self-destruction mechanisms, sperm cells may be even more susceptible to genetic damage than somatic cells [ 27 , 43 , 44 ]. Besides, some gene mutations are paradoxically enriched because they confer a selective advantage to the spermatogonial cells in which they arise [ 45 , 46 ]. In conclusion, germ cells are apparently not protected from mutagenesis more reliably than somatic cells are. If so, one can hypothesize that the killer program (i.e., cancer) is unleashed in a somatic cell when its vital ("cancer-related") genes are damaged to such an extent that testifies to intolerably high mutation frequency in body tissues and, most importantly, in germ cells ("in mutant soma a mutant semen" principle). This may endanger the genetic stability of the population. Therefore, if at the cellular level cancer cells look like aggressive "cheats" [ 23 ], at the body level the process may be an altruistic suicide to remove mutant alleles from the genetic pool. While in the case of sporadic tumors the notion that cancer is a local manifestation of generalized mutagenesis is only a more or less validated assumption, it is a truism in the case of hereditary tumors [ 47 ]. Carcinogenesis in such an individual has significant "odds" compared to wild-type individuals [ 11 ] because all his cells are mutant (the trigger, in other words, has been moved one position ahead from the very outset). The association between the evident threat of germinal mutations to the population, on one hand, and their extremely high cancer risk, on the other, seems not to be by chance. The life-span risk of getting cancer for persons with germinal mutations in the suppressor genes reaches 50–80%, and their tumors, often multiple, appear at a reproductive age [ 11 ]. Owing to an extremely strong selection pressure, the alleles that predispose to cancer are very rare (ca. 1:1000 or less), suggesting that the mechanism is efficient. The most convincing argument for the hypothesis that malignant tumors have an altruistic function comes from hereditary forms of cancer in which the association "mutant semen in a mutant soma" is most evident. The altruism here is that the carriers of mutant alleles die at a reproductive age. It is germinal mutations arising in a population with a certain frequency that could have been the driving force for cancer evolution. It is evident that the greater the number of pre-cancerous stages, the more strongly the individual is protected against cancer. A computational model of cancer progression was elaborated recently to show that the appearance of an extra stage and the additional buffering, which arises as a result, reduce the impact of any single hereditary mutation and therefore allow the accumulation of more nonlethal mutations in the population [ 48 ]. Because natural selection cannot purge mutations that are mostly hidden by robust pathways, mutations will continue to accumulate until their consequences become sufficiently deleterious that they are balanced by natural selection. An additional protection from cancer by extra stages thereby leads to the evolution of partially decreased cancer mortality and significantly increased genetic variability in the population as a whole [ 49 ]. This point of view is in accordance with the assumption that if such purging mechanism as cancer was absent, deleterious mutations would be widespread in the population. There are many germinal mutations with phenotypic expression but just a few of them (affecting a small group of ~50 genes [ 50 , 51 ]) are linked with hereditary cancer. Probably, only mutations in key genes that present the greatest threat to the population are prevented by this mechanism from being spread. Many other germinal mutations are unable to pass the "sieve" of embryonic development because they induce early abortions [ 27 ]. There are a number of barriers to prevent spreading of mutant alleles in a population, and cancer seems to be only one of them. A favorable trait is retained only if it manifests itself during the reproductive period. So, the fact that cancer is predominantly a disease of the elderly would seem to be at conflict with a supposition of its evolutionary significance. This is perhaps an apparent conflict: the incidence of cancer among the young may be relatively low exactly because of the efficient selection against the adverse alleles that constantly appear in the population and exist as an inevitable background. As regards the high incidence of cancer at the old age, which seems to have no evolutionary significance, this can be explained from the viewpoint of the evolutionary theory of antagonistic pleiotropy [ 52 ]. A genetic program that has played a positive role in youth continues to be active in the older age, despite its possible counter-productive effects, simply "from force of inertia" because no correction mechanisms are available [ 53 ]. This, together with an increased mutation and weaker repair, results in the exponential growth of cancer incidence in old age [ 10 , 35 ]. Germinal mutations in functionally important genes are a strong stimulus for development of a mechanism to prevent their spread in the population. Apart from these highly penetrant rare alleles with a strong hereditary predisposition to cancer, which are merely eradicated from the population, there are many alleles that determine a weak predisposition [ 49 ] (their cumulative effect can be very significant [ 11 , 54 , 55 ]). The frequency of such alleles in a population seems to be negatively correlated with the cancer risk they contribute. There is another apparent contradiction between the thesis that tumor appearance manifests generalized mutagenesis and the numerous cases when a tumor is clearly linked with a local exposure – inflammation, bacterial infection, or UV-irradiation. The occupational cancer, on one hand, and experimentally induced tumors in animals, on the other hand, makes this association unquestionable. This linkage is probably a side effect of the evolutionary mechanism described here, in which the tumor cell plays a dual role, the mutagenesis sensor and the death program executor. In such a mechanism, a local fluctuation can unleash a process even when the overall mutation level is low (similarly, a sensor designed to respond to whole system temperature is activated by a local heating). In carcinogenesis, the hypothetical sensor/executor functions under the conditions of continuous interference and noise, which are generated by numerous external and internal locally acting mutagens. From this, many false actions ensue. If one takes the hypothesis that cancer is a means of protection, one must admit that in humans this mechanism is hyperactive and operates beyond the originally set objectives. Both the high cancer incidence in old age and the multitude of cancer cases resulting from local exposure to carcinogens are examples of this hyperactivity that obscure the true evolutionary nature of the phenomenon. Evolution hypotheses that attempt at explaining the appearance of cooperation and altruistic behavior are based on the ideas of kin or group selection [ 56 , 57 ]. Their weakness is sometimes seen as an inequality between gross individual losses and relatively small population benefits, thereby questioning the validity of the proposed mechanisms. However, in the case of germinal mutations affecting important genes, the threat of genetic imbalance in the population is perhaps so high (see [ 48 ]) that it justifies the individual losses due to such a protection means as cancer. Here again one can perceive an analogy with cell suicide, which is likewise hyperactive in "forestalling" the potentially hazardous consequences of genetic defects (a weakening of such a preventive defense is undesirable [ 58 ]). Maintaining DNA integrity is one of the main priorities of living organisms. Depending on the extent of DNA damage, three outcomes are possible: (i) small damage induces repair which restores the initial state; (ii) strong damage launches apoptosis thus preventing cell-to-cell transfer of damaged DNA; (iii) accumulating lesions, when apoptosis is impossible, trigger cancer thus preventing individual-to-individual transfer of damaged alleles of vitally important genes. From this point of view, apoptosis is not a protection means against cancer as generally believed, but rather they are the first and second lines of defense against genome instability, respectively. Testing the hypothesis A striking discovery was made recently that a tumor can survive, propagate, and spread in the body only through the unnatural help coming from normal tissues [ 6 , 13 , 59 ]. One more step further in elucidation of tumor-host relationships is yet to be made, namely, a discovery of a mechanism of killer function. Although it is this feature that imparts so much significance to malignant growth, the current paradigm of carcinogenesis does not envisage the killer function as some special property; as a result, this function does not attract the attention it deserves. Meanwhile, there are possibilities to unveil the mechanism of tumor malignancy at present. The modern DNA array technology is capable of revealing gene expression profiles responsible for killer function. This can be done by comparison of (i) malignant tumors having different expression of this trait and (ii) benign and malignant tumors. Besides, identification of genes responsible for killer function is to be supplemented, using the same technology, with serial analyses of expression profiles of various organs and tissues of tumor-bearing animals at various stages of tumor progression. This can help to identify those specific targets within the body that are most strongly affected by the tumor growth. Implications of the hypothesis Recognition of killer function as a crucial capability of cancer cells suggests not only new avenues of cancer research (see above), but also principally new therapeutic strategy. Achievement of better understanding of mechanisms of body death may help to pinpoint new targets for therapy, such as some factors presumably emitted by cancer cells (unusual cytokines, for example), that exert the deadly effect. These factors are likely to be cancer-specific, so their elimination would not entail severe side effects. The present day cancer therapy is based, without much success, on the imperative to "exterminate the evil" (i.e., the cancer cells). The essence of an alternative strategy which may turn out to be more effective is not to kill the cancer cells, but to neutralize them. Competing interests The author(s) declare that they have no competing interests.

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549608

EGFR Inhibition in Non-Small Cell Lung Cancer: Resistance, Once Again, Rears Its Ugly Head

Most patients with non-small cell lung cancer who initially respond to gefitinib or erlotinib (tyrosine kinase inhibitors) ultimately develop resistance and disease relapse. What is the mechanism for this resistance?

Kinase Inhibition for Treatment of Cancer Uncontrolled proliferation of tumor cells is a hallmark of cancer. In many types of cancer, mutations in genes that activate cellular signal transduction pathways contribute to enhanced proliferation and survival of cancer cells. One well-characterized example is mutation in tyrosine kinases, enzymes that regulate the growth and survival of cells. Tyrosine kinase activity is tightly regulated in normal cells, but is dysregulated due to mutation in some cancers, including lung cancer, resulting in enhanced proliferation and survival of cancer cells. The tyrosine kinases are attractive candidates for molecularly targeted therapy in cancer, because cancers become dependent on growth signals from the mutant tyrosine kinases. Tyrosine kinases require ATP for their enzymic activity, and thus small molecules that mimic ATP can bind to mutant kinases and inactivate them. The paradigm for tyrosine kinase inhibition as treatment for cancer using small-molecule inhibitors was first established in the context of chronic myelogenous leukemia (CML) associated with the BCR-ABL gene rearrangement [ 1 ]. Imatinib (Gleevec), a 2-phenylaminopyrimidine, is a competitive inhibitor of ATP binding to the ABL kinase, thereby inhibiting the constitutively activated BCR-ABL tyrosine kinase. Imatinib induces complete remission in most patients with CML in stable phase [ 1 ], and also has activity in CML that has progressed to blast crisis [ 2 ]. Imatinib is also a potent inhibitor of the ARG, KIT, PDGFRA, and PDGFRB tyrosine kinases. As a consequence, there have been additional dividends from the United States Federal Drug Administration approval of imatinib for treatment of BCR-ABL-positive CML. For example, imatinib is effective in treatment of chronic myelomonocytic leukemia with gene rearrangements that constitutively activate PDGFRB [ 3 ], of hypereosinophilic syndrome with activating mutations in PDGFRA [ 4 ], and of gastrointestinal stromal cell tumors associated with activating mutations in KIT [ 5 ] (all reviewed in [ 6 ]). More recently, this paradigm has been extended to treatment of non-small cell lung cancer (NSCLC). Several mutations have been identified in the context of epidermal growth factor receptor (EGFR) in patients with NSCLC that are associated with clinical response to the small-molecule EGFR inhibitors gefitinib (Iressa) or erlotinib (Tarceva) [ 7 , 8 , 9 ], including in-frame deletions such as del L747–E749;A750P in exon 19, or L858R in exon 21. Although responses are often dramatic, most responding patients ultimately develop clinical resistance and relapse of disease [ 7 , 8 , 9 ]. The basis for resistance had not been known, in part owing to the difficulty in obtaining tissue from re-biopsy at time of relapse. Resistance to Small-Molecule Tyrosine Kinase Inhibitors As might have been anticipated in treatment of cancer with any single agent, resistance to small-molecule tyrosine kinase inhibitors has emerged as a significant clinical problem. This was first appreciated in patients with CML treated with imatinib whose tumors developed resistance, and has been most extensively studied in that context. Although there are many potential mechanisms for development of clinical resistance, most cases of imatinib-resistant CML are due to point mutations in the BCR-ABL kinase domain itself, including T315I [ 10 , 11 ]. Similar mutations in the homologous residues of the kinase domains of PDGFRA (T674I) and KIT (T670I) account for imatinib resistance in some patients with hypereosinophilic syndrome and gastrointestinal stromal cell tumors, respectively [ 4 , 12 ]. These findings suggest strategies to overcome resistance that include the use of alternative small-molecule inhibitors. Indeed, about three years after the recognition of imatinib resistance mutations in BCR-ABL-positive CML, new drugs are now in clinical trials that are potent inhibitors of imatinib-resistant BCR-ABL mutants [ 13 , 14 ]. A Basis for Resistance to Small-Molecule EGFR Inhibitors in NSCLC In an elegant new study in PLoS Medicine , Pao and colleagues have identified acquired mutations in patients with NSCLC that appear to explain clinical resistance to gefitinib or erlotinib [ 15 ]. The mechanism of resistance in three patients was acquisition of a T790M substitution in EGFR that was not present at time of diagnosis, but was detected with progression of disease after initial response to gefitinib or erlotinib. T790M in the context of either transiently expressed wild-type EGFR or the mutant alleles del L474–E749;A750P or L858R impairs inhibition by gefitinib or erlotinib as assessed by autophosphorylation. Furthermore, the NSCLC cell line H1975 harbors both the L858R and T790M mutations, and is resistant to inhibition by gefitinib or erlotinib, unlike cell lines that express the L858R allele alone. In the H1975 cell line, it was possible to obtain adequate quantities of RNA to confirm that the L858R and T790M mutations are present on the same allele, as would be predicted if T790M confers resistance to inhibition of the L858R allele. Structural models of EGFR provide structural insights into these biological data. A ribbon structure of erlotinib bound to the EGFR kinase domain ( Figure 1 ) shows the threonine residue at position 790 in green and the positions of the exon 19 and L858R gain-of-function mutations. Substitution of methionine for threonine at position 790 would be predicted to result in steric hindrance of erlotinib binding to EGFR ( Figure 2 ). Figure 1 Erlotinib Bound to the EGFR Kinase Domain Schematic representation of the wild-type EGFR tyrosine kinase domain (cyan) bound to erlotinib (orange) from the Protein Data Bank ( http://www.rcsb.org/pdb/ ) entry 1M17. The threonine 790 side chain is shown in green. The positions of the phosphate-binding loop (P-loop), the αC-helix, and the activation loop (conserved structural features in kinase domains) are shown for reference. Sites of common lung-cancer-associated drug-sensitive mutations (exon 19 deletion [del] and L858R) are also depicted. (Figure: Nikola Pavletich, Structural Biology Program, Memorial Sloan-Kettering Cancer Center) Figure 2 Structural Models of EGFR Showing the T790M Resistance Mutation (A) Space-filling representation of the wild-type kinase active site (cyan) with the viewer looking down the vertical axis. The structure above the plane of the figure is omitted for clarity. The threonine 790 side chain is green, and erlotinib's molecular surface is shown as a yellow net. (B) The threonine 790 side chain is replaced by the corresponding methionine side chain from the structure of the insulin receptor tyrosine kinase (Protein Data Bank entry 1IRK). The EGFR and insulin receptor have a similar structure in this region of the active site. The methionine side chain would sterically clash with erlotinib, as shown, as well as with the related kinase inhibitor gefitinib (not shown). (Figure: Nikola Pavletich, Structural Biology Program, Memorial Sloan-Kettering Cancer Center) These observations provide convincing evidence that, at least in some patients with NSCLC, resistance to gefitinib or erlotinib can be attributed to acquisition of a T790M mutation in the context of EGFR. However, three additional patients with clinical resistance to gefitinib or erlotinib did not have the T790M mutation, nor did they have mutant KRAS alleles that have previously been shown by these same authors to confer resistance to these inhibitors [ 9 ]. Thus, mechanisms of resistance are heterogeneous. Next Steps, and Lessons Learned It will be important to identify alternative small-molecule inhibitors for the T790M resistance mutation. Structural data suggest that one compound, lapatinib, may subserve this purpose [ 16 ], but it has not been tested for biological activity in this context. New chemical screens and/or rational drug design to identify alternative inhibitors is warranted. In addition, only half of this small cohort of patients with NSCLC with clinical resistance to gefitinib or erlotinib had the T790M substitution. Efforts to identify alternative mechanisms for resistance may be guided by experience with imatinib resistance in the context of BCR-ABL, and should include full-length sequencing of EGFR to identify other resistance mutations, and analysis for evidence of gene amplification, as well as investigation of other well-characterized mechanisms of drug resistance such as drug efflux or increased drug metabolism. Pao and colleagues' superb study also highlights several important points that may guide development of kinase-targeted therapies in the future. It is clear that, to the extent that small-molecule kinase inhibitors are effective as single agents in treatment of cancer, resistance will develop. Furthermore, based on previous experience, some of these patients are likely to harbor acquired point mutations in the target kinase that confer resistance. Resistance mutations identified via in vitro screens have shown a high degree of correlation with those that develop in vivo, as shown in screens for imatinib-resistant BCR-ABL mutants [ 11 ] and PKC412-resistant FLT3 mutants [ 17 ], as well as the T790M resistance mutation to gefitinib in the context of EGFR [ 18 ]. Thus, in vitro screens for mutations that confer resistance to kinase inhibitors are warranted, followed by efforts to identify drugs that overcome resistance. This proactive approach should shorten the time frame for new drug development. These findings also emphasize the critical need for re-biopsy of patients with cancer treated with molecularly targeted therapies at time of relapse. Tissue acquisition is more challenging in solid tumors than for hematopoietic malignancies, and may entail risk. Nonetheless, it is clear that data derived from such analyses will be essential to inform approaches to improving therapy for NSCLC and other solid tumors.

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548516

Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a Containment Level-III Laboratory as part of a Laboratory Risk Assessment Program

Background In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating /chemical fixation) may not consistently kill MTB organisms. Methods An inclusive viability study ( n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks. Results Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80°C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again ( n = 18), resulting in 0% viability. Conclusions This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures.

Background Mycobacterium tuberculosis (MTB), the causative organism of tuberculosis, has the distinction of repeatedly being ranked within the top five most commonly laboratory-acquired infections (LAIs) [ 1 - 3 ]. In 1976, Robert Pike prepared an extensive summary based on both published reports and surveys of 3921 LAIs that included both M. tuberculosis and other pathogens as the infectious agent [ 3 ]. He reported that laboratory and mortuary workers that are exposed to tubercle material have a TB incidence rate three times higher than that of the general population and indicated that only 18% of infections could be traced back to a known event. In 1987, a 25 year review at the National Animal Disease Center (NADC) described while only 35% of infections at the strict Biological Laboratory at Fort Detrick, MD, had a reportable, documented cause, the NADC could not account for 73% of LAIs occurring at its own facility [ 2 ]. With these reported statistics, it is negligent not to consider aerosol exposure in the absence of a known infecting episode, such as a needle prick [ 2 ]. A more recent report from 2003 demonstrated rates from 2 to 6.6 % of TB conversion among heath care workers (HCWs) in New York [ 4 ], in spite of current knowledge on precautions and safety measures in place. Furthermore, surveys suggest actual incidence of LAIs with MTB is greater than the amount of reported cases illustrate: these occurrences are likely underestimated due to the nature and length of the disease progression (i.e. workers move or retire before becoming symptomatic), and underreported to the social stigma attached [ 1 , 3 , 5 ]. Due to the nature of this organism, containment level three (CL3) laboratory operational and physical requirements have been recommended for manipulation of the live organism in North America [ 6 ]. Therefore, one would hypothesize that working in a CL3 with personal protective equipment including a respirator would be adequate to protect the worker. However, since conversions are still occurring, it is appropriate to consider the possibility that the procedure one is using to deactivate and extract material from the organism is not 100% efficient. Currently, the application of molecular methodologies for rapid diagnostics of MTB, such as nucleic -acid amplification based identification and subtyping schemes, in addition to extensive genomic and proteomic research in this area, necessitates the removal of material derived from this organism out of a CL3 laboratory to perform the work in a less restrictive containment level 2 (CL2) rated area. Commonly, due to both limited CL3 space, costs and preventative maintenance needs, high-tech equipment such as liquid handling robots and sequencers are shared and can be housed in a central "DNA core" CL2 laboratory. To consider the biosafety impact of removing organism material from a CL3 and manipulating it in a CL2, part of a risk assessment undertaken included the review of current literature on decontamination verification and viability testing of M. tuberculosis . The existing literature is limited in regards to viability testing of material derived from MTB with respect to safe manipulation outside of a Biological Safety Cabinet (BSC). To date, no conclusive study has confirmed that this organism is noninfectious after theoretical 'deactivation' steps. A few reports concerning survival after different heating kill treatments for DNA extractions [ 7 - 9 ], heat fixing of smears [ 10 , 11 ] and chemical fixation [ 12 ] were found. Since, by definition, a risk assessment is based on, but not limited to, the properties of the agent used, personal risk factors, manipulation techniques, and the training and experience of staff, it is necessary to develop a method encompassing these factors to validate the safe removal of material derived from MTB from a CL3 laboratory for use in other laboratory areas. This approach was applied to all our methodologies and took into consideration variables such as interpersonal technique, culture load, temperature fluctuations and statistical significance. Methods The current viability study consisted of the evaluation of a total of 226 material extracts, consisting of 208 initial extracts and 18 extracts tested after revision of faulty protocols (figure 1 ). For each test performed, laboratory staff members sub-cultured a proportion (100 uL) of the resulting material to Bactec 12B radiometric medium (Becton Dickinson, Oakville, ON) that was kept for eight weeks to observe for growth. The vials were incubated at 37°C and the growth index (GI) was read weekly on BACTEC 460 machines. All vials with positive GIs were sub-cultured to tryptic soy agar (TSA) with 5% sheep blood, Middlebrook 7H10 agar and stained with the Kinyoun method for the presence of acid-fast bacilli (AFB). Those positive for AFB were examined for the presence of MTB using a DNA probe specific for M. tuberculosis complex, Accuprobe (GenProbe, San Diego, CA). Methods that resulted in viable MTB were revised and retested. All work completed was performed in a CL3 environment, using Class II Type B2 biosafety cabinets (BSCs), and personal protective equipment (PPE) including N100 particulate respirators, double gloves, and protective gowns. The following describe the protocols used in our laboratory at the start of this study. Figure 1 Overall study flow chart with results after eight weeks incubation of material at 37°C DNA extraction for IS6110 fingerprinting This was performed according to the standard protocol [ 13 ]. A total of 125 Bactec 12Bs were inoculated with 0.1 mL of lysate materials. For initial heat deactivation steps, 1.5 mL screw-cap tubes were placed in a water-bath maintained at 80°C. The tubes were not submerged. Three technicians normally performing the procedure inoculated 12B media at different steps of the lysate protocol. Twenty-four samples were processed up to the point of lysozyme and proteinase K addition, prior to CTAB addition, and tested for viability. In 30 other samples the protocol was continued with the addition of choroform:isoamyl alcohol and subsequently centrifuged into organic and aqueous phases. For 10 samples, the organic (bottom) layer that is normally discarded was inoculated into 12B vials and the remaining 20 samples had their aqueous (top) layer planted. Sixty-one samples were processed to completion and inoculated into 12B vials. Finally, 10 extracts from frozen storage (-20°C) were retroactively tested by inoculation to 12B media. Crude lysate preparation A total of 45 lysates used for PCR testing were tested for viability. Thirty-six lysates were prepared from actively growing cultures by three technicians that normally perform the testing procedure. Briefly, a loopful of culture is placed in a pyrex glass bottle containing 1 mL distilled water and glass beads and vortexed in a BSC. Generally this suspension has a turbidity of > 1 McFarland. This suspension is transferred to a screw-capped vial and is placed in a boiling water bath for 10 minutes followed by transfer into a tube containing 0.5 mm silica beads and mechanically lysed for 2 minutes with a Mini-8-Beadbeater (BioSpec Products, Bartlesville, OK). The resulting lysate is spun down for removal of debris and the supernatant transferred to a new tube to be used for PCR. Lysates were processed in duplicate and the position in water bath was noted (i.e. periphery or centre). A proportion of lysates (100 uL) were planted pre and post bead-beating. An additional nine previously frozen lysates stored at -20°C were also inoculated into 12B media ( n = 45 lysates). Smear preparation Slides were prepared in duplicate according to our standard protocol: a loopful of organism is suspended in water with beads, vortexed and one drop added to a glass slide containing one drop of 0.5% phenol serum (made in-house). The slides were allowed to dry and were placed on a 95°C slide-warmer (Lab-Line Instruments, Melrose Park, IL) for 15, 30, 45 minutes, 1, 1.5 and 2 hours ( n = 12 slides). The heat fixed slide material was emulsified using a sterile swab and sterile distilled water. The suspension was transferred to a pyrex glass bottle containing 1 mL sterile water with beads, vortexed and 100 μl was inoculated into a 12B media vial. Protein extraction Twenty-six extractions of M. tuberculosis organism were processed as follows. A volume of 25 mL of Middlebrook 7H9 broth was inoculated and incubated at 37°C for 20 days. The culture was centrifuged at 1900 × g for 15 minutes at room temperature and the pellet was resuspended with 2 ml of ice-cold phosphate buffer solution (PBS) pH 6.8 supplemented with 1% Tween-80. The sample was centrifuged as before at 4°C and the pellet washed twice in ice-cold 1% Tween-80 in PBS. Silicon beads (0.5 mm) and 500μl lysis buffer (2% CHAPS, 2% Triton X-100, 9.5 M urea and 1% DTT/TBP in water) were added to the pellet. The sample was mechanically lysed for 30 seconds and cooled on ice for 30 seconds, repeated eight times. The sample was then filter sterilized with a PES membrane, 0.22 μm Millipore filter unit (Millipore, Etobicoke, ON) into a 2 ml microcentrifuge tube on ice and stored at -80°C. Results In total, 226 samples were tested for viability. As described below, 21 samples grew within the 8-week period, with 16 of those samples from three separate procedures yielding positive growth for M. tuberculosis (figure 1 ). Of the 125 RFLP lysates tested, 2 of 24 sample materials that were tested for viability before addition of CTAB remained viable for M. tuberculosis . Following the complete extraction protocol as described [ 13 ], cultured samples yielded 0% viability for M. tuberculosis . Four of ten 12B media vials planted with lysates from frozen storage did show growth, and upon further investigation were determined to be contaminants (Table 1 ). All other vials had negative GI readings after 8 weeks of incubation. Table 1 Results of positive Bactec 12B vials ( n = 21). Description of material Growth on TSA + 5% SBA @ 48 hrs Growth on Middlebrook 7H11 Kinyoun Stain direct from positive 12B Probe Conclusion RFLP lysate – before CTAB addition (2 vials) - + , MTB morphology AFB+ (4+), serpentine cording ND Viable MTB 1 RFLP lysate from frozen storage #1 - + , KS of growth AFB - 4 AFB/slide, morph not consistent with TB MTB- contaminant present – ubiquitous mycobacteria RFLP lysate from frozen storage #2 - + , KS of growth AFB - 15 AFB/slide, morph not consistent with TB MTB- contamination present – ubiquitous mycobacteria RFLP lysate from frozen storage #3 & #4 - + , KS of growth AFB - No AFB contamination present Boiled lysate - + , Smooth colonies not consistent with MTB AFB+ 5–10/field, clumping, no obvious serpentine cording MTB- ubiquitous mycobacteria Smear, 1 h at 95°C & Smear, 2 h at 95°C 2 - + , MTB morphology AFB+ (4+), serpentine cording MTB+ Viable MTB Protein extracts (2) - +, MTB morphology AFB+ (4+), serpentine cording MTB+ Viable MTB 1 Accuprobe was not performed on these isolates, as MTB was expected due to morphology observed in addition to data from prior studies [7]. 2 All 12Bs from smears ( n = 12) were positive, only 2 were chosen as representative for further testing Definitions: KS = Kinyoun stain, AFB = acid-fast bacilli, MTB = M. tuberculosis Forty-five vials of TB PCR lysates were tested for viability. With the exception of one sample that was positive for growth and was later shown to be a contaminant (Table 1 ), all were negative for viable M. tuberculosis . All vials containing suspensions from slide material that had been incubated for less than 1 hour on the slide-warmer exhibited identical growth rates, with a positive 12B vial in two weeks. Vials containing suspensions from slides that were incubated greater than 1 hour exhibited identical growth rates and had a positive 12B vials in 3–4 weeks. All 12 12B vials were positive for growth at 4 weeks incubation. Two representative vials were chosen for further analysis (1 hour and 2 hour slide incubations). These 12B culture vials were confirmed positive for M. tuberculosis complex using Accuprobe, in addition to displaying colony morphology consistent with TB on subculture to Middlebrook 7H10 agar and being AFB positive with the Kinyoun stain. Two of 25 vials tested for viability from protein extractions became positive. Confirmation of viable MTB in these test vials was confirmed as described above. Based on these results, the methods for slide fixation and protein extraction were modified. Slides were fixed prior to staining using 5% phenol in ethanol according to Chedore et al . [ 10 ], and protein extractions samples were centrifuged at 4°C after lysis steps to rid the supernatant of cell debris before filter sterilization. These modified tests were planted again for growth of MTB, resulting in 0% organism viability for every test performed by each laboratory worker (Table 1 ). To date, ongoing viability testing of these tests have not shown any growth (data not shown). Discussion There are few publications that review the efficacy of methodologies that render material extracts from a M. tuberculosis culture non-viable [ 7 - 10 ]. One such study by Bemer-Melchior et al . was prompted by a case of pulmonary tuberculosis acquired by a laboratory technician performing the standard method for IS6110-RFLP in a CL3 mycobacteriology [ 7 ]. In this study, placing the organisms in 80°C for 20 minutes gave breakthrough growth, which was also observed in our laboratory. Conflicting information exists which claims 100% loss of viability using this method, however, this report outlines potential reasons for the difference, such as the complete submersion of sample tubes, or volume and density of the suspension [ 9 ]. Bemer-Melchior et al . also found that submerging the culture at 100°C for 5 minutes rendered the sample completely non-infectious; data that was supported by work previously done in 1994 by Zwadyk et al. [ 8 ]. Although these studies were the first of its kind in verifying the safety of laboratory protocols and addressed issues regarding temperature, timing, cell density and sample volume in deactivating MTB cultures, they clearly demonstrated that certain methods of heating may not be efficient in complete sterilization of a MTB culture. With the unique cell wall characteristics and ability of MTB to clump (or cord), variables to be considered include cell mass density, actual temperature reached and actual time exposed to the heat source. Zwadyk et al. showed that using a 95°C heat block failed to completely inactivate the culture, and in fact, using an internal temperature probe demonstrated that the tube did not reach the intended temperature even after 20 minutes [ 8 ]. Another study issued a caution in removing MTB fixed with 0.5 – 1% glutaraldehyde from a CL3 as the process used to sterilize the culture failed [ 12 ]. Again, both the efficacy of rendering MTB material non-viable as well as the effect of the clumping or cording factors not being known was questioned. Therefore, testing all deactivation methods was recommended for all preparations in this laboratory [ 12 ]. In preparing a risk assessment for our CL3, it became evident that this was a necessary course of action to reduce the risk of LAIs for both CL3 and CL2 mycobacteriology staff as well as other non-mycobacteriology laboratory staff in the shared CL2 environment. Genomic extractions, the most commonly performed test in our laboratory, are conducted using the standardized method outlined in 1997 by Van Embden et al for the purpose of RFLP typing [ 13 ]. Prior viability testing of this method in our lab showed that lysates contained viable MTB with the initial deactivation steps of this protocol, heating for 20 minutes at 80°C with addition of lysozyme and proteinase K, and thus could not be removed from containment until DNA extraction with CTAB. It is presumable that the small rate of viability observed (2 of 24), which is similar to what was seen by Bemer-Melchior et al ., resulted from incompletely submerging sample tubes or cell density within the tube [ 9 ]. However, phenol-chloroform extraction such as the CTAB method and heating of specimens should be lethal to mycobacteria since phenolic-based disinfectants have been shown to be tuberculocidal [ 14 ], and to date our results reflect this fact. To both preserve the integrity of genomic DNA for the use of fingerprinting and allow the sample to be further processed safely outside a CL3 laboratory, it is recommended to complete DNA extractions with CTAB as per the standard protocol to confirm the complete inactivation of M. tuberculosis . Further sampling of the DNA extractions performed in our laboratory (10% of each lysate batch) is ongoing for quality assurance purposes, i.e. to confirm integrity of reagents as well as to monitor staff performance and adherence to protocol. In addition, continuous sampling attaches statistical significance to the claim that this method ensures that the extracted material is 100% non-viable and guarantees staff safety. For procedures that do not require intact, high quality DNA such as PCR testing, our laboratory depends on the much more expedient lysate method of boiling culture at 100°C for 10 minutes, followed by mechanical lysis for 2 minutes to release DNA. Although crude, this procedure is adequate for our PCR testing needs, has been shown to completely destroy live organism in our laboratory, and is consistent with other studies [ 7 , 8 ]. The study by Zwadyk et al. concluded that inactivating mycobacteria by heat lysing at a temperature of 100°C for 30 minutes did not inhibit its ability to be amplified by PCR or strand displacement amplification [ 8 ]. Furthermore, this study has shown that inoculating the boiled lysate alone without mechanical lysis was adequate in rendering the sample non-viable. The viability testing of the two methods outlined above for DNA extractions were performed by various technicians who routinely follow these procedures, lending interpersonal variability to the study. It was demonstrated that the small nuances to procedure, such as the varying density of culture used, did not affect the method employed to deactivate the organism. The last routine test assessed for organism viability was the slide preparation. While phenolics are known to be tuberculocidal, the use of phenol serum alone or in conjunction with heat in slide preparation is not sufficient for killing of the live organism. Flaming of the slide was not an option due to facility requirements that prohibit open flames inside a BSC, despite this, flaming of smear material was found in prior studies to unsuccessfully inactivate smear material [ 10 ]. The primary method for slide fixation was a 2-hour incubation at 95°C on the slide-warmer, which was assumed adequate for staining purposes. Slides were then transported to an area where respirator usage was not mandatory. It was discovered that every slide preparation was positive for viable TB. As a consequence of the viability testing and subsequent risk analysis, the protocol was altered to include chemical fixation with a 5% fixative of phenol:ethanol [ 10 ]. This allows lab staff to safely remove slides from a CL3 area and examine slides under a microscope without interference by the need to wear a bulky respirator. Examining laboratory protocols for staff and building safety should be an integral part of any CL3 laboratory program. Implementation of a policy to test all procedures used routinely for viability in addition to new methodologies is highly recommended. As an example, applying this policy to a new procedure being utilized in the research arm of our laboratory proved valid: a protein extraction method being developed initially showed that of 2/26 tested samples yielded viable MTB. The researcher concluded that it was due to a clogging of the filter from large particles in the lysed culture used to isolate the proteins and revised the protocol to centrifuge the mixture after final lysis steps to remove cellular debris before filtration. This has proven successful so far, and continuous sampling of extracts is ongoing before removing the material from the CL3. To date, all have been negative. Conclusions It is imperative to evaluate and record the actual rate of viability of DNA lysates from deactivated MTB cultures in individual laboratory settings [ 12 ]. This includes all material removed from the CL3 area. This process is vital to establish biosafety validated procedures and practices to protect laboratory workers conducting these procedures [ 2 ]. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KB carried out the studies involving genomic DNA extractions and drafted original and final manuscripts. TB tested all smear preparations, compiled viability data and assisted with the writing of the manuscript. MS contributed by testing protein extractions and writing the method used. CT contributed by testing of crude lysates and editing of manuscript. MS contributed by testing protein extractions and writing the method used. AK and JW conceived of the study, and participated in its design and ongoing coordination. All authors read and approved the final manuscript Pre-publication history The pre-publication history for this paper can be accessed here:

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548516.xml

544853

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli .

Background The production of purified proteins is important for several experimental approaches aimed to assign gene function including antibody generation for immunocytochemistry and immunoprecipitation studies [ 1 - 3 ], in vitro mapping of protein – protein, protein – DNA or protein – RNA interactions [ 4 , 5 ] and structure determination [ 6 ]. The availability of proteins is also important for biomedical applications such as small molecule drug discovery and the production of therapeutic proteins and vaccines. In these situations it is essential to be able to reliably express the proteins in a heterologous system and purify them so that they possess the same folds and structure as they would in a natural in vivo state. To achieve this on a whole proteome scale a generic approach must be taken to the expression of protein families, unlike the traditional approach of protein chemistry in optimising the isolation of individual proteins on a case by case basis. E. coli has been the expression system of choice for the majority of laboratories engaged in high-throughput, multi-plexed cloning, expression and purification of proteins for structural genomics [ 7 ]. The advantages of E. coli as an expression host include well studied physiology, genetics and availability of advanced genetic tools [ 8 - 10 ], rapid growth, high-level protein production rates achieving up to 10–30% of total cellular protein, ease of handling in a standard molecular biology laboratory, low cost and the ability to multiplex both expression screening [ 11 ] and protein production [ 12 ]. There are however several disadvantages, particularly for eukaryotic proteins, of expression in a prokaryotic system. The lack of eukaryotic chaperones, specialised post-translational modifications, ability to be targeted to sub-cellular locations or to form complexes with stabilising binding partners can result in protein mis-folding and aggregation. For example, when 2078 randomly selected C. elegans full-length genes were cloned and expressed in E. coli only 11 % yielded soluble protein [ 13 ]. Similarly for 44 cloned human proteins, 12 were expressed solubly and 4 purified to homogeneity [ 14 ]. With the exception of full-length membrane proteins, the property of protein solubility has been shown to be a good indicator of correct folding as determined by functional binding [ 15 , 16 ] or enzymatic [ 17 ] assays. Purification of inclusion bodies and in vitro refolding has been used in a number of cases, but refolding conditions are highly protein specific and so unlikely to be useful for high-throughput protein expression. There are several fall-back strategies for expression of correctly folded eukaryotic proteins in E. coli one of which is to truncate long multi-domain proteins into separate domains, as has been performed for the Ephb2 receptor [ 15 , 18 , 19 ]. Reducing translation rates so that proteins have an increased chance of folding into a native state prior to aggregating with folding intermediates, can be successful by lowering the temperature after induction [ 20 ] or inducing with lower concentrations of IPTG [ 21 ]. Alternate approaches include: co-expressing stabilising binding partners (see review [ 7 ]) or chaperones [ 22 ]; the induction of chaperones by heat shock [ 23 ] or chemical treatment [ 24 ]; or the use of genetically modified host-strains that can conduct oxidative protein folding in the cytoplasm [ 25 , 26 ], over-express rare tRNAs [ 27 ] or lipid rafts [ 28 ]. Perhaps one of the most successful generic strategies to enhance the expression of soluble proteins is the fusion with solubility enhancing tags, such as maltose binding protein (MBP), thioredoxin (Trx) and glutathione-S-transferase (GST) [ 29 - 31 ]. The aim of this work was to ask if it is possible to derive some general conclusions regarding which expression strategy would most likely result in the expression of soluble, functionally active mammalian protein on a family-by-family or domain-by-domain basis. A deep-mining approach was taken to maximise the chances of successful expression by examining the soluble expression of 30 different proteins using 14 different expression vectors. This study allowed us to make several conclusions regarding the best strategies to adopt for the soluble expression of different mammalian proteins in bacteria. The conclusions were tested by the expression of an additional 95 mammalian proteins. Results Expression clone construction The 30 proteins chosen for this expression study are listed in Table 1 . With the exception of GFP, they are all human or mouse proteins, and represent several diverse protein families with extra-cellular, cytoplasmic and nuclear cell locations. The list includes a mixture of full-length and truncated proteins expected to be easy or more challenging to express in a bacterial system. Protein truncations were designed to express individual domains annotated from the SwissProt [ 32 ] or Pfam [ 33 ] databases or following previous examples of successful expression [ 15 ]. The genes were isolated from cDNA using a nested PCR strategy [ 34 ] or provided by the FlexGene Consortium and sequence confirmed. A recombinational cloning strategy was employed termed "GATEWAY" cloning [ 35 , 36 ] based on a modification of the phage lambda site-specific recombination system [ 37 ]. Primers were designed using the nearest neighbour algorithm [ 38 ] and open reading frames (ORFs) were PCR amplified from first strand cDNA with 5' attB1 and 3' attB2 linkers and then recombined with pDONR221 (Invitrogen) to give a set of entry clones which were sequence confirmed and then recombined with various destination vectors to give the expression constructs. Two sets of clones for each ORF were generated with and without stop codons for expression with N or C-terminal tags respectively. Recombinational cloning was useful in this study where the same set of ORFs could be cloned into a large set of different expression vectors without the requirement to check for compatible restriction sites in each vector or their absence within the ORFs. For this study a set of destination vectors were constructed by modifying pET-DEST42 (see Materials and Methods). The T7 promoter was chosen over other promoters commonly used for bacterial expression because of the high specificity and processivity of T7 RNA polymerase and the wide choice of expression strains currently available. Briefly, multicloning sites were created either 5' of the attR1 or 3' of the attR2 recombination sites for insertion of DNA inserts encoding N or C-terminal tags respectively. The expression vectors contained a T7lac promoter [ 39 ] for improved control of basal expression. The N-terminal tag expression vectors contained a sequence at the translational start site to provide a partial match with the down-stream box (ATG AAT CAC CAT), shown to provide enhancement of translation [ 40 ] and a decahistidine (H10) tag for enhanced affinity for Nickel resins compared with hexahistidine (H6) tags (data not shown). A fusion partner was inserted between the H10 tag and recombination sites to examine the effect on soluble protein expression. Unlike previous tag comparisons [ 29 - 31 ] here the same promoter and 5'-UTR sequence was employed so that any expression differences observed would be purely due to the presence the fusion partner. A vector was also included in this study (pDEST17) with a T7 promoter and no downstream lac operator, which would add a H6 tag at the N-terminus (Figure 1 ). Effect of different N-terminal fusions on expression Expression plasmids generated by recombination reactions were used to transform E. coli BL21(DE3), an expression strain containing chromosomally integrated T7 RNA polymerase gene (λDE3 lysogen) under the control of the lacUV5 promoter. To handle a large number of expression experiments (420 total) and associated manipulations to screen for total and soluble expression in E. coli, the recombinational cloning, transformation, growth of expression cultures and cell lysis and filtration separation of insoluble protein were performed in 96-well plate format. Figure 2 shows Western blots for total and soluble protein expression 2 hours after induction with 1 mM IPTG as described in Materials and Methods. The method for separating total from soluble proteins was based on that of Knaust and Nordlund [ 11 ] and consisted of detergent lysis of harvested cells followed by filtration through a 0.65 μm 96-well filter plate, which separates larger inclusion bodies from the soluble fraction. The filtration method agrees well with traditional centrifugation methods to separate soluble from insoluble protein [ 11 , 41 ] and has the advantage that multiple samples can be processed in parallel. Quantitation was achieved by separating the proteins by SDS-PAGE, electro-blotting onto PVDF membranes and detecting His tagged proteins with an anti-His5 monoclonal antibody followed by probing with an anti-mouse Cy-5 labelled antibody. The advantage of expression analysis by Western blot, compared to dot-blots, is that this allows one to quantitate the expression levels of full-length constructs and eliminate the contribution from cleaved protein tag. It was found that Western blots based on fluorescence detection [ 42 ] gave a greater dynamic range of detection compared with detection based on enzymatic amplification such as horse radish peroxidase (data not shown). A His-tagged protein molecular weight ladder was used for normalisation to eliminate any blot to blot variation. Table 2 shows the results of this analysis, quantitating expression yields in terms of mg expressed protein per litre of induction media for total and soluble expression. Expression yields greater than 2 mg/l are highlighted in bold. Looking first at the results for total (soluble and insoluble) expression, no clear patterns emerge for the various expression vectors used. With the exception of CASP2, CDKN2A, Trp53, EGFR(TK), FOS and CD44 most proteins expressed well across all expression vectors. Interesting differences are apparent however when one looks at the production of soluble protein. Using decahistidine green fluorescent protein (H10-GFP) or decahistidine glutathione-S-transferase (H10-GST) as fusion partners at the N-terminus gave poor yields of soluble intact product. This may not be because they were poor at promoting soluble expression but because they were prone to proteolysis during cell lysis reducing the yield of full-length soluble protein. A set of proteins (GFP, RAF1(Ras-bd), HRAS, mdm2(p53-bd), Ephb2(TK) and CCND2) gave high soluble expression levels in the baseline N-terminal decahistidine vector, which was not improved when expressed as decahistidine thioredoxin (H10-Trx) or decahistidine maltose binding protein (H10-MBP) fusions. The molecular weight of these proteins ranged from 9 – 35 Kda and averaged 22.8 Kda. These proteins are all expressed in the cytoplasm, have an average of 1 low-complexity region, 3.8 contiguous hydrophobic amino acids (hp_aa), pI of 6.6, grand average of hydropathicity index (termed GRAVY[ 43 ] where increased positive number indicates increased hydrophobicity) of -0.32, 2.6% cysteine residues and no coiled-coil structures. A second grouping of proteins was observed where soluble expression was improved when expressed as H10-Trx or H10-MBP fusions compared with the H10 tag alone. This grouping included GRB2, Efnb2(EC1 or 2), MAD, MAX, Efna1 (FL and EC). The molecular weight of these proteins ranged from 16 – 25 Kda and averaged 20.5 Kda. These proteins were a mixture of those expressed in the cytoplasm, nucleus and extra-cellular, have an average of 0.71 low-complexity regions, 3.6 contiguous hydrophobic amino acids (hp_aa), pI of 6.8, GRAVY score of -0.79 and 1.7% cysteines. A third set of proteins resulted in almost undetectable soluble expression with a H10 tag but good expression with H10-Trx or H10-MBP fusions. These included CDK2, FLI1, CDKN-1B, mdm2, GATA2, Ephb2(LB) and CASP2 with molecular weights ranging from 22.5 – 54.5 Kda, with an average molecular weight of 40.4 Kda. These proteins were also a mixture cytoplasmic, nuclear and extra-cellular proteins, have an average of 2 low-complexity regions, 5 contiguous hydrophobic amino acids (hp_aa), pI of 6.9, GRAVY score of -0.55 and 2.3% cysteines. Finally a set of proteins was grouped (MMP1, FOS, EGFR(TK), Trp53, CD44) where very low (< 1 mg/l) soluble full-length expression was observed, even when expressed as MBP or Trx fusions. Here the molecular weight ranged from 40.7 – 81.6 Kda and averaged 51.4 kDa. These proteins were a mixture of those expressed in the cytoplasm, nucleus and extra-cellular, have an average of 3 low-complexity regions, 5.6 contiguous hydrophobic amino acids (hp_aa), pI of 5.7, GRAVY score of -0.50 and 1.8% cysteine content. Comparing the 20 mammalian proteins where there are examples in all 6 expression vectors the average yields of soluble protein for the H10, H10-GFP, H10-GST, H10-Trx and H10-MBP tags are 3.3, 1.0, 1.4, 6.0 and 5.8 mg per litre of culture. This ranks the ability of the tag fusions to produce full-length soluble protein as H10-Trx ~ H10-MBP > H10 > H10-GST > H10-GFP. The pDEST17 vector (which encodes a H6 tag) was dramatically poorer at expressing soluble protein compared with the vector pN110 (which encodes a H10 tag), with average soluble expression yields of 0.8 and 3.3 mg per litre of culture respectively. Both vectors contain T7 RNA polymerase promoters, but pN110 also contains a lac operator (lacO) downstream of the promoter and the gene encoding the lac repressor (lacI) for tighter control of gene expression. This may result in a faster rate of transcript synthesis, after induction with IPTG, and hence translation rates (due to an increased concentration of mRNA) for pDEST17 compared with pN110. If translation rate exceeds the rate of protein folding, then increased production of insoluble protein would occur. Effect of different C-terminal fusions on expression A similar study was performed where the 30 ORFs were cloned into 8 different C-terminal tag expression vectors shown in Figure 1 . C-terminal fusions studied here included V5-H6 or H10 or protein fusions MBP, GST, Trx, murine or human dihydrofolate reductase (Dhfr or DHFR respectively), all with H10 at the C-terminus. The expression screen and quantitation of total and soluble protein expression was performed as for the N-terminal tag study. Figure 3 shows the fluorescence western blots for this C-terminal tag study. Here a greater number of constructs were observed with either undetectable or low levels of expression compared with the N-terminal tag study. Table 3 quantitates the Western blot data for the intact fusion products, with expression yields greater than 2 mg/l in bold. The last row of the table describes the average expression yield for each C-terminal fusion partner. For total protein expression levels there are large expression level differences observed between the various C-terminal tags. The C-terminal decahistidine tag was particularly poor here with an average total expression yield of only 0.7 mg/l compared with 7.3 mg/l when this tag was fused to the N-terminus. In contrast the C-terminal MBP-H10 tag resulted in an average total expression yield of 20.2 mg/l. The ranking of the C-terminal fusion partners in promoting total expression was MBP-H10 > GST-H10 > V5-H6 > Trx-H10 > Dhfr-H10 > DHFR-H10 > GFP-H10 > H10. MBP-H10 was the most effective tag at the C-terminus to promote protein solubility with an average construct full-length soluble yield of 5.0 mg/l, which compares well with an average of 5.8 mg/l when this tag is fused at the N-terminus. The order of C-terminal tags to promote soluble expression was similar for total expression: MBP-H10 > GST-H10 > V5-H6 > Dhfr-H10 ~ GFP-H10 ~ Trx-H10 > H10 ~ DHFR-H10. Thioredoxin was not as effective a solubility enhancing tag when fused at the C-terminus with an average soluble yield of only 0.7 mg/l compared with 6.0 mg/l when fused to the N-terminus. Several correlations with protein features are seen when one groups the MPB fusions according to soluble protein expression levels. For the first group, where soluble expression levels were in the range of 5 – 50 mg/l, the average molecular weight, pI and GRAVY score were 20.6 KDa, 5.9 and -0.58 respectively. The average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 3.1, 0.56 and 0.22 respectively. The second group displayed soluble expression levels between 1 – 5 mg/l. Here, the average molecular weight, pI and GRAVY score were 25.1 KDa, 7.9 and -0.39 respectively and the average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 4.3, 0.71 and 0 respectively. The last group displayed soluble expression levels between 0 – 1 mg / l. Here the average molecular weight, pI and GRAVY score were 41.1 KDa, 6.2 and -0.51 respectively and the average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 5, 2.43 and 0.21 respectively. There were representatives of nuclear, cytoplasmic and extra-cellular proteins in all three groupings. Expression of a test set of 95 mammalian proteins A diverse set of proteins were chosen to test the conclusions of this study (Table 4 ). They range from proteins that are well annotated, some of which have been expressed in E. coli previously (Nfkb1), to those that contain no PfamA domains and have not been expressed in E. coli previously (Maat1, BC031407, Ttyhl, 1500001H12RIKEXT2, Ext2, KIAA1136, G2 and KIAA1549). They included 24 proteins not annotated as PfamA domains, with unknown function. All cDNAs were amplified from a primary cDNA library, cloned into pDONR221 and sequence confirmed prior to transfer to pDEST-N112-MBP (Figure 1 ) for expression as N-terminal H10-MBP fusions. In some cases primers were designed to clone protein fragments to express particular PfamA domains or minimise the molecular weight or numbers of low complexity (LC) regions or contiguous hydrophobic amino acids (hp_aa). For proteins with no PfamA annotations, such as BC031407, SMART sequence analysis [ 44 ] was performed to identify the low complexity regions of the protein and truncations performed accordingly. Protein expression and quantitation of intact soluble fusion protein product was performed as for the N- and C-terminal tag comparison study. The total and soluble expression levels (mg of protein per litre culture) are listed in the last column of Table 4 together with selected protein features. 63 of the 95 proteins yielded soluble expression levels of greater than 1 mg/l and the average molecular weight, number of LC regions and hp_aa for these proteins was 24.4 kDa, 0.9 and 3.7 respectively. For the 32 proteins that failed to give soluble product of the correct size, the average molecular weight, number of LC regions and hp_aa was 37.1 kDa, 1.8 and 4.5 respectively. Discussion Correlation between protein properties and solubility To guide future expression strategies for new proteins, particularly regarding the choice of expressing a full-length protein in a bacterial or eukaryotic system and also where to truncate multi-domain containing proteins, it is interesting to investigate if the proteins expressed in a soluble form in this study share any common properties. Recently Goh et al. [ 45 ] used data generated by a structural genomics consortium to examine the ability of proteins to progress from cloning to expression and purification to crystallisation. The data used was very large, consisting of 27,000 targets from over 120 organisms and a number of important features were inferred that correlated with success including percentage composition of charged residues, occurrence of hydrophobic patches and length. Although a large study, there was a problem with interpretation of all the data-sets as it was unclear whether targets were simply waiting in the pipeline or had failed. Also structural genomics targets are often initially biased in favour of easy to express proteins, not representative of the whole proteomes of these organisms. The present study, focused on mammalian proteins from several diverse families, examined the relationship between successful soluble expression with various protein properties. Several protein features were identified in this study to correlate with soluble expression, which had not previously been shown experimentally. For both the N and C-terminal tag expression studies it was observed that the presence of several features did not correlate with successful expression including protein pI, grand average of hydropathicity index (GRAVY) [ 43 ], sub-cellular location, the cysteine content as a percentage of the total number of amino acids and the number of coiled-coils. Protein pI has been linked to sub-cellular location [ 46 ] with a bimodal distribution observed in bacterial and archaeal genomes and trimodal pattern in eukaryotes. Proteins are thought to be less soluble at a pH environment near their pI. GRAVY simply calculates overall hydrophobicity of the linear polypeptide sequence with increasing positive score indicating greater hydrophobicity, but no account is taken of the way the protein folds in three dimensions or the percentage of residues buried in the hydrophobic core of the protein. In a recent study Luan et al. [ 47 ] tested the soluble expression of 10,167 full-length C. elegans ORFs and found that protein hydrophobicity was an important factor for an ORF to yield a soluble expression product. This different result may be attributable to the fact that the C. elegans study included a greater proportion of membrane proteins. Therefore the lack of correlation between GRAVY score and soluble expression we observed may be true for non-membrane proteins or for proteins where the trans-membrane domain has been deleted. There was a strong correlation between successful soluble expression and molecular weight of the protein. Small proteins with an average molecular weight of 22.8 KDa did not require to be fused with solubility enhancing proteins for soluble expression whereas proteins that required to be fused with N-terminal MBP or Trx for soluble expression had an average molecular weight of 40.4 KDa and those where the addition of a N-terminal fusion could not rescue soluble expression had an average size of 51.4 KDa. The same pattern also emerged in the C-terminal fusion study. The decreasing probability of successful soluble expression of mammalian proteins with increasing molecular weight is likely due to increasing protein complexity, perhaps requiring specialised eukaryotic chaperones for folding or stabilising binding partners. The majority of proteins solubly expressed in this study contained single domains and as fusion proteins were either capable of self-folding or were folded with the aid of prokaryotic chaperones. Braun et al. found a similar relationship with their set of 32 human proteins with 4 different N-terminal fusions [ 30 ]. A correlation in this study was observed between increasing numbers of contiguous hydrophobic amino (hp_aa) acids (AILFWV) and soluble expression. This ranged from an average of 3.8 hp_aa for those proteins not requiring a N-terminal fusion for high level soluble expression to 5 hp_aa for proteins requiring a N-terminal fusion for successful expression and 5.6 hp_aa where expression failed under the conditions described here. This pattern was also repeated in the C-terminal fusion study where good expression proteins had an average of 3.1 hp_aa whereas poor expression proteins had an average of 5 hp_aa. In a study of the sequences of 2753 non-membrane proteins it was found that the sequences of three or more consecutive hydrophobic residues are suppressed in globular proteins [ 48 ]. Low complexity regions of proteins are regions of a protein of biased composition containing a small number of amino acids [ 33 ] and can have a disordered structure important for protein function [ 49 ]. Here we found that the greater the number of low complexity regions contained within the target protein, the less likely soluble expression would be achieved. This was true for both the N- and C-terminal fusion protein studies with 0.6 – 1 low complexity regions for proteins easy to express in a soluble form to 2.4 – 3 low complexity regions for proteins difficult to express. Low complexity regions are less common in bacterial proteins and these may be targets for proteolytic degradation in vivo . Some interesting conclusions were drawn when soluble expression was measured for an additional set of 95 mammalian proteins expressed as H10-MBP fusions (Table 4 ). In several cases (ELF1, Fli1, Ldb1, BC031407, Nfkb1 and RelA-p65) truncating the proteins to minimise the molecular weight and the numbers of low complexity regions and contiguous hydrophobic amino acids made the difference between failed expression and good soluble protein expression. For proteins such as BC031407, with no annotated PfamA domains, it was found that truncating at low complexity regions was a good method to identify a fragment that could express in a soluble form of the correct size (protein 81). Although we found that successful soluble expression of the 95 protein set correlated with lower molecular weight, number of low complexity regions and contiguous hydrophobic amino acids compared with proteins that failed to express solubly with the correct size, validating our earlier conclusions, there were some exceptions. For example Elf1 and Gata1 both expressed well despite having 4 and 6 low complexity regions respectively and molecular weights of 66 and 42.5 kDa, whereas some smaller proteins such as the PDZ domains of Dlgh3 and Grip1 failed to express. It may be that there are additional protein features, such as the ability to form a stabilising interaction with a binding partner, that are also important for soluble expression. Also ensuring correct protein domain boundaries may be important since the annotated Pfam domain boundaries, based on sequence alignment, do not always match the structural or folding domain boundaries. Protein fusions that enhance protein solubility There have been three comparative studies recently where sets of proteins were cloned into several expression vectors and the effects of the fusion partner on total and soluble expression yield were examined. Hammarstrom et al. [ 29 ] cloned 27 human proteins (MW < 20 Kda) into various expression vectors and ranked the tags ability to promote soluble expression as Trx ~ MBP ~ Gb1 > ZZ > NusA > GST > His6. Another study ranked tags in terms of increased expression and yield after purification as GST ~ MBP > CBP > His6 when comparing the expression of 32 human proteins where the molecular weight varied from 17 – 110 kDa.[ 30 ] Here GST was preferred because of the weak affinity between MBP and amylose resin. In a third study of 40 different proteins (10 mammalian, 3 plant and 2 insect) with 8 different tags MBP gave the best overall results in terms of total and soluble expression [ 31 ]. However, these studies used different combinations of promoter and fusion partner, so it was unclear whether the observed effect was purely due to expression with the fusion partner or variable rates of transcript synthesis that would also affect translation rates. In this study it was found that, on average, N-terminal fusion partners are preferable for optimal protein expression. When proteins are expressed with their native N-terminus, as in our C-terminal fusion proteins, total expression levels can be more variable than when expressed with a constant N-terminal tag. This may be because of variable RNA secondary structures in the region around the start codon which could interfere with ribosome binding. An additional explanation is that during translation the expressed protein emerges from the ribosome first and initiates an incorrect, irreversible, folding pathway before the soluble fusion partner has been translated and folded. The mis-folded protein would be ubiquitin labelled and targeted to the proteasome for degradation resulting in lower total expression levels. This scenario is more likely when expressing mammalian proteins in a bacterial system which lacks specific eukaryotic chaperone proteins. It has been shown previously that proteins prone to mis-folding and aggregation can arrest GFP folding when fused at the C-terminus [ 17 ]. However, when the soluble protein is fused at the N-terminus, this would be translated first and perhaps increase the solubility of the downstream protein domain folding intermediates, increasing their half lives prior to irreversible aggregation. This would allow greater reversibility in the individual steps along the folding pathway and increase the probability that the protein would eventually reach the lowest free energy native conformation. It was found that Trx and MBP were the best N-terminal protein fusions to promote protein solubility. The best C-terminal fusion to promote protein solubility was MBP and this may be acting as a true intra-molecular chaperone [ 50 ], able to promote folding of the N-terminal protein fusion. The mechanism could be due to direct binding to folding intermediates [ 51 ], allowing stabilisation prior to correct folding and inhibition of aggregate formation. The observation that MBP was effective at enhancing soluble expression when fused at the C-terminus, in contrast to thioredoxin, suggests that MBP can actually reverse the process of incorrect folding that would have started prior to the translation of the downstream MBP. This property was not observed for thioredoxin when fused to the C-terminus suggesting either that, in three-dimensions, different proximal faces of the fusion partners have different solubility enhancing properties or that thioredoxin does not posses any chaperone properties and acts only as a solubility enhancer. Alternatively, the folding of thioredoxin may be more prone to inhibition than MBP. Also there are examples where MBP fusions can form soluble inclusion bodies [ 52 , 53 ], and this cannot be ruled out as a possibility here, although there are also several examples where MBP fusion proteins are fully functionally active [ 50 , 52 , 54 , 55 ]. It must be stressed here that although protein solubility is a useful indicator of correct folding, additional measurements need to be performed to give supporting evidence for correct folding. These may include removing the protein fusion with a protease and analysis of the cleaved protein of interest by a variety of biophysical and functional assays such as analysis of monodispersity by light scattering [ 52 ], NMR [ 56 , 57 ], CD spectropolarimetry, bis-ANS binding [ 53 ], ligand binding or enzymatic activity. In this study a protease cleavage site was not included in the vector constructs because the main use of the proteins generated in our laboratory will be in high-throughput antibody production where the cleavage of the fusion partner is unnecessary. GFP did not significantly enhance soluble protein expression when fused to the C-terminus of the proteins in this study, supporting the use of this tag as an indicator of soluble protein expression of fused ORFs.[ 17 , 41 ] The observation that the V5-His6 tag resulted in a higher average soluble expression level than the His10 tag (1.7 compared with 0.3 mg/l) indicates that the identity of the peptide tag can also affect overall solubility of expressed proteins. Conclusions What guidelines have emerged from this study in developing a strategy for the production of soluble mammalian proteins in E. coli ? If the protein has a molecular weight of less than 30 KDa and contains 1 or less low complexity regions and less than 4 contiguous hydrophobic amino acids expression of the full-length protein in E. coli should give good levels of soluble protein. As a generic strategy we would recommend expressing the protein with a fusion partner and found MBP and Trx to be the best fusions to enhance protein solubility as N-terminal tags with MBP being superior as a C-terminal fusion. C-terminal fusions are desirable for proteins such as the P450s where N-terminal tags can inhibit functional activity. When fused to an optimal fusion partner, nuclear, cytoplasmic and extra-cellular domains were equally likely to be expressed solubly. For larger proteins over 50 KDa, truncations should be considered to express specific protein domains and to minimise the molecular weight, number of low complexity regions and contiguous hydrophobic amino acids. In conclusion, this study will help enable a systematic expansion in the number mammalian proteins and domains that can be successfully expressed in E. coli as soluble product, and also predict which are best targeted for a eukaryotic expression system. Methods Materials Oligonucleotides were synthesised by Qiagen-Operon (Cologne, Germany) or Sigma-Genosys (Haverhill, UK). All restriction enzymes were from New England Biolabs (Hitchin, UK). The vectors pET-DEST42, pDEST17 and pDONR201 and E. coli DB3.1 and BL21(DE3)Star pLysS, Gateway BP and LR clonase enzyme mix, pre-cast 4–12 % NuPAGE Bis-Tris gels and PVDF membranes (0.45 μm pore size) were all from Invitrogen (Paisley, UK). Entry plasmids in both open (minus stop codon) or closed format (plus stop codon) containing the full-length genes for GRB2, HRAS, JUN, FOS, MAD, MAX, CDK2, CDK4, CDKN1B, CASP2, MMP1, CDKN2A and CD44 were provided by Pascal Braun and Josh LaBaer (Harvard Institute of Proteomics, Cambridge, USA). A full length clone containing the full-length human EGFR ORF was provided by the RIKEN BioResource Center (Tsukuba, Japan) and Efna1 from the Mammalian Gene Collection (MGC) archived at the Wellcome Trust Sanger Institute (Hinxton, UK). First strand synthesis human and mouse cDNA was from BD Biosciences (Oxford, UK). Plasmid, gel extraction and PCR purification kits and 6xHis protein ladder were purchased from Qiagen (Crawley, UK). The expression strain BL21(DE3), BugBuster protein extraction reagent and His tag monoclonal antibody was from Merck Biosciences (Nottingham, UK). The 96-well multiscreen-DV durapore filter plate with 0.65 μm pore size was from Millipore (Watford, UK) and Cy5-labelled goat anti-mouse IgG from Amersham Biosciences (Little Chalfont, UK). Europium labelled antibodies and DELFIA reagents were from Perkin Elmer (Beaconsfield, UK) and all other chemicals unless otherwise stated were from Sigma-Aldrich (Gillingham, UK). N-Terminal fusion GATEWAY destination vector construction To prepare pET-DEST42-MCS, a multi-cloning site was inserted into pET-DEST42 (Invitrogen) at nt396, between the shine-dalgarno sequence and the attR1 recombination site, encoding the recognition sequences for NdeI, KpnI, DraIII and BfrBI. Inverse or whole plasmid PCR was performed on pET-DEST42 with 5'-phosphorylated PAGE purified primer pairs 20 (5' TACCCACGAAGTGATGCATACAAGTTTGTACAAAAAAGCTGAACG 3') and 21 (5' CCCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAG 3') in a 20 μl reaction containing 10 ng pET-DEST42, 0.3 μM primers 20 and 21, 20 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 200 μM each of dATP, dCTP, dGTP and dTTP, 1 mM MgSO 4 , and 0.5 unit KOD hot start DNA polymerase (Novagen). PCR cycling conditions were: 94°C – 2 mins followed by 15 cycles of 94°C – 15 s, 59°C – 30 s, 68°C – 9 mins. The 7468 bp PCR product was purified using a PCR purification spin column (Qiagen) and eluted with 30 μl of 10 mM Tris-HCl (pH8.5), digested with 20 units of DpnI enzyme at 37°C for 4 hrs, to remove methylated plasmid DNA, purified by spin column and an intramolecular ligation reaction performed using 16 ng of linear PCR product and 5 units T4 DNA ligase and the buffers from the rapid ligation kit (Roche). The ligated PCR product was used to transform E. coli DB3.1 and the resultant pET-DEST42-MCS plasmid DNA prepared and sequence confirmed. Insert 1, encoding a decahistidine tag with a 5'-NdeI site and blunt 3' end, was prepared by PCR with primer pairs 22 (5' GGAATTCCATATGAAUCAC 3') and 24 (5' pGTGATGGTGATGGTGATGGTGATGGTGATTCATATGGAATTCC) and insert 2 encoding a decahistidine tag flanked by a 5'-NdeI site and 3'-KpnI site was prepared with primer pairs 22 and 26 (5' CGGGGTACCATGGTGATGGTGATGGTGATGGTGATGGTGATTCATATGGAATTCC 3'). PCR reactions were as above except the annealing temperature dropped to 44°C, extension time to 10 s and 12 cycles employed. Insert size was checked by 10 % TBE-PAGE and purified by a nucleotide removal kit (Qiagen). Expression vectors (b) pDEST-N110 and pDEST-N112 (Figure 1 ) were prepared by digestion of inserts 1 and 2 with NdeI only or NdeI and KpnI combined respectively, purified by spin column and ligated in a 1:1 ratio to NdeI, BfrBI or NdeI, KpnI digested pET-DEST42-MCS respectively prior to transformation of E. coli DB3.1. Inserts encoding MBP, GFP, GST or Trx flanked by a 5' DraIII site and a 3' blunt end were generated by PCR amplification from the plasmids pMALc2 (New England Biolabs), pET41a or pET32 (Novagen) respectively The primer pairs for MBP were 78 (5' TTATTACACGAAGTGAAAATCGAAGAAGGTAAACTGGTAATC 3') and 79 (5' pGTTCGAGCTCGAATTAGTCTGCGCGTCTTTC), for GFP 84 (5' TTATTACACGAAGTGGCTAGCAAAGGAGAAGAACTTTTCACTGGAG 3') and 85 (5' pTTTGTAGAGCTCATCCATGCCATGTGTAATC 3'), for GST 86 (5' TTATTACACGAAGTGTCCCCTATACTAGGTTATTGGAAAATTAAGGG 3') and 87 (5' pATCCGATTTTGGAGGATGGTCGCCACC 3') and for Trx 88 (5' TTATTACACGAAGTGAGCGATAAAATTATTCACCTGACTGAC 3') and 89 (5' p CAGGTTAGCGTCGAGGAACTCTTTC 3'). The inserts were digested with DraIII and ligated with DraIII, BfrBI cut pDEST-N112 vector to create the GATEWAY destination vectors pDEST-112-MBP, pDEST-112-GFP, pDEST-112-GST, pDEST-112-Trx. C-Terminal fusion GATEWAY destination vector construction pDEST-C101 was designed to insert a decahistidine encoded sequence between the attR2 recombination site and T7 transcription termination region. pDEST-C102 is as C101 except a DraIII, BfrBI site was inserted downstream of the attR2 recombination site. Inverse PCR was performed as described above with primer pairs 1 (5' pCACCATCACCATCATCACCATCACCATTGAGTTTGATCCGGC) and 2 (5' pATGCACCACTTTGTACAAGAAAGCTGAAC) to generate pDEST-C101 and primer pairs 1 and 3 (5' pATGCATACCACTCACTTCGTGCACCACTTTGTACAAGAAAGCTGAAC) to prepare pDEST-C102. Murine and human dihydrofolate reductase (Dhfr and DHFR respectively) inserts flanked by a 5' DraIII site and blunt end at the 3' were amplified from MGC clones using the primer pairs 82 (5' TTATTACACGAAGTGCGACCATTGAACTGCATCGTCGCCGTG) and 83 (5' pGTCTTTCTTCTCGTAGACTTCAAACTTATAC 3') for Dhfr and 80 (5' TTATTACACGAAGTGGGTTCGCTAAACTGCATCGTCGCTGTG) and 81 (5' pATCATTCTTCTCATATACTTCAAATTTG) for DHFR. The DraIII digested inserts were ligated with DraIII, BfrBI digested pDEST-C102 vector to create pDEST-C102-MBP, GFP, GST, Trx, Dhfr and DHFR as shown in Figure 1 . cDNA isolation and expression clone generation A nested PCR strategy was used to isolate protein encoding ORFs directly from cDNA adapted for GATEWAY cloning from the method described by J. E. Collins et al. [ 34 ]. Briefly 2 sets of primer pairs were designed, the first pair of optimised primers binding 1 – 200 bp 5' and 3' of the ORF using DS-Gene software (Accelerys) and a second set of primers targeted to the beginning and end of the ORF. All primers were designed with melting temperatures around 60°C. PCR 1 contained 50 pg of either human universal QUICK-clone II cDNA (Clontech) or 50 pg of a mixture of mouse brain, heart, kidney, liver, smooth muscle, spleen, testis and 7, 11, 15 and 17-day embryo QUICK-clone cDNA (Clontech), 0.25 μM primers, 20 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 200 μM each of dATP, dCTP, dGTP and dTTP, 1 mM MgSO 4 , and 0.5 unit KOD hot start DNA polymerase (Novagen) in a total volume of 20 μl. The PCR reaction consisted of 94°C – 2 mins, and 30 cycles of 94°C – 15 s, 55°C – 30 s, 68°C – 2.5 mins followed by 68°C – 5 mins. A 50-fold dilution of the PCR 1 reaction was made for the second 30 cycle PCR containing the ORF specific primers. Linkers were added to these primers encoding half the attB1 and attB2 sites for forward and reverse primers respectively. For entry clone generation to be transferred to N-terminal tag expression vectors the 5'-linkers for the forward and reverse primers were 5' AAAAAGCAGGCTCT 3' and 5' AGAAAGCTGGGTTCTA 3' respectively with the reverse primer adding a stop codon. For inserts destined to the C-terminal tag expression vectors the forward and reverse primers were 5' AAAAAGCAGGCTTCGAAGGAGATAGAACCATGG 3' and 5' AGAAAGCTGGGTT 3' respectively with the forward primer encoding the shine-dalgarno and kozak sequences and start codon. PCR 2 products were analysed by 1 % TBE-agarose electrophoresis[ 58 ] and correct size fragments were then subjected to an adapter PCR step to complete the flanking attB1 and attB2 sites. This consisted of a PCR reaction as described above using 1 μl of a 50-fold dilution of the PCR 2 reaction in a total volume of 20 μl and primer pair 113 (5' GGGGACAAGTTTGTACAAAAAAGCAGGCT 3') and 114 (5' GGGGACCACTTTGTACAAGAAAGCTGGGT 3') except that the annealing temperature was 45°C, only 12 cycles were used and extension time was 2 mins. The products of the adapter PCR were purified by a 96-well PCR clean-up kit (Qiagen), eluted in 100 μl 10 mM Tris-HCl (pH8.5) and had an average concentration of 40 ng /μl. Recombinational cloning of attB flanked PCR products with an attP containing pDONR vector to generate a set of entry plasmids was as described previously [ 35 ] except that pDONR221 (Invitrogen) was used. The ORFs within sequence confirmed attL containing entry plasmids were then recombined the various attR destination vectors described above to generate sets of expression plasmids. The LR recombination reactions [ 35 ] were used to transform E. coli DH5α cells, miniprep plasmid DNA prepared and this used to transform the various BL21(DE3) expression strains used in this study. Expression screening and quantitation All BL21(DE3) transformants were selected and propagated in the presence of 100 μg/ml ampicillin. A single antibiotic resistant colony was used to inoculate 0.5 ml 2xYT media in a 96-deep well block containing the appropriate antibiotics and shaken at 210 rpm at 37°C. When the average OD 600 had reached 1 (3 hrs for BL21(DE3)), 60 μl was transferred to 1.2 ml 2xYT media in a 96-deep-well block containing the appropriate antibiotics, placed on a shaking incubator at 37°C and when the OD 600 reached 0.5 (2 hrs for BL21(DE3)) IPTG added to a final concentration of 1 mM and shaking continued at 25°C for 12 hours. Total protein was analysed by transferring a 20 μl aliquot of the induced culture to a 96-well PCR plate containing 20 μl of 2 × NuPage LDS loading buffer (Invitrogen), 0.1 M DTT, heated to 95°C for 10 mins and cooled on ice prior to loading 10 μl on a 17-well 4–12 % NuPAGE Bis-Tris gels with a multi-channel gel loading syringe (Hamilton). Soluble protein was extracted by transferring 290 μl of induced culture to a shallow well plate, centrifugation at 3000 g for 5 mins, supernatant removed and cells were resuspended in 58 μl BugBuster containing 1.4 units of benzonase and 58 units of recombinant lysozyme (Novagen). For the C-terminal tag and expression strain comparison this buffer was also supplemented with 0.58 μl protease inhibitor cocktail set III 10-fold diluted in DMSO (Novagen). The cell-pellets were resuspended with a multi-channel pipette and incubated with slow shaking for 20 mins at room temperature prior to transfer to 96-well multiscreen-DV durapore filter plates with 0.65 μm pore size (Millipore). The filter plate was placed on top of a shallow 96-well plate and centrifuged at 1000 g for 2 mins. 4 μl of the filtrate was then added to a 96-well plate containing 5 μl of 4 × NuPage LDS loading buffer (Invitrogen), 11 μl of 182 mM DTT, the plate heated at 95°C for 5 mins and loaded onto a 17-well 4–12 % NuPAGE Bis-Tris gel. A His-tagged molecular weight ladder (Qiagen) was also loaded onto each gel. Gel electrophoresis and electro-transfer to PVDF membrane was as described.[ 58 ] Blots were blocked with 3 % Marvel milk powder in PBS-Tween (PBS with 0.1% Tween) either 1 hour at room temperature or over-night at room-temperature, washed with PBS-Tween and incubated with 40 ng/ml anti-His5 tag monoclonal antibody (Novagen), 3 % Marvel, PBS-Tween for 1 hr, washed 3 × PBS-Tween, incubated with 1 μg/ml Cy5 labelled goat anti-mouse in 3% Marvel, PBS-Tween for 1 hr, washed 3 × PBS-Tween and 2 × PBS and blots dried at 37°C for 10 mins between blotting paper. The blots were scanned on a Typhoon 8600 variable mode imager (Amersham) with fluorescence scan mode, 633 nm excitation laser, 670 nm emission filter, 600 V PMT and 200 μm / pixel scan resolution. The integrated fluorescence intensity volumes of bands on the gel were quantitated using ImageQuant TL software (Amersham). Conversions to protein yield were made by using a calibration curve of purified His-tagged single chain antibody (scFv). Differences between the molecular weight (MW) of the scFv (31 KDa) and each expressed fusion protein were taken into account by multiplying each protein quantitation by the ratio MW construct (KDa) / 31. The numbers were normalised to eliminate blot to blot variation using a His-tagged molecular weight ladder (Qiagen). Authors' contributions MRD performed the molecular biology, participated in the bioinformatics, expression screening, quantitation, experimental design and drafted the manuscript. SPS and RLP participated in the expression screening and quantitation. KJV helped with the bioinformatics (database searching, protein domain annotation and primer design). JM participated in the experimental design, coordination and helped to draft the manuscript. All authors approved the final manuscript.

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544853.xml

548264

Psychological distress among Bam earthquake survivors in Iran: a population-based study

Background An earthquake measuring 6.3 on the Richter scale struck the city of Bam in Iran on the 26th of December 2003 at 5.26 A.M. It was devastating, and left over 40,000 dead and around 30,000 injured. The profound tragedy of thousands killed has caused emotional and psychological trauma for tens of thousands of people who have survived. A study was carried out to assess psychological distress among Bam earthquake survivors and factors associated with severe mental health in those who survived the tragedy. Methods This was a population-based study measuring psychological distress among the survivors of Bam earthquake in Iran. Using a multi-stage stratified sampling method a random sample of individuals aged 15 years and over living in Bam were interviewed. Psychological distress was measured using the 12-item General Health Questionnaire (GHQ-12). Results In all 916 survivors were interviewed. The mean age of the respondents was 32.9 years (SD = 12.4), mostly were males (53%), married (66%) and had secondary school education (50%). Forty-one percent reported they lost 3 to 5 members of their family in the earthquake. In addition the findings showed that 58% of the respondents suffered from severe mental health as measured by the GHQ-12 and this was three times higher than reported psychological distress among the general population. There were significant differences between sub-groups of the study sample with regard to their psychological distress. The results of the logistic regression analysis also indicated that female gender; lower education, unemployment, and loss of family members were associated with severe psychological distress among earthquake victims. Conclusion The study findings indicated that the amount of psychological distress among earthquake survivors was high and there is an urgent need to deliver mental health care to disaster victims in local medical settings and to reduce negative health impacts of the earthquake adequate psychological counseling is needed for those who survived the tragedy.

Background An earthquake measuring 6.3 on the Richter scale struck the city of Bam in Iran on the 26th of December 2003 at 5.26 A.M. It was devastating, the city was destroyed, left over 40,000 dead and around 30,000 injured, as well as destroying approximately 20,000 homes, leaving more than 45,000 people homeless [ 1 ]. The profound tragedy of thousands killed has caused emotional and psychological trauma for tens of thousands of people who have survived. In general earthquakes are known to be related to increased psychological symptoms among survivors and in particular it has been shown that earthquakes might cause post-traumatic stress disorder (PTSD). Several studies on psychological morbidity have been reported following earthquakes in different parts of the world [ 2 - 5 ], but there is no report from Iran. Iran is known to be a country of frequent severe earthquakes causing thousands of deaths in recent years. Therefore for the first time a study was conducted to measure psychological distress among the survivors of Bam earthquake in Iran. The objective was to indicate prevalence of psychological distress among survivors and to specify factors associated with psychological symptoms and thus provide information for interventions needed in Bam and in similar conditions in the country. In addition it was thought this might add to a small body of existing literature on the topic from a different population. Methods This was a population-based study of psychological distress among Bam earthquake survivors aged 15 years and over at five months after the event. The pre-earthquake population of Bam was reported to be 97,000 and surprisingly it was increased to around 120,000 at the post-earthquake period (listing from the authorities). This was due to the fact that because of worry many people living in rural areas were moved to the city of Bam after earthquake. At the time of screening about 80% of people stayed inside their homes and the remaining 20% were moved to four specially designated sites with around 4000 tents. However, to select a representative sample of individuals living in Bam a stratified multi-stage area sampling was applied. The stratification was used because people living in the southern region of the city were less affluent and exposed to more damage as compared to those living in the northern part of the city. Every household or tent within the city had the same probability to be sampled. For the purposes of the first stage, units (census sections) were randomly selected after stratifying by region and size of residence. Then, within each census section the homes and tents to be sampled were selected using the procedure of random routes. Finally the last stage sampling units (the individuals) were selected randomly from all persons living in the same home or tent. Random sampling was based on a list of all households (N = 23000) derived from the electricity bills. A sample of 1150 households (5%) was selected as explained. However, we were actually able to screen 1000 households, giving a success rate of 87%. The main reason for the failed attempts for contact was due to the fact that the targeted house was uninhabited because the residents had moved out and there was no possibility to trace the survivors. In addition considering logistic difficulties we did not replace them with other households. A team of trained interviewers collected data in a week and all participants were interviewed in their homes or designated camps. However, the data were collected in a disorganized environment and thus data gathering strategy was based on offering help to survivors. Since there was no validated measure of PTSD in the Iranian language, only psychological distress was measured using the Iranian version of the 12-item General Health Questionnaire, GHQ-12 [ 6 ]. The scale examines whether the respondent has experienced a particular symptom or behavior recently. Each item is rated on a four-point scale (less than usual, no more than usual, rather more than usual, or much more than usual). The GHQ-12 is brief, simple, easy to complete, and its application in research settings as a screening tool is well documented, and there is evidence that the GHQ-12 is a consistent and reliable instrument when used in general population samples [ 7 ]. The study used the original scoring method. In this method response categories score 0, 0, 1, and 1 respectively. This gives scores ranging from 0 to 12 and higher values indicate more psychological symptoms [ 8 ]. In addition, we collected information about demographic and the number of family members that died due to the earthquake. To analyze data Student's t-test and one-way analysis of variance were used to find out differences in psychological distress among different sub-groups of the study sample and the logistic regression analysis was performed to investigate factors associated with distress. For the purpose of the logistic regression analysis we used the population mean score on the GHQ-12 as cut-off point as recommended [ 9 ]. Results Data were collected from 1000 randomly selected households (600 homes and 400 tents). In total 999 survivors were interviewed, and data for 916 respondents were complete, giving a response rate of 91.7%. The mean age of the respondents was 32.9 years (SD = 12.4), and mostly were male (53%), married (66%) and had secondary school education (50%). The mean score of the survivors on the GHQ-12 was 8.7 (SD = 3.2). The findings showed that 58% of the respondents suffered from severe psychological distress as measured by the GHQ-12. Forty-one percent of the respondents reported that they lost three to five members of their family in the earthquake. The characteristics of the respondents are shown in Table 1 . Table 1 The characteristics of the study sample (n = 916) No. % Age ≤ 20 126 14 21–30 352 38 31–40 205 23 41–50 148 16 > 50 85 9 Mean (SD) 32.9 (12.4) Gender Male 486 53 Female 432 47 Marital status Single 257 28 Married 600 66 Divorced/widowed 59 6 Educational level Illiterate 158 17 Primary 181 20 Secondary 459 50 College/university 118 13 Employment status Employed 233 25 Housewife 277 30 Student 97 11 Unemployed 275 30 Retired 34 4 Number of family loss None 185 20 < 3 261 29 3–5 377 41 > 5 93 10 GHQ score Mean (SD) 8.7 (3.2) < mean score 385 42 > mean score 531 58 To examine differences in psychological distress among sub-groups of the study sample Student's t-test and one-way analysis of variance were performed. The findings showed that there were significant differences between people with different characteristics. People with old age, females, less educated individuals, divorced or widowed and unemployed respondents, and those with loss of more family members in the earthquake reported more severe psychological distress. The results are shown in Table 2 . Table 2 The GHQ-12 score by demographic characteristics (n = 916) Mean (SD) P Age groups 0.006 ≤ 20 7.8 (3.2) 21–30 8.6 (3.3) 31–40 8.9 (3.0) 41–50 8.9 (3.1) > 50 9.3 (3.0) Gender 0.04 Male 8.5 (3.2) Female 8.9 (3.1) Marital status < 0.0001 Single 8.1 (3.3) Married 8.9 (3.1) Divorced/widowed 9.9 (2.5) Educational level < 0.0001 Illiterate 9.7 (2.7) Primary 8.7 (3.2) Secondary 8.5 (3.2) College/university 8.3 (3.1) Employment status < 0.0001 Employed 8.4 (3.2) Housewife 8.8 (3.4) Student 7.0 (3.2) Unemployed 9.3 (2.9) Retired 9.0 (3.2) Number of family loss < 0.0001 None 7.7 (3.5) < 3 8.7 (3.2) 3–5 9.1 (2.8) > 5 9.3 (2.7) To investigate factors associated with severe psychological distress all the significant findings from the univariate analysis were entered into the logistic regression analysis. The analysis indicated that being female (OR = 2.73, 95% CI 1.19–6.26, P = 0.02), illiterate (OR = 3.36, 95% CI 1.11–10.2, P = 0.03), unemployed (OR = 4.39, 95% CI 1.56–12.4, P = 0.005), and the loss of more family members (OR for loss of more than five family members = 2.11, 95% CI 1.22–3.61, P = 0.007) were associated with more severe psychological distress. The results are shown in Table 3 . Table 3 The results of logistic regression analysis OR (95% CI) P Age 0.98 (0.95–1.01) 0.25 Gender Male 1.0 (ref.) Female 2.73 (1.19–6.26) 0.02 Marital status Single 1.0 (ref.) Married 1.56 (0.71–3.41) 0.26 Divorced/widowed 2.17 (0.50–9.37) 0.30 Educational level College/university 1.0 (ref.) Secondary 1.09 (0.51–2.31) 0.82 Primary 1.02 (0.43–2.43) 0.96 Illiterate 3.36 (1.11–10.2) 0.03 Employment status Housewife 1.0 (ref.) Employed 2.29 (0.85–6.18) 0.10 Student 1.90 (0.57–6.32) 0.29 Unemployed 4.39 (1.56–12.4) 0.005 Retired 1.43 (0.33–6.17) 0.63 Number of family loss None 1.0 (ref.) < 3 1.76 (1.21–2.55) 0.003 3–5 1.98 (1.32–2.98) 0.001 > 5 2.11 (1.22–3.61) 0.007 Discussion This was a population-based study measuring psychological distress among survivors of the Bam earthquake in Iran. Psychological morbidity was higher among females, less educated respondents and unemployed individuals. Perhaps such observation might relate to the fact that these groups of people were exposed to a relatively homogenous set of psychological stressors, leading to relatively homogenous mental health outcomes [ 10 , 11 ]. In addition although in the univariate analysis there were significant differences among sub-groups of the study sample with regard to their GHQ-12 mean scores, in the logistic regression analysis age and marital status no longer remained significant. This is consistent with other research findings where female gender, lower education, and lower socio-economic status were found to be related to higher PTSD and depression among earthquake survivors [ 12 , 13 ]. However, recent findings on the topic showed a differential predictor pattern for PTSD and depression among earthquake survivors indicating that although certain factors (e.g. grater fear during the earthquake and female gender) relate to PTSD, lower education and loss of family members tend to relate to depression and not to PTSD [ 13 , 14 ]. This suggests that when interpreting the study results one might relate female gender to possible PTSD which we did not measure, and the other variables to depression which was measured using the GHQ-12. Most notably it was found that people who lost family members reported significant severe psychological distress compared to those who did not. Also there was a distinct pattern of psychological distress among those who lost their family members in the earthquake showing a dose-response relationship between loss and the risk of more psychological distress. One might argue that the relationship between loss of relatives and psychological distress may be a reflection of the relationship between severity of trauma exposure and psychopathology. Since no other variables reflecting trauma-exposure (e.g. fear during the earthquake, level of damage to home, injury, etc.) were entered in the regression equation, 'loss of relatives' was the only variable tapping that, as there is high correlation between loss of first degree relatives (with whom survivors often share the same roof) and exposure to serious damage or collapse of the house. In addition studies have indicated that loss is a strong determinant of PTSD among earthquake survivors and thus it is argued the observation that the risk of PTSD is linked to the amount of loss is an important issue that needs to be incorporated in the development of any effective preventive strategy [ 15 ]. As discussed earlier such finding has been challenged and studies have shown that PTSD and depression are distinct issues and loss only relates to depression whereas PTSD is linked to factors relating to exposure to threat or fear for life [ 14 ]. However, since in the present study only the GHQ-12 was used, there is no way of knowing which factors related to PTSD and which to depression. The GHQ-12 has items tapping depression but little is known as to how sensitive is to picking up PTSD. Psychological distress among earthquake survivors alongside experience of other problems could be considered a serious issue for people's health status living in such difficult conditions. Evidence suggests that severe earthquakes even can cause long-standing morbidity [ 12 ]. However, past psychiatric illness also might contribute to this situation [ 15 , 16 ]. Unfortunately one of the shortcomings of the present study was that we did not measure previous psychiatric conditions among survivors and thus it was not possible to comment on this further. It is recommended that the mean GHQ score for the whole population of respondents provide a rough guide to the best cut-off threshold [ 9 ]. Thus considering people who scored above the mean, the findings from the present study indicated that 58% of the respondents showed an indication of severe mental health problems. Comparing the figure with the national data this was found to be three times higher than reported mental disorders in the general population [ 17 ]. A study using the GHQ-28 measuring prevalence of psychiatric disorder following the China earthquake showed relatively a similar result where the rate of psychological morbidity for earthquake survivors was found to be 51% [ 18 ]. Thus it is argued that there is an urgent need to deliver mental health care to disaster victims in local medical settings and health care professionals who work with the earthquake victims need to be promptly and efficiently trained in mental health crisis interventions [ 19 ]. The results reported here is an estimate for the overall population experiencing the earthquake. Studies have shown that there is variation in psychological morbidity among earthquake survivors by epicenter proximity and rate of property damage [ 12 , 18 ]. However one should be aware that these variables are not the only information that is needed for studying the relationship between psychological distress and the impact of the earthquake. There is also need to obtain information from earthquake survivors on the extent of damage to their homes, experience of being buried under rubble, participation in rescue operations, and witnessing grotesque sites [e.g. see [ 13 , 14 ]]. The present study had certain methodological limitations. Firstly, the results were based on the GHQ-12 scores and we did not measure PTSD symptoms. In this respect also it is important to acknowledge that the GHQ is a general measure of mental health and it is not a measure of diagnostic depression. Thus, since depression is also common in earthquake survivors, our study is limited in not including a validated diagnostic measure of depression. Secondly, no information was obtained on important trauma-exposure variables such as extent of fear or perceived life-threat during the earthquake, rubble experience, disability or injury, et cetera. Thirdly, data on important demographic variables such as past psychiatric illness, and family psychiatric illness were not collected. However in spite of these limitations, the results from this study are useful. This study is the only paper addressing reports on the psychological consequences of the most disastrous earthquakes of the last 50 years. In addition, to our knowledge the international literature does not contain any study on the psychological status of Iranian earthquake survivors, despite the fact that Iran is an earthquake-prone country. It seems that the future research should carefully assess the psychological distress and disruption experiences of the survivors in order to implement necessary interventions. Given that Iran is a country that suffers catastrophic earthquakes relatively frequently, there is need to develop and validate standard instruments such as PTSD measures to include in the future research. Conclusions In conclusion the findings from this study indicated that the survivors of Bam earthquake suffer from psychological distress three times higher than the normal population. In addition female gender, lower education, unemployment, and loss of family members were found to be associated with more severe psychological morbidity among survivors. This suggests that to reduce negative health impacts of the earthquake adequate psychological counseling is needed for those who survived the tragedy. Abbreviations GHQ-12: The 12-item General Health Questionnaire; PTSD: Post-traumatic stress disorder; SD: Standard deviation; OR: Odds ratio, CI: Confidence interval. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AM was the principle investigator and contributed to the study design, the data analysis and wrote the paper. All other investigators contributed to the study design, the data collection and analysis. All authors reviewed and contributed to the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548264.xml

548270

A homology model of restriction endonuclease SfiI in complex with DNA

Background Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified. Results SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis. Conclusions The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses.

Background Type II restriction endonucleases (REases) comprise one of the major families of endonucleases and one of the largest groups of experimentally characterized enzymes (comprehensively reviewed in: [ 1 ]). The "orthodox" Type IIP REases are dimeric, they recognize a short (4–8 bp) palindromic sequence of double-stranded DNA and in the presence of Mg 2+ , catalyze the hydrolysis of phosphodiester bonds at precise positions within or close to this sequence, leaving "blunt" or "sticky" ends (with a 5' or 3' overhangs). The enzymes that do not fit this definition or exhibit certain structural and functional peculiarities, have been classified into several subtypes (review: [ 2 ]). REases coupled with DNA methyltransferases (MTases) of similar specificity form restriction-modification (RM) systems, which are ubiquitous among Bacteria and Archaea [ 3 ]. While cleavage at specific sequences provides efficient means of destroying foreign DNA, methylation of these sequences in the prokaryotic chromosome renders them resistant to REase and thereby protects the own DNA from cleavage. Because cleavage of the chromosomal DNA in unmodified sequences would be deletorious for the cell, the REases must maintain extremely high specificities, tightly coupled with that of the methyltransferase. A change in just one base pair of the "cognate" site can reduce the ratio k cat / K m for DNA cleavage by a factor ≥10 6 [ 4 ]. To date, only crystal structures of 15 REases have been solved, compared to over 3000 biochemically characterized enzymes (review: [ 1 ]; see also [ 3 ] for updates). It was found that they share a characteristic structural core and a very weakly conserved catalytic motif (P) D -X n -( D/E )-X- K (where X is any amino acid), together with a number of non-specific and structure-specific nucleases, suggesting that these proteins are evolutionarily related despite the absence of overall sequence similarity (review: [ 5 ]). The comparison of crystal structures of members of this so-called "PD-DxK" superfamily suggested that the catalytic and DNA-binding regions are major determinants of structural stability of these proteins [ 6 ]. Structural comparisons revealed also two major branches or classes, α and β, whose archetypal members were the enzymes that cleave DNA to generate 4 nt long 5' "sticky ends", for instance EcoRI (α-class), and those that generate "blunt" ends after the cleavage, for instance EcoRV (β-class) [ 7 - 9 ]. Using the experimentally solved structures as templates, bioinformatics methods such as iterative sequence database searches and protein fold-recognition have been used to predict the active site in some REases [ 10 - 13 ]. From these analyses it was learned that certain functional peculiarities, like the requirement of a binding of an uncleaved effector site for cleavage of another site characteristic for Type IIE enzymes, evolved independently in the α and β branches of the PD-DxK superfamily [ 11 , 13 - 15 ]. It was also found that other REases belong to completely unrelated superfamilies, with different three-dimensional folds and catalytic sites (review: [ 9 ]): BfiI is a member of the phospholipase D (PLD) superfamily [ 16 ], Eco29kI belongs to the GIY-YIG superfamily [ 17 ], and KpnI and a few other REases belong to the HNH superfamily [ 17 - 19 ]. While the structural information is essential to infer the molecular basis of sequence specificity in REases, the lack of overall sequence conservation in these enzymes, the absence of invariable residues even in the active site and the presence of several alternative folds make structure prediction and classification extremely difficult. SfiI is a REase isolated from Streptomyces fimbriatus . It recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3', where N denotes any base, and cleaves it as indicated by "^", leaving 3' extensions 3 nt long [ 20 ]. SfiI is a prototype of Type IIF enzymes, which function as tetramers that bind simultaneously to two recognition sites and cleave both sites concertedly [ 21 ]. However, a structural model of SfiI, which could be used as a platform to study its sequence-function relationships, is not yet available. Thus, despite the availability of a large body of biochemical data on how SfiI interacts with the DNA substrate (mainly on the kinetics of protein-DNA interactions with different substrates and the geometry of DNA looping [ 22 - 25 ], but not on the "residue-level" details thereof), the identity of amino acid residues important for dimerization, tetramerization, DNA binding and cleavage remains completely unknown. We have therefore carried out bioinformatics analyses of SfiI that allowed to identify its closest relative amongst REases with known structure and use this information to construct a tertiary model of SfiI in complex with its target DNA. Results and discussion In the absence of experimentally determined protein structures, homology-based models may serve as working models for the investigation of sequence-structure-function relationships between diverged enzymes [ 26 ]. Homology-modeled structures may be of too low resolution to characterize the protein-protein or protein-DNA contacts at the atomic level, but they can suggest which sequence regions or individual amino-acids are essential components of the binding surfaces. In particular, identification of amino acids potentially involved in protein-DNA contacts may guide mutagenesis experiments aimed at the engineering protein variants with novel specificities. However, homology modeling requires a homologous template structure to be identified and the sequence of the protein of interest (a target) to be correctly aligned to the template. Identification of the three-dimensional fold of SfiI The sequence of SfiI showed no significant similarity to any other protein sequences. Also among the proteins reported by BLAST with sub-optimal scores, there were no proteins of known structure and no nucleases (data not shown) that could hint at potential relationships of SfiI to any previously characterized protein superfamily. Thus, in order to identify a template structure for modeling of SfI we used the threading approach, which allows to assess the compatibility of the target sequence with the available protein folds based not only on the sequence similarity but also on the structural considerations (match of secondary structure elements, compatibility of residue-residue contacts, etc.) (reviews: [ 27 , 28 ]). The SfiI sequence was therefore submitted to the GeneSilico protein fold-recognition metaserver [ 29 ]. As expected, fold-recognition methods that rely only on sequence similarity (PDB-BLAST, and FFAS) failed to identify any significant matches between SfiI and proteins with known structrues. However, several threading methods that explicitly use the structural information from the templates reported a match between SfiI and the structure of a Type II REase BglI [ 30 ], a member of the PD-DxK superfamily of nucleases (FUGUE [ 31 ]: 4.25, INBGU [ 32 ]: 3.8, SAM-T02 [ 33 ]: 0.13, 3DPSSM [ 34 ]: 4.4; note that these scores are not normalized as each server uses a different evaluation system; see the individual references for details). Additionally, FUGUE reported a match (low score 3.17) between SfiI and the structure of another REase, EcoRV [ 35 ]. Despite the scores reported by the individual threading methods (except FUGUE for BglI) were hardly significant, the consensus server Pcons5 [ 36 ] assigned a significant score (1.35) to the BglI structure as a potential modeling template. Homology modeling of the SfiI monomer A homology model of SfiI was constructed based on the alignments reported by threading methods, using the "FRankenstein's Monster" approach [ 37 ] (see Methods). Since the PD-DxK nuclease fold was selected by Pcons as the only reasonable template and no other nuclease folds were identified by the FR methods, only alignments between SfiI and the PD-DxK superfamily members BglI and EcoRV were used. The final model was constructed by iterating the homology modeling procedure (initially based on the raw FR alignments), evaluation of the sequence-structure fit by VERIFY3D, merging of fragments with best scores, and local realignment in poorly scored regions. Local realignments were constrained to maintain the overlap between the secondary structure elements found in the bglI structure used as the modeling template, and predicted for SfiI. This procedure was stopped when all regions in the protein core obtained acceptable VERIFY3D score (>0.3) or their score could not be improved by any manipulations, while the average VERIFY3D score for the whole model could not be improved. The final model, comprising residues 13–240 obtained the average VERIFY3D score of 2.6. The alignment between SfiI and BglI is shown in Figure 1 , the corresponding final model of the monomer is shown in Figure 2 . Figure 1 Alignment between SfiI and structurally characterized REases. A) Fold-recognition alignment between full-length sequences of SfiI and BglI. Amino acids are colored according to the physico-chemical properties of their side-chains (negatively charged: red, positively charged: blue, polar: magenta, hydrophobic: green. Pairs of residues conserved between SfiI and BglI are highlighted. Putative catalytic residues are indicated by "#", putative DNA-binding residues are indicated by "*". Secondary structure elements of BglI are shown below the alignment. Numbers of amino acid residues at the N-terminus of each panel are shown. B) Structure-based sequence alignment of the conserved core, corresponding to the PD-(D/E)XK motif, including SfiI, BglI, and other selected REases from the β-class. Conserved residues of the active site are highlighted. Figure 2 Homology model of the SfiI monomer. A) Superposition of the BglI template structure (red) and the SfiI model (blue). The CCGG half-site of the DNA target is shown in green. The Ca 2+ ions from the BglI structure are shown as white dots. B) SfiI model colored according to the sequence conservation: residues identical between SfiI and BglI are shown in blue, residues with physico-chemically similar side chains are in green, dissimilar residues are in yellow and red. The putative conserved active site is shown in the wireframe representation. Modeling of the SfiI dimer in complex with the DNA BglI belongs to the "EcoRV-like" β-class of PD-DxK nucleases. The most typical features of REases from this class are: antiparallel orientation of the 5 th strand of the common β-sheet and recognition of the DNA by an additional β-sheet formed by extended loops between the common secondary structure elements [ 5 , 7 , 9 ]. Most of β-class PD-DxK REases (including EcoRV) exhibit a similar mode of dimerization, which results in positioning of the two active sites as to cut the pair of the opposite phosphodiester bonds in the middle of the recognition sequence and thereby produce the "blunt" ends. BglI is exceptional in that its mode of dimerization is completely different, which leads to a different arrangement of the active sites and the sequence-recognition loops, resulting in the recognition of an interrupted sequence 5'GCCNNNN^NGGC3' and cleavage in the position indicated by "^" that yields 3' ends 3 nt long. SfiI also recognizes an interrupted sequence 5'GGCCNNNN^NGGCC3' and cleaves it in the same manner and therefore can be regarded as a more specific variant of BglI. These striking functional similarities, together with the results of the threading analysis, suggest that SfiI is indeed closely related to BglI and that both enzymes interact with their substrate DNA in a similar manner. Thus, we modeled the structure of the SfiI dimer in complex with the DNA based on the available crystal structure of BglI [ 30 ]. Briefly, the SfiI monomer model was duplicated and each of the copies was superimposed onto the corresponding monomer in the BglI dimer. A few minor steric clashes between the side-chains of residues at the protein-protein interface were removed by choosing alternative rotamers for the respective amino acids. The DNA duplex (sequence 5'ATCGCCTAATAGGCGAT3') was copied from the BglI co-crystal structure (1 dmu) [ 30 ] and "mutated" to 5'AT G GCCTAATAGGC C AT3' using HyperChem 7.1 (Hypercube, Inc.), followed by local geometry optimization. One of the adenine residues in the mismatched A/A base pair in the middle of the DNA molecule was "mutated" to T/A. The curvature of the DNA remained unchanged. Essentially, the global structure of the protein-DNA complex for SfiI remains exactly as in the BglI structure, as reliable modeling of macromolecular interactions remains beyond the capabilities of the existing methods. The model of SfiI dimer is shown in Figure 3 and is available for download from . Figure 3 Model of the SfiI dimer. Individual subunits are shown in yellow and blue. The modeled DNA sequence is shown in green, the specifically recognized CCGG half-sites are in red. Model-based identification of amino acid residues important for catalysis, DNA-binding and dimerization of SfiI In the proposed model of SfiI, the spatial configuration of the catalytic residues is typical for an active site architecture conserved among PD-DxK nucleases. We predict that the active site of SfiI comprises residues: E55, D79, D100 and K102, which superimpose well on the catalytic residues of BglI: E87, D116, D142 and K144, respectively (Figure 4 ). We predict that the DNA-binding mode of SfiI will be very similar to that of BglI, with the side chains of residues S210 and R218 (homologs of D268 and R277 in BglI) involved in the recognition of the inner C/G base pair (Figure 5a ), backbone oxygen and the side chain of K208 (a homolog of K266 in BglI) recognizing the middle C/G base pair (Figure 5b ), and the side chain of R220 (a homolog of R279) recognizing the G of the middle G/C base pair (Figure 5c ). The specificity of SfiI towards the outer G/C base pair, not discriminated by BglI, can be explained by the development of new contacts made by residues from a divergent loop adjacent to the REase active site and comprising residues 104–110 of SfiI and 146–155 of BglI. In BglI, D150 makes specific contacts to the middle C/G base pair and the G/C base pair [ 30 ], however this residue is not conserved in SfiI (Figure 1 ). Instead, we predict that the changes of the loop length and the amino acid substitutions lead to a different conformation of the corresponding loop in SfiI, which allows R109 (not present in BglI) to make a specific contact to the G of the outer G/C base pair (Figure 5d ). Other residues from the same loop, such as K107 may also contribute to the specific sequence recognition by SfiI by making contacts to either of the two G/C base pairs. It is noteworthy that according to our model of SfiI, the majority of specific contacts are achieved by three Arg residues (R109, R218, and R220). These predictions can be tested by site-directed mutagenesis of the respective residues to Ala and testing whether the mutant proteins are proficient in DNA cleavage and/or binding. Figure 4 Superposition of SfiI and BglI structures. The predicted active site of SfiI (in blue) superimposed onto the BglI structure (red) Individual subunits are shown in yellow and blue. The Ca 2+ ions from the BglI structure are shown as cyan spheres. Only the two nucleotides adjacent to the scissile phosphodiester bond are shown. Figure 5 Predicted specific protein-DNA contacts. A) Recognition of the inner C/G pair B) Recognition of the middle C/G pair. C) Recognition of the middle G/C pair. D) Recognition of the outer G/C pair. Interestingly, our model suggests that SfiI lacks the counterpart of a loop corresponding to aa 63–80 in BglI used by this enzyme to interact with the target site from the minor groove side. Thus, SfiI appears to recognize its target solely from the major groove side and to use fewer specific contacts than BglI to recognize its cognate site. This suggests that SfiI may be an easier target for the engineering of REases with new sequence specificities. The dimerization interface of SfiI is comparable to that of BglI. We predict that the following residues may be important for monomer-monomer interactions: Q59, Y60, E63, E66, R73, F74, G76 and that mutating them to change the volume of the side chain (for instance G76R) or introducing (or reversing) the charge (E63R, E66R, R73D, F74R) could disrupt the formation of the SfiI dimer and destroy the REase activity. Prediction of the dimer-dimer interaction surface in the SfiI tetramer SfiI is a Type IIF enzyme, i.e. a tetramer that binds simultaneously to two recognition sites and cleaves both sites concertedly [ 21 ], while BglI is an orthodox IIP enzyme, i.e. a dimer that acts on single sites [ 38 ]. Therefore, the crystal structure of BglI cannot be used to model the tetrameric structure of SfiI. To date, the only Type IIF enzymes, for which crystal structures have been solved, are NgoMIV [ 39 ], Cfr10I [ 40 ] and Bse634I [ 41 ], which are all relatively closely related to each other and exhibit similar mode of interactions between two dimers within the tetramer. These enzymes, however, even at the level of a dimer exhibit a completely different arrangement of monomers, compatible with the generation of 5' overhangs 4 nt long (compared to 3' overhangs 3 nt long in the case of SfiI and BglI). Therefore, it is impossible to obtain a meaningful superposition of the BglI or SfiI dimer onto any pair of subunits in the NgoMIV, Cfr10I or Bse634I tetramer. However, it is tempting to speculate that SfiI may tetramerize in a similar manner to these enzymes, i.e. to use surface regions on the opposite sites of the molecule to the protein-DNA and protein-protein binding. A highly speculative model of SfiI tetramer obtained by manual docking of two dimers is shown in Figure 6 . Based on this model, we predict that the dimer-dimer interface will be composed mostly of hydrophobic and polar residues (and very few charged ones), involve the following segments of the amino-acid sequence, corresponding to loops on the surface of the dimer: 26–34, 67–69, and 86–93. The putative dimer-dimer interactions involve contacts between hydrophobic regions (aa 86–93 from different subunits) as well as hydrophilic ones (aa 26–34). It is possible that the C-terminal region of the SfiI sequence, which could not be modeled (aa 241–269) may also participates in tetramerization. Figure 6 Putative structure of the SfiI tetramer. Individual subunits are shown in yellow, green, red, and magenta. The two DNA substrates are shown in white. Conclusions Implications for the evolutionary history of different (sub) Types of REases Comparative analysis of nucleases from the PD-DxK superfamily suggests that they can be classified into two remotely related lineages: α (EcoRI-like) and β (EcoRV-like) [ 7 - 9 ]. It was proposed that extant REases evolved independently from non-specific or structure-specific nucleases from both lineages (review: [ 5 ]). Interestingly, the phylogenetic tree of the PD-DxK superfamily revealed intriguing cases of convergent evolution. So far, it was found that Type IIE enzymes that bind two copies of the recognition site (the actual target of cleavage and the non-cleaved allosteric effector), evolved independently at least three times: EcoRII is an α-lineage member that apparently evolved from IIP enzymes similar to SsoII or PspGI by acquisition of an N-terminal effector-binding domain [ 12 , 42 ]. NaeI is a β-lineage member remotely related to IIP enzymes EcoRV and HincII that acquired a C-terminal effector-binding domain unrelated to that of EcoRII [ 7 ]. Finally, Sau3AI is a β-lineage member that apparently evolved by a duplication of a catalytic domain closely related to a DNA repair enzyme MutH, followed by the loss of catalytic residues in the C-terminal domain, thereby adapted to function as an effector-binding domain [ 14 , 15 ]. Another type of evidence for convergent evolution is provided by the finding that the specificity for the GATC sequence appeared independently in the α-lineage (MboI and its close homologs [ 5 , 13 ]) and in the β-lineage (Sau3AI and its close homologs [ 14 , 15 ]). Our results strongly suggest that the archetypal Type IIF enzyme SfiI is closely related to a β-lineage member, an "orthodox" Type IIP REase BglI. Another well-characterized group of Type IIF REases comprises α-lineage members for which crystal structures were solved (NgoMIV, Cfr10I, Bse643I) [ 39 - 41 ]. Thus, our analysis provides evidence that the ability of Type IIF REases to tetramerize and cut two target sites in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily. Different Type IIF REases appear to have evolved independently from the simplest, "orthodox" Type IIP enzymes, like previously found for Type IIE REases. It was previously demonstrated that deletion of the effector-binding domain converts the Type IIE REase EcoRII to function as a Type IIP enzyme [ 43 ]. However, tetramerization seems to be important for the catalytic activity of the Type IIF enzyme Cfr10I, since the DNA cleavage activity of the dimeric W220A mutant of this REase is <0.1% of that of the wild-type enzyme [ 44 ]. In the absence of a high resolution co-crystal structure of SfiI in complex with DNA, our model will serve as a convenient platform to study sequence-structure-function relationships in this enzyme. In particular, it will facilitate the mutagenesis of residues potentially involved in tetramerization, dimerization, DNA-binding and catalysis. Methods Structure prediction Sequence searches of the non-redundant (nr) database and of the putative translations from finished and unfinished microbial genomes were carried out at the NCBI using PSI-BLAST [ 45 ]. Secondary structure prediction and tertiary fold-recognition was carried out via the GeneSilico meta-server gateway [ 29 ]. Secondary structure prediction was predicted using PSIPRED [ 46 ], PROFsec [ 47 ], PROF [ 48 ], SABLE [ 49 ], JNET [ 50 ], JUFO [ 51 ], and SAM-T02 [ 33 ]. Solvent accessibility for the individual residues was predicted with SABLE [ 49 ] and JPRED [ 52 ]. The fold-recognition analysis (attempt to match the query sequence to known protein structures) was carried out using FFAS03 [ 53 ], SAM-T02 [ 33 ], 3DPSSM [ 34 ], INBGU [ 32 ], FUGUE [ 31 ], mGENTHREADER [ 54 ], and SPARKS [ 55 ]. Fold-recognition alignments reported by these methods were compared, evaluated, and ranked by the Pcons server [ 36 ]. Homology modeling The alignments between the sequence of SfiI and the structures of selected templates (members of the fold identified by Pcons) were used as a starting point for modeling of the SfiI tertiary structure using the "FRankenstein's Monster" approach [ 37 ], comprising cycles of model building by MODELLER [ 56 ], evaluation by VERIFY3D [ 57 ] via the COLORADO3D server [ 58 ], realignment in poorly scored regions and merging of best scoring fragments. The positions of predicted catalytic residues and secondary structure elements were used as spatial restraints. This strategy has previously helped us to build accurate, experimentally validated models of other REases, such as SsoII [ 11 ], PspGI [ 12 ], MboI [ 13 ], and KpnI [ 19 ]. List of abbreviations aa, amino acid(s); bp, base pair(s); nt, nucleotide; e, expectation; REase, restriction endonuclease; MTase, methyltransferase; ORF, product of an open reading frame, RM, restriction-modification; Authors' contributions JMB carried out the fold-recognition analysis for SfiI, built the preliminary models, drafted the manuscript and coordinated the whole study. AC built the final models and identified functionally important residues. KS participated in interpretation of the data and writing the manuscript. All authors have read and accepted the final version of the manuscript.

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543457

Evaluation of the chicken transcriptome by SAGE of B cells and the DT40 cell line

Background The understanding of whole genome sequences in higher eukaryotes depends to a large degree on the reliable definition of transcription units including exon/intron structures, translated open reading frames (ORFs) and flanking untranslated regions. The best currently available chicken transcript catalog is the Ensembl build based on the mappings of a relatively small number of full length cDNAs and ESTs to the genome as well as genome sequence derived in silico gene predictions. Results We use Long Serial Analysis of Gene Expression (LongSAGE) in bursal lymphocytes and the DT40 cell line to verify the quality and completeness of the annotated transcripts. 53.6% of the more than 38,000 unique SAGE tags (unitags) match to full length bursal cDNAs, the Ensembl transcript build or the genome sequence. The majority of all matching unitags show single matches to the genome, but no matches to the genome derived Ensembl transcript build. Nevertheless, most of these tags map close to the 3' boundaries of annotated Ensembl transcripts. Conclusions These results suggests that rather few genes are missing in the current Ensembl chicken transcript build, but that the 3' ends of many transcripts may not have been accurately predicted. The tags with no match in the transcript sequences can now be used to improve gene predictions, pinpoint the genomic location of entirely missed transcripts and optimize the accuracy of gene finder software.

Background The definition of transcription units within a finished genome sequence in higher eukaryotes is challenging and relies on genome mapping of cDNAs and ESTs backed up by theoretical gene finder algorithms. An increasing number of gene sequences from model organisms have made the prediction of well conserved ORFs easier, but less conserved coding and untranslated regions are difficult to detect. The best way to unambiguously define transcription units is full length cDNAs, but large scale projects are expensive in terms of labor and costs. One also needs to bear in mind that some cDNAs elude detection, because unusual secondary structure or toxicity inhibits reverse transcription or cloning. SAGE investigates the transcription profile of a given cell sample by large scale sequencing of short cDNA tags derived from the bulk mRNA [ 1 , 2 ]. Whereas tag mapping to the cDNA and genome databases of the organism indicates the type of expressed genes, the prevalence of individual tags within the library reflects their relative levels of expression. Since SAGE tags are only short sequences, they can be collected more easily in higher numbers than ESTs and full length cDNA sequences. The potential of SAGE to discover new or better define already known transcription units is particularly advantageous in situations where the entire genome sequence of an organism has been determined, but gene predictions based on theoretical algorithms and the mapping of a relatively small number of EST and cDNA sequences remain tentative. LongSAGE generates longer tags of 21 bases as compared to the classical SAGE protocol and is therefore better suited for the unambiguous assignments of tag to genome sequences [ 3 , 4 ]. Cellular and molecular features of early B cell development [ 5 ] and lymphoma formation [ 6 , 7 ] have been extensively studied in the chicken. Gene expression signatures of primary bursal B cells, pre-neoplastic and neoplastic lymphoma cells were collected by microarray hybridizations in a first attempt to identify genes up- or down-regulated during myc-induced B cell lymphoma development [ 8 ]. The whole chicken genome including a genome scale transcript build from Ensembl [ 9 ] and a collection of bursal full-length cDNAs [ 10 ] have recently been released. We describe here the mapping of large collections of SAGE tags from bursal lymphocytes and DT40 to these reference datasets to evaluate the quality of the transcript build. Furthermore, the transcription profiles of bursal cells and DT40 as defined by this first SAGE analysis in the chicken should lead to a better understanding of B cell transformation and facilitate the selection of candidate genes for disruption in DT40 [ 11 ]. Results and Discussion Generation of SAGE tag libraries and SAGE tags collections Two SAGE libraries, named busage and dt40sage, were made from the bursa of Fabricius and DT40 cells using the LongSAGE technique which generates tags of 21 nucleotides in length and therefore decreases the likelihood of ambiguous matches [ 3 , 4 ]. Of the 129,568 tags collected, about equal numbers were derived from the busage and the dt40sage libraries respectively (Table 1 ). In total 38,212 unitags were derived from the SAGE tags of both libraries. The library from bursal cells and the DT40 cell line seem to be similar with regard to the number of extracted unitags and the average counts of matching SAGE tags. Underlying a standard binomial model, one would expect to find a special Unitag among the busage tags or dt40 tags with a probability of 95% at least once if the relative abundance of this unitag among all busage tags or dt40 tags is at least 4.55 * 10 -5 or 4.69 * 10 -5 respectively. Table 1 SAGE and unitag collections SAGE tags Unitags Average frequency of matching SAGE tags within library Average count of SAGE tag per unitag Busage 65,798 24,064 2.73 4.63 Dt40sage 63,770 21,308 2.99 4.97 Total 129,568 38,212 3.39 Tag to gene assignment using bursal cDNAs, the Ensembl transcript build and the genome sequence Successful mapping of SAGE tags to reference sequences is influenced by the quality of the sequences, the complexity of the reference sequence datasets and the prevalence of polymorphisms within the tag sequences. It was therefore decided to first search for matches within a bursal cDNA collection which represents the best possible reference dataset, as it was derived from the same tissue and genetic background as the busage library. Subsequently, unitags were mapped to the Ensembl transcript build and finally the chicken genome sequence. Unitags found in a previous dataset were not searched for any more in the next. To facilitate the searches, candidate tags starting with the CATG tetra-nucleotide were extracted from each reference dataset prior to analysis. As expected the highest rate of matching to total candidate tags was found for the bursal cDNA collection (3,030 of 26,044 candidate tags matched unitags), followed by the Ensembl transcript build (2,934 of 208,048) and the genome (14,505 of 9,091,924) (Table 2 ). Some unitags mapped more than once within a dataset making an unambiguous assignment difficult. In comparison to the complexity of the dataset, multiple hits occurred more frequently in the bursal cDNA (33/26,044; 0.0012%) and Ensembl dataset (637/208,048, 0.0031%) than in the genome (1,003/9,091,924; 0.0001%). This can be explained if these transcript collections are not completely normalized or if there is bias for certain sequence motifs within gene transcripts. Manual analysis of the bursal cDNAs revealed that most of the multiple unitag matches were due to alternative processing of transcripts originating from the same locus (data not shown). A relatively large fraction of unitags (17,743/38,212; 46.4%) did not match to any reference dataset. It is currently impossible to analyze this in more detail, but sequencing errors, polymorphisms and positions of tags on exon/exon boundaries are likely to explain the missed hits [ 12 ]. Non-matching unitags have a significantly lower average count of SAGE tags than matching unitags (2.1 versus 4.65) suggesting that they either over-represent lowly expressed genes or are artifacts of the SAGE technique. Table 2 Unitag mapping to reference datasets Dataset matches of unitag Unitags Candidate tags Average count of SAGE tags per unitag Bursal cDNA 3,030 26,044 6.89 1 2,997 6.90 > 1 33 5.15 Ensembl transcript build 2,934 208,048 9.93 1 2,275 10.60 > 1 659 7.63 Genome 14,505 9,091,924 2.84 1 13,427 2.83 > 1 1,078 2.91 Total matching 20,469 4.45 Non-matching 17,743 2.17 SAGE tags are expected at the position of the NlaIII site closest to the polyA tail of the transcript, but alternative transcript processing as well as incomplete NlaIII digestion or internal priming can produce upstream tags. Indeed, when the positions of the matching candidate tags were analyzed for bursal cDNA transcripts, about 40% of the tags matched to non-last positions (data not shown). Mapping of tags to the genome Most interesting from the perspective of gene discovery are the 13,427 unitags without transcript match, but with a single match in the genome (Table 3 ). When the positions of these tags within the genome were correlated with the positions of the Ensembl transcripts, 1,637 fell within annotated transcript boundaries indicating that they are located on missed or incomplete exons. To see whether the remaining tags were located in the neighborhood of already identified transcripts, the numbers of tags falling within regions of defined length upstream and downstream of the Ensembl transcripts were determined. Indeed many tags map very close to annotated transcripts with a strong preference for the region downstream of the transcript, as would be expected, if the tag matches the missed 3' end of an annotated Ensembl gene. Since not all tags are derived from the most 3' transcript position, the tags matching immediately upstream of transcripts might indicate missed 5' exons. Some of the tags mapped close to upstream and downstream transcripts (12 at the 500 base distance limit), perhaps indicating that these transcripts belong together. At a distance limit of 5000 bases, 7,169 tags mapped into the neighborhood of annotated transcripts; 5,627 downstream, 669 upstream and 1,061 both upstream and downstream. When the distance limit was extended to 10,000 bases, the number of downstream matching tags was only marginally increased to 5,627 whereas the number of dual positioned tags more than doubled to 2,101. This indicates that at distances over 5000 bases the tag assignment to the neighboring transcripts is becoming increasingly ambiguous, and the tags might in fact correspond to entirely missed genes. Table 3 Locations of unitags having a single match in genome but no transcript match Unitags Bases searched next to annotated Ensembl transcripts Matching unitags Matches only downstream of Ensembl transcripts Matches only upstream of Ensembl transcripts Matches upstream and downstream of Ensembl transcripts Total 13,427 Within Ensembl transcript boundaries 1,637 Outside Ensembl transcript boundaries 11,177 100 409 362 46 1 200 732 668 64 2 500 1,651 1,496 143 12 1,000 2,896 2,553 262 81 5,000 7,169 5,439 669 1,061 10,000 8,639 5,627 911 2,101 Relationship of genome mapping unitags to Ensembl transcripts To further investigate those unitags mapping close to the 5' boundary of Ensembl transcripts or within transcript boundaries to the genome, the bursal EST database [ 11 ] was searched for ESTs matching the tags in the sense strand orientation. These ESTs were then aligned to the chicken genome sequence and the neighboring Ensembl gene predictions. As many ESTs linked the SAGE tags to the Ensembl transcripts, this provided independent experimental evidence that these tags are indeed derived from non-annotated parts of these transcripts (Table 4 ). Table 4 Analysis of unitags mapping 5' of or within Ensembl transcript boundaries. # Unitag Ensembl ID BLAST result ## Supporting bursal EST Unitag relationship to Ensembl transcript ### Unitags mapping 5' CATGCTGCTCGCACGAGCCCT ENSGALT00000002525.1 Q9W7P7 riken1_17l12r1 Upstream 5' exon CATGGCGGGGTTCCCGGGGCA ENSGALT00000005092.1 PEF protein with a long N-terminal hydrophobic domain riken1_18i20r1 Upstream 5' exon (EST supports two additional 5' exons) CATGCTCCTGCTGCTGGCTGG ENSGALT00000009521.1 LAC_CHICK dkfz426_24a5r1 Upstream 5' exon CATGAGGCACCTCCTGTTGGC ENSGALT00000001476.1 GR78_CHICK riken1_25c14r1 5' upstream/Exon1 (EST supports one additional 5' exon) CATGGCCGCCCAAGGAGAGCC ENSGALT00000004055.1 RAN_CHICK riken1_25b20r1 5' upstream/Exon1 (EST supports one additional 5' exon) Unitags mapping within transcript boundaries CATGTACTGGTTGTCTGTTTT ENSGALT00000025884 HG14_CHICK dkfz426_13h16r1 Intron 4–5 CATGCATAGAGGCTTTATTGC ENSGALT00000021336 Aldo-keto reductase family 1 member dkfz426_3h12r1 Intron 8–9 CATGTTGGGACTCACCACTCT ENSGALT00000000504 No description dkfz426_13d22r1 Intron 5–6/Exon6 CATGGTCACCCTAGTAAATAG ENSGALT00000009677 Protein kinase C, beta type dkfz426_38f16r1 Intron 14–15 CATGTAAAGTGTTAGCTGTAC ENSGALT00000006857 ITF2_CHICK dkfz426_14i24r1 Intron 8–9 CATGTTACCTGCAACCTGCTG ENSGALT00000021577 Centromeric protein E dkfz426_17a21r1 Intron 28–29 CATGGGATATACTGAAAATCT ENSGALT00000009956 T-cell activation leucine repeat-rich protein dkfz426_41d20r1 Intron 1–2 CATGGGCTGGTTGGTTTTTGT ENSGALT00000028428 No description dkfz426_43g3r1 Intron 2–3 CATGGTCAAGTACAACTCTTA ENSGALT00000022583 Bcl-2-associated transcription factor dkfz426_12n7r1 Intron 8–9 # Only a few representative examples are shown ## BLAST results are abbreviated ### Unitag aligns within an intron or exon or lies across an intron/exon or upstream sequence/exon boundary To confirm that the distribution of the tags downstream of Ensembl transcripts is statistically significant, their positions were compared to the positions of simulated tags generated by randomly selecting 21 bp sequences in the genome beginning with the 'CATG' tetra-nucleotide. This comparison shows that the real tags map closer to the 3' end of the Ensembl predicted coding sequences (CDS) than the simulated tags providing strong evidence that most of the closely positioned tags are indeed related to the predicted transcripts (Figure 2 ). Figure 2 Mappings of SAGE unitags downstream of Ensembl transcripts compared to simulated genomic tags. The number of tags falling within windows of 10 bp is plotted on the y-axis whereas the distance from the 3' end of the nearest predicted Ensembl transcript is plotted on the x-axis. Sage unitags coordinates are indicated by crosses and randomly selected tag coordinates by diamonds. If one summarizes the unitag to transcript mappings, 5,964 unitags map directly to transcripts, 1,637 map to not annotated sequences within the limit of the Ensembl transcripts and 7,169 map to within 5,000 bases of annotated transcript boundaries (Table 5 ). This leaves about 20% (4,621 out of 19,391 total) bona fide unitags unaccounted for which might be taken as an estimate for the percentage of genes present in the released genome sequence, but absent from the Ensembl transcript collection. Nevertheless, one needs to bear in mind that this calculation includes a number of uncertainties. It is for example possible that the 5000 base limit is too large, since only 5% of 3' UTR sequences in the human transcriptome are reported to be over 2,000 bps according to NCBI's AceView database or that both SAGE and the gene predictions have missed a substantial number of lowly expressed transcripts. In these cases, the estimate of the percentages of missed genes would increase. Table 5 Unitag mapping to transcripts Unitag Match to annotated transcript Match to genome within boundaries of annotated transcript Match next to annotated transcript using 5000 base cut-off Match distant from annotated transcript Total 38,212 Without match 17,743 With only multiple genome matches 1,078 With match to annotated transcripts or single genome match 19,391 5,964 1,637 7,169 4,621 Significant gene expression differences between bursal cells and the DT40 One of the goals of this SAGE analysis was the identification of differentially expressed transcripts between the two libraries and the significance of count differences for the busage and the dt40sage tags were calculated for each unitag. In total 629 unitags showed p values below 0.01 suggesting that the corresponding transcripts are differentially expressed in bursal cells and DT40. In contrast to this, the false discovery rate (FDR) controlling procedure of Benjamin & Hochberg would admit the first 229 genes at an FDR of 5% [ 13 ]. Twenty-five of the most significant unitags mapping to bursal cDNAs are listed in Table 6 . Table 6 List of genes differentially expressed in bursal cells and DT40 Unitag Busage DT40sage Significance Sequence ID # Best BLAST result ## CATGGCAGGGGGCGGAAACCT 4 45 2.83E-10 riken1_2o24 (AAH61765) Hypothetical protein CATGGTGAGCCAAGGTGTTGT 24 82 2.06E-9 riken1_4m1 (AAH69219) Cold inducible RNA-binding protein CATGCAGAAATAAGCTTCTCC 45 109 4.09E-8 riken1_7b15 (Q7ZUR6) Similar to muscle-specific beta 1 integrin binding protein CATGAGCGGGGGCAGCACTTG 118 203 5.75E-7 riken1_25p23 (Q90YW7) Ribosomal protein L4 CATGCTGGAAGAAAGAATAAC 46 114 1.92E-8 riken1_32c11 (Q9YGQ1) Peptide elongation factor 1-beta CATGCGCTCTCCTTTTAAAAG 9 41 2.67E-6 riken1_15l3 (CAA31409) Chinese hamster asparagine synthetase CATGGATGGCCAGCAAGTGTT 29 4 1.17E-5 riken1_4k19 (P13796) L-plastin (Lymphocyte cytosolic protein 1) CATGTCCGTGGCATCCTTTGA 0 16 1.18E-5 riken1_24e23 (Q8BGQ8) Heterogeneous nuclear ribonucleoprotein K CATGGCTTTGGAATATTTGAC 25 3 2.90E-5 riken1_2f9 (AAH46152) Selenoprotein P precursor CATGGAGTCCATAACACGGCG 21 2 6.88E-5 riken1_34m12 (Q96CJ1) Testosterone regulated apoptosis inducer and tumor suppressor CATGCAAAGTGCCCTTGGCTT 17 1 1.46E-4 riken1_10g19 (P30281) G1/S-specific cyclin D3 CATGTAAGCCAATTCTGAACC 19 1 4.09E-5 riken1_33a18 (Q8JHJ4) TNF family B cell activation factor CATGTTGTACACACGGGCACT 11 0 5.79E-4 riken1_5g12 (Q90YB0) FEN-1 nuclease CATGTGCCCGTGACCCCCATC 2 16 6.12E-4 riken1_4n15 (Q13200) 26S proteasome non-ATPase regulatory subunit 2 CATGTCGTGCTCTGTGCCTCC 5 26 9.28E-5 riken1_2i9 (Q90W60) XNop56 protein CATGCTTTCTGCTTTGACTTT 21 4 9.42E-4 riken1_12p16 (P22794) Ecotropic viral integration site 2A protein CATGTTTGTGCATAGCTGTCC 5 28 1.17E-5 riken1_30e3 (Q91XC8) Similar to death-associated protein CATGGCCGGGCGCCCCACCAG 0 15 2.41E-5 riken1_15i13 (Q99P44) Leucine aminopeptidase CATGGGACCAACAAATAAAGC 19 4 0.0027 riken1_4o10 (P97440) Histone RNA hairpin-binding protein CATGAAAATGTACTGTGCTAA 2 13 0.0036 riken1_20p3 (P34022) Ran-specific GTPase-activating protein CATGTATACAGAACTGCTGGA 8 0 0.0044 riken1_2i24 (Q9UMR2) ATP-dependent RNA helicase DDX19 CATGGCCAAATTAGAGGAGTG 1 10 0.0051 riken1_32c11 (Q9YGQ1) Peptide elongation factor 1-beta CATGCTACGCTGTGTCTGCCA 11 1 0.0062 riken1_2m14 (AAQ20009) Heterogeneous nuclear ribonucleoprotein H1-like protein CATGCTCTCCGGTGGTACAAT 0 7 0.0070 riken1_32c11 (Q9YGQ1) Peptide elongation factor 1-beta CATGTTGATTCCTATGCTAAA 7 0 0.0087 riken1_3a6 (Q9H165) B-cell lymphoma/leukemia 11A # only unitags matching bursal cDNAs are listed ## BLAST results are abbreviated To verify the validity of the SAGE data, semi-quantitative PCR was performed using primers close to the tags for 27 transcripts (Figure 3 ). This confirmed the expression pattern suggested by SAGE tag counts in the majority (21 out of 27) of the cases. Certainly, these PCR results could not be explained by the statistical variation in the SAGE data alone (FDR below 5% vs. FDR of 22% indicated by PCR). Although more analysis is needed to find out which differentially expressed genes are related to differences in the behavior of bursal B cells and DT40, the freely available SAGE repository will be a good resource to select candidates for more detailed investigations. Figure 3 Confirmation of differential gene expression using semi-quantitative PCR. Primers derived from reference genes for SAGE tags were used for the amplification of cDNA from bursal cells and DT40 employing different cycle numbers as indicated on top of the lanes. Based on the SAGE tag counts, the reference genes were classified as likely to be equally expressed (left part), higher expressed in bursal cells (middle part) or higher expressed in DT40 (right part). The size of the expected PCR product is indicated by a bar adjacent to the gel image. The numbers of tags found for the busage and dt40sage libraries as well as the calculated significance for differential expression are indicated in brackets under the gene names. Conclusions The mapping of the SAGE tags to the recently released cDNA collections and the chicken genome has been useful to assess the completeness and accuracy of the current transcript catalog. On the positive side, it appears that the transcript build may have missed only a low percentage of genes, since relatively few tags map to genome regions far away from annotated transcription units. On the downside, fewer than 6,000 of over 19,000 tags with matches to reference sequences could be mapped to transcripts. The majority of the tags missed in transcripts are positioned downstream of annotated transcripts with a minority mapping upstream or within the genomic boundaries of transcripts. The most straightforward explanation for this is that many transcripts in the current version of the chicken transcriptome do not accurately reflect the 3' and the 5' ends of transcripts. This proposition is independently supported by the comparisons of the bursal full length cDNAs to the Ensembl transcript build which detected discrepancies to Ensembl annotated transcripts for approximately 50% of the cDNAs [ 10 ]. Another explanation for at least part of the missing transcript matches is variability in poly-adenylation and splicing, which seems to account for substantial variety in the human transcriptome [ 12 ]. Accurate definitions of the transcribed parts of the chicken genome is highly desirable not only to ascertain the correct ORFs, but also to identify transcription and translational control sequences often located in 5' and 3' untranslated regions. It should be interesting to use the genomic positions of the missed transcript tags in combination with current gene finder algorithms to improve transcript coverage. Many of the missed tags are close to already annotated exons facilitating this task. It should also be possible to use promising tag sequences to screen cDNA libraries for clones whose sequence will identify missed genes or exons. The riken1 bursal cDNA library is of excellent quality and should be suitable for this purpose. Although the presented SAGE data provides valuable information about the expression levels of many genes in bursal cells and the DT40 cell line, the full potential of SAGE for gene expression profiling could not be exploited due to the difficulties in tag to gene assignment. Nevertheless, this first SAGE analysis in the chicken lays the basis for further studies. SAGE has the advantage that data from different experiments and laboratories are easily comparable as the tag sequences serve as a common standard. Accumulation of additional data will increasingly facilitate the interpretation of results because bona fide tags will be distinguished from artifacts by being replicated and even polymorphic tags will eventually be defined and assigned to their corresponding transcripts. Methods LongSAGE library construction Total RNA from bursal tissue of chicken 20 day old CB-inbred chicks and from DT40 Cre1 cells [ 14 ] was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. PolyA RNA was isolated using the mRNA DIRECT kit from Dynal . The RNA bound to oligo(dT) 25 magnetic beads was immediately used for the construction of a LongSAGE library [ 1 , 3 ] following a modified protocol as described previously [ 15 ]. High fidelity PfuUltra (Stratagene) polymerase was used for the PCR amplification step. The SAGE libraries from bursal tissue and DT40 were named busage and dt40sage respectively. For each library, distinct Linker/Primer combinations were used to exclude accidental amplification of ditags from the other library. Sequencing of SAGE library clone inserts The pZero-1 (Invitrogen) plasmids containing SAGE ditags as multimeric inserts were transformed into E. coli. Zeocin resistant colonies transformed by the plasmids were grown at low density on agar plates, picked and directly suspended in 50 microliters of H 2 O. This suspension was heated at 95°C for 10 minutes and stored at -20°C until further processing. The PCR amplification used primers from the plasmid backbone, M13 forward and reverse. Sequencing was performed using the Big Dye v3.1 ready reaction mix (Applied Biosystems) and a nested primer (SSP2) from the plasmid poly-linker. Reactions were analyzed on an ABI 3730 DNA Analyzer (Applied Biosystems). The raw sequencing files were processed as described previously [ 16 ]. Ditag, tag and unitag definition The library insert sequences were searched for ditags in which the flanking CATG tetra-nucleotides are separated by a spacer sequence of more than 31 and less than 37 bases. Ditags of identical sequence were entered only once for each library to avoid the possibility of entering PCR amplification artifacts. The ditags were then divided into two SAGE tags of 21 bases including the CATG tetra-nucleotides. The combined SAGE tag collections of both libraries were normalized to generate a collection of unitags possessing unique tag sequences. A low number of tags (197 of 129,568 total tags) were found to be identical to the sequences of the linker tags used for the library construction and therefore were removed. Care was taken to minimize the possibility of tag sequence errors by using a high fidelity polymerase for the PCR amplification step of the library construction and by rejecting any ditag sequences which contained even a single ambiguous base call or a PHRED score lower than 10. It is possible that some unitags are due to sequencing errors, but these artificial tags are unlikely to match transcript or genome sequences. Tag-to-gene mapping To map the unitags to reference sequences, candidate tags were extracted from i) full length bursal cDNA sequences [ 10 ], ii) the Ensembl transcript build and iii) the chicken chromosome sequences . Candidate tags in the transcript datasets were extracted only in the sense orientation whereas both strands of the chromosome sequences were searched. The SAGE tags, unitags and candidate tags together with relevant information concerning their positions and frequencies were entered into tables of a relational database to facilitate further analysis. Unitag matches were sequentially searched for in the bursal cDNA collection, the Ensembl transcript build and the Genome. Once a match had been identified, that tag fell out of the remaining search process and only matches of identical sequences were accepted. To relate the position of matching unitags in the genome sequence to the Ensembl transcripts, the chromosome coordinates of the Ensembl transcripts and their orientation were extracted from their headers. The database table structure, all tabulated entries as well as the FOUNTAIN software [ 17 ] used for the analysis is freely available for download under and . Calculation of the significance of SAGE count differences To evaluate the significance of SAGE tag count differences between the libraries for each unitag, we used Fishers exact test [ 18 ] since it is most easy to use, has exact size and does not require specifying hyper-parameters like for a Bayesian approach. As usual, no method to account for multiple testing was used, so p-values were just used as a convenient tool to rank the unitags. Semi-quantitative PCR cDNA was synthesized from bursal tissue and DT40 Cre1 cell line using the SuperScript Preamplification System (Invitrogen). Primers were designed to amplify a region of a few hundred base pairs encompassing the SAGE unitag sequence of the reference transcript. PCR amplification was performed using the Expand Long Template PCR System (Roche) under the following conditions: 2 min initial incubation at 93°C; 20, 25, 30 and 35 cycles consisting of 10 sec at 93°C, 30 sec at 65°C and 5 min at 68°C with 20 sec elongation per cycle. Authors' contributions JMB and MBW conceived the project. MBW, RBC, HA and JMB participated in the design of the study and its coordination. MBW constructed the libraries. MBW, RBC, NH, CJ, MS, MC and YDW performed clone management, sequencing and data analysis. HA performed confirmation PCR analysis. MBW, RBC, AMK, EE, VL and JMB performed bioinformatics and statistical analysis. AMK and JMB programmed the FOUNTAIN software package to include the SAGE analysis modules. MBW, RBC, AMK, HA, VL and JMB helped draft the manuscript. All authors read and approved the final manuscript. Figure 1 Outline of SAGE tag production and reference gene assignment.

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534807

Allergic conditions and risk of hematological malignancies in adults: a cohort study

Background Two contradictory hypotheses have been proposed to explain the relationship between allergic conditions and malignancies, the immune surveillance hypothesis and the antigenic stimulation hypothesis. The former advocates that allergic conditions may be protective against development of cancer, whereas the latter proposes an increased risk. This relationship has been studied in several case-control studies, but only in a few cohort studies. Methods The association between allergic conditions and risk of developing leukemia, Hodgkin's disease, non-Hodgkin's lymphoma and myeloma was investigated in a cohort of 16,539 Swedish twins born 1886–1925. Prospectively collected, self-reported information about allergic conditions such as asthma, hay fever or eczema was obtained through questionnaires administered in 1967. The cohort was followed 1969–99 and cancer incidence was ascertained from the Swedish Cancer Registry. Results Hives and asthma tended to increase the risk of leukemia (relative risk [RR] = 2.1, 95% Confidence Interval [CI] 1.0–4.5 and RR = 1.6, 95% CI 0.8–3.5, respectively). There was also an indication of an increased risk of non-Hodgkin's lymphoma associated with eczema during childhood (RR = 2.3, 95% CI 1.0–5.3). Conclusion In contrast to most previous studies, our results do not indicate a protective effect of allergic conditions on the risk of developing hematological malignancies. Rather, they suggest that allergic conditions might increase the risk of some hematological malignancies.

Background An association between allergic conditions and cancer risk has been the subject of several epidemiological studies. Both positive and negative associations have been observed and two hypotheses have been formulated to explain such relationships. First, the immune surveillance hypothesis, which proposes that allergic conditions may lead to a decreased risk of malignancy by enhancing the ability of the immune system to detect and eliminate malignant cells [ 1 ]. Evidence from some previous studies of hematological malignancies in relation to allergic conditions primarily supports this hypothesis [ 2 - 4 ]. Second, the antigenic stimulation hypothesis states that immune-stimulating conditions lead to an increased risk of malignancy, which for hematological malignancies is supported by some studies [ 5 , 6 ]. This would be caused by a mechanism where the chronic stimulation induced by the activated cells of the immune system eventually lead to randomly occurring pro-oncogenic mutations in actively dividing cells. There are also a number of studies where no associations between different allergic conditions and hematological malignancies were found, or where inconsistent results were obtained. It is plausible that the association between allergic conditions and cancer risk is complex and that the risk of developing cancer could depend on the specific malignancy and also could be influenced by the type of allergic condition. Most previous studies are retrospective case-control studies. Recall bias may have influenced the results of retrospective case-control studies that have asked for past allergic conditions after diagnosis of malignancy [ 2 - 5 , 7 , 8 ], a problem that partly remains after confirmation of the information on medical history in medical records [ 9 - 11 ] and also when a combination of self-reported information and information from hospital notes and general practioner notes was used [ 12 ]. There are only a few cohort studies that have investigated the relationship between allergic conditions and hematological malignancies [ 6 , 13 - 19 ]. Four of the studies primarily lend support to the antigenic stimulation hypothesis for hematological malignancies, as increased risks, or tendencies toward increased risks, were found for a history of allergy [ 6 , 15 , 18 , 19 ], whereas two of them support the immune surveillance hypothesis by showing decreased risks [ 16 , 17 ]. Four of the studies concerned asthma only [ 13 , 14 , 16 , 17 ]. The purpose of the present study was to investigate the influence of allergic conditions on the risk of developing leukemia, malignant lymphoma and myeloma in a well-established cohort of Swedish twins, and to see whether the results support the immune surveillance hypothesis or the antigenic stimulation hypothesis. An important strength of this study is that information about allergic conditions and confounding factors has been collected prospectively, and therefore the exposure is not subject to differential misclassification. Methods The study base consists of a cohort of twins followed by the Swedish Twin Registry, which was established in 1961 when questionnaires were sent to all 25,778 individuals in a same-sexed twin pair who were born between 1886 and 1925 and were both living in Sweden in 1961. A response from both twins in a twin pair was required for inclusion in the Swedish Twin Registry (21,870 individuals) [ 20 ]. After 1961 they were followed-up as individuals, irrespective of their twin-sibling's status. Out of the responders to the 1961 questionnaire, a new questionnaire was sent to 21,863 eligible subjects in 1963 (response-rate 85.1 %), and in 1967 an additional questionnaire was sent to 20,576 eligible individuals (response rate 81.5 %). The twin cohort was used as a population-based cohort without considering twin status. The present study includes the 16,539 individuals (7,167 men and 9,372 women), who responded to the questionnaire mailed in 1967, and who were still alive and not previously diagnosed with a hematological malignancy January 1, 1969. The median follow-up time was 23 years for men and 27 years for women. The median age at baseline was 56 years (10 th and 90 th percentiles; 46 and 71 years, respectively). The cohort was followed from January 1, 1969 until diagnosis of a hematological malignancy, death or end of the study (December 31, 1999), whichever came first. Cancer incidence and date of death were ascertained by record linkage to the Swedish Cancer Registry and the Swedish Cause of Death Registry, respectively. In the Swedish Cancer Registry, leukemia is coded according to ICD-8 during the investigated period, while all other malignancies are coded according to ICD-7. We identified 324 subjects with hematological malignancies; 10 cases of HD (ICD-7 201), 112 cases of NHL (ICD-7 200.0–200.3, 202.0–202.4), 75 cases of myeloma (ICD-7 203) and 134 cases of leukemia (ICD-8 204.0–207.9). The leukemias consist of 3 cases of ALL, 67 cases of CLL, 31 cases of AML, 7 cases of CML and 26 cases of unspecified leukemia. Exposure assessment Assessment of exposure is based primarily on the 1967 questionnaire, in which questions were asked about the allergic conditions asthma, hay fever, eczema and hives, considering both present and past conditions. The questions were posed as "Have you ever had asthma? (No/Yes), "Have you ever had hay fever, rose fever or allergic rhinitis (characterized by running nose, watery and itching eyes when you do not have a cold)?" (No/Yes), "Did you have eczema when you were a baby?" (No/Yes), "Did you at times later in life have eczema-like skin conditions?" (No/Yes), Do you know the name of the skin lesion you have or have had? (No/Yes, psoriasis/Yes, hives (urticaria) allergic rash/Yes, contact eczema/Yes, eczema in knee or elbow fold/Yes, allergic eczema/Yes, others: specify name). There were no questions about dates when symptoms first started or ended, and no information about treatments used. In the questionnaire from 1963 responses to two questions about asthma and eczema were included. The subjects could mark if they had had asthma or eczema from a list of 17 diseases or the alternative that they hadn't had any of the given diseases. The answer from 1963 was used only if a subject had failed to answer the corresponding question in the 1967 questionnaire. Each medical condition was analyzed separately. In addition we combined the different conditions in order to achieve larger numbers of individuals and thereby obtain more precise results. First, a variable for eczema was created, requiring at least one positive answer for childhood eczema or allergic eczema. Then, a general group of allergic conditions was created, combining the positive answers for eczemas with positive answers on the questions of ever having had asthma or hay fever. Confounders and effect modifiers All analyses were adjusted for age at enrolment and sex. We have controlled for confounding from alcohol consumption (g/month), level of education, and smoking habits (non-smokers, former smokers, current smokers). Adjustment for these factors did not affect the risk estimates in the majority of analyses, and changed the magnitude of the effect at the most 6% in a few instances. Therefore, the presented results are only adjusted for age and sex. Statistical methods We estimated the RR and its 95% CI of each hematological malignancy through Cox's Proportional Hazards Model, (SAS program PHREG, SAS Institute, Cary, North Carolina). To ensure that confidence intervals were not erroneously narrowed due to dependencies within twin pairs we performed analyses that adjusted variance estimates for correlated outcomes. We accomplished this through the use of a SAS macro that stems from the same theoretical background [ 21 - 24 ] and yields the same results as the published Fortran program of D.Y. Lin [ 24 ]. In simple terms, variance estimates are increased in magnitude proportional to the degree of extra correlation within twin pairs. Thus, adjusted confidence intervals are more conservative than unadjusted. If correlations within twin pairs are not different from what is observed between unrelated individuals in the cohort with respect to cancer risk, adjusted and unadjusted variance estimates are identical. Relative risk estimates are not altered by this procedure. Results Our results showed either increased risks or risks close to unity for hematological malignancies following allergic conditions. For leukemia, we found an increased risk associated with hives, and an indication of elevated risk associated with asthma, although with wide confidence intervals (Table 2 ). For leukemia, excluding CLL, the increased RR associated with hives was further elevated. Table 2 Age- and sex-adjusted relative risks for leukemia among subjects with allergic conditions. Leukemia Leukemia, excluding CLL Exposure N e N o RR 95% CI N e N o RR 95% CI N e N o RR 95% CI Asthma, hay fever or hives 31 94 1.4 (0.9–2.1) 21 43 2.0 (1.2–3.4) 10 51 0.8 (0.4–1.6) Hay fever 21 105 1.1 (0.7–1.8) 14 50 1.5 (0.8–2.8) 7 55 0.7 (0.3–1.6) Asthma 7 126 1.6 (0.8–3.5) 3 64 1.3 (0.4–4.2) 4 62 1.9 (0.7–5.3) Hives 7 120 2.1 (1.0–4.5) 6 58 3.6 (1.6–8.5) 1 62 0.6 (0.1–4.3) Eczema * 8 115 0.9 (0.5–1.9) 3 59 0.7 (0.2–2.2) 5 56 1.2 (0.5–3.1) Allergic conditions ** 30 87 1.1 (0.8–1.7) 18 42 1.4 (0.8–2.4) 12 45 0.9 (0.5–1.7) N e = No. of exposed cases, N o = No. of unexposed cases. * At least one positive answer for eczema during childhood or allergic eczema. ** At least one positive answer for asthma, hay fever, eczema during childhood or allergic eczema. The risk estimates for myeloma were generally close to or below unity (Table 3 ). The number of cases with HD was small, with few exposed cases in all analyses making results difficult to interpret (data not shown). For NHL, the risk associated with having had eczema during childhood was increased. Table 3 Age- and sex-adjusted relative risks for myeloma and non-Hodgkin's lymphoma among subjects with allergic conditions. Myeloma non-Hodgkin's Exposure N e N o RR 95% CI N e N o RR 95% CI Asthma, hay fever or hives 10 58 0.7 (0.4–1.4) 22 83 1.1 (0.7–1.8) Hay fever 10 62 0.9 (0.5–1.7) 21 87 1.3 (0.8–2.2) Asthma 0 75 - 0 11 - Hives 1 67 0.5 (0.1–3.9) 1 10 0.4 (0.0–2.6) Eczema * 3 67 0.6 (0.2–2.0) 8 87 1.3 (0.6–2.6) Eczema during childhood 1 69 0.5 (0.1–3.7) 6 89 2.3 (1.0–5.3) Allergic conditions ** 12 55 0.7 (0.4–1.4) 27 69 1.3 (0.8–2.0) N e = No. of exposed cases, N o = No. of unexposed cases. * At least one positive answer for eczema during childhood or allergic eczema. ** At least one positive answer for asthma, hay fever, eczema during childhood or allergic eczema. Discussion Our results suggested that allergic conditions are risk factors for hematological malignancies, and gave support to the antigenic stimulation hypothesis. Thus, the results are in concordance with most previous cohort studies [ 6 , 15 , 18 , 19 ]. A major strength of the present cohort study is that information about allergic conditions and confounding factors has been collected prospectively. Therefore the exposure is not subject to differential misclassification, which is in contrast to retrospective case-control studies where recall bias may be a problem [ 2 , 3 , 9 ] and where separating the effects of prior allergic conditions from the effect of malignancy per se on the immune system may be difficult. This study has focused specifically on how allergy influences the risk of developing hematological malignancies. Another strength is that the study is based on the Swedish Twin Registry, which is a unique resource allowing for an unusually long period of follow-up. In our study, 31 years of follow-up was possible. The cohort has been followed continuously in the Population Registry and the Cause of Death Registry during the study period, and therefore loss to follow-up is unlikely to be a problem. The Swedish Twin Registry is considered a study base representative of the general population of Sweden and has been used in many epidemiological studies [ 25 - 27 ]. Another strength is the completeness of the Swedish Cancer Registry, to which it is required by law to report all incident cancer cases in Sweden. New cases of cancer are reported by physicians in hospitals and other establishments as well as by pathologists. The two independent notifications systems ensure a high coverage. In addition, we could adjust for more confounding factors than in previous cohort studies [ 6 , 13 - 19 ]. One limitation of the study is the small number of exposed cases, and therefore random variation cannot be excluded as an explanation for our findings and for the same reason no stratification for calendar time was performed. Another limitation in the study is that there may be non-differential misclassification of the malignancies. The study period covers 31 years, and during this time diagnostic practices may have changed. In particular, some cases previously diagnosed as HD are now likely to be classified as NHL [ 28 ]. This type of error would bias the effect estimates towards unity. Differential misclassification of exposure is unlikely as there is no reason to believe that reporting exposure should differ between subjects subsequently (years later) diagnosed with a cancer, and those who are not. Non-differential misclassification of exposure is likely to affect the results, but cannot explain increased risks since it would dilute the effect estimates towards unity. The allergic conditions are self-reported and not diagnosed by a physician. However, these self-reported conditions have been used in an earlier study of brain tumors, where some support for the postulated hypothesis that allergic conditions are associated with a decreased risk of developing glioma was found [ 29 ]. Also, the validity of the allergic conditions has been investigated in a group of subjects from the Swedish Twin Registry [ 30 ]. In general, a good agreement was found between the self-reported conditions and an allergologist's diagnosis. Follow-up starts in 1969 and continues until the end of 1999. During 31 years it is possible to develop an allergic condition, but these individuals will still be considered as unexposed members of the cohort. However, the youngest individuals in our cohort were 42 years old when the questionnaire was sent out, which means that this bias have not at all affected childhood eczema, and hay fever and allergic asthma only to a small extent, as these conditions usually present earlier in life. We found an increased risk of NHL among individuals with eczema during childhood. In the literature, there are only few studies concerning an association between eczema and NHL. In one study, a history of eczema was associated with an increased risk of NHL [ 10 ]. In several studies, elevated risks for different hematological malignancies among persons with eczema have been found, e.g. [ 7 , 11 ]. On the other hand, eczema has also been observed to decrease the risk of NHL in two studies [ 3 , 9 ]. Comparisons between these other studies and our study are difficult, however, since the other studies have investigated general eczema while we have focused on eczema of allergic origin (i.e. allergic eczema and eczema during childhood). When using the general definition for eczema many non-allergic forms will be included and while these may influence the risk of developing malignancies, the mechanisms involved are probably different from the ones active in allergic conditions. Thus, these eczemas are not included in the present study. In our material subjects with hives showed an increased risk of leukemia, especially after exclusion of CLL. Several other studies have also found an increased risk of AML [ 12 ] and other hematological malignancies associated with hives [ 6 , 9 , 18 ]. In contrast, some other studies did not show this association [ 8 ]. A number of studies have found a protective effect of asthma on the risk of developing lymphatic leukemia and leukemia, respectively [ 16 , 17 ]. This relationship between asthma and leukemia was not confirmed in our study. If anything, our results support the antigenic stimulation hypothesis. In a recent cohort study, an increased risk of leukemia was indicated [ 19 ]. On the other hand, most studies of hematological malignancies in relation to a history of asthma have shown risks close to unity [ 8 , 9 ]. Clearly, these conflicting results indicate that this area needs to be investigated further. Allergic conditions, like asthma and hay fever, are increasing and it is of great importance to clarify if and how they are connected to hematological malignancies. The contradictory findings may have many explanations, e.g. that different immunological mechanisms may be involved in different types of asthma, that the pathogenesis is likely to be different even in seemingly similar hematological malignancies, and that new forms of pharmacological therapy may influence not only the outcome of asthma but also the risk of developing cancer. To solve these problems, large prospective epidemiological studies on individuals with clinically strictly defined allergic conditions, including data on pharmacological treatment and severity of disease, need to be combined with information about morphologically defined hematological malignancies, including subtyping with techniques from modern molecular biology. Conclusions In summary, findings from our cohort study suggest that chronic antigenic stimulation from allergic conditions might increase the risk of some hematological malignancies. Abbreviations ALL = Acute lymphoblastic leukemia; AML = Acute myeloid leukemia; CLL = Chronic lymphocytic leukemia; CML = Chronic myeloid leukemia; HD = Hodgkin's disease; NHL = Non-Hodgkin's lymphoma; RR = Relative risk; CI = Confidence Interval. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KCS has been the principal investigator, contributed to the planning of the study, performed the statistical analysis and drafted and coordinated the writing of the manuscript. LH participated in the planning of the study and the writing of the manuscript. JS contributed to the writing of the manuscript. MF carried out the study design and contributed to the writing of the manuscript. All authors contributed to the interpretation of results, have read and approved the final manuscript. Table 1 Self-reported allergic conditions among 16,539 subjects. Self-reported allergic conditions Number of respondents Number reporting condition % reporting condition Asthma, Hay fever or Hives 15,168 3,022 19.9 Hay fever 15,546 2,428 15.6 Asthma 16,376 604 3.7 Hives 15,379 430 2.8 Eczema * 14,803 1,033 7.0 Eczema during childhood 14,816 400 2.7 Allergic conditions ** 14,294 3,430 24.0 * At least one positive answer for eczema during childhood or allergic eczema. ** At least one positive answer for asthma, hay fever, eczema during childhood or allergic eczema. Pre-publication history The pre-publication history for this paper can be accessed here:

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479046

Patterning the Face

Vertebrates come in a dazzling array of shapes and sizes, from blue whales to pygmy bats, their overt morphology determined largely by the skeleton. The head skeleton in particular has undergone remarkable diversification, as is beautifully illustrated in Darwin's examination of beak morphology in Galapagos finches. It is now appreciated that a large part of the facial skeleton is derived from a newly identified, vertebrate-specific population of cells, called the cranial neural crest, that has its origins at the border of the dorsal neural plate (the future brain). Vertebrates develop from three germ layers—the endoderm, mesoderm, and ectoderm—which each give rise to distinct elements in the emerging body plan, and interactions between these layers are a common feature of embryogenesis. For example, early in development, cranial neural crest cells migrate to positions along the bottom (ventral side) of the future head, where they form a series of developmental intermediate structures called pharyngeal arches. The arches facilitate interactions between crest cells (derived from ectoderm) and neighboring tissues (such as endoderm and surface ectoderm), which induce specific bone and cartilage patterns in the face. Recent chick studies showed that head endoderm, which contributes to the lining of the pharynx and gills, can pattern the facial skeleton. But the question remained, by what mechanism does endodermal signaling induce specific patterns of cartilage and bone? In this issue of PLoS Biology , Justin Crump, Mary Swartz, and Charles Kimmel study the patterning of a jaw-support cartilage called the hyosymplectic in the larval zebrafish and find a “hierarchy of tissue interactions” at work. In zebrafish mutated for a gene called integrin α 5 , the authors report, a specific region of the hyosymplectic cartilage fails to develop. The loss of this cartilage region correlates with the loss of the first endodermal pouch. Pouches are outpocketings of the head endoderm that fuse with the skin to form the gill slits later in development. By labeling individual crest cells with fluorescent dye and making time-lapse recordings of these cells in transgenic fish, Crump et al. show that the hyosymplectic cartilage regions lost in the integrin α 5 mutant are normally derived from crest cells directly adjacent to the first pouch. Pharyngeal development in a zebrafish embryo Integrins are transmembrane receptors that promote cell adhesion and signaling. Although integrins function in crest cell migration, Crump et al. show that the Integrinα5 receptor is required in endoderm for hyosymplectic cartilage development and appears to promote development of the first pouch. The first pouch in turn acts as a template, by promoting both the survival and local clustering of crest cells, to pattern a specific region of the hyosymplectic cartilage. But the pouch may have more far-reaching effects. Since integrin α 5 mutants also have region-specific defects in cranial muscles and nerves, the first pouch may serve to organize an entire functional unit in a region of the head. As the hyosymplectic element has undergone considerable change during evolution—from a jaw-support element in fish to a tiny, sound-conducting bone called the stapes in mammals—Crump et al. speculate that such a local, interconnected strategy of development would facilitate evolution of the vertebrate head. Changes in endodermal signaling would allow a particular skeletal element to vary in shape or size, in coordination with the muscles and nerves that move the skeletal element and independent of other regions of the head. It will be interesting to determine, the authors note, whether this hierarchical organization applies to other skeletal elements in the head. But for now, these results will inform efforts to understand the specificity of interrelated defects seen in human craniofacial syndromes such as DiGeorge Syndrome, whose underlying causes lie in the development of the endoderm.

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534813

Aerial Surveys Give New Estimates for Orangutans in Sabah, Malaysia

Great apes are threatened with extinction, but precise information about the distribution and size of most populations is currently lacking. We conducted orangutan nest counts in the Malaysian state of Sabah (North Borneo), using a combination of ground and helicopter surveys, and provided a way to estimate the current distribution and size of the populations living throughout the entire state. We show that the number of nests detected during aerial surveys is directly related to the estimated true animal density and that a helicopter is an efficient tool to provide robust estimates of orangutan numbers. Our results reveal that with a total estimated population size of about 11,000 individuals, Sabah is one of the main strongholds for orangutans in North Borneo. More than 60% of orangutans living in the state occur outside protected areas, in production forests that have been through several rounds of logging extraction and are still exploited for timber. The role of exploited forests clearly merits further investigation for orangutan conservation in Sabah.

Introduction The two orangutan species, Pongo abelii in Sumatra and Pongo pygmaeus in Borneo, are threatened with extinction in the near future [ 1 , 2 ]. A prerequisite for conserving great apes in their natural habitat is good knowledge of population distribution, density, and size. However, precise information is still lacking for many orangutan populations living in Borneo, hindering the design of wise strategies for their long-term conservation [ 3 ]. Densities of orangutans and other great apes are usually estimated from nest censuses along ground line transects [ 4 , 5 ]. In order to obtain final estimates of great ape population sizes, these densities are extrapolated to large forest blocks identified from maps as being “suitable habitat” for apes. In most surveys, however, the size of the area actually sampled is very small, and the estimates may not be representative of the population status and/or the variety of habitats and human disturbances (such as logging or mining) existing in the entire range of the population [ 6 ]. In addition, recent land-use changes (such as poaching), and ecological catastrophes (such as those caused by El Niño) or disease outbreaks do not appear in published maps [ 1 ]. The latest estimates available for orangutan populations in the Malaysian state of Sabah (North Borneo) range from 20,000 [ 7 ] to less than 2,000 orangutans [ 1 ]. Recent land transformation renders these estimates out-of-date [ 8 ], and in order to gain precise, up-to-date information, we developed an aerial methodology to assess the entire range of the species in the state precisely. Although some preliminary work using orangutan nest counting from a helicopter was conducted in the past in Sabah and Sarawak [ 9 ], this is the first time that aerial surveys have been used to determine population estimates for a great ape species at a state level. This methodology is likely to be useful for documenting the status of great ape populations living in fragmented and exploited forests in Asia and possibly in some parts of Africa. Results Correlation between Ground and Aerial Nest Densities Ground densities estimated with Distance 3.5 and aerial densities predicted with our model are given in Table 1 . Ground and aerial densities showed a positive correlation with data recorded by the first observer ( R 2 = 0.86, n = 13, p < 0.001), by the second observer ( R 2 = 0.70, n = 13, p < 0.001), by both observers ( R 2 = 0.58, n = 26, p < 0.001), and with the average value obtained for both observers at each site ( R 2 = 0.83, n = 13, p < 0.001). Table 1 Estimated Ground and Aerial Orangutan Nest Densities (Number of Nests/km 2 ) at 13 Different Sites Surveyed during the Orangutan General Census of Sabah LKWS, Lower Kinabatangan Wildlife Sanctuary; FR: forest reserve Orangutan Distribution in Sabah We recorded 2,708 orangutan nests during ground surveys (225 km of line transects and 300 km of recce walks) and 6,936 nests from the helicopter (1,963 km of aerial lines). The size of the sampling areas ranged from 0.001% to 1% (ground survey) and from 1.8% to 16.9% (helicopter survey, assuming an average strip width of 300 m) of the total size of each forest surveyed ( Table 2 ). Table 2 Area Name and Size of Habitat Occupied by Orangutans, Aerial Indexes, Nest and Orangutan Densities, and Final Population-Size Estimates for the 16 Major Orangutan Populations Identified during the Surveys in Sabah, Malaysia, Borneo Old: exploitation older than 15 y; recent: exploitation less than 15 y; active: ongoing exploitation (less than 1 y) a Data from Payne, 1987 [9] b Data from SWD c Data from Ancrenaz et al. 2004 [6] d Confidence intervals are obtained by bootstrapping CL, conventional logging; DLDF: dry lowland dipterocarp forest (<500 m asl); HDF: hill dipterocarp forest (500–1,000 m asl); LMF: lower mountain forest (1,000–1,500 m); n.a.: not available; NP: nonprotected; OU: orangutan; P: protected; SF: swamp forest; SIMLDF: semi-inundated mixed lowland dipterocarp forest (<500 m); SL: sustainable logging; UMF: upper mountain forest (>1,500 m); UBF: ultrabasic forest DOI: 10.1371/journal.pbio.0030003.t002 Our surveys confirmed that orangutans were patchily distributed throughout their range in Sabah [ 7 ], occurring mainly in the eastern and central parts of the state ( Figure 1 ). Only two significant small and isolated populations were found in the western and northern parts of the state, in Crocker Range National Park (population 7) and Mount Kinabalu National Park (population 1; see Table 2 and Figure 1 ). Figure 1 Distribution and Size of the 16 Major Orangutan Populations Identified during the Surveys in Sabah, Malaysia, Borneo The highest nest abundances were recorded in lowland forests below 300 m asl, although we recorded a few nests as high as 1,300 m asl, which appeared to be the upper altitudinal limit for the species in Sabah. The highest orangutan densities (more than six individuals/km 2 locally) were identified in the semi-inundated lowland forests of Kinabatangan (population 13) and Segama floodplains (population 16), Kulamba (population 12), and Tabin (population 14). Most of these forests were highly disturbed, fragmented, and located at the edge of newly established oil palm plantations. Extensive areas of dry lowland dipterocarp forests found in the commercial forest reserves located in the central parts of Sabah (populations 11, 15, and 16) yielded higher orangutan densities in old exploited areas and in areas that were exploited under sustainable logging practices (1.2–2.7 individuals/km 2 , n = 4) than in areas where more conventional practices were implemented (0.1–2.0 individuals/km 2 , n = 11): Mann–Whitney U test, U = 5.5, p = 0.03. Hunting pressure was low in all these forests ([ 7 ]; Kinabatangan Orangutan Conservation Project [KOCP], unpublished data). Orangutan Numbers in Sabah Our surveys showed that about 11,000 orangutans (95% confidence interval: 8,000 to 18,000) were present in Sabah at the time of our surveys ( Table 2 ). Two major orangutan populations were found in logged commercial forest reserves: the Segama forests (population 16, included within the Sabah Foundation forest concession) with about 4,500 individuals, and on the north side of the upper Kinabatangan River (population 15) with about 1,700 individuals (see Table 2 and Figure 1 ). Four significant populations occurred in isolated protected areas: Tabin Wildlife Reserve (population 14; about 1,400 individuals), Kinabatangan Wildlife Sanctuary (population 13; 1,100 individuals), Kulamba Wildlife Reserve (population 12; 500 individuals), Danum Valley Conservation Area (part of population 16; 500 individuals). The remaining populations were of smaller size, scattered, and isolated. Discussion Aerial surveys are widely used for estimating animal abundance and population trends in open and semi-open landscapes [ 22 ]. In Sabah, we report that helicopters can also be used for a forest-dwelling species for (1) directly assessing orangutan distribution, and (2) estimating orangutan population size if aerial surveys are conducted in conjunction with a precalibrating stage based on ground-nest surveys. Aerial nest counts increase the size of the sampling areas significantly, provide a way to survey remote areas that are not accessible from the ground, are faster, and require a lower human investment than classical ground censuses. Nest detectability from the helicopter depends on observers and canopy structure. Ideally, specific models for deriving nest densities from aerial indexes (number of nests detected per kilometer of flight) should be designed for different human observation skills and for different habitat types. However, observer bias can be avoided if the same team of skilled people conducts the entire survey. The second source of bias could be overcome with the design of several habitat-specific models. Before these types of models are designed, ground-truthing must be conducted in different habitat types in order to validate a baseline model and to determine habitat-specific correction factors when necessary. Nest parameters used for obtaining the final orangutan density estimates (nest decay rate, daily rate of nest construction) are a major source of inaccuracy in aerial and ground nest surveys [ 23 ], and there is a need to investigate interpopulation differences in nest life-span estimates further to produce more precise estimates of orangutan densities [ 18 ]. Our survey shows that there are currently about 11,000 orangutans present in Sabah, making the state the main stronghold for the P. p. morio subspecies [ 24 ]. However, this represents a minimum 35% decline over the past 20 years [ 7 ]. This decline is mainly due to habitat loss resulting from the recent conversion of extensive tracts of lowland forests to agriculture [ 1 , 8 ]. The current network of protected areas in Sabah harbors about 4,000 orangutans, representing about 40% of the total number found in the state. About 60% of the total number of orangutans survives in commercial forest reserves subjected to timber extraction, and these forests harbor the largest unfragmented population of the subspecies P. p. morio found in Borneo (population 16). The impacts of forest exploitation on ape abundance and ecology depend on several factors, such as (1) the forest types that existed initially and the quality of the regrowth forest [ 25 ], (2) type of habitat exploitation [ 26 , 27 ], (3) hunting pressure [ 28 ], and (4) species ecology [ 29 ]. Our results tend to indicate that the mosaic of habitats found in the semi-inundated mixed dipterocarp forests that were originally occurring in the floodplains of east Sabah could potentially still harbor a significant number of animals following high disturbance levels (populations 12, 13, and 14). However, we can assume that the very high orangutan densities documented in some of the areas located close to oil palm plantations partly result from the influx of newcomers following recent land conversion to agriculture [ 7 , 30 ]. We can also assume that the response of the forests to logging will directly impact the susceptibility of orangutans to habitat exploitation [ 7 ]. Less diverse habitats (dry lowland dipterocarp forests) located in the interior of the state appear to maintain fewer orangutans, particularly following conventional, nonsustainable logging practices (populations 15 and 16). In the extensive tracts of dry lowland dipterocarp forests exploited for timber (populations 15 and 16), our data suggest that, when hunting pressure is low, orangutan abundance is directly related to the degree of logging and associated damage. For these two populations, the highest orangutan densities were identified in Deramakot, a commercial forest reserve (part of population 15) implementing sustainable logging practices [ 10 ], suggesting that more conventional, uncontrolled logging activities have a negative impact on orangutan abundance. Possible inter- and intraspecific differences in general ecology and feeding behavior of orangutans may also influence population responses to habitat disturbances [ 31 ], and the results documented in Sabah for P.p. morio are not necessarily valid for other Bornean orangutan subspecies and for the Sumatran species [ 32 , 33 , 34 , 35 ]. All great ape species require large forest areas to survive. An ecological network combining protected areas with seminatural landscape elements and production forests could be seen as an option to conserve biodiversity, while also providing opportunities for the sustainable use of natural resources [ 36 , 37 ]. However, there is a need for in-depth field studies investigating further the impacts of logging and associated human activities (such as illegal killing) on great ape ecology and survival in order to assess the role of nonprotected forests for ape conservation. Finally, aerial nest surveys may also be of use in Africa, although it may be difficult to detect nests of African great apes from a helicopter because they tend to be lower in the canopy and it may be impossible to distinguish between gorilla and chimpanzee nests in those areas where the two species are sympatric [ 4 ]. Materials and Methods Study area: Sabah Sabah covers about 76,000 km 2 in the northern portion of the island of Borneo. It is one of the 13 states in the federation of Malaysia. Approximately half of the total land mass is covered with forests ( Figure 2 ). Commercial forest reserves are designated for timber extraction and represent 76% of all forests in Sabah [ 8 ]. Sustainable logging practices (proper forest management plan and precise extraction planning, selective and reduced-impact logging) are currently implemented in Deramakot Forest Reserve (part of population 15) and are in the process of being generalized to other commercial forest reserves where more conventional practices were still implemented in the recent past [ 10 ]. The remnant forests have various protection statuses, but most of them have been logged using conventional forestry practices at least once in the past ( Table 2 ). Figure 2 Location of Ground and Aerial Surveys during the Orangutan Census in Sabah, Malaysia, Borneo Aerial transects are not shown for the Lower Kinabatangan forests. Helicopter census. All major forest blocks in Sabah were identified from 1:50,000 vegetation maps, and these were divided into 16 different blocks. For each block, we determined a systematic stratified sampling using equidistant parallel line transects, the location of the first line being randomly selected ( Figure 2 ). Because the specific topographical features (steep slopes and high altitudes) encountered over Crocker Range and Kinabalu National Parks prevented the helicopter from following a random pattern of transects, the location of our aerial lines followed valleys in these two blocks. Aerial censuses were carried out with a small-type Bell 206 Jet ranger helicopter. Helicopter speed and height were kept constant at about 70 km/h and 60–80 m above the forest canopy. The copilot recorded the precise flight path location with a Global Positioning System every 30 s and monitored altitude, forest type (semi-inundated vs dry), signs of human activities, and forest disturbance continuously. Four types of disturbances were distinguished during aerial surveys: (1) no disturbance: tall and large trees; rather closed canopy; no sign of human exploitation; (2) old exploited forests (timber extraction was conducted more than 15 y prior to the survey): logging roads and stamping areas colonized by pioneer tree species such as Macaranga sp. (crown shape and color easily distinguishable from other tree species), some emergent trees; (3) recently exploited forests (timber extraction was conducted less than 15 y ago): logging roads not entirely colonized by pioneer tree species; few emergent trees; (4): active exploitation: logging activities were ongoing at the time of the survey. From the back seats, two observers looked for orangutan nests from either side of the helicopter. All visible nests were recorded. It was impossible to estimate the impact of nest age on nest detection, and the observers acknowledged that a few days-old fresh nests (still green in color) and nests at their latest stages of decay (just a few branches visible) were difficult to detect in the canopy. These nest categories are likely to have been underdetected. The two observers indicated all sightings to a nest recorder seated between them. The nest recorder noted the number of nests detected by the observers per each 30-s period. All crew members were in constant radio contact during the flights. After the flight, data collected by the copilot and the nest recorder were matched in order to determine the location of all sightings along the aerial line transect precisely. The same team of observers conducted all aerial surveys in order to avoid the observer bias. For technical reasons, it was impossible to fit external devices to the helicopter to estimate the distance of the nests to the aerial transects. This prevented us from determining the detection function from our data alone [ 11 ]. Trailing tapes placed on the aircraft window limited the observers' field of view to a strip of approximately 150 m wide on either side of the aircraft. However, fluctuations in canopy's height prevented the direct determination of the exact width of the sampling area. Ground censuses. Because the proportion of the actual nest population existing in the forest that was detected from the helicopter was unknown, it was impossible to directly estimate nest densities from our aerial results [ 9 ]. We thus designed a calibration function relating nest density estimated from the ground to the number of nests detected per kilometer of flight (aerial nest index) by comparing the aerial results with results from extensive ground surveys carried out in 13 patches of old and recently disturbed forests located in the Kinabatangan Wildlife Sanctuary [ 6 ], and Deramakot, Kalumpang, and Segaliud forest reserves. Nest densities and their variances were estimated by ground line transects using distance sampling [ 11 , 12 ]. A set of line transects was randomly selected and the perpendicular distance of each nest to the transect was carefully recorded [ 6 ]. Densities were computed using the software Distance 3.5 [ 13 ]. For each transect, the truncation level was set following identification of outliers from box plots (outliers being values higher than 1.5 box-lengths from the 75 th percentile). Heaping was assessed from histograms, and data were grouped where necessary [ 11 , 14 ]. The probability of nest detection was estimated with models combining density functions (uniform, half-normal, and hazard-rate) with adjustments (cosine, simple, Hermite polynomials). The model with the lowest Akaike's Information Criterion was selected for each site [ 15 ]. The adequacy of the selected model to the perpendicular distances was assessed by a chi-square goodness-of-fit test on grouped data [ 11 ]. Finally, we estimated the variance of nest density using nonparametric bootstrapping to handle sources of variation, such as model selection uncertainty [ 11 ]. Results are given in Table 1 , and are extensively described in [ 6 ]. Estimation of nest density from aerial indexes. The calibration function relating absolute nest density to aerial nest index stipulated that the logarithm of the orangutan nest density D^ was a linear function of both the logarithm of the aerial index AI and the observer effect obs , plus their interaction, in order to include possible differences between observers. We weighted the general regression by the estimated variances of ground nest densities, thus giving a greater emphasis to precise density estimates. The least squares method was used for model fitting by incorporating weights 1/σ^ log(D^ i ) where σ^ log(D^ i ) was the estimated standard error of the estimated nest density logarithm in forest area i, given by Then, assuming that the densities were log-normally distributed, the overall regression model was conveniently written with a matrix notation as where D^ was a 26 × 1 vector of the orangutan nest densities (13 points per observers), and X was the matrix of covariates: β was a 4 × 1 vector of parameters to be estimated, and ɛ was a 26 × 1 vector of errors with multivariate normal distribution N 26 (0,Σ) , where Σ was a 26 × 26 matrix with σ 2 · diag (var(logD^ i )) in the diagonal and zeroes elsewhere. To simplify, (2) was D^ rewritten using the quantities log ( D^ ) w = W · log( D^ ), X w = W · X , and ɛ w = W · ɛ, with W = diag (σ^ −1 log(D^ i ) as Unlike ɛ, ɛ w has a more familiar distribution N 26 (0, σ 2 · I 26 ), allowing the use of linear regression tools to estimate model parameters via least square theory. We used the backward model selection procedure [ 15 ] to select between models. The first regression model to be tested included all covariates. Covariates with the highest p value and greater than a 10% cutoff were then removed one by one, and each new model was retested until all p values of the remaining covariates were less than the cutoff value. We assessed the goodness of fit of the best model by computing the coefficient of determination R 2 . The best model supported by the data considered only the aerial index effect ( R 2 = 0.9587, F 23 2,0.05 = 3.42 , p < 0.001, on the logarithmic scale) with Using this model (5), we predicted an orangutan nest density from any new aerial index values, AI 0 , recorded during helicopter flights, as ( Figure 3 ). This model was applied to all the forests that were surveyed only by helicopter. A 95% confidence interval for the predicted orangutan nest density was built up on the logarithmic scale as Figure 3 Graph Showing the Predicted Orangutan Nest Density as a Function of Aerial Indexes The plain line is the fitted line via model (5), and dashed lines are prediction intervals; n = 13 sites, 2 observers. with and X , the matrix defined above, once the observer effect had been removed, s 2 the residual mean square up to a constant that was an estimate of σ 2 , and t 23,0.025 the appropriate two-sided t -distribution percentile [ 16 ]. Following [ 11 ], this interval was then back-transformed to obtain a final confidence interval for the predicted orangutan nest density as where and A numerical application gave These intervals are shown in Figure 3 . Correction factors and habitat types. Model (5) was obtained with results from the old and recently exploited semi-inundated mixed lowland dipterocarp forests of Kinabatangan. We tested its validity by comparing nest densities predicted from aerial data and estimated from ground line transects at several sites. We found no significant differences for five sites of old and recently exploited dry lowland dipterocarp forests ( t -test, n = 5, t = −1.738, p = 0.157; 95% confidence interval of the difference: −110 to 25; ratio between ground and aerial nest densities = 0.94). This result showed that nest detectability was similar in forests that had been exploited for timber in Sabah and in Kinabatangan. Thus, we used the baseline model without any correction for all recent and old exploited forests of the state. Exploited swamp forests had a very open canopy, and predicted aerial nest densities were higher than estimated ground densities, although the difference was not significant ( t -test, n = 3, t = −3.331, p = 0.08; 95% confidence interval of the difference: −384 to 49; ratio = 0.54). However, in order to not overestimate the final densities, we applied a correction factor of 0.54 to aerial indexes obtained in two areas of extensive exploited swamp forests (parts of populations 12 and 14). In primary lowland dipterocarp forest, the predicted aerial nest density was lower than the estimated ground density in the only site that was tested ( n = 1; 392 nests/km 2 vs 592 nests/km 2 ; ratio = 1.5). Aerial indexes obtained for Danum (part of population 16), the only site of primary lowland forest with a significant orangutan population assessed during our survey, were multiplied by a correction factor of 1.5. Estimation of orangutan density from nest density. The actual orangutan density D^ ou was estimated using with D^ * O the predicted nest density, p^ the estimated proportion of nest builders, t^ the estimated nest decay rate, and r^ the estimated daily rate of nest production [ 4 ]. The proportion of nest builders has been estimated as 0.9 for orangutans [ 5 , 17 , 18 ]. The daily rate of nest production is currently available for only two Bornean orangutan populations: 1.005 in Kinabatangan [ 18 ] and 1.163 in Gunung Palung [ 17 ]. In order to take into account interpopulation variability in orangutan nesting behavior and to obtain more conservative estimates of orangutan densities in Sabah, we used an average value of 1.084 for our survey (with an associated coefficient of variation of 0.063). Nest decay rate varies with forest type [ 5 , 18 ], and the most reliable estimates are obtained via direct monitoring of the survival of a sufficient number of nests [ 19 ]. Such estimates for t^ are available for only two sites in Borneo: Gunung Palung, with 399 d and 258 d in mixed semi-inundated lowland and dry lowland forests, respectively [ 17 ]; and Kinabatangan with 202 d [ 18 ]. Since specific nest decay rates were not available for the different forests surveyed in Sabah, we considered an average t^ value of 286.3 d (coefficient of variation: 0.373). Using the δ-method [ 20 ], a 95% confidence interval for the estimated D^ ou was built up as with cv as the coefficient of variation and the other quantities as already defined (see above). This interval was then back-transformed to obtain a confidence interval for D^ ou as where The numerical application gave with Estimation of orangutan population size. The results of the ground and aerial surveys were processed with a geographic information system (Arcview 4.1; ESRI, Redlands, California, United States), using a combination of administrative maps and satellite images. When necessary, we stratified each forest block according to (1) disturbance type: no disturbance, old or recently exploited forests, ongoing exploitation; (2) altitude: lowland, below 500 m above sea level (asl); upper land, 500–1,000 m asl; lower mountain, 1,000–1,500 m asl; mountain, above 1,500 m asl; and (3) habitat type: swamp forests, semi-inundated mixed lowland dipterocarp forests, dry lowland dipterocarp forests. We then determined the percentage of habitat actually occupied by orangutans as the ratio between the total length of aerial transects and the length flown over areas with no visible orangutan nests (large areas with no trees, such as grasslands, large forest gaps, rivers, and oxbow lakes). This percentage was applied to the total size of each forest block determined from maps in order to estimate the final size of “habitat occupied by orangutans.” We then multiplied the estimated orangutan densities by the estimated size of orangutan habitat occupation to obtain overall population estimates. A confidence interval for the population estimate was computed via parametric bootstrapping for the whole population in Sabah [ 21 ]. We assumed a normal distribution for population sizes extracted from the literature, and we assumed a log-normal distribution for other populations, with parameters given by formula (4). Values were sampled from their appropriate distribution and summed to obtain the whole population size. We repeated these two steps 1,000 times to obtain the bounds of a 95% bootstrapped confidence interval as the 25th and 975th largest values. We adopted a similar procedure for the subpopulations constituting populations 9, 11, 15, and 16. Survey efforts. Over a 2-y period (2002–2003), ground surveys (using a combination of line transects and recce walks for a total effort of 1,100 “man days” of fieldwork) and aerial surveys (72 “man days”) were conducted in all major forests of the state ( Figure 2 ). Recce walks were conducted to assess the presence/absence status of orangutans in areas with harsh topographical features or with extremely low orangutan abundance. Results from recce walks were not used to estimate nest densities.

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434152

Harnessing DNA-Based Technology for Drug Discovery

Traditionally, developing small molecules for research or drug treatments has been a painstaking enterprise. Drugs work largely by binding to a target protein and modifying or inhibiting its activity, but discovering the rare compound that hits a particular protein is like, well, finding a needle in a haystack. With a specific protein target identified, scientists typically either gather compounds from nature or synthesize artificial compounds, then test them to see whether they act on the target. Small-molecule evolution The birth of combinatorial chemistry in the early nineties promised to revolutionize this laborious process by offering a way to synthesize trillions of compounds at a time. Though molecules still had to be evaluated one by one, high-throughput screening technology could manage up to a million molecules a day. Despite these technological advances, few drugs have emerged from combinatorial chemistry approaches, leaving the promise largely unfulfilled. Another strategy looks to nature as a model. The immune system fights disease and infection by generating billions of antibodies, each primed to recognize a specific pathogen. Antibodies recognize antigens (protein fragments of pathogens) with an exacting specificity that develops through an iterative process. The body first produces a diverse, random collection of antibodies. Every antibody is encoded with a unique DNA blueprint in a B-cell. Antibodies that happen to bind to a pathogen are “selected” to pass on their blueprints: successful binding stimulates cell division, during which blueprints are copied, varied by mutation, and used to create a new generation of antibodies. Specificity is refined over multiple generations. Over the past fifteen years, biologists have developed techniques to recreate this process in a test tube. Today, it's common practice to “evolve” collections of as many as a quadrillion different proteins or nucleic acids to bind a molecular target. These techniques are called molecular breeding, because like traditional livestock and crop breeding techniques, they combine sets of genotypes over generations to produce a desired phenotype. Molecular breeding has so far been restricted only to applications that involve materials encoded by DNA. Drugs produced by conventional synthetic organic chemistry, for example, cannot be bred. In a series of three articles in this issue of PLoS Biology , David Halpin et al. describe a strategy that addresses this limitation. By inventing a genetic code that acts as a blueprint for synthetic molecules, the authors show how chemical collections of nonbiological origin can be evolved. In the first article, Halpin et al. present a method for overcoming the technical challenge of using DNA to direct the chemical assembly of molecules. In the second, they demonstrate how the method works and test its efficacy by creating a synthetic library of peptides (protein fragments) and then showing that they can find the “peptide in a haystack” by identifying a molecule known to bind a particular antibody. The third paper shows how the method can support a variety of chemistry applications that could potentially synthesize all sorts of nonbiological “species.” Such compounds, the authors point out, can be used for drug discovery or as molecular tools that offer researchers novel ways to disrupt cellular processes and open new windows into cell biology. While medicine has long had to cope with the evolution of drug-resistant pathogens, it may now be possible to fight fire with fire. To learn more about the DNA display method described here, see the primer “Translating DNA into Synthetic Molecules,” also in this issue.

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517953

Characterization of the microheterogeneity of transthyretin in plasma and urine using SELDI-TOF-MS immunoassay

Background It has been shown that transthyretin (TTR) exists in different molecular variants. Besides point mutations associated with different diseases such as amyloidosis, other posttranslational modifications occur that might be of diagnostic interest. Results TTR levels as determined by ELISA in plasma and urine of healthy individuals were 489 ± 155 μg/ml plasma and 46 ± 24 ng/g creatinine, respectively. Average levels in urine of pregnant women were 45 ± 65 μg/g creatinine. The molecular heterogeneity of TTR was analyzed using a high-throughput mass spectrometric immunoassay system. TTR was extracted from plasma or urine onto an antibody-coated (via protein A) affinity chip surface (PS20) using the surface-enhanced laser desorption/ionization (SELDI) technique. Subsequently samples were subjected to time-of-flight mass spectrometry (TOF-MS). In healthy individuals, TTR in plasma occurred rather consistently in two variants of 13732 ± 12 and 13851 ± 9 Da for the native and S-cysteinylated forms and at a smaller signal of 14043 ± 17 Da for the S-glutathionylated form. In urine of pregnant women, various signals were observed with a dominant signal at 13736 ± 10 Da and a varying number of smaller immunoreactive fragments. These fragments are possibly the consequence of metabolism in plasma or kidney. Conclusion This chip-based approach represents a rapid and accurate method to characterize the molecular variants of TTR including protein or peptide fragments which are either related to TTR or have resulted from its catabolism. These molecular variants may be of diagnostic importance as alternative or novel biomarkers due to their predominant relation to the TTR metabolism both in healthy and diseased individuals.

Background Transthyretin (TTR, formerly called prealbumin) belongs to a group of proteins including thyroxine-binding globulin and albumin which bind and transport thyroid hormones in the blood. It is a single polypeptide chain of 127 amino acids (14 kDa) and is present in the plasma as a tetramer of non-covalently bound monomers. The major sites of TTR synthesis are the liver and choroid plexus [ 1 - 3 ]. Under physiological conditions, the macromolecular complex plays an important physiological role in vitamin A homeostasis because it binds the specific transport protein for retinol, the lipocalin retinol-binding protein (RBP) [ 4 , 5 ]. This reduces the glomerular filtration of the low molecular weight transport protein (21 kDa) in the kidneys. Any TTR or RBP molecules that are filtered are rapidly bound to megalin, the multiligand receptor expressed on the luminal surface of the renal proximal tubules and therefore internalized. Thus, under physiological conditions, TTR and RBP are present in urine if at all, only in trace amounts [ 6 ]. The TTR variants described thus far have mostly been associated with variable degrees of cardiac and/or neural tissue amyloid deposits [ 7 , 8 ]. Therefore, mutations of the amino acid sequence of TTR are of clinical interest [ 9 ]. In general, mutations appear to be distributed randomly within the molecule and most of these mutations lead to the synthesis of TTR molecules which have the tendency to form insoluble protein aggregates. These so-called amyloid deposits accumulate extracellularly in various organs. Although the role of amyloid deposits in the pathogenesis of the disease is not clear, preventing their formation or promoting their disaggregation is necessary to control the development of clinical symptoms [ 10 , 11 ]. With regard to nutrition, TTR is a so-called visceral protein that is synthesized in the liver in response to nutritional supply. TTR plasma levels have thus been proposed as sensitive biochemical parameters of subclinical protein malnutrition, because both the adequacy and levels of protein as well as energy intakes are reflected in plasma levels. Plasma levels of TTR, however, are as well affected by acute and chronic diseases associated with an acute-phase response. Under these conditions, liver activity is converted to the synthesis of acute-phase response proteins, resulting in a dramatic drop in visceral proteins, despite nutritional support [ 1 - 3 ]. This study was conducted to establish a sensitive and reproducible high-throughput SELDI-TOF-MS immunoassay for characterizing TTR variants in plasma and urine arising from amino acid substitutions, posttranslational modifications and/or products of protein degradation or proteolysis. Results TTR levels in plasma and urine TTR levels in plasma determined by ELISA in 10 healthy individuals were 489 ± 155 μg/ml. TTR levels in urine of the healthy individuals were one thousand times lower than in the 40 pregnant women with 46 ± 24 ng/g creatinine and 45 ± 65 μg/g creatinine, respectively. TTR microheterogeneity in plasma and urine In Figure 1 the TTR variants found in serum and in urine of pregnant women and non-pregnant controls were compared using the Western blot technique. The results obtained with this method showed that TTR was present in plasma of both groups and in urine of pregnant women but not in the urine of non-pregnant individuals. The intensity of the immunoreactive band in urine of pregnant women was substantially lower than that in plasma. In all investigated TTR-positive plasma and urine samples TTR was present as a single band indicating a molecular weight of 14 kDa. Figure 1 Western blot of TTR in paired plasma (A) and urine (B) of seven (1–7) healthy individuals and in seven urine samples (C) of healthy pregnant women. S = TTR standard SELDI-TOF-MS immunoassays showed that in healthy non-pregnant and pregnant females as well as in males TTR in plasma occurred rather consistently in two major variants of 13732 ± 12 and 13851 ± 9 Da, of which generally the 13851 Da variant is the dominant one. The mass difference between these two variant was 120 ± 9 Da. In addition to the two major signals a minor one at 14043 ± 17 Da was observed in plasma. The mass difference to the signal at 13851 Da was 192 ± 11 Da. In urine, traces of TTR were detected in samples from non-pregnant females and from males. In the urine of pregnant women however strong mass signals were observed. As shown in Figure 2c , the variant of 13736 ± 10 Da also present in plasma, as well as lower molecular immunoreactive species were observed at 12847 ± 29, 12984 ± 7, 13202 ± 16, 13349 ± 15 and 13575 ± 15 Da. In urine, although the TTR pattern was different to the one in plasma, there were similarities in all samples in which TTR was present. Figure 2 Mass spectra resulting from SELDI-TOF-MS immunoaffinity analysis of three (1–3) paired plasma (A) and urine (B) samples obtained from healthy individuals (a) and three urine samples (C) from healthy pregnant women. Discussion The SELDI platform can be readily adapted for developing an immunoassay format. This approach, using antibodies as an affinity capture device has been successfully used to detect and quantify different proteins by MS in complex biological mixtures [ 12 ]. Unlike current ELISA technology that uses an indirect detection mechanism by "sandwiching" the targeted protein with antibodies, the SELDI-based immunoaffinity assay allows for direct detection without tags. The specificity of the mass spectrometry immunoassay is unsurpassed because there is the combined discriminating power of both the antibody and the high molecular weight accuracy of the detector. In terms of sensitivity, the detection limit can approach that of current assays for TTR. The antibody used as bait recognizes an epitope in different isoforms of TTR. This direct sampling is a unique advantage over the more traditional ELISA methods in which the resulting signal is a weighted average of all the bound species. The discriminating power of the assay is shown in the direct comparison between Western blotting (Figure 1 ) and SELDI-TOF-MS immunoassay (Figure 2 ). The small mass differences observed with mass spectrometry can not be resolved by the combination of electrophoresis and immunological detection. The results support and confirm previous studies with regard to molecular variants of TTR in plasma and urine [ 13 - 15 ]. As in these studies, TTR in plasma was dominant in two variants. The 120 Da larger variant is the S-cysteinylated form of TTR. The inconsistently present smaller signal at 14043 Da can be attributed to the S-glutathionylated form of TTR [ 13 , 16 , 17 ]. Other forms due to dehydration, or phosphorylation were not observed. The reported isoforms of TTR result when the Cys10 residue makes a mixed disulfide with the amino acid cysteine, the peptide glutathione or the peptide cysteinyl-glycine. Recent studies show that the most prevalent modification of TTR renders it substantially more amyloidogenic than the non-cysteinylated form at pH 5 [ 18 ]. The possible importance as a risk factor for the onset of senile systemic amyloidosis remains to be elucidated. Additionally, the S-homocysteinylation of TTR has been described in plasma of humans with hyperhomocysteinemia [ 19 ]. These two aspects might support the importance of a diagnostic approach to characterize isoforms of TTR more easily. Apart from the case of absence, some modifications might not be detected with this method due to limitations in resolution. Both methods, ELISA and SELDI immunoassay are sensitive enough to observe only trace amounts of TTR in body fluids as in the case of urine of healthy individuals. These amounts were not detected using immunoblotting. Despite the small signal it was obvious that additional variants to the ones in plasma are present in urine. Contrary to a pervious study using a different method [ 14 ] we were not able to observe the cysteinylated form of TTR in urine despite being the dominant one in paired plasma samples. Major differences to this method are the possibility of one step on chip-enrichment and the analysis of much smaller sample amounts but a lower mass resolution. This latter aspect however, can not explain these differences. No study is available in which TTR has been described quantitatively in the urine of healthy individuals. In this study we are able to show for the first time that levels of TTR in urine of pregnant women are 1000-fold higher compared to the levels in urine of healthy non-pregnant individuals. The excretion of substantial amounts of TTR has only been described in individuals with different kidney diseases indicating disturbed glomerular filtration and/or insufficient tubular reabsorption [ 13 ]. The involvement of the megalin receptor in the tubular reabsorption of filtered TTR has been shown in patients with Dent's disease [ 6 ]. Interestingly, as shown by us also for the first time, TTR in the urine of pregnant women is present in different molecular variants. In contrast to plasma, the S-cysteinylated form dominant in plasma was if at all present only in trace amounts. It remains to be determined if this is limited to the excretion of TTR in pregnancy or if under other circumstances such as in different kidney diseases similar molecular variants occur as well. The lower molecular weight immunoreactive variants found in urine of pregnant women may arise from limited proteolysis. At this point it is not possible to determine location and extent of proteolysis within the molecule. The immunoreactive fragments might arise from a breakdown within the plasma or the kidney structures. Conclusions The SELDI immunoaffinity-isolation of TTR in combination with mass spectrometry offers a rapid, highly reproducible and cost-effective system for the determination of molecular variants of microheterogenous proteins and peptides. This is of importance in "second phase" proteomics which is characterized by the repetitive investigation of the same protein to validate the protein phenotype in large population based studies. This provides the basis for a substantial progress in the diagnostic with regard to personalized medicine [ 20 , 21 ]. With regard to TTR, this approach might not only be of importance in the diagnosis of TTR related amyloidosis but may also have the potential as alternative or new biomarkers related to the metabolism of TTR for use in nutrition related disease as well as in the diagnosis of kidney function. Methods Participants Paired plasma and urine samples from 10 healthy individuals were obtained. The plasma was prepared by centrifugation of the blood (1500 × g , 10 min, 4°C) within 1–3 h after acquisition. Additionally, urine samples of the ten individuals and of healthy pregnant women (n = 40) undergoing routine medical examination, were collected and immediately mixed with protease inhibitors (Sigma, Deisenhofen, Germany). Hematuria was tested using a routine dipstick method (Combur 9 Test, Roche, Basel, Switzerland). Cells and other non-soluble material were cleared from the sample by brief centrifugation (1500 × g , 2 min). Aliquots of centrifuged urine were stored at -80°C and processed as soon as possible. The study protocol was approved by the hospitals and University of Potsdam Ethics Committee. Informed consent was obtained from each participant. Determination of TTR using ELISA and Western blotting Polyclonal antibodies to TTR were produced in rabbit and affinity purified (Dako Diagnostics, Hamburg, Germany). TTR in plasma and urine was quantitatively determined by an enzyme-linked immunoassay (ELISA) method developed in our laboratory. To further assess the presence of TTR quantitatively and qualitatively, we performed a SDS-polyacrylamide gel electrophoresis (PAGE) immunoblot analysis as has been described [ 22 ]. Immuno SELDI-TOF-MS Protein A (Sigma, Deisenhofen, Germany) at 0.1 mg/ml in PBS was added (5 μl) to the spots of a pre-activated ProteinChip® Array (PS20; Ciphergen Biosystems Inc., Palo Alto CA, USA). The PS20 array consists of a surface with epoxy groups that dock proteins by covalently reacting with their amine groups. The arrays were incubated for 1 h at 25°C in a humidity chamber. After blocking residual active sites with 5 μl blocking buffer (0.5 M ethanolamine in PBS at pH 8.0) for 25 min, the array was washed three times in a 15 ml conical tube with PBS (pH 7.4). TTR antibody (4.1 μg/μl) was added to individual spots (2 μl) and incubated in a humidity chamber for 1.5 h with mixing. The unbound antibodies were removed by washing the array three times in a 15 ml conical tube with PBS (pH 7.4). Before incubation urine was diluted 1:1 (v:v) in PBS and plasma 1:100 (v:v) in PBS. A total volume of 10 μl was applied to each spot and incubated for 1.5 h at 25°C in a humidity chamber. Samples were washed on the spot with 5 μl PBS three times and finally with 5 μl H 2 O. After drying, 0.6 μl of a saturated energy-absorbing molecule (EAM) solution (5 mg sinapinic acid dissolved in 75 μl acetonitrile and 75 μl 1% trifluoroacetic acid) was applied to the spot surface, and the sample was allowed to dry. After insertion of the ProteinChip Array into the ProteinChip Reader, a laser pulse was focused on the sample under vacuum. The array was analyzed under the following settings: laser intensity 250/220, detector sensitivity 8, mass focus, molecular mass range 10 to 18 kDa, position 20–80 and a 150–200-shot average per sample. CytochromeC (equine cardiac; 12360.1 MW), myoglobin (equine cardiac; 16951.5 MW), GAPDH (rabbit; 35688 MW), albumin (bovine serum; 66433MW) and β-galactosidase (E. coli; 116351MW) were used as calibrators. Mass resolution (defined as m/Δm) is routinely achieved below 300 and mass accuracy was within 0.1%. Peaks with amplitudes at least 3 times greater than the average background noise level were considered. The reproducibility was tested with different aliquots of the same sample on eight different spots of the protein chip array. Competing interests None declared. Authors' contributions FJS participated in the conception, design, data analysis and the writing of the manuscript, KW in data analysis and writing of parts of the manuscript. JR participated in the conception, data analysis and the writing of the manuscript. All authors have read and approved the last version of the manuscript.

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515177

A 4q35.2 subtelomeric deletion identified in a screen of patients with co-morbid psychiatric illness and mental retardation

Background Cryptic structural abnormalities within the subtelomeric regions of chromosomes have been the focus of much recent research because of their discovery in a percentage of people with mental retardation (UK terminology: learning disability). These studies focused on subjects (largely children) with various severities of intellectual impairment with or without additional physical clinical features such as dysmorphisms. However it is well established that prevalence of schizophrenia is around three times greater in those with mild mental retardation. The rates of bipolar disorder and major depressive disorder have also been reported as increased in people with mental retardation. We describe here a screen for telomeric abnormalities in a cohort of 69 patients in which mental retardation co-exists with severe psychiatric illness. Methods We have applied two techniques, subtelomeric fluorescence in situ hybridisation (FISH) and multiplex amplifiable probe hybridisation (MAPH) to detect abnormalities in the patient group. Results A subtelomeric deletion was discovered involving loss of 4q in a patient with co-morbid schizoaffective disorder and mental retardation. Conclusion The precise region of loss has been defined allowing us to identify genes that may contribute to the clinical phenotype through hemizygosity. Interestingly, the region of 4q loss exactly matches that linked to bipolar affective disorder in a large multiply affected Australian kindred.

Background The isolation of unique DNA probes from the sub-telomeric regions of all chromosomes has opened up a field of cytogenetics research that was previously inaccessible to conventional karyotyping protocols [ 1 ]. Since then a number of studies have shown that cryptic structural abnormalities (deletions, duplications etc.) in the subtelomeric regions are relatively commonly found in groups of individuals with idiopathic mental retardation (UK; learning disability; LD). The biological attributes of these chromosomal regions may explain this interesting link. The frequency of meiotic recombination is at its highest at the ends of chromosomes (recently confirmed in the Icelandic microsatellite map of the human genome [ 2 ]). Therefore errors in this process should randomly result in a greater frequency of unbalanced chromosome rearrangement products at telomeres. There also appears to be a greater density of genes at the ends of some chromosomes, especially those with non-staining R-bands. Thus, any telomeric copy number change is likely to affect several genes; potentially resulting in clinical features typical of a contiguous gene syndrome – dysmorphisms, developmental delay and mental retardation. A number of reports have now shown that 0.5%–23% of idiopathic mental retardation cases are associated with cryptic translocations in the vicinity of chromosome telomere (see [ 3 - 17 ] and [ 18 ] for a recent review). FISH, using a commercially available set of subtelomeric probes is the most commonly used screening technique [ 19 , 20 ]. Variations on the theme of FISH (e.g. SKY and CGH) have also been employed. More recently, methods that rely on the detection of copy number changes at subtelomeric loci have been described. MAPH [ 21 - 24 ] is one such technique in which probes are representatively amplified by the polymerase chain reaction following hybridisation to a patient's genomic DNA sample to generate a quantitative profile of subtelomeric sequence copy number. Psychiatric disorders such as schizophrenia (SCZ) and bipolar affective disorder (BPAD) are relatively common in the general population and there is much evidence for a genetic component to susceptibility (for a review see [ 25 ]). However, it is clear from the lack of consistent findings from linkage mapping and association studies that they are likely to be complex and aetiologically heterogeneous disorders. For example, several genes might act simultaneously (oligogenic action) or interact (epistasis) to produce the clinical phenotype in any individual, and those genes might be different in different individuals (locus heterogeneity). An alternative to cohort based linkage and association approaches uses cytogenetic abnormalities as direct pointers to candidate gene loci and this has been successfully applied to patients with psychiatric disorders resulting in the identification of a number of candidate susceptibility genes including DISC1/DISC2 [ 26 ], DIBD1 [ 27 ] and GRIA3 [ 28 ]. The chromosome abnormalities that disrupted these genes were reciprocal translocations visible by standard cytogenetic methods. The risk of schizophrenia and affective disorders in patients with idiopathic mild mental retardation is significantly raised and it is well established that schizophrenia is three times more common in this group than the general population and that there is a strong familial element [ 29 ]. Both bipolar illness and major depressive disorder have also been described as of increased prevalence in the population with mild mental retardation. The study also revealed a previously undetected complex re-arrangement between chromosomes 2 and 11, and a case of trisomy X, but did not address subtelomeric changes. It strongly suggested however that the co-association between mental retardation and schizophrenia is highly familial with greater rates of both schizophrenia and co-morbid schizophrenia/mental retardation occurring in the families of co-morbid probands compared to families of probands with schizophrenia alone or with mental retardation alone. Limbic system (amygdalo-hippocampal) neuropathology is especially pronounced in this group [ 30 ]. We have formed the hypothesis that patients who are co-morbid for severe psychiatric illness and mental retardation may be homogenous in their pathophysiology and that, in addition to large-scale structural chromosomal abnormalities, they may harbour as yet undetected cryptic telomeric changes. To test this we have screened a series of 69 patients co-morbid for mental retardation and psychiatric illness using fluorescence in situ hybridisation (FISH) and multiplex amplifiable probe hybridisation (MAPH). Methods Patient Cohort Local research ethics permission was obtained for this study. The patients were initially ascertained through computerised psychiatric clinical case-registers that allowed us to identify adults with dual diagnosis of psychosis and mental retardation. A specific psychiatry service exists in Scotland to meet the needs of patients with mental retardation who also suffer from psychiatric disorders and initial clinical diagnoses were confirmed by consultation between the relevant specialist clinician involved and the research team member who is also a specialist in the psychiatry of mental retardation (WM). Confirmation that IQ fell within the mild range of mental retardation was obtained from case records. 69 patients with mild mental retardation (IQ 70 to around 50) and a referral diagnosis of co-existing schizophrenia or major affective disorder were studied. Parental samples were not available in many cases due to the age of the probands. This cohort is a subset of 74 originally ascertained subjects: 5 were removed because of aneuploidy (2 cases of 47(XXX)) or after more thorough psychiatric evaluation. One discounted subject with only mental retardation possessed a 6q subtelomeric deletion as determined by several MAPH probes (data not shown). The lifetime version of the Schedule for Affective Disorders and Schizophrenia (SADS-L [ 31 ]) along with extensive case record review, and interviews with key carers and relatives was used to gather the information needed to make a diagnosis of schizophrenia or affective disorder according to the Diagnostic and Statistical Manual 4 th Edition (DSM-IV [ 32 ]). Diagnosis was finalised by consensus between two experienced psychiatrists (DB, WM) one of whom specialises in the psychiatry of mental retardation (WM). SADS-L has previously been successfully used in people with mild mental retardation [ 29 ] to establish psychiatric diagnoses. Overall 49 subjects met the DSM-IV criteria for definite schizophrenia, 3 for schizoaffective disorder, 11 for Bipolar I Disorder, 1 for recurrent Major Depressive Disorder (unipolar depression). In addition, 5 subjects were diagnosed as having a unspecified functional psychosis (DSM-IV 298.9, Psychotic disorder NOS). None had co-existing Down Syndrome or Fragile X disorder. A breakdown of the patients into their clinical categories and methodology of screening is presented in table 1 . Table 1 Subject classification and analysis Breakdown of subjects into their diagnostic categories and applied experimental methodology. MR; mental retardation, SCZ; schizophrenia, BP1; bipolar affective disorder I, SCAFF; schizoaffective disorder, UFP; unspecified functional psychosis, UPR; unipolar depression. Clinical category MAPH alone MAPH and FISH FISH alone MR/SCZ 34 7 8 MR/BP1 6 3 2 MR/SCAFF 1 2 0 MR/UFP 3 0 2 MR/UPR 1 0 0 TOTAL SCREEN 45 12 12 DNA extraction DNA was extracted from venous blood samples (10 mls) of all patients by standard methods using Nucleon BACC2 kits (Nucleon Biosciences). 1 mg/ml dilutions were prepared for MAPH. MAPH All 57 MAPH samples were tested in triplicate using the subtelomeric screening set described previously [ 23 ]. All samples were anonymised prior to MAPH analysis. Each sample was tested three times, and putative positives identified by a univariate method (standard hypothesis testing against a normal distribution) and multivariate methods employed by the software SYSTAT 8.0 (Bivariate scattergraphs and Hadi outlier analysis). Four putative positives were divided into one confident (univariate analysis, p < 0.01, corrected for multiple observations) and three possible (univariate analysis, p < 0.05, corrected for multiple observations, Hadi outlier distance >4, all three results reporting a consistent change: either all >1.0 or <1.0) positive results. The three "possible" positives have since been discounted since they involved gain of the 20p telomeric probe ST18E1, which from experience with normal control subjects has shown to have unacceptably high measurement error. This probe has been replaced in more recent formulations of the subtelomeric probe set. Subtelomeric FISH Blood samples from a subset of the patients were cultured in Peripheral Blood Medium (Sigma) for 72 hours. After colcemid treatment for one hour, lymphocytes were lysed and fixed in methanol:acetic acid (3:1). Fixed metaphase material was dropped onto microscope slides. Each slide was hybridised by three fluorescently labelled probe mixes (ToTelVysion, Vysis Inc.) under separate coverslips. All 15 mixes covering every subtelomeric region for a patient could thus be analysed on 5 slides. Images were captured on a Zeiss Axioskop2 microscope coupled to a Macintosh G4 computer running SmartCapture2.1 software (Digital Scientific). Five metaphases were scored for each probe mix. Results A subtelomeric deletion identified in one subject Complete accord was seen in the 12 instances where both screening methodologies were used. The FISH approach did not detect any subtelomeric abnormalities (including balanced translocations, which would not be observable by MAPH). However, MAPH identified a subject with a loss of one copy of the 4q subtelomeric region (p < 1 × 10 -3 , corrected for multiple observations). It is unlikely that this copy number change (4q - ) defined by MAPH represents an irrelevant polymorphism; no similar change was found on analysis of 83 unrelated control individuals, giving an upper (95% confidence) limit of 1.6% for the frequency of this variant. Precise definition of 4q loss Additional MAPH probes were designed to determine the extent of the 4q deletion (fig. 1 ). The results show that the proximal boundary of the subtelomeric deletion is between the FAT gene and the proximal end of clone 713c19 (Genbank accession number AC108073). P values for boundary probes (Ho, value = 1.00), p < 5 × 10 -5 for deletion and p > 0.05 for normal dosage. This 4q deletion encompasses a region of annotated genomic DNA of approximately 3 Mb. The transcript map of this region is not yet completely defined (see Table 2 ) but contains at least 10 transcriptional units with varying levels of authenticity/experimental evidence attached to each one. Figure 1 Subtelomeric region of chromosome 4q Only annotated, unique chromosome sequence is shown, derived from the November 2002 version of the UCSC Human Genome assembly (subtelomeric repeats would extend to the right of the diagram). A scale bar and the gene content (see Table 2) of the region are shown. The positions of the MAPH markers are also shown which allowed the maximum and minimum extents of the deletion to be defined (black bar). Above the chromosome region is shown the result of duplicate analysis from each MAPH probe (mean +/- 95% CI), together with the 3 standard deviation threshold and the results from the other control probes (mean +/- 95% CI). Table 2 Gene content of 4q deletion Genes/putative transcriptional units within the deleted region on chromosomes 4q. ESTs a-f are represented in figure 1. An attempt to gauge the approximate expression levels of each gene was based on the number of EST clones present in the UCSC Human Genome Browser (Nov.2002/Apr.2003 releases). A brief summary of gene function and a representative accession number, where informative, is also included. TUBB4Q (4q35) is omitted from this list because it is a confirmed pseudogene. 4q35 GENE EST exp. Function/comments FAT ++++ Cadherin-related tumor suppressor homologue precursor EST a + (BE856720) Novel. EST b + (BM806339) Novel. Contains 5 1/2 copies of 34aa repeat motif EST c ++ (AI917275) Novel. No obvious ORF ZFP42/FLJ32157 + (AK056719) Similar to transcriptional repressor protein YY1 FLJ25801 + (AK098667) Protein contains SMC (chromosome segregation ATPase) domain and PRY/SPRY domains (unknown function). EST d +++ (BU571187) Novel. EST e + (BC033535) Novel. EST f + (BC029568) LOC256307 novel predicted gene FRG1 ++++ Facioscapulohumeral muscular dystrophy region gene 1 DUX4 + Homeobox protein, multiple copies. Discussion Unlike simple chromosomal translocations, large deletions associated with a certain condition can present many candidate genes for further study. In the context of psychiatric disorders velo-cardio-facial syndrome (VCFS or del22q11 syndrome) offers a model of how a cryptic deletion associated with schizophrenia has highlighted several candidate genes for future study [ 33 - 35 ]. We used two methods to screen for subtelomeric changes in our cohort of patients. The MAPH technique has proved a fast and accurate method for determining copy number changes in the human genome and represents a cost-effective route to the screening of large numbers of patients. In addition, the disorders that can be studied by this approach are limited only by the design of suitable primers so that screening for both single and contiguous gene disorders is feasible. MAPH has also been proved to be a simple method to map deletion breakpoints with greater resolution than FISH. The subtelomeric FISH approach is a more demanding approach because of the patient sample preparation, the requirement for specialised microscopy equipment, the cost of the commercial probe sets and the labour involved. Nevertheless, FISH has some advantages: the FISH approach is the only way to detect balanced chromosome rearrangements such as inversions and translocations. Other studies of subtelomeric regions in mental retardation subjects have identified such chromosomal aberrations. In addition, FISH has been a vital technique for identifying disrupted genes such as DISC1 in psychiatric patients with (non-subtelomeric) chromosomal rearrangements. Therefore, the selection of the screening technique should be determined by the number of cases to be studied and the nature of the abnormalities expected. We have identified a subtelomeric deletion within our cohort of 69 patients using the MAPH and FISH methodologies. This 4q deletion is associated with a co-morbid phenotype of schizoaffective disorder and mild mental retardation (fullscale IQ between 60 and 70). Consent was not forthcoming to determine whether the deletion was of parental or de novo origin. However, psychiatric illness has not been diagnosed in other members of the family. We cannot, therefore, formally link the presence of the deletion with mental retardation and/or psychiatric illness in the patient. The annotation of transcripts at 4q35.2 is currently an active area of research (see fig. 1 and table 2 ) because of good linkage evidence (LOD score of 3.2 for microsatellite marker D4S1652) from an extended Australian kindred multiply affected with bipolar affective disorder [ 36 - 38 ]. Importantly, the principal linkage region almost exactly matches the deletion interval observed in our patient. The 4q deletion patient has been diagnosed with schizoaffective disorder (DSM-IV 295.7) – with periods of psychotic depression but also mood incongruent hallucinations and delusions. This is in contrast to the clear bipolar affective disorder diagnosed for members of the described linkage family. However, it has been repeatedly observed that schizoaffective disorders and bipolar affective disorders overlap clinically and are indeed often difficult to separate. One postulated explanation for the now frequently reported linkage overlaps between bipolar illness, schizoaffective disorders and schizophrenia is that the inherited susceptibility is for psychosis rather than a specific disorder (reviewed in [ 39 ]). The subtelomeric region of 4q is also interesting because it contains a candidate gene, FRG1 , for facioscapulohumeral muscular dystrophy. The 4q patient does not show any of the typical features of this disorder but this can be explained by the fact that copy number does not appear to be critical for the onset of the disorder [ 40 ]. Rather, the proximity of the gene to a variable number telomeric repeat sequence (D4Z4) seems to be the chief determinant of pathology [ 41 ]. Of the 69 patients with clear co-morbidity, one (1.4%) possessed a single copy subtelomeric deletion. This frequency is in line with those from studies of individuals with mental retardation alone [ 18 ]. As more studies examine chromosomal integrity in people with mental retardation or other conditions we hope that replication of subtelomeric abnormalities will be observed, perhaps leading to the eventual clinical definition of range of 'subtelomeric syndromes' such as that recently described for the subtelomeric deletion of 1q [ 42 ]. Conclusions The identification of the precisely delimited 4q deletion may contribute to the mapping of the susceptibility gene for psychiatric illness at this locus. The finding of bipolar affective disorder linkage to this region suggests that, in this case at least, the schizoaffective and mental retardation components to the co-morbid phenotype may be discrete and genetically separable in the manner of other contiguous gene disorders. If it is assumed that either component of the clinical phenotype is caused by haploinsufficiency then examining the comparative expression levels of candidate genes in normal, bipolar-linked and 4q deleted lymphoblastoid cell lines might provide a quick route to the identification of causative genes. Alternatively, gene association studies may be required to identify the candidate psychiatric illness gene at 4q35. The high rate of recombination in subtelomeric DNA means that a higher density of genetic markers will be required to establish linkage or association reliably; conversely, once detected, the high rate of recombination will allow high resolution fine mapping of significant loci. Competing interests None declared. Authors' contributions BSP participated in the design of the study and the FISH analysis and drafted the manuscript. EJH and JALA carried out all MAPH assays and associated statistical analyses. MPM carried out blood culture and FISH analysis. WJM conceived the study and was responsible for the generation of all clinical data, and DHRB and DJP participated in the study design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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555553

JTE-522, a selective COX-2 inhibitor, inhibits growth of pulmonary metastases of colorectal cancer in rats

Background Epidemiological studies have shown that individuals who regularly consume NSAIDs have lower rates of mortality associated with colorectal cancer. Because COX-2 inhibitors prevent tumor growth through some mechanisms, we assessed the effect of JTE-522, a selective COX-2 inhibitor, on pulmonary metastases of colon cancer in a rat model. Methods A suspension of 5 × 10 6 RCN-9 (rat colon cancer cells) was injected into the tail vein of 24 anesthetized male F344/DuCrj rats. Oral JTE-522 (0, 3, 10, or 30 mg/kg/day) was administered from the day before RCN-9 injection until the end of the study. Twenty-four days later, the lungs were removed from sacrificed rats and weighed. Pulmonary metastatic tumors were microscopically evaluated in the largest cross sections. We also performed immunohistochemical staining for both COX-2 and VEGF. Results JTE-522 dose-dependently decreased lung weight (p = 0.001) and the size of pulmonary metastatic tumors (p = 0.0002). However, the differences in the number of metastatic tumors among 4 groups were insignificant. Significant adverse effects of JTE-522 were undetectable. Immunohistochemical staining showed high levels of both COX-2 and VEGF in pulmonary metastatic tumors. Conclusion JTE-522 dose-dependently decreased the size, but not the number of pulmonary metastases. COX-2 inhibitors might block metastatic tumor growth, but not actual metastasis. Selective COX-2 inhibitors might be useful as therapeutic agents that inhibit the growth of metastatic tumors, as well as the tumorigenesis of colorectal cancer.

Background Colorectal cancer is a widespread fatal malignancy in the West [ 1 ] and its incidence in Japan is increasing [ 2 ]. Epidemiological studies have shown that regular doses of nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with lower rates of colorectal polyps, colorectal cancer, and colorectal cancer-associated mortality. Waddell and Loughry initially reported that sulindac decreased the number of polyps in patients with familial adenomatous polyposis (FAP) [ 3 ]. The largest study of aspirin and colon cancer was undertaken by Thun et al . in 1991 [ 4 ]. They found that colon cancer mortality decreased among those who used aspirin, and that the protective effect was increased by increasing doses. NSAIDs inhibit prostaglandin production mainly by inhibiting cyclooxygenase (COX) that is expressed as COX-1 and COX-2 isoforms. Cox is a key enzyme, which converts arachidonic acid to prostaglandins. COX-1 is constitutively expressed in most tissues and is thought to be responsible for homeostatic functions. On the other hand, COX-2 expression is induced by various factors including cytokines, growth factors, and tumor promoters [ 5 , 6 ]. Many studies have indicated that COX-2 expression is associated with both the carcinogenesis and growth of colon cancer. COX-2 is overexpressed in 85% of human colorectal cancers [ 7 ]. Oshima et al . disclosed that COX-2 gene knockouts and a COX-2 inhibitor decreased the number of polyps of apcΔ716 knockout mice [ 8 ]. In addition, several studies have shown that the selective inhibition of COX-2 reduces colorectal tumorigenesis in various models of carcinogenesis. Reddy et al . found that the COX-2 inhibitor, SC-58635 (celecoxib), significantly suppressed colonic aberrant crypt foci and crypt multiplicity in rats [ 9 ]. This supports the hypothesis that COX-2 inhibitors are chemopreventive. Kawamori et al . showed that celecoxib inhibited both the incidence and multiplicity of colon tumors in a rat model [ 10 ], and another study showed that COX-2 expression in colon cancer cells increased metastatic potential [ 11 ]. We reported that increased COX-2 levels in human colorectal carcinomas correlated with both the size and invasiveness of tumors [ 12 ]. Tomozawa et al . demonstrated a correlation between COX-2 overexpression and the recurrence of colorectal cancer [ 13 ]. It is well known that colorectal metastasis occurs primarily in the liver and secondarily in the lung [ 14 ]. A laparotomy is necessary for making experimental model of liver metastasis in rats, but it is also a stressful event. On the other hand, it is less invasive to make lung metastasis by injection of tumor cells into the tail vein of rats. Thus, we considered that the experimental model of pulmonary metastasis was appropriate for the evaluation of the real effect of JTE-522, a selective COX-2 inhibitor, on hematogenous metastasis of colon cancer in rats. The aim of this study was to examine the effect of JTE-522 on hematogenous metastasis of colon cancer in a rat model. Methods Preparation of JTE-522 The selective COX-2 inhibitor, JTE-522 [ 15 ], was suspended in 0.5% carboxymethyl cellulose sodium salt (Wako Life Science Reagents, Osaka, Japan). Animals Twenty-four 4-week-old male F344/DuCrj rats, weighing 50 to 70 g purchased from SLC (Shizuoka, Japan) were acclimatized for one week before use in this study. The rats were maintained in an environment with a controlled temperature (22 to 24°C), humidity (40 to 50%), and lighting (12 hours light-dark cycle) with free access to tap water and standard rodent chow (CE-2, Nihon Clea, Tokyo, Japan). Tumors The RIKEN CELL BANK donated the rat colon cancer cell line, RCN-9, which was established by Inoue et al . after inbred F344/DuCrj rats were subcutaneously injected with neutralized 1, 2- dimethylhydrazine (DMH) [ 16 ]. The tumor cells were stored at -80°C. After thawing, tumor cells were washed in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 (Sigma Chemical Co., St. Louis, MO) medium containing 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 0.05% penicillin-streptomycin (Sigma). After repeated subculture, cell viability was assessed by Trypan blue dye exclusion and the cells were counted using a hemocytometer. Induction of lung metastases F344 male rats were anesthetized with ether and then a tumor suspension containing 5 × 10 6 RCN-9 cells in 0.4 ml of PBS was injected into the tail vein. Pulmonary metastases of colon cancer in rats Twenty-four rats were divided into four groups. Group 1 (control) was not given JTE-522. The other three groups all orally received daily doses of 3, 10 and 30 mg/kg JTE-522 (groups 2, 3 and 4, respectively) by gavage from the day before RCN-9 injection until the end of the study. The rats were sacrificed 24 days after injection and the lungs were removed and weighed. After 3-day formalin fixation, lungs were embedded in paraffin. Paraffin-embedded sections were cut into 3-μm serial sections, which stained with hematoxylin and eosin to evaluate the pharmacological effects of JTE-522 on pulmonary metastases. The number and the largest diameter of metastatic tumors were examined microscopically in the largest sagittal cross section. The largest metastatic tumor was selected after measuring the diameter of 10 tumors in each slide. Immunohistochemistry Slides were immunohistochemically examined using the Universal Immuno-enzyme Polymer (UIP). Sections of 3 μm thick were cut from formalin-fixed and paraffin-embedded blocks of rat tissues. The slides were deparaffinized in xylene, rehydrated through a graded series of ethanols and washed with distilled deionized water. Antigen was retrieved by incubating the sections in 10 mmol/L citrate buffer (pH 6.0) in an autoclave (120°C) for 5 minutes. Thereafter, slides were cooled to room temperature and then washed with distilled deionized water. To quench endogenous peroxidase activity, slides were immersed in 3% H 2 O 2 in methanol for 20 minutes. After a short rinse in phosphate-buffered saline (PBS), non-specific binding was blocked by incubating the sections in 10% normal goat serum for 10 minutes at room temperature. Thereafter, the slides were incubated with primary antibodies directed against COX-2 (rabbit polyclonal, Cayman Chemical, Ann Arbor, MI; dilution 1:250) for 2 hours or vascular endothelial growth factor (VEGF) (rabbit polyclonal, VEGF A-20: sc-152; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:200) for 1 hour at room temperature. The slides were washed in PBS (3 × 5 minutes) and incubated with secondary antibody (N-Histofine ® Simple Stain MAX PO, Nichirei, Tokyo, Japan) for 30 minutes at room temperature. After washing in PBS, color was developed using 3, 3'-diaminobenzidine tetrahydrochloride (Sigma) in 50 mmol Tris-HCl (pH 7.5) for 5 minutes and counterstaining with 1% Mayer's hematoxylin. Sections known to show intense immunostaining for COX-2 and VEGF were included in each experiment, having been incubated with either primary antibody or PBS as positive and negative controls, respectively. The positive controls were always obviously stained and negative controls were never stained. Evaluation of immunostaining The specimens immunostained for COX-2 and VEGF were independently scored by two blinded investigators. Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative fashion. Sections were scored, according to the method of Yukawa et al [ 17 ]. Both the number of positive cells and the intensity of staining were evaluated as follows. The number of positive cells: none, 0; focal (one third of cells stained), 1; multifocal (two third of cells stained), 2; and diffuse (most cells stained), 3. The intensity of staining: none, 0; mild (between 0 and 2), 1; and strong (clearly identified by × 40 magnification), 2. The score for distribution and intensity were added and graded as follows: 0, 1, and 2: negative; and 3, 4, and 5: positive. Statistical analysis The effects of JTE-522 on pulmonary metastasis were analyzed by the Kruskal-Wallis test and are expressed as means ± standard deviation. A correlation between COX-2 and VEGF expression was analyzed by Spearman's rank test. In all instances, statistical significance was established at p < 0.05. Results Pulmonary metastases Each group consisted of 6 rats. JTE-522 significantly and dose-dependently decreased the weight of the lungs (p = 0.0001, Figure 1A ). Cross sections contained many metastatic tumors that were not visible on the surface (Figures 2 ). The size of the metastatic tumors was significantly and dose-dependently decreased (p = 0.0002, Figure 1B ), although the number of metastases did not differ (Figure 1C ). JTE-522 appeared to have no significant adverse effects. Immunohistochemistry Immunohistochemical staining showed that both COX-2 and VEGF were expressed at high levels in pulmonary metastatic tumors of all groups (Figures 3 ). The large airways and vascular structures were stained for both proteins, as were tumor cells in all groups. However, there was no correlation between COX-2 score and VEGF score (p = 0.60, Figure 4 ). The degree of COX-2 and VEGF immunohistochemical staining in tumor cells did not significantly differ among groups (Table 1 ). Discussion JTE-522 dose-dependently reduced the weight of the lung with metastases and the size of metastatic tumors. These observations are consistent with those of other studies of selective COX-2 inhibitors. In the present study, however, the number of metastatic lesions determined microscopically was not reduced by JTE-522, which conflicts with the earlier findings. Masferrer et al . described the inhibition of tumor growth and lung metastasis of Lewis Lung carcinoma in mice by continuous dietary celecoxib (160–3200 ppm) supplied from the date of implantation [ 18 ]. Celecoxib at a dose of 160 ppm did not affect the number and size of lung metastases, but doses between 480 and 3200 ppm reduced the number of metastatic nodules by >50%. Histopathological analysis revealed that celecoxib dose-dependently reduced the size of the metastatic lung tumors. Masferrer et al . evaluated the pharmacological effects of celecoxib on lung metastasis by counting the metastatic tumors using a stereomicroscope and by histochemical analyses of consecutive lung sections. Tomozawa et al . investigated the effect of JTE-522 on lung metastasis of colon cancer in mice [ 19 ]. Their study showed that JTE-522 significantly reduced the number of metastatic nodules and lung weight. They macroscopically counted the metastatic tumors on the lung surface. Our study using cross sections disclosed that many nodules were not visible on the surface and could not be seen by the naked eye. These observations may indicate that JTE-522 blocks the growth of metastatic tumors, but does not inhibit the process of metastasis. On the other hand, a recent study in vitro, using cancer cell lines preincubated with celecoxib, suggests that COX-2 is involved in adhesion of cancer cells to vascular endothelium [ 20 ]. If the cancer cells had been pretreated with JTE-522 for an appropriate period in the present study, it might reduce the number of the lung metastases. Here, we immunohistochemically stained COX-2 and VEGF because some reports indicate that prostaglandins regulate VEGF expression [ 21 , 22 ] and that COX-2 inhibitors directly affect angiogenesis [ 23 ]. Other recent studies have confirmed the notion that tumor growth and metastasis are dependent on angiogenesis and require the development of new vessels [ 24 - 26 ], and that COX-2 is related to tumor angiogenesis. COX-2 modulates the production of angiogenic factors derived from colon cancer cells, while COX-1 regulates angiogenesis in endothelial cells [ 27 ]. COX-2 is also related to tumor angiogenesis in human colorectal cancer. Cianchi et al . identified a significant correlation between COX-2 and VEGF, the latter of which might be one of the most important mediators of the COX-2 angiogenic pathway [ 28 ]. Immunohistochemical staining in the present study revealed parallel COX-2 and VEGF expression that was mainly localized in the cytoplasm of tumor cells, epithelial cells of the airways, and vascular structures. These findings may indicate that COX-2 is involved in the production of VEGF. Furthermore, the present study supports our previous report that JTE-522 interferes with the growth of metastatic tumors of colon cancer, due to inhibition of neovascularization [ 29 ]. Our results showed that high levels of COX-2 were expressed in the groups administered with JTE-522 as well as in controls. This is consistent with the fact that JTE-522 suppresses not the production, but the activity of COX-2 in prostaglandin biosynthesis [ 15 ]. VEGF was also highly expressed in all groups. One of the reasons may be hypoxia induced by multiple pulmonary metastases. Christou et al . reported that hypoxic animals have significantly higher VEGF concentrations compared with normoxic controls [ 30 ]. On the other hand, these findings may indicate that VEGF is produced via a pathway, which does not include COX-2, as well as through a COX-2 pathway. We reported that COX-2 is related to human colorectal tumorigenesis. COX-2 mRNA expression is obviously increased in sporadic adenomas compared with normal colorectal mucosa and is significantly greater in adenomas with larger diameters [ 31 ]. Additionally, COX-2 expression and degree of dysplasia are significantly associated in adenomas [ 32 ]. COX-2 levels in human colorectal carcinomas are significantly higher in larger tumors and in those that invade more deeply [ 12 ]. We therefore postulated that COX-2 might correlate with metastasis as well as primary colorectal tumor, because hematogenous metastasis is the key for prognostic factor in colorectal cancer. The advances in the treatment for metastasis would improve the prognosis of the patients with colorectal cancer. The present study found that JTE-522 dose-dependently suppressed metastatic tumor growth, although the function to prevent cancer cells from metastasizing might not be so strong. Our recent report has revealed that administration of rofecoxib, a selective COX-2 inhibitor, decreased the number and size of rectal polyps in familial adenomatous polyposis patients, whereas the relapse of rectal polyps was seen 3 months after completion of the treatment [ 33 ]. This phenomenon might indicate that COX-2 inhibitors make colorectal polyps invisible, although atypical cells still exist microscopically. This is consistent with the result of the present study. Recent reports have shown the inhibitory effect of rofecoxib on liver metastases. Yao et al . demonstrated that rofecoxib decreased the growth and metastatic potential of colorectal cancer in mice through multiple mechanisms [ 34 ]. Fenwick et al . reported that refecoxib negatively regulated angiogenesis in human colorectal cancer liver metastases [ 35 ]. It is considered that COX-2 inhibitors can be used as therapeutic agents for hematogenous metastases of colorectal cancer. Conclusion In conclusion, JTE-522, a selective COX-2 inhibitor, dose-dependently inhibited the growth of pulmonary metastasis from colorectal cancer in a rat model. Selective COX-2 inhibitors may constitute useful therapeutic agents that can interfere with the growth of hematogenous metastasis, as well as the tumorigenesis of colorectal cancer. Competing interests The author(s) declare that they have no competing interests. Authors' contributions HK carried out the animal experiment and drafted the manuscript. HK and TH performed immunostaining and its evaluation. HU performed the statistical analysis. ME and KS participated in the design of the study and its coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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350664

Sequence-Specific Inhibition of Small RNA Function

Hundreds of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified from both plants and animals, yet little is known about their biochemical modes of action or biological functions. Here we report that 2′ -O- methyl oligonucleotides can act as irreversible, stoichiometric inhibitors of small RNA function. We show that a 2′ -O- methyl oligonucleotide complementary to an siRNA can block mRNA cleavage in Drosophila embryo lysates and HeLa cell S100 extracts and in cultured human HeLa cells. In Caenorhabditis elegans , injection of the 2′- O -methyl oligonucleotide complementary to the miRNA let-7 can induce a let-7 loss-of-function phenocopy. Using an immobilized 2′ -O- methyl oligonucleotide, we show that the C. elegans Argonaute proteins ALG-1 and ALG-2, which were previously implicated in let-7 function through genetic studies, are constituents of a let-7 -containing protein–RNA complex. Thus, we demonstrate that 2′ -O- methyl RNA oligonucleotides can provide an efficient and straightforward way to block small RNA function in vivo and furthermore can be used to identify small RNA-associated proteins that mediate RNA silencing pathways.

Introduction The endoribonuclease Dicer produces two types of small regulatory RNAs that regulate gene expression: small interfering RNAs (siRNAs) and microRNAs (miRNAs) ( Bernstein et al. 2001 ; Grishok et al. 2001 ; Hutvágner et al. 2001 ; Ketting et al. 2001 ; Knight and Bass 2001 ). In animals, siRNAs direct target mRNA cleavage ( Elbashir et al. 2001 b, 2001 c), whereas miRNAs block target mRNA translation ( Lee et al. 1993 ; Reinhart et al. 2000 ; Brennecke et al. 2003 ; Xu et al. 2003 ). Recent data suggest that both siRNAs and miRNAs incorporate into similar, perhaps even identical, protein complexes and that a critical determinant of mRNA destruction versus translation regulation is the degree of sequence complementary between the small RNA and its mRNA target ( Hutvágner and Zamore 2002 ; Mourelatos et al. 2002 ; Zeng et al. 2002 ; Doench et al. 2003 ; Saxena et al. 2003 ; Zeng et al. 2003 ). Target RNA cleavage directed by siRNA is called RNA interference (RNAi). RNAi is a powerful method for the study of gene function in animals and plants and has even been proposed as a therapy for treating genetic disorders and viral infections. Biochemical studies in Drosophila S2 cells ( Bernstein et al. 2001 ; Hammond et al. 2001 a; Caudy et al. 2002 ; Liu et al. 2003 ) and affinity purification ( Martinez et al. 2002 ) or immunoprecipitation ( Hutvágner and Zamore 2002 ) from cultured human HeLa cells have identified protein components of the RNAi effector complex, the RNA-induced silencing complex (RISC). Genetic mutations that disrupt RNAi in Caenorhabditis elegans , Drosophila , green algae, fungi, and plants have likewise identified proteins required for RNAi ( Cogoni and Macino 1997 , 1999 a, 1999 b; Ketting et al. 1999 ; Tabara et al. 1999 , 2002 ; Catalanotto et al. 2000 , 2002 ; Dalmay et al. 2000 , 2001 ; Fagard et al. 2000 ; Grishok et al. 2000 ; Ketting and Plasterk 2000 ; Mourrain et al. 2000 ; Wu-Scharf et al. 2000 ; Grishok and Mello 2002 ; Tijsterman et al. 2002 a, 2002 b). Key steps in the RNAi pathway have also emerged from studies of RNAi reconstituted in cell-free extracts ( Tuschl et al. 1999 ; Zamore et al. 2000 ; Hammond et al. 2001 b; Nykänen et al. 2001 ; Martinez et al. 2002 ; Schwarz et al. 2002 ; Tang et al. 2003 ). Recently, hundreds of miRNAs have been identified in animals and plants ( Lagos-Quintana et al. 2001 , 2002 ; Lau et al. 2001 ; Lee and Ambros 2001 ; Reinhart et al. 2002 ; Ambros et al. 2003 ; Aravin et al. 2003 ; Brennecke and Cohen 2003 ; Lim et al. 2003 ). Of these, the biological functions of only four animal miRNAs are known. In C. elegans , the miRNAs lin-4 ( Lee et al. 1993 ; Olsen and Ambros 1999 ) and let-7 ( Reinhart et al. 2000 ) regulate developmental timing, whereas the Drosophila miRNAs bantam and miR-14 control cell survival by repressing translation of proapoptotic genes ( Brennecke et al. 2003 ; Xu et al. 2003 ). Computational approaches promise to identify the mRNA targets of other miRNAs ( Enright et al. 2003 ; Lewis et al. 2003 ; Stark et al. 2003 ), but these proposed miRNA/target mRNA pairs will require experimental confirmation. Despite the widespread use of RNAi to “knock down” gene function, the RNAi pathway itself remains poorly understood. Similarly, despite intensive efforts to identify all miRNAs in vertebrates, worms, and flies, the mechanisms underlying miRNA function remain mysterious and no biological function has been ascribed to the vast majority of miRNAs. Here we show that 2′- O -methyl oligonucleotides are potent and irreversible inhibitors of small RNA-directed RNA silencing in vivo and in vitro. Our experiments using 2′- O -methyl oligonucleotides also demonstrate that the acquisition of a target RNA by an siRNA-programmed RISC is far more efficient than the binding of an antisense oligonucleotide to the same region of the target. To demonstrate the utility of 2′- O -methyl oligonucleotides in probing RNA silencing pathways, we show that 2′ -O- methyl oligonucleotides efficiently block siRNA-directed RISC activity in cell extracts and in cultured human HeLa cells. When injected into C. elegans larvae, a let-7 -complementary 2′- O -methyl oligonucleotide can efficiently suppress lin-41 translational repression by the let-7 miRNA. Finally, we use a tethered 2′- O -methyl oligonucleotide to demonstrate association of the C. elegans Argonaute proteins ALG-1 and ALG-2 with let-7 . Results Inhibition of RNAi by 2′- O -Methyl Oligonucleotides Although RNAi has proved a straightforward and cost-effective method to assess the function of protein-coding mRNAs ( Fire et al. 1998 ; Caplen et al. 2000 , 2001 ; Carthew 2001 ; Elbashir et al. 2001 a) and even some noncoding RNAs ( Liang et al. 2003 ), no comparable method allows the sequence-specific inactivation of the siRNA or miRNA components of the RISC. The ideal inhibitor of RISC function would be a nucleic acid that would be recognized by the RISC by nucleotide complementarity, but be refractory to RISC-directed endonucleolytic cleavage or translational control. In theory, such a molecule would titrate out RISCs containing a complementary siRNA or miRNA, but not affect the function of RISCs containing guide RNAs unrelated in sequence. Such a RISC inhibitor should also resist degradation by cellular ribonucleases so that it persists long enough to bind RISC and block its function. Finally, the ideal inhibitor of small RNA function would act at concentrations unlikely to elicit nonspecific responses to the inhibitor itself, i.e., in the low nanomolar range, the same concentration at which siRNAs themselves are effective. At micromolar concentration, DNA antisense oligonucleotides may block miRNA function in Drosophila embryos ( Boutla et al. 2003 ), but the poor stability of DNA oligonucleotides in vivo may limit their utility. Phosphorothioate-substituted DNA oligonucleotides, which show good in vivo stability, do not inhibit RISC function in vitro (data not shown). 2′- O -methyl oligonucleotides are also highly resistant to cellular ribonucleases ( Inoue et al. 1987 ). To test whether 2′- O -methyl oligonucleotides can act as RISC inhibitors, we asked whether a 2′- O -methyl oligonucleotide, tethered to streptavidin paramagnetic beads via a 5′ biotin linkage, could be used to deplete siRNA-programmed RISC from the reaction. Drosophila embryo lysate was programmed with a synthetic siRNA duplex directed against a firefly ( Photinus pyralis ) luciferase ( Pp -luc) mRNA target ( Figure 1 A). Then, a tethered 31-nt 2′- O -methyl oligonucleotide complementary to the 21-nt siRNA antisense strand was added. Finally, the beads were removed from the solution using a magnet, and the supernatant was tested for siRNA-programmed RISC activity. Under these conditions, the 2′- O -methyl oligonucleotide completely depleted the reaction of the RISC programmed with the antisense strand of the siRNA, but not of RISC programmed with the sense strand ( Figure 1 B). Thus, depletion occurred only when the siRNA strand contained within RISC was complementary to the tethered oligonucleotide. Figure 1 A 2′ -O- Methyl RNA Oligonucleotide Inhibits RNAi In Vitro in Drosophila Embryo Lysate (A) Sequences of the sense and antisense Pp -luc target RNAs (black), the siRNA (red, antisense strand; black, sense strand), and the sense and antisense 2′ -O- methyl oligonucleotides (blue) used. (B) Sequence-specific depletion of RNAi activity by immobilized 2′- O -methyl oligonucleotides from Drosophila embryo lysate programmed with siRNA. siRNA was incubated with lysate to assemble RISC; then, immobilized 2′- O -methyl oligonucleotide was added. Finally, the beads were removed from the supernatant, and either sense or antisense 32 P-radiolabeled target RNA was added to the supernatant to measure RISC activity for each siRNA strand. Symbols and abbreviations: Ø, target RNA before incubation with siRNA-programmed lysate; T, total reaction before depletion; unbound, the supernatant after incubation with the immobilized antisense (AS) or sense (S) 2′- O -methyl oligonucleotides shown in (A). The absence of 5′ cleavage product demonstrates that the sense oligonucleotide depleted RISC containing antisense siRNA, but not sense siRNA, and the antisense oligonucleotide depleted the sense RISC, but not that containing antisense siRNA. Bi, 5′ biotin attached via a six-carbon linker. We extended this method to measure the amount of RISC formed in the in vitro reaction at different concentrations of the siRNA duplex. An siRNA duplex in which the antisense strand was 5′- 32 P-radiolabeled was incubated in the reaction; then, the tethered 2′- O -methyl oligonucleotide was added to deplete the reaction of antisense siRNA-programmed RISC. The beads were then washed and the fraction of 32 P-siRNA bound to the beads determined. Depletion was verified by testing the supernatant for RISC activity. Formally, the amount of 32 P-siRNA retained on the beads for a given concentration of siRNA duplex places an upper limit on the concentration of RISC formed. However, our results using this assay are, within error, identical to the amount of RISC measured by two independent methods: the accumulation of single-stranded siRNA from functionally asymmetric siRNA duplexes ( Schwarz et al. 2003 ), and the magnitude of the burst of target cleavage measured by pre-steady-state kinetics (B. Haley and P. D. Zamore, unpublished data). The simplest explanation for our results is that this assay directly measures siRNA incorporated into RISC. Figure 2 A shows the results of this assay for six different concentrations of siRNA duplex (5 nM, 15 nM, 25 nM, 50 nM, 100 nM, and 200 nM siRNA). First, the data show that RISC assembly in vitro is inefficient; the majority of siRNA duplexes do not contribute to RISC production. Second, RISC assembly is saturable, suggesting that some component of RISC itself is limiting. Figure 2 2′- O -Methyl Oligonucleotides Act as Stoichiometric, Irreversible Inhibitors of RISC Function (A) The immobilized sense 2′- O -methyl oligonucleotide was used to determine the concentration of 32 P-radiolabeled antisense siRNA assembled into RISC in Drosophila embryo. The 2′- O -methyl oligonucleotide and siRNA duplex are shown in Figure 1 A. (B–G) Inhibition of RNAi was measured using free 2′- O -methyl oligonucleotide and 1.3 nM (B), 4.6 nM (C), 9.3 nM (D), 14.5 nM (E), 18 nM (F), and 23.5 nM (G) RISC. The concentration of 2′- O -methyl oligonucleotide required for half-maximal inhibition (IC 50 ) was calculated by fitting each dataset to a sigmoidal curve using a Hill coefficient of 1. (H) A plot of IC 50 versus RISC concentration suggests that each 2′- O -methyl oligonucleotide binds a single RISC. The data suggest that binding is essentially irreversible. To understand better the mechanism by which the 2′- O -methyl oligonucleotide interacted with RISC, we measured the concentration of free 2′- O -methyl oligonucleotide required for half-maximal inhibition of RISC activity (IC 50 ; Figure 2 B– 2 G) at the six different RISC concentrations determined in Figure 2 A. The IC 50 for inhibition by free 2′- O -methyl oligonucleotide is shown for each RISC concentration in Figure 2 H. The IC 50 for the 2′- O -methyl oligonucleotide was remarkably close to half the RISC concentration, suggesting that a single 31-nt 2′- O -methyl oligonucleotide binds each RISC and blocks its function. Consistent with this apparent 1:1 stoichiometry, the data for the 2′- O -methyl oligonucleotide titrations fit well to sigmoidal curves, with a Hill coefficient of 1 ( Figure 2 B– 2 G). The sequence specificity of 2′- O -methyl oligonucleotide inhibition of RISC function clearly shows that inhibition reflects binding of the oligo to the RISC. Our data are most easily explained if the concentration of the 2′- O -methyl oligonucleotide required for inhibition in these experiments is much greater than the K D for binding; i.e., the experiments were conducted in a stoichiometric binding regime. Under a stoichiometric binding regime, inhibition by the 2′- O -methyl oligonucleotides would be essentially irreversible. In theory, the 2′ -O -methyl oligonucleotide might act by displacing the passenger (sense) strand of the siRNA duplex, thereby blocking incorporation of the guide (antisense) strand into RISC ( Elbashir et al. 2001 c). We can exclude this possibility because a 5′ tethered 31-nt 2′- O -methyl oligonucleotide complementary to the passenger strand of the siRNA did not deplete the guide-strand RISC activity (see Figure 1 B). Similarly, an antisense sequence 2′- O -methyl oligonucleotide cannot pair with antisense RISC, but can pair with a sense target mRNA. We anticipated that this antisense 2′- O -methyl oligonucleotide would pair with the sense target mRNA and occlude the antisense RISC from the target. Surprisingly, the antisense 2′- O -methyl oligonucleotide was a poor inhibitor of antisense RISC function when it was used to bind the target site, requiring 300 nM for half-maximal inhibition in a reaction containing 14.5 nM RISC and 3 nM sense target RNA ( Figure 3 A). By contrast, the same antisense 2′- O -methyl oligonucleotide was highly effective in blocking the activity of the sense RISC, to which it is complementary, acting with an IC 50 of 8.2 nM in a reaction containing 16.8 nM sense-strand RISC and 3 nM antisense target RNA ( Figure 3 B). (In this experiment, sense-strand RISC was generated by changing the first nucleotide of the sense strand from C to U, thereby reversing its functional asymmetry [ Schwarz et al. 2003 ].) Figure 3 RISC Does Not Act through an Antisense Mechanism (A) Inhibition of sense target cleavage by an antisense 2′ -O- methyl oligonucleotide requires an approximately 40-fold higher concentration than by a sense oligonucleotide. The antisense oligonucleotide can pair completely with the sense target RNA, but not with the antisense siRNA-programmed RISC. The IC 50 value and the RISC concentration are indicated. Also shown are the sequences of the sense Pp -luc RNA target (black), the siRNA (red, antisense strand; black, sense strand), and the 2′ -O- methyl oligonucleotide (blue). (B) The same antisense 2′- O -methyl oligonucleotide is an effective competitor of antisense target cleavage. In this experiment, inhibition occurs via binding of the antisense oligonucleotide to the sense siRNA-programmed RISC, not the target RNA. The IC 50 value and the RISC concentration are indicated. Also shown are the sequences of the Pp -luc antisense RNA target (black), the siRNA (red, antisense strand; black, sense strand), and the 2′ -O- methyl oligonucleotide (blue). The G:U wobble in the siRNA duplex in (B) acts to direct the sense strand into RISC and improving its efficacy in target cleavage. Thus, the interaction of 2′- O -methyl oligonucleotide with RISC is dramatically different from the interaction of 2′- O -methyl oligonucleotide with target RNA; RISC has a more than 40-fold greater affinity for the 2′- O -methyl oligonucleotide than the oligonucleotide has for an RNA target (compare Figures 2 E and 3 A). These data imply that the interaction of RISC with target is more complex than simple nucleic acid hybridization. Inhibition of the siRNA-programmed RISC by a 2′- O -methyl oligonucleotide with the sequence of the target RNA is more effective than inhibition mediated by binding of an oligonucleotide to the target RNA itself. Thus, the RISC is more adept at finding or remaining bound (or both) to the target RNA than a 2′- O -methyl oligonucleotide. These data suggest that specific proteins in the RISC facilitate either target finding, target binding, or both. Consistent with this idea, inhibition of RISC function was incomplete using 21-nt 2′- O -methyl oligonucleotides (data not shown). Thus, target sequence flanking the site of complementarity to the siRNA guide strand may play a role in target RISC binding. Perhaps an active mechanism that involves target sequences flanking the siRNA facilitates the search for the target sequence; future studies will clearly be needed to test this idea. Inhibition of RNAi in Cultured Human Cells Our data show that 2′ -O- methyl oligonucleotides are stoichiometric, irreversible, sequence-specific inhibitors of siRNA function in RNAi reactions using Drosophila embryo lysate. Can 2′- O -methyl oligonucleotides block siRNA function in vivo? To address this question, we carried out sequential transfection experiments using 1 nM, 5 nM, 10 nM, or 25 nM siRNA duplex. siRNA was transfected on the first day; then, reporter and control plasmids were cotransfected together with various amounts of 2′- O -methyl oligonucleotide on the second day. Silencing of Pp -luc, relative to the sea pansy ( Renilla reniformis ) luciferase ( Rr -luc) control, was measured on the third day. For each siRNA concentration, we determined the concentration of 2′- O -methyl required for half-maximal inhibition of RNAi ( Figure 4 A– 4 D). Increasing amounts of the 2′- O -methyl oligonucleotide gradually extinguished the ability of the siRNA to silence Pp -luc in all four experiments. The inhibition of silencing in the cultured cells cannot be a consequence of the 2′- O -methyl oligonucleotide displacing the sense strand of the siRNA duplex, because assembly of siRNA into RISC occurred a full day before the oligonucleotide was introduced. When 10 nM siRNA was used in the transfection, approximately 1 nM 2′- O -methyl RNA was required for half-maximal inhibition of RNAi ( Figure 4 C and 4 E). At 25 nM siRNA, approximately 1.1 nM 2′- O -methyl RNA was required to inhibit half the RNAi activity ( Figure 4 D and 4 E). In Figure 4 E, we plot the siRNA concentration versus the amount of 2′- O -methyl oligonucleotide required for half-maximal inhibition of silencing (IC 50 ). The data fit well to a sigmoidal curve, consistent with the idea that, at these concentrations, increasing amounts of siRNA do not produce a corresponding increase in RISC activity. Higher concentrations of siRNA were not examined because they produce sequence-independent changes in gene expression ( Persengiev et al. 2003 ; Semizarov et al. 2003 ). We conclude that both cells and extracts have a limited capacity to assemble RISC on exogenous siRNA. Our data suggest that the use of siRNA concentrations greater than that required to produce the maximum amount of RISC will lead to the accumulation of double-stranded siRNA in vivo and may thus contribute to the undesirable, sequence-independent responses sometimes observed in cultured mammalian cells ( Persengiev et al. 2003 ; Semizarov et al. 2003 ; Sledz et al. 2003 ). Figure 4 A 2′- O -Methyl Oligonucleotide Is a Potent Inhibitor of RNAi in Human Cultured HeLa Cells (A–D) HeLa cells were transfected with 1 nM (A), 5 nM (B), 10 nM (C), or 25 nM (D) siRNA-targeting Pp- luc mRNA. The next day the cells were cotransfected with Rr- luc - and Pp -luc-expressing plasmids together with various amounts of a 31-nt 2′ -O- methyl oligonucleotide complementary to the antisense strand of the siRNA. The half-maximal concentration of 2′- O -methyl oligonucleotide required to inhibit (IC 50 ) was determined by fitting the data to a sigmoidal curve using a Hill coefficient of 1. (E) IC 50 plotted as a function of the concentration of transfected siRNA. Inhibition of miRNA Function In Vitro and In Vivo In animal cells, miRNAs are thought predominantly to function as translational regulators. Nonetheless, a growing body of evidence suggests that they function through a similar, if not identical, RISC as siRNAs ( Hutvágner and Zamore 2002 ; Zeng et al. 2002 , 2003 ; Doench et al. 2003 ; Khvorova et al. 2003 ; Schwarz et al. 2003 ). Because 2′- O -methyl oligonucleotides block siRNA function in vitro and cultured human cells, we asked whether these oligonucleotides might likewise disrupt the function of a specific miRNA in vitro and in vivo. An ideal candidate for such an miRNA is let-7 . Classical genetic mutations in C. elegans let-7 produce well-characterized, readily scored phenotypes. Furthermore, human HeLa cells express multiple let-7 family members, and endogenous let-7 is present naturally in RISC ( Hutvágner and Zamore 2002 ; Zeng and Cullen 2003 ). We tested whether a 31-nt 2′ -O- methyl oligonucleotide complementary to let-7 could block target cleavage guided by the endogenous let-7 -programmed RISC present in HeLa S100 extract ( Figure 5 A). (Our assay detects the target-cleaving activity of let-7 ; we have not examined endogenous human mRNA targets whose translation may be repressed by let-7 .) As a control, we also tested whether the oligonucleotide could block the activity of a let-7 -containing RISC assembled in vitro in Drosophila embryo lysate. Addition of this 2′- O -methyl oligonucleotide efficiently blocked target RNA cleavage directed by the endogenous let-7 -programmed RISC in the HeLa S100 extract and by the RISC programmed with exogenous let-7 siRNA duplex in Drosophila embryo lysate ( Figure 5 C). Figure 5 A Complementary 2′ -O- Methyl Oligonucleotide Blocks Endogenous let-7 -Containing RISC Function (A) Sequence of the let-7 -complementary site in the target RNA (black), of the siRNA (red, antisense strand; black, sense strand), and of the let-7 -complementary 2′ -O- methyl oligonucleotide (blue). (B) Schematic representation of the target RNA, which contained both Pp -luc and antisense let-7 sequences. (C) Drosophila embryo lysate (left) was programmed with let-7 siRNA; then, the target RNA and the 2′ -O- methyl oligonucleotide were added together. Target RNA and 2′- O -methyl oligonucleotide (right) were added to HeLa S100 extract, which contains endogenous human let-7 -programmed RISC. (D) An RNA target containing both Pp -luc and antisense let-7 sequence can be simultaneously targeted by Pp -luc siRNA and endogenous let-7 in HeLa S100 lysate. The let-7 -complementary 2′- O -methyl oligonucleotide blocks let-7 -programmed, but not Pp -luc siRNA-programmed, RISC function. The bottom panel shows the same samples analyzed separately to better resolve the let-7 5′ cleavage product. (E) Drosophila embryo lysate was programmed with let-7 siRNA and then incubated with biotinylated 2′- O -methyl oligonucleotide tethered to paramagnetic streptavidin beads. The beads were removed and the supernatant tested for RNAi activity. Symbols and abbreviations: Ø, target RNA before incubation with siRNA-programmed lysate; T, total reaction before depletion; unbound, the supernatant after incubation with the paramagnetic beads. “Mock” indicates that no oligonucleotide was used on the beads; “ let-7 ” indicates that the beads contained the let-7 -complementary oligonucleotide shown in (A). In addition to containing endogenous let-7 -programmed RISC, HeLa S100 can be programmed with exogenous siRNA duplexes ( Martinez et al. 2002 ; Schwarz et al. 2002 ). The target RNA used in Figure 5 B also contains sequence from the Pp -luc mRNA and can therefore be targeted by a Pp -luc-specific siRNA duplex (see Figures 1 A and 5 C). We incubated the Pp -luc siRNA duplex with the human HeLa S100 to form Pp -luc-directed RISC, then added the let-7 -complementary 2′- O -methyl oligonucleotide and the target RNA. The oligonucleotide blocked cleavage by the endogenous let-7 -programmed RISC, but had no effect on cleavage directed by the exogenous Pp -luc siRNA in the same reaction ( Figure 5 D). When tethered to a paramagnetic bead, this oligonucleotide could also quantitatively deplete the let-7 -programmed RISC from the Drosophila embryo lysate ( Figure 5 E), demonstrating that, again, the interaction between the 2′- O -methyl oligonucleotide and the RISC was apparently irreversible. The 2′- O -methyl oligonucleotide was a specific and potent inhibitor of target cleavage directed by a naturally occurring, miRNA-programmed RISC. Furthermore, our data demonstrate that individual RISCs act independently even when they target the same RNA. Next we asked whether 2′- O -methyl oligonucleotides can inhibit miRNA function in vivo. Translational repression directed by miRNAs occurs in C. elegans , where both the lin-4 and let-7 miRNAs have been shown to block translation of their target mRNAs without altering mRNA stability ( Wightman et al. 1993 ; Ha et al. 1996 ; Moss et al. 1997 ; Olsen and Ambros 1999 ; Reinhart et al. 2000 ; Seggerson et al. 2002 ). The genetics of lin-4 and let-7 function are well-characterized in worms, where they are required during larval development to control the timing and pattern of cell division in the hypodermis ( Lee et al. 1993 ; Reinhart et al. 2000 ). First, we tested whether injection into the germline of wild-type adult hermaphrodites of 2′ -O- methyl oligonucleotides complementary to either lin-4 or let-7 could block lin-4 or let-7 function during the larval development of the resulting progeny. Although the 2′ -O- methyl oligonucleotides were not toxic and when coinjected with an unrelated DNA transformation reporter did not prevent the uptake and expression of the coinjected DNA, we did not observe inhibition of lin-4 or let-7 activity (data not shown). This finding suggests that single-stranded 2′ -O- methyl oligonucleotides are not efficiently transmitted to the progeny of injected animals. To circumvent this problem, we next injected 2′ -O- methyl oligonucleotides directly into larvae and examined the phenotypes of the injected animals. The lin-4 miRNA functions in L1/L2 larvae, and we have found that, in our hands, L1 larvae do not survive microinjection (data not shown); thus, it was not possible to assay for inhibition of lin-4 function by direct injection. In contrast, let-7 functions during the L4 stage, and we found that L2 and L3 larvae survive the microinjection procedure ( see Materials and Methods ). Loss of let-7 function causes worms to reiterate the L4 larval molt and inappropriately produce larval cuticle at the adult stage. Loss-of-function let-7 phenotypes include weak cuticles prone to bursting at the vulva, defects in egg-laying, and loss of adult-specific cuticular structures that run the length of the animal's body, the alae ( Reinhart et al. 2000 ). After larvae were injected with the let-7 -specific 2′ -O- methyl oligonucleotide, 80% of the adult worms lacked alae; 77% lacked alae and also exhibited bursting vulvae ( Figure 6 A). In contrast, animals injected with an unrelated control 2′ -O- methyl oligonucleotide displayed no abnormal phenotypes ( Figure 6 A). Figure 6 Injection of a 2′- O -Methyl Oligonucleotide Complementary to let-7 miRNA Can Phenocopy the Loss of let-7 Function in C. elegans (A) Wild-type and lin-41(ma104) L2-stage C. elegans larvae were injected with either a 2′ -O- methyl oligonucleotide complementary to let-7 miRNA ( Figure 5 A) or an unrelated Pp -luc 2′- O -methyl oligonucleotide. Absence of alae and presence of bursting vulvae were scored when the injected animals reached adulthood. (B) Isolation of let-7 -associated proteins with a tethered 2′- O -methyl oligonucleotide. Northern blot analysis of let-7 miRNA remaining in the supernatant of the worm lysate after incubation with the let-7 -complementary ( let-7 ) or Pp -luc (unrelated) oligonucleotide. Input represents the equivalent of 50% of the total extract incubated with tethered oligonucleotide. (C) Western blot analysis of the GFP-tagged ALG-1 and ALG-2 proteins associated with let-7 . The upper band corresponds to GFP-tagged ALG-1 and the lower to GFP-tagged ALG-2. Extracts from a transgenic strain expressing the tagged proteins was incubated with the indicated tethered 2′- O -methyl oligonucleotide; then, the beads were washed and bound proteins were fractionated on an 8% SDS-polyacrylamide gel. Western blots were probed using anti-GFP monoclonal or anti-RDE-4 polyclonal antibody. The RDE-4-specific band is marked with an asterisk ( Tabara et al. 2002 ). (D and E) Analysis of let-7 miRNA in ALG-1/ALG-2 complexes (D). Extracts prepared from mixed-stage wild-type worms (N2) or from GFP::ALG-1/ALG-2 transgenic worms were immunoprecipitated using anti-GFP monoclonal antibodies. The unbound and immunoprecipitated RNAs were analyzed by Northern blot hybridization for let-7 (D), and 5% of the immunoprecipitated protein was analyzed by Western blotting for GFP to confirm recovery of the GFP-tagged ALG-1/ALG-2 proteins (E). All of the phenotypes associated with injection of the let-7 -complementary 2′ -O- methyl oligonucleotide are consistent with a loss of let-7 activity. let-7 represses translation of lin-41 mRNA by binding to a partially complementary site in the lin-41 3′-untranslated region ( Reinhart et al. 2000 ; Slack et al. 2000 ; Vella et al. 2004 ). Consequently, many of the phenotypes associated with the loss of let-7 reflect overexpression of LIN-41 protein; let-7 mutants are partially suppressed by mutations in lin-41 . We reasoned that if the phenotypes observed in the injected larvae reflect a loss of let-7 activity, then they should likely be partially suppressed by a lin-41 mutation ( Reinhart et al. 2000 ; Slack et al. 2000 ). To test this possibility, we injected the let-7 -specific 2′- O -methyl oligonucleotide into the lin-41(ma104) strain and compared the penetrance of the phenotypes with those observed for injection into wild-type . Consistent with the idea that the injected oligonucleotide specifically inactivates let-7 , the absence of alae- and vulval-bursting phenotypes were both suppressed in the lin-41(ma104) mutant strain ( Figure 6 A). The number of worms lacking alae was reduced from 80% to 16%, and worms with bursting vulvae were dramatically reduced (74% in wild-type compared to 3.8% in the lin-41(ma104) strain). The observed suppression (64%) was nearly identical to that reported for a let-7, lin-41 genetic double mutant (70%; Reinhart et al. 2000 ; Slack et al. 2000 ). Together, our data support the idea that 2′ -O- methyl oligonucleotides are potent inhibitors of miRNA function that can be used to probe the function of specific miRNAs in C. elegans . Isolation of Protein–miRNA Complex Using a Tethered 2′-O-Methyl Oligonucleotide Our in vitro experiments suggest that both siRNA- and miRNA-containing RISCs are stably bound by 2′ -O- methyl oligonucleotides. In theory, tethered 2′- O -methyl oligonucleotides could be used to isolate cellular factors associated with specific miRNAs. In human cells, miRNAs such as let-7 are in a protein complex that contains Argonaute proteins ( Hutvágner and Zamore 2002 ; Mourelatos et al. 2002 ; Dostie et al. 2003 ). In C. elegans , the Argonaute protein-encoding genes alg-1 and alg-2 are required for the biogenesis or function (or both) of the miRNAs lin-4 and let-7 ( Grishok et al. 2001 ), but it has not been shown whether ALG-1 and ALG-2 proteins are directly associated with let-7 . We prepared extracts from wild-type adult worms carrying a transgene expressing GFP-tagged ALG-1 and ALG-2 proteins. The extracts were then incubated with the let-7 -complementary 2′ -O- methyl oligonucleotide tethered by a 5′ biotin to streptavidin-conjugated paramagnetic beads. As a control, the experiment was performed in parallel using an oligonucleotide not complementary to let-7 . The let-7 -complementary, but not the control, oligonucleotide depleted nearly all the let-7 miRNA from the extract ( Figure 6 B). Western blotting using an anti-GFP antibody revealed that both GFP-tagged ALG-1 and ALG-2 protein copurified with the let-7 -complementary oligonucleotide, but not the control oligonucleotide ( Figure 6 C). In contrast, the RNA-binding protein RDE-4, which is required for RNAi but not for miRNA function in C. elegans , did not copurified with the let-7 -complementarity oligonucleotide, providing further support for the specificity of the let-7 :ALG-1/ALG-2 interaction ( Figure 6 C). Finally, we used a coimmunoprecipitation assay to examine the interaction between let-7 and ALG-1/ALG-2. In this assay, a monoclonal anti-GFP antibody was used to coimmunoprecipitate ALG-1/ALG-2 and small RNAs from the GFP::ALG-1/GFP::ALG-2 strain, which expresses GFP::ALG-1/ALG-2 fusion proteins. Northern blot analysis of the immune complex showed that it contained mature 22-nt let-7 miRNA ( Figure 6 D). No detectable let-7 was recovered with the anti-GFP antibody from the N2 wild-type strain. By comparing the fraction of let-7 associated with GFP::ALG-1/ALG-2 with the unbound fraction of let-7 miRNA, we estimate that approximately 30% of the 22-nt let-7 RNAs coimmunoprecipitate with GFP::ALG-1 and GFP::ALG-2. These data support a model in which that ALG-1 and ALG-2 form a complex, in vivo , that contains a substantial fraction of the mature let-7 miRNA. Discussion Our studies indicate that 2′- O -methyl oligonucleotides bind efficiently and essentially irreversibly to RISC by basepairing with the small guide RNA. These findings provide a rapid and reliable method to measure programmed RISC concentration in vitro and to identify the in vivo functions of small RNA and the identities of their associated proteins. The ability to measure RISC concentration should enable detailed kinetic studies of the enzymatic activity of RISC, an essential step in understanding RISC function. In fact, this method was recently put to use in analyzing the molecular basis of asymmetry in siRNA function ( Schwarz et al. 2003 ). In this study, we have used a tethered 2′- O -methyl oligonucleotide to demonstrated the association of ALG-1/ALG-2, two C. elegans Argonaute proteins, with the endogenous worm miRNA let-7 . Our in vitro and in vivo studies using 2′- O -methyl oligonucleotides demonstrate that cells and extracts have a limited capacity to assemble RISC on exogenous siRNA. Our in vitro experiments suggest that inhibition of RISC by 2′- O -methyl oligonucleotides is stoichiometric and essentially irreversible. Using a sequential transfection protocol in cultured cells, we find that the half-maximal amount of 2′- O -methyl oligonucleotide required to inhibit silencing (IC 50 ) is less than the amount of siRNA transfected. These data suggest that only a fraction of the transfected siRNA forms RISC. Furthermore, the data are consistent with stoichiometric and irreversible binding of the 2′- O -methyl oligonucleotide to RISC in vivo. Our data hint that recognition of the 2′- O -methyl oligonucleotide by RISC and, by inference, recognition of target RNA by RISC are qualitatively different from the simple binding of two complementary nucleic acids by basepairing. We observed that RISC function was far more readily inhibited by binding a 2′- O -methyl oligonucleotide to RISC than by binding the same 2′- O -methyl oligonucleotide to the site of RISC recognition on a target RNA. A clear implication of this finding is that RISC does not acquire its RNA target by a passive basepairing mechanism that zippers together 21 nt of complementary RNA. Thus, RNAi is not merely a form of antisense inhibition in which the antisense strand is stabilized in a duplex. Rather, an active mechanism—perhaps involving target sequences flanking the region of complementarity—underlies the specificity and efficiency of RISC targeting. Finally, we have shown the utility of 2′ -O- methyl oligonucleotides to probe miRNA function in vivo. Injection of a 2′- O -methyl oligonucleotide complementary to the let-7 miRNA into C. elegans larvae phenocopied a let-7 loss-of-function mutation, demonstrating that 2′ -O- methyl oligonucleotides can disrupt the function of a single miRNA in vivo. These data, combined with our studies in vitro and in cultured cells, show the promise of 2′- O -methyl oligonucleotides as a tool for dissecting the function of the numerous miRNAs found in a wide range of organisms. In this regard, 2′- O -methyl oligonucleotides provide a tool similar in practice, but mechanistically distinct from, RNAi itself and thus may facilitate the study of small RNA function in cases in which classical genetic mutations in miRNA genes are unavailable. Materials and Methods General methods Drosophila embryo lysate preparation, in vitro RNAi reactions, and cap-labeling of target RNAs were as described elsewhere ( Haley et al. 2003 ). Target RNAs were used at approximately 3 nM concentration. Cleavage products of RNAi reactions were analyzed by electrophoresis on 5% or 8% denaturing polyacrylamide gels. Gels were dried, exposed to image plates, and then scanned with a FLA-5000 phosphorimager (Fuji Photo Film Company, Tokyo, Japan). Images were analyzed using Image Reader FLA-5000 version 1.0 (Fuji) and Image Gauge version 3.45 (Fuji). Data analysis was performed using Excel (Microsoft, Redmond, Washington, United States) and IgorPro 5.0 (Wavemetrics, Lake Oswego, Oregon, United States). siRNA and 2′- O -methyl oligonucleotides Synthetic siRNA (Dharmacon, Lafayette, Colorado, United States) was deprotected according to the manufacturer, annealed ( Elbashir et al. 2001 b, 2001 c), and used at 50 nM final concentration unless otherwise noted. 2′- O -methyl oligonucleotides (either from IDT, Santa Clara, California, United States, or from Dharmacon) were 5′-CAU CAC GUA CGC GGA AUA CUU CGA AAU GUC C-3′ and 5′-Bio-CAU CAC GUA CGC GGA AUA CUU CGA AAU GUC C-3′ (complementary to the Pp -luc siRNA sense strand); 5′-GGA CAU UUC GAA GUA UUC CGC GUA CGU GAU G-3′ and 5′-Bio-A CAU UUC GAA GUA UUC CGC GUA CGU GAU GUU-3′ (complementary to the Pp -luc antisense strand); and 5′-Bio-UCU UCA CUA UAC AAC CUA CUA CCU CAA CCU U-3′ (complementary to let-7 ); 5′ biotin was attached via a six-carbon spacer arm. Immobilized 2′- O -methyl oligonucleotide capture of RISC Biotinylated 2′- O -methyl oligonucleotide (10 pmol) was incubated for 1 h on ice in lysis buffer containing 2 mM DTT with 50 μl of Dynabeads M280 (as a suspension as provided by the manufacturer; Dynal, Oslo, Norway) to immobilize the oligonucleotide on the beads. To ensure that the tethered oligonucleotide remained in excess when more than 50 nM siRNA was used, 20 pmol of biotinylated 2′- O -methyl oligonucleotide was immobilized. For RISC capture assays, siRNA was preincubated in a standard 50 μl in vitro RNAi reaction for 15 min at 25°C. Then, the immobilized 2′- O -methyl oligonucleotide was added to the reaction and incubation continued for 1 h at 25°C. After incubation, beads were collected using a magnetic stand (Dynal). The unbound supernatant was recovered and an aliquot assayed for RISC activity as previously described ( Elbashir et al. 2001 b; Nykänen et al. 2001 ) to confirm that RISC depletion was complete. The beads were then washed three times with ice-cold lysis buffer containing 0.1% (w/v) NP-40 and 2 mM DTT, followed by a wash without NP-40. To determine the amount of RISC formed, input and bound radioactivity was determined by scintillation counting (Beckman Instruments, Fullerton, California, United States). To isolate let-7 -containing complexes from C. elegans adults, we incubated 20 pmol of immobilized 2′- O -methyl oligonucleotide with 1 mg of total protein. Sequential transfection HeLa S3 cells were transfected in a 24-well plate (200 mm 2 per well) using Lipofectamine 2000 (GIBCO, San Diego, California, United States) according to the manufacturer's protocol, first with various concentrations of siRNA targeting Pp- luc mRNA. After 6 h, the cells were washed with PBS and the media replaced. On the next day, the cells were cotransfected with Rr -luc-expressing (0.1 μg/well) and Pp -luc-expressing (0.25 μg/well) plasmids and 2′- O -methyl oligonucleotides using Lipofectamine 2000 (GIBCO) according to the manufacturer's protocol. The luciferase activity was measured 24 h later with the Dual Luciferase assay kit (Promega, Madison, Wisconsin, United States) using a Mediators Diagnostika (Vienna, Austria) PhL luminometer. Worm injection For in vivo inhibition of let-7 function, 1 mg/ml let-7 -complementary 2′- O -methyl oligonucleotide in water (100 μM) was injected into either wild-type (N2) or lin-41(ma104) L2 larvae. Injection of L2 larvae was essentially as described elsewhere ( Conte and Mello 2003 ). The 2′- O -methyl oligonucleotide solution was injected into the body cavity of the larvae using the low flow and pressure setting to prevent animals from dying. Despite these precautions, approximately 60% of the animals do not survive injection, irrespective of the oligonucleotide injected. let-7 phenotypes were also observed at 10 μM oligonucleotide, but were less penetrant. Phenotypes were scored after the injected animals survived to adulthood. Other methods Synchronized transgenic animals carrying GFP::ALG-1, GFP::ALG-2 were harvested at adulthood and homog-enized in ice-cold buffer (25 mM HEPES–NaOH [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% [v/v] glycerol, 0.5% [v/v] Triton X-100, 2% [v/v] SUPERaseIn [Ambion, Austin, Texas, United States]) and Mini Complete Protease Inhibitor cocktail (1 tablet/10 ml solution) (Roche, Basel, Switzerland) using a stainless-steel Dounce homogenizer (Wheaton Incorporated, Millville, New Jersey, United States). The homogenized extract was clarified by a centrifugation at 13,817 × g for 10 min at 4°C. To recover the proteins associated with the let-7 miRNA, the beads were boiled for 10 min in 20 μl of SDS loading buffer (10 mM Tris–HCl [pH 6.8], 2% [w/v] SDS, 100 mM DTT, and 10% [v/v] glycerol). Proteins were resolved by SDS-PAGE on an 8% gel and transferred to Hybond-C membrane (Amersham Biosciences, Little Chalfont, United Kingdom). To detect GFP-tagged ALG-1, ALG-2, and RDE-4 proteins, the membrane was incubated overnight at 4°C with either monoclonal anti-GFP (Roche) or an affinity-purified polyclonal anti-RDE-4 antibody ( Tabara et al. 2002 ) diluted 1:1000 into TBST-milk solution (100 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.1% [v/v] Tween-20, and 5% [w/v] dried milk), incubated 1 h at room temperature with either anti-mouse (GFP-tagged ALG-1/ALG-2) or anti-rabbit (RDE-4) HRP-conjugated secondary antibody (Jackson Laboratory, Bar Harbor, Maine, United States) diluted 1:5,000 in TBST and then visualized by enhanced chemulinescence (New England Nuclear, Boston, Massachusetts, United States). Immunoprecipitation of GFP-tagged ALG-1/ALG-2 complexes was performed by preclearing worm extract with 50 μl of protein G–agarose beads (Roche) per 5 mg of total protein for 1 h at 4°C. The cleared extract was then incubated with 10 μg of monoclonal antibody anti-AFP 3E6 (Qbiogene, Montreal, Quebec, Canada) for 1 h at 4°C followed by 50 μl of protein G–agarose. The agarose beads were then washed three times with ice-cold homogenization buffer. Depletion of let-7 miRNA was monitored by Northern blotting. RNA was eluted from the immobilized 2′- O -methyl oligonucleotide by digestion with 1 mg/ml proteinase K in 200 mM Tris–HCl (pH 7.5), 25 mM EDTA, 300 mM NaCl, 2% (w/v) SDS at 50°C for 30 min, followed by extraction with phenol–chloroform, and recovered by precipitation with ethanol. Recovered RNA was resuspended in 10 μl of formamide-loading buffer (98% [v/v] deionized formamide, 10 mM EDTA, 0.025% [w/v] xylene cyanol, 0.025 % [w/v] bromophenol blue), heated to 100°C for 2 min. RNA was resolved on a 15% denaturing polyacrylamide gel, transferred to Hybond-N membrane (Amersham Biosciences), and detected by Northern blot analysis using a 5′- 32 P-radiolabeled antisense let-7 RNA probe (UAU ACA ACC UAC UAC CUC AUU) as described elsewhere ( Hutvágner and Zamore 2002 ). Supporting Information Accession Numbers The GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession number for Photinus pyralis is X65324 and for Renilla reniformis is AF025846. Rfam ( http://www.sanger.ac.uk/Software/Rfam/index.shtml ) accession numbers for the let-7 family members are MI0000060–MI0000068, MI0000433, and MI0000434. The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) ID numbers for the genes discussed in this paper are alg-1 (181504), alg-2 (173468), bantam (117376), let-7 (266954), lin-4 (266860), lin-41 (172760), miR-14 (170868), and rde-4 (176438).

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC350664.xml

549191

Transient expression analysis of allelic variants of a VNTR in the dopamine transporter gene (DAT1)

Background The 10-repeat allele of a variable number tandem repeat (VNTR) polymorphism in the 3'-untranslated region of the dopamine transporter gene ( DAT1 ) has been associated with a range of psychiatric phenotypes, most notably attention-deficit hyperactivity disorder. The mechanism for this association is not yet understood, although several lines of evidence implicate variation in gene expression. In this study we have characterised the genomic structure of the 9- and 10-repeat VNTR alleles, and directly examined the role of the polymorphism in mediating gene expression by measuring comparative in vitro cellular expression using a reporter-gene assay system. Results Differences in the sequence of the 9- and 10- repeat alleles were confirmed but no polymorphic differences were observed between individuals. There was no difference in expression of reporter gene constructs containing the two alleles. Conclusions Our data suggests that this VNTR polymorphism may not have a direct effect on DAT1 expression and that the associations observed with psychiatric phenotypes may be mediated via linkage disequilibrium with other functional polymorphisms.

Background The dopamine transporter (DAT) mediates uptake of dopamine into presynaptic neurons, and is a major target for various pharmacologically active stimulants such as cocaine. Genetic association studies provide considerable evidence that a variable number tandem repeat (VNTR) polymorphism in the 3'-untranslated region (UTR) of the dopamine transporter gene ( DAT1 ) is associated with a range of psychiatric phenotypes. In particular, the 10-repeat allele of this polymorphism has been widely associated with attention deficit hyperactivity disorder (ADHD) (e.g. [ 1 , 2 ]), although there have been several non-replications reported (e.g. [ 3 , 4 ]). The mechanism for this association is not yet understood, although several lines of evidence implicate variation in gene expression [ 5 ]. The VNTR polymorphism consists of a 40 bp sequence that most frequently occurs as 9 or 10 tandem repeat units, although 3 through to 11 repeats are also observed. Its location within the transcribed 3'-UTR is interesting since these regions have been shown to play an important role in the regulation of transcription efficiency, mRNA stability or mRNA sub-cellular localization [ 6 ]. Several in vivo studies using single photon emission computed tomography (SPECT) show an increased density of DAT in ADHD probands compared to controls [ 7 - 10 ], although such findings are not ubiquitous [ 11 ]. In addition, several studies suggest there may be an association between VNTR genotype and DAT density [e.g. [ 12 , 13 ]]. Furthermore, methylphenidate, which is used as a treatment for ADHD, has been shown to lower levels of DAT in the brain [ 9 ]. Finally, the most thorough investigation of methylphenidate response in relation to DAT1 genotype suggests that the 10-repeat allele is associated with a positive response to the drug [ 14 ] as would be expected if the density of the DAT, to which methylphenidate binds, is increased in individuals with the 10-repeat allele. Two smaller studies also report an effect of the DAT1 VNTR on methylphenidate response, although they find poor response is associated with the 10-repeat allele [ 15 , 16 ]. Previously, we measured DAT1 messenger RNA (mRNA) levels in cerebellum, temporal lobe and lymphocytes and observed that increased levels of DAT1 expression were associated with the number of 10-repeat alleles [ 17 ]. These data suggested that the VNTR or another polymorphism in linkage disequilibrium (LD) with the VNTR is involved in regulating expression of this gene. A number of groups have investigated the functional role of the DAT1 VNTR in vitro , although the results from these studies are inconclusive. Michelhaugh et al demonstrated that the human DAT1 9-repeat VNTR enhances transcription in SN4741 cells, obtained from a mouse-embryonic substantia nigra-derived cell line [ 18 ]. However, their study did not compare the effect of the 10- versus the 9-repeat. Furthermore, their constructs contained the repeat region inserted upstream of the reporter gene promoter, and not in the 3'UTR of the gene itself. Thus the position of the repeat did not mirror that seen in the wild-type DAT1 gene. It has been postulated that large repeat motifs in the 3'UTR of genes may alter mRNA stability, but such effects would not be detected in the research presented by Michelhaugh et al. Furthermore, it has been shown that the enhancing effect of sequence elements is largely determined by their relative location [ 19 ]. Fuke et al examined the effect of the VNTR polymorphism on gene expression using the luciferase reporter system in COS-7 cells [ 20 ]. They found that luciferase expression was significantly higher in cells transfected with vectors containing the 10-repeat allele compared to the 7-repeat or 9-repeat alleles. However, they used a monkey cell-line that may not be representative of endogenous human cells and did not employ an internal transfection control for the numerous forms of experimental variability that could affect their data. Miller and Madras concluded that the 9-repeat allele was correlated with increased expression in HEK-293 cells compared with the 10-repeat, but that expression was further mediated by a SNP also located in the 3'UTR of DAT1 [ 21 ]. Again, this study did not utilise the dual-luciferase assay system and thus their data could be skewed by experimental variability. Inoue-Murayama et al investigated the functional effect of DAT1 VNTR alleles from several primate species transfected into the human neuroblastoma cell-line, SK-N-SH [ 22 ]. They found that the VNTR sequences of nonhuman primates show higher reporter-gene activity compared to human alleles, and a general trend for longer VNTR alleles to reduce transcription. Finally, Greenwood & Kelsoe found no effect on transcription of the 9- and 10-repeat alleles in SN4741 cells, but found that introns 9, 12, and 14 may contain enhancer elements capable of increasing expression ~2-fold [ 23 ]. They conclude that it may be the particular combination of polymorphisms in a haplotype across the gene that ultimately effects DAT1 gene expression. The aim of this study was to examine directly the role of the VNTR polymorphism in mediating gene expression by measuring comparative in vitro cellular expression implementing a reporter-gene assay system in which the VNTR has been inserted in an appropriate 3' configuration, and using human cell-lines known to express DAT1 . Analysis was limited specifically to the VNTR sequence itself, so that any functional effect detected could be attributed solely to this candidate polymorphism. Another aim of this study was to fully characterise the genomic structure of the DAT1 VNTR. It is possible that any functional effects of the VNTR are not mediated by repeat length, but instead by variation in the actual sequence of the repeat units. Recent work on the DRD4 exon III VNTR sequence has demonstrated that there can be considerable variation within the sequence of large VNTRs [ 24 ]. Such variation, if present, could confound analyses of gene expression and negate associations with disease based solely on repeat length. Results Sequence analysis of the DAT1 VNTR Differences were found between repeat motifs (the individual repeat units making up the total VNTR), and nine variants (labelled A-I) were detected in total (see Figure 1 ). Interestingly, although differences were found between the orders of motifs in 9- and 10-repeat alleles, they were identical for all examples of each allele size. The order of motifs in the 10-repeat VNTRs was found to be A-A-B-C-D-E-F-D-G-H, and the order in 9-repeat alleles was found to be A-A-B-C-D-I-F-D-H. These sequences are identical to those reported by Fuke et al in a Japanese sample [ 20 ]. Furthermore, aligning these sequences up against those available on the NCBI database (accession numbers NM_001044 and AF119117.1) also suggested a lack of between-individual polymorphisms within the VNTR. Interestingly, the 'I' motif (seen in the 9-repeat VNTR but not the 10-repeat) appears to be a combination of the 'E' and 'G' motifs (seen in the 10-repeat VNTR but not in the 9-repeat). The 'F' motif, which occurs in both the 9- and 10-repeat alleles, is also interesting in that it contains 45 base-pairs rather than 40. Furthermore, almost all the between-motif variation occurs within the first (1–24 bp) section of each sequence, before the location of the 5 bp insertion in motif 'F'. Figure 1 Alignment of the repeat motifs in the 9 and 10 repeat VNTR alleles. Blue letters highlight deviations from the 'A' sequence motif. These sequences are identical to those observed in a Japanese population by Fuke et al [20]. Luciferase activity from transiently transfected cells Table 1 shows the luciferase transcriptional activity of the seven transfected pGL3 plasmid constructs in SH-SY5Y and HEK-293 cells. Plasmids containing the VNTR insert produced slightly less transcriptional activity than the original pGL3-promoter and pGL3-control plasmids in both cell-lines, although this difference was not statistically different. No statistical difference in transcriptional activity was detected between constructs containing the 9 or 10-repeat DAT1 VNTR sequence in either the SH-SY5Y cells or the HEK-293 cells, although a slight decrease in expression was noticed with increasing repeat length (no insert > 9-repeat insert > 10-repeat insert). Some slight differences were noted between the two cell lines: in both SH-SY5Y cells and HEK-293 cells, the pGL3-basic plasmid, which contains neither promoter nor enhancer sequences, generated the lowest transcriptional activity as expected. In the SH-SY5Y cells, control plasmids containing both an enhancer and promoter showed significantly higher levels of transcriptional activity than the promoter plasmids, although this difference was not seen in the HEK-293 cells. Table 1 Luciferase transcriptional activity of the DAT1 VNTR constructs in SH-SY5Y and HEK-293 cells. pGL3 Construct Luciferase/ Renilla ratio (95%CI) SH-SY5Y HEK-293 pGL3-Basic 0.0033 (0.0026–0.0041) 0.1811 (0.1197–0.2424) pGL3-Control 1.0407 (0.8464–1.2350) 1.7432 (1.1692–2.3172) pGL3-Control-9rpt 0.7400 (0.6018–0.8781) 1.4963 (1.0416–1.951) pGL3-Control-10rpt 0.5557 (0.4598–0.6515) 1.2098 (0.8637–1.5559) pGL3-Promoter 0.1624 (0.0853–0.2396) 2.3461 (1.246–3.4462) pGL3-Promoter-9rpt 0.1350 (0.1053–0.1646) 1.5967 (1.0963–2.0972) pGL3-Promoter-10rpt 0.1172 (0.0929–0.1416) 1.2916 (0.925–1.6583) Discussion The aims of this study were twofold: first to characterise the genomic structure of the DAT1 VNTR; and second to investigate the effect of the 9- and 10-repeat VNTR alleles on levels of transcription. It is possible that any effects, both on behavioural phenotype or levels of DAT1 expression, commonly associated with the VNTR are due to polymorphisms within the repeat rather than the actual length of the VNTR itself. This theory has often been postulated to explain cases of non-replication of association studies with complex disorders. Recent work on the DRD4 exon III VNTR polymorphism has highlighted the existence of both within individual and between individual variation in repeat motif sequence [ 24 ]. In other words the 1 st and 2 nd repeat motifs within a certain length DRD4 VNTR allele may differ in sequence in a single individual, but there is also variation within the 1 st motif between different individuals. We sequenced the DAT1 VNTR in individuals homozygous for either the 9- or 10-repeat allele. Differences were found between repeat motifs in both the 9- and 10-repeat alleles, and nine variants were detected in total. Interestingly, although differences were found between the 9- and 10-repeat alleles, the order of the motifs was identical for all examples of each allele size. Given that not one variation in these sequences was observed in the 60 chromosomes sequenced, it can be concluded that any between individual variations that do occur are extremely rare and unlikely to be the cause of common disorders such as ADHD. Previously, Ueno et al screened the DAT1 3'UTR for novel polymorphisms in a Japanese population, but only detected a G>A SNP located upstream of the VNTR at position 2319 [ 25 ]. Fuke et al have also sequenced the DAT1 VNTR in a small number of Japanese subjects with results identical to those we describe [ 20 ]. Miller et al report a SNP that abolishes a Dra I restriction site, which they claim to be within the 10-repeat allele [ 26 ]. Closer inspection of their paper, however, shows that this polymorphism is located outside of the repeat region and thus their conclusion that they have found a 'novel variant of the 10-repeat allele' is technically incorrect. It is possible, however, that either this polymorphism or that discovered by Ueno et al [ 25 ] may be the real risk variant, and associations reported for the VNTR polymorphism may be a result of LD relationships with either of these SNPs. The lack of between-individual variation in the sequence of the DAT1 VNTR is perhaps surprising given the number of polymorphisms seen in other large VNTRs (e.g. [ 24 ]). VNTRs are often mutation hotspots with a high level of genetic recombination due to misaligned repeat units that can cause variations in sequence as well as length [ 27 - 29 ]. Furthermore, according to several estimates of SNP frequency across the genome (e.g. [ 30 ]), the average total repeat length of ~400 bp might be expected to contain at least one SNP, especially as it is not located in a highly-conserved coding-region of the gene. The fact that there appears to be no between-individual variations within the 9- and 10-repeat VNTR sequences suggests that it may be subjected to some form of selective pressure, and perhaps have an important functional role. A clue to the importance of the DAT1 VNTR may come from its location within the 3'UTR of the gene. Sequence motifs in the 3'UTR have been shown to have important roles in translation, mRNA stability, subcellular localization, and polyadenylation [ 6 ]. Alternatively, given that the between-motif variations seen within the VNTR are ubiquitous, it is possible that they are relatively ancient and have been pushed towards fixation within the population via demographic and random genetic drift. The second aim of this study was to examine in vitro possible functional consequences of the DAT1 VNTR polymorphism. We made constructs containing the 9- and 10-repeat alleles of the DAT1 VNTR and the luciferase reporter gene, and transiently transfected them into SH-SY5Y and HEK-293 cells. Constructs containing the VNTR alleles gave slightly lower levels of luciferase activity compared to vectors without the inserts. Although these differences were not statistically significant, they appear to go against the conclusions of a previous study in which the VNTR sequence acted as a strong enhancer of transcription [ 18 ]. However, the trend of our results do agree with data presented by Fuke et al who found that vectors containing the VNTR polymorphism gave lower luciferase expression levels compared to positive control vectors having no VNTR inserted [ 20 ]. Furthermore, Greenwood & Kelsoe found no evidence to suggest that the VNTR sequence acts as a transcriptional enhancer [ 23 ]. The discrepancies between these results may be explained by the different methodological strategies employed. Michelhaugh et al utilised GFP as a reporter gene [ 18 ], which does not have the sensitivity or specificity associated with the luciferase reporter system, and is generally used in qualitative detection assays. Furthermore, they cloned the VNTR sequence upstream of the SV40 promoter, in a location not analogous to the 3'UTR location of the polymorphism in the DAT1 gene. The importance of the location of regulatory elements has been well-documented [ 19 ] and so it is possible that the enhancing effect reported by Michelhaugh et al [ 18 ] is specific to the location of the VNTR in their particular experimental design. Additionally, it is possible that the VNTR polymorphism has an important role in mediating processes such as mRNA stability – such effects will have been missed by Michelhaugh et al, as their insert is not transcribed. We found no significant differences in luciferase activity between constructs containing the 9- or 10-repeat DAT 1 VNTR alleles. Other transient expression studies that have compared the VNTR alleles have provided mixed results. Fuke et al found that luciferase expression was significantly higher in cells transfected with vectors containing the 10-repeat allele compared to the 7-repeat or 9-repeat alleles [ 20 ]. Their study used a different cell-line, COS-7, which is derived from African Green Monkey kidney. Miller and Madras, on the other hand, concluded that the 9-repeat allele was correlated with increased expression in HEK-293 cells, but that expression was further mediated by a SNP also located in the 3'UTR of DAT1 [ 21 ]. Finally, our data is in agreement with that of Greenwood & Kelsoe, who also found no effect on transcription of the 9- and 10-repeat alleles in SN4741 cells, but found that introns 9, 12, and 14 may contain enhancer elements capable of increasing expression ~2-fold [ 23 ]. Therefore, our data suggest that the VNTR polymorphism itself may not be functional. Unlike the studies of Fuke et al and Miller & Madras [ 20 , 21 ], our inserts contained no flanking sequence and were restricted to specifically the VNTR itself. There is considerable evidence that there is a functional polymorphism in the vicinity of the 3'UTR of the DAT1 gene. Genetic association studies with ADHD, SPECT brain imaging studies, and correlations with levels of DAT protein and DAT1 mRNA all suggest that a variant associated with DAT1 expression is present in this region. Given that in each of these associations, the VNTR has been nominated as the causative polymorphism, it is likely that the real risk variant is in strong LD with it. Ueno et al and Miller et al both report novel SNPs located within the 3'UTR and close to the VNTR [ 25 , 26 ]. It is possible that either of these SNPs, or another polymorphism yet to be characterised, is mediating expression of DAT1 and is the real risk variant. There are several obvious limitations to this study. First, it is not known how well in vitro studies of gene expression reflect patterns seen in vivo . Future work could employ animal models to characterise more realistically the effect of the VNTR on DAT1 expression. Second, while we ensured that we used cell-lines that naturally express DAT1 , and inserted the VNTR into the correct 3'UTR location of the luciferase reporter gene, our constructs could have been improved by using a homologous DAT promoter. Third, we only cloned a very small portion of the DAT1 gene. Even though this was necessary to examine functional effects specific to the VNTR, the fact that the majority of the DAT1 gene was absent means that it is likely that several cis -acting regulatory elements were not present in the constructs and thus the observed expression may not reflect the actual regulation of the gene. Finally these studies have only analysed DAT1 gene expression in the basal state, and complexities such as the induction of expression by factors such as cellular signals would thus be missed. Future work should focus on systematically characterising the remainder of the 3'UTR to discover the functional effects of other polymorphisms in this candidate region. Conclusion In this study we have characterised the genomic structure of the 9- and 10-repeat DAT1 VNTR alleles, and directly examined the role of the polymorphism in mediating gene expression by measuring comparative in vitro cellular expression using a reporter-gene assay system. No expression differences were observed between the 9- and 10-repeat alleles suggesting that this polymorphism may not have a direct effect on DAT1 function. Methods Sequence analysis of the DAT1 VNTR polymorphism Before cloning the DAT1 VNTR and performing expression analyses it was imperative to characterise fully its structure. If there was variation within the VNTR between individuals who have repeats of the same length, then these variants could confound expression analyses based simply upon length. Thirty individuals of predominantly Caucasian ethnicity, homozygous for either the 9- or 10-repeat allele of the VNTR, were selected for sequencing. The VNTR region was amplified on an MJ PTC-225 thermal cycler (MJ Research, Massachusetts, USA) using the primers 5'- TGT GGT GTA GGG AAC GGC C-3' and 5'- CAT TCG CAA ACA TAA AAA CTG TTG T-3' using a standard PCR protocol with a proofreading polymerase and an annealing temperature of 58°C. PCR products were run on a 2% agarose gel stained with ethidium bromide, and then purified using the QIAquick Gel Extraction Kit (Qiagen, Crawley, UK). Purified fragments were sequenced using an ABI BigDye Terminator (v3.0) Cycle Sequencing kit (PE Applied Biosystems, Foster City, CA, USA) and electrophoresed on an ABI 3100 Genetic Analyzer (PE Applied Biosystems). Sequencing traces were analysed using Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA) and multiple samples aligned to aid mutation detection. Cloning the DAT1 VNTR into pGL3 gene expression vectors The pGL3 expression vector family (Promega, UK) contains an Xba I restriction site (TCTAGA) immediately downstream from the luciferase reporter gene, enabling inserts to be cloned into the 3'UTR. Primers were designed to directly flank the DAT1 VNTR and to both of these an Xba I restriction site, along with three extra bases of DAT1 sequence, were added. The primer sequences used were: 5'-TGT TCT AGA TTG TGG TGT AGG GAA CGG C-3' and 5'-AGG TCT AGA AGA GTG TTG GTC TGC AGG CT-3'. The aim of this project was to concentrate solely on the functional significance of the VNTR, so flanking regions around the VNTR were kept as small as possible. Using these primers, the VNTR was amplified in individuals homozygous for the 9- and 10-repeat alleles using standard PCR conditions with an annealing temperature of 55°C. The PCR products were isolated from a 2% agarose gel as described above. The purified fragments were cloned into pCRII TA cloning vectors (Invitrogen, UK), which were then transformed into TOP10F E. Coli cells (Invitrogen, UK) following the manufacturers protocol. Colonies containing recombinant plasmids were identified using X-gal and IPTG. DNA from colonies containing recombinant plasmids was prepared using a Qiagen Midi-prep kit (Qiagen, UK), and the presence of insertions was verified using Xba I restriction enzyme digestion and further checked using direct plasmid sequencing. The Xba I digested 9- and 10-repeat VNTR inserts were run on a 2% agarose gel and purified as described above. The fragments were cloned into pGL3-Control and pGL3-Promoter vectors. The pGL3-Control vector contains both SV40 promoter and enhancer sequences whereas the pGL3-Promoter vector contains only an SV40 promoter. Additionally the pGL3-Basic vector which lacks eukaryotic promoter and enhancer elements was tested on its own without any insert, for transcriptional activity and served as a negative control. Five colonies from each of the four cloning reactions (pGL3-Control-9rpt, pGL3-Promoter-9rpt, pGL3-Control-10rpt, and pGL3-Promoter-10rpt) were selected. Following plasmid DNA preparation, the presence and orientation of the inserts was again verified by Xba I digestion and fluorescent sequencing. New DNA stocks for each of the 8 plasmids to be used in subsequent transfection experiments (pGL3-Control, pGL3-Promoter, pGL3-Basic, pRLSV40, pGL3-Control-9rpt, pGL3-Promoter-9rpt, pGL3-Control-10rpt, and pGL3-Promoter-10rpt) were prepared, and tested again by Xba I digestion. Cell culture, transient transfections, and luciferase assays SH-SY5Y and HEK-293 cells were purchased from ATCC (VA, USA). The SH-SY5Y cell line is a thrice-cloned subline of the neuroblastoma cell line SK-N-SH. The HEK-293 cell line is derived from human embryonic kidney. A problem with many in vitro studies of gene expression using reporter genes is that the cell-lines used do not naturally express the gene of interest (i.e. DAT1 ). Before we transfected our constructs into the SH-SY5Y and HEK-293 cell lines they were tested for DAT1 expression using quantitative RT-PCR. Both cell lines were found to naturally express DAT1 making them suitable for our experiments (data not shown). In addition previous in vitro studies have shown regulation of the DAT1 gene in both the HEK-293 and SH-SY5Y cell lines [ 31 , 32 ], suggesting endogenous expression of the gene in these cell types. Both cell lines were cultured in 6-well tissue culture plates containing Minimun essential medium Eagle (ATCC) supplemented with 10% foetal bovine serum (Invitrogen, UK). Cells were grown at 37°C in a humidified atmosphere containing 5% CO 2 . Transfection of the SH-SY5Y cells was carried out using a calcium phosphate transfection kit (Invitrogen, UK). The two different transfection methods used were found to be optimal for each line respectively. The HEK-293 cell line was transiently transfected using Lipofectamine 2000 (Invitrogen, UK). Transfection efficiencies were normalised by the co-transfection of the Renilla vector, pRL-SV40 (Promega, UK). Following transfection, the cells were allowed to grow for 48 hours. The cells were then washed and 500 μl cell lysis buffer (Invitrogen, UK) was added to each well. The firefly luciferase and Renilla luciferase assays were carried out using the Dual-Luciferase Assay System (Promega, UK) following the manufacturers protocol. All transfections were done in triplicate, and repeated at least three times. All comparisons between constructs were analysed by one-way analysis of variance (ANOVA), followed by Tukey post-hoc analysis for pairwise comparisons between specific plasmids. Authors contributions JM carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. UD participated in the overall design and co-ordination of the study with supervision of cell culture techniques. PA/IC contributed to the interpretation of findings and general points of the experimental design. All authors read and approved the final manuscript

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517947

Fusion of green fluorescent protein to the C-terminus of granulysin alters its intracellular localization in comparison to the native molecule

The engineering of green fluorescent protein (GFP) fusion constructs in order to visibly tag a protein of interest has become a commonly used cell biology technique. Although caveats to this approach are obvious, literature reports in which the chimeric molecule behaves differently than the native molecule are scant. This brief report describes one such case. Granulysin, a small lytic and antimicrobial protein produced by cytotoxic lymphocytes, traffics to the regulated secretory system and is subsequently released from cells upon proper stimulus. In an attempt to elucidate mechanisms by which it accumulates in and is released from cytolytic granules, GFP was fused to the C-terminus of granulysin and expressed in an NK cell line. A control construct expressing the native protein was similarly expressed. The data demonstrate that, while the fusion protein is expressed and secreted, its subcellular localization is altered in comparison to native granulysin. Thus, the addition of GFP to the C-terminus of granulysin obscures the signal(s) that cytotoxic lymphocytes use to sort it to the regulated secretory pathway despite its normal biosynthesis and secretion. This example is offered as a cautionary account for other researchers who contemplate using this technology.

Background The intrinsically fluorescent protein from the jellyfish Aequoria victoria , termed green fluorescent protein (GFP), can be used to visualize dynamic processes in live cells in real time [ 1 ]. A fusion between a molecule of interest and GFP is supposed to localize fluorescence to the normal intracellular locale of the target protein. This technology has been used to study the intracellular location and dynamics of many different proteins in many different organisms. Included in this group are proteins that traffic to and ultimately are released from regulated secretory compartments [ 2 ]. In this report an attempt was made to use a GFP fusion protein strategy to study the regulated secretory compartment of cytotoxic lymphocytes. Vertebrate organisms have developed a system of contact-dependent cytotoxicity in order to control tumors and infection. Specialized cytotoxic lymphocytes, T cells and natural killer (NK) cells, function as effector cells in this system [ 3 ]. Cell surface receptors on these cells recognize changes to cell surface molecules of transformed or infected cells. Signaling through these receptors initiates target cell destruction. A major method used by these cells to kill targets is the regulated exocytosis of cytolytic granules [ 4 ]. The active components of these organelles and mechanisms by which they lead to target cell death have been well studied. However, the underlying molecular mechanisms governing their biogenesis and release remain less well understood. Adaptation of GFP tagging technology to analyze these processes might therefore be of considerable value in elucidating the underlying molecular mechanisms. Thus, GFP was fused to granulysin, a small secreted protein that sorts to and accumulates in cytolytic granules [ 5 , 6 ], and expressed in the functional human NK cell line YT [ 7 ]. The YT cell line was utilized in this study because it had previously been stably transfected with native granulysin and shown to properly produce and accumulate the processed product in its regulated secretory compartment [ 6 ]. Findings Stable transfectant lines for native granulysin, GFP-tagged granulysin, and non-fused GFP were derived using G418 selection. The GFP proteins were first characterized by immunoblot analysis of lysates and cell supernatants probed with antisera to GFP (figure 1a ). The cell line transfected with the granulysin-GFP expression construct produces a doublet of proteins in the correct molecular weight range for the fusion protein, with both the cell lysate and supernatant media containing immunoreactivity. No explanation presently exists as to the difference between the two proteins of the doublet. The cell line expressing non-fused GFP, which does not contain a signal sequence, displayed an immunoreactive protein only in the cell lysate and not in the extracellular media. Thus, the granulysin-GFP fusion construct correctly directs the biosynthesis of the chimeric molecule into the secretory pathway. A previous publication demonstrated that a native granulysin transfectant also expresses protein, detectable by anti-granulysin sera, in both lysate and extracellular media fractions [ 6 ]. Next, the intracellular localization of native granulysin and granulysin-GFP was analyzed by two color immunofluorescent confocal microscopy using a polyclonal antisera reactive to granulysin and a monoclonal antibody reactive to perforin, a well characterized constituent of cytolytic granules [ 4 ] (figure 1b ). Significant overlap in the dual staining, as evidenced by the abundant yellow signal, demonstrates that transfected native granulysin co-localizes with perforin in granules. On the contrary, the granulysin-GFP fusion protein displays very little overlap in staining with perforin, indicating that the chimera is altered in its subcellular distribution in comparison to the native molecule. Conclusions to be drawn from these data regarding the mechanism(s) of sorting to cytolytic granules are limited but could suggest that altering the overall biophysical properties of granulysin by the addition of the relatively large GFP moiety negates the information necessary to gain entrance into the correct secretory pathway. However, of perhaps broader scientific significance, the data serve as a striking demonstration of an obvious but seldom published limitation of using GFP fusion proteins as substitutes for the native molecules. Figure 1 Granulysin-GFP fusion protein is expressed and secreted but doesn't colocalize with perforin. a) Immunoblot analysis using anti-GFP sera was performed on cell lysate and cell media supernatant samples of YT cells expressing granulysin-GFP fusion protein (YT.granGFP) and native GFP (YT.GFP). b) Confocal immunofluorescence staining for granulysin (green) and perforin (red) was performed for YT expressing native granulysin (YT.granulysin) and granulysin-GFP fusion protein (YT.granGFP). Materials and Methods Cells The NK cell line YT was transfected via electroporation with the linearized expression construct plasmids of full-length native granulysin-pcDNA3.1, full-length granulysin (res. 1-145)-pGFP and control non-fused pGFP. Stable transfectants were selected and grown in RPMI 1640 + 10% fetal bovine serum containing 1 mg/ml G418. Immunoblots One ml liquid aliquots of log-phase cells were pelleted (supernatant saved), washed in PBS, and lysed in 100 μl of reducing sample buffer. The supernatant was diluted 1:1 (v:v) in 2X sample buffer containing DTT. Ten μl of lysate sample (10% of total) and 10 μl of supernatant sample (0.5% of total) were loaded into wells of a 10% PAGE-SDS gel. After electrophoretic separation, proteins in the gel were transferred to nitrocellulose using a semi-dry blot apparatus. Blots were probed with rabbit antisera specific for GFP followed by peroxidase-conjugated anti-rabbit antibodies. Reactive protein bands were revealed by chemiluminescent detection. Confocal microscopy Log-phase cells were immobilized in wells of a poly-L-lysine coated printed glass slide. After fixation with 4% (w/v) paraformaldehyde in PBS, the cells were permeabilized for 30 minutes in staining buffer [10% (v/v) normal goat serum, 1% (w/v) nonfat dry milk powder, 0.1% (w/v) saponin, in PBS]. Next, samples were incubated for 30 minutes with primary antibodies to granulysin (rabbit antisera) and perforin (mouse mAb) diluted in staining buffer. After washing 3X with staining buffer, samples were incubated 30 minutes with species-specific fluorescent Alexa 488 goat anti-rabbit and Alexa 568 goat anti-mouse secondary antibodies (Molecular Probes). Cells were then washed 3X with staining buffer, then twice with PBS, and glass coverslips mounted using 50% (v/v) glycerol in PBS. Images were collected using a BioRad MRC 1024 confocal imaging system mounted on a Nikon Diaphot inverted microscope. Authors' contributions DAH designed the study, carried out all experimental procedures, and drafted the manuscript. SFZ consulted on the experimental design, provided the resources that allowed the study to be conducted and edited the manuscript. Both authors read and approved the final manuscript.

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539304

Validity of self reported male balding patterns in epidemiological studies

Background Several studies have investigated the association between male pattern baldness and disease such as prostate cancer and cardiovascular disease. Limitations in the lack of standardized instruments to measure male pattern baldness have resulted in researchers measuring balding patterns in a variety of ways. This paper examines the accuracy and reliability of assessment of balding patterns by both trained observers and men themselves, using the Hamilton-Norwood classification system. Methods An observational study was carried out in Western Australia with 105 male volunteers aged between 30 and 70 years. Participants completed a short questionnaire and selected a picture that best represented their balding pattern. Two trained data collectors also independently assessed the participant's balding pattern using the same system and the men's self assessment was compared with the trained observer's assessment. In a substudy, observers assessed the balding pattern in a photo of the man aged 35 years while the man independently rated his balding at that age. Results Observers were very reliable in their assessment of balding pattern (85% exact agreement, κ = 0.83). Compared to trained observers, men were moderately accurate in their self-assessment of their balding status (48–55% exact agreement, κ = 0.39–0.46). For the substudy the exact agreement between the men and the observers was 67% and the agreement within balding groups was 87%. Conclusions We recommend that male balding patterns be assessed by trained personnel using the Hamilton-Norwood classification system. Where the use of trained personnel is not feasible, men's self assessment both currently and retrospectively has been shown to be adequate.

Background Male pattern baldness is the most common form of baldness observed in human beings[ 1 ]. The two main types of balding are: frontal balding in which the hair recedes bilaterally from the forehead region backwards; and vertex balding in which a bald spot appears on the top back of the head. Total balding may be due to continued spread of frontal balding, or a joining up of frontal recession and vertex balding[ 1 , 2 ]. Whilst human hair growth is affected by a number of factors, androgens are the most obvious regulators of normal hair growth and are a prerequisite for male pattern baldness[ 1 ]. Serum levels of total and free testosterone, sex hormone binding globulin, and dihydrotestosterone may be important especially given the strong association between free testosterone level and baldness[ 3 ]. Several studies have been conducted investigating the association between male pattern baldness (as a proxy for testosterone levels) and various health issues such as prostate cancer [ 3 - 7 ] and cardiovascular disease[ 6 , 8 , 9 ]. Different methods have been used to assess male pattern baldness in these studies, including the Hamilton-Norwood classification system[ 2 ]. This study was undertaken in order to determine the accuracy and reliability of assessment of balding patterns by both trained observers and men themselves, using the Hamilton-Norwood classification system[ 2 ]. Methods Subjects were male volunteers between the ages of 30–70 years recruited from sporting clubs, shopping centres, universities, the central business district and other public areas throughout the metropolitan area of Perth, the capital city of the state of Western Australia, during March 2003 to August 2003. Of the 105 volunteers recruited, two were less than 30 years of age, and therefore were ineligible for the study. Participants completed a questionnaire providing minimal information regarding demographic characteristics, including age, ethnicity, and level of education. Included in the questionnaire was an unlabelled Hamilton-Norwood classification scale[ 2 ] (Figure 1 ), which participants were asked to examine and to select the picture they believed best depicted their own balding pattern. Figure 1 Hamilton Norwood Classification Scale (OT Norwood, 1975) Whilst the subjects completed the questionnaire, two data collectors with some training in recognising different balding patterns independently assessed the participant's balding pattern using the same classification system. On recruitment, participants were told the study was to help with medical research, however the role of the data collectors with regard to their independent assessment of the participant's balding pattern was not revealed until after the questionnaire was complete. Where possible, (sporting clubs and universities) volunteers were asked to bring a photograph of themselves at age 35 on the day on which the study would be done. The photographs were used by the observers to independently assess the men's balding patterns at age 35 while the participants completed the questionnaire. These participants were also asked to select the picture from the Hamilton-Norwood scale which best depicted their balding pattern at age 35, and were not permitted to look at the photographs while completing the questionnaire. For data analysis, we compared the assessments of the two observers with each other, and the assessment of the participants with the assessments of the observers. Initially, each of the 12 reference pictures included in the Hamilton-Norwood classification system[ 2 ] were considered separately and the percentage of cases with exact agreement on these was determined between each of the observers and the subjects, and between the two observers. The classifications were then arranged into four groups according to overall balding pattern: no balding (A and B in Figure 1 ); vertex balding only (C); combination of frontal and vertex balding (D to H); and frontal balding only (I to L). Percent agreement within group was then calculated for each observer versus the subjects and between the two observers. The percent of agreement was compared by: age groups (younger than 50 years, and 50 years or older); self-reported ethnicity ("Australian" versus other); and education. Where possible, kappa statistics were also calculated. Results There were 69 participants (67%) between the ages 30–49 years, and 34 (33%) were between the ages 50–70 years. Whilst 70% of subjects considered themselves of "Australian" ethnicity (Caucasian born in Australia), the remainder was made up of representatives from Europe (20.4%), Asia (4.9%), New Zealand (3.9%) and South America (1%). A third of subjects had trade education or equivalent (30.1%); 22.3% had completed high school but not gone onto further education; 12.6% had completed junior high school only; and, because of recruitment through universities, 19.4% of subjects had an undergraduate degree and a further 15.5% had completed post-graduate study. Overall, trained observers were found to be highly reliable at analysing balding patterns with an exact agreement of 85.4% and an agreement within balding pattern groups of 90.3% (Table 1 ). Compared to the observers, men were found to be moderately accurate in their ability to describe their current balding pattern with an exact agreement percentage ranging from 48.5 to 55.3% and agreement as to balding group around 70%. Table 1 Reliability and validity of assessment of balding patterns Exact Balding groups % agreement κ (p) % agreement κ (p) Observer 1 vs. Observer 2 85.4 0.828 (p < 0.001) 90.3 0.858 (p < 0.001) Observer 1 vs. subjects 48.5 0.386 (p < 0.001) 68.0 0.520 (p < 0.001) Observer 2 vs. subjects 55.8 0.463 (p < 0.001) 73.8 0.599 (p < 0.001) In regards to how different demographic characteristics affect men's ability to predict their balding pattern (Table 2 ), the characteristic with the most influence appeared to be age, with men aged 50 or above being more accurate (exact agreement 56–62%) than men aged less than 50 years (exact agreement 45–52%). Men who finished high school were the most accurate at assessing their balding status followed by either those that had studied at technical college or university. The least accurate were men who had completed year 10 at high school or less, with men who had completed post-graduate studies also performing fairly poorly. The effect of ethnicity on ability to assess balding patterns (between Australian and non-Australian men) appeared to be of little significance. Table 2 Validity of assessment of balding group by age, ethnicity and education. Observer 1 vs subjects Observer 2 versus subjects % agreement κ (p) % agreement κ (p) Age group <50 years 65.2 0.46(<0.001) 72.5 0.56(p < 0.001) >50 years 73.5 0.62(<0.001) 76.5 0.66(p < 0.001) Education Junior high school 46.2 0.29 (p = 0.058) 61.5 0.48 (p = 0.003) Senior high school 91.3 0.86 (p < 0.001) 87.0 0.79 (p < 0.001) Trade school 64.5 0.46 (p < 0.001) 74.2 0.58 (p < 0.001) Undergraduate 70.0 - 70.0 - Postgraduate 56.2 0.37 (p = 0.013) 68.7 0.57 (p < 0.001) Ethnicity "Australian" 66.7 0.50 (p < 0.001) 75.0 0.61 (p < 0.001) Other 71.0 0.57 (p < 0.001) 71.0 0.57 (P < 0.001) There were 15 subjects who provided photos of themselves aged approximately 35 years and both observers examined 13 of these. The inter-observer reliability for exact match was 81.8% (κ = 0.766, p < 0.001) and agreement within balding pattern groups increased to 100% (κ = 1, p < 0.001). Observer 2 only examined 13 subject, so for the 15 subjects examined by Observer 1, agreement between the men and the observer was 66.7% for exact match and 86.7% for agreement within the balding pattern groups. Discussion In this study we have shown that trained observers are very reliable in assessing men's balding patterns. Our data also show that, when compared to the trained observers, men themselves can assess their balding patterns quite well. In particular, men are accurate in assessing which balding pattern group they have. This result is important due to previous research suggesting that it is the overall pattern of hair loss rather than extent of balding that determines whether men are at an increased risk of developing negative health outcomes including prostate cancer[ 3 , 4 , 7 ]. There have been several studies investigating the link between male pattern baldness and prostate cancer[ 3 - 5 , 7 , 10 ], as well as other health issues such as cardiovascular disease[ 6 , 8 , 9 ]. In these studies, balding patterns have been assessed using different techniques, some more complex than others. There has been controversy over the use of some of the more simplistic methods of assessment[ 6 ], as little research has been performed regarding their accuracy and their ability to discriminate between the types of balding (vertex, frontal, and combination of vertex and frontal). In a study performed by Hererra et al [ 11 ] assessment of subject's balding pattern involved counting the total number of bald spots found on the head. In a repeat assessment performed six years later on the same subjects, there was actually a decreased level of baldness in 12% of study participants. This apparent reversal of baldness was unable to be attributed to regrowth from treatment or other means, and so it must be concluded that the methods used to assess baldness in these participants were unreliable. Other methods for assessing baldness have been used in clinical situations including reference grids used with standardized photographs of the scalp or in vivo[ 12 ]; and videomicroscopic[ 13 ] and macrophotographic[ 14 ] techniques in which the individual hairs are counted. While these techniques may be used in well-funded clinical trials with the aim of assessing change in balding, they are not appropriate for epidemiological studies in which often the only requirement is to classify men as to their type of balding. The majority of studies[ 4 , 7 , 10 , 15 ] have used variations on the Hamilton scale as modified by Norwood in 1975[ 2 ]. This method allows for the grading of baldness in terms of severity and pattern. The scale can be used either by independent observers, or by men themselves, but no official instructions for use or training manuals are available. No previous studies have been performed to assess the accuracy and reliability of either trained independent observers or the participants themselves in the assessment of balding patterns. The strengths of our study included the recruitment of volunteers from a broad cross-section of the population thus allowing for extrapolation of the results back to the wider community. As our research was performed as an observational study, care was taken to avoid the data collectors influencing the results. This included not informing participants that the observers would be assessing their balding pattern until after the questionnaire was complete. The data collectors also refrained from giving advice to participants when asked to help assess their balding pattern. Older subjects appeared to be better at assessing their balding group than men less than 50 years. This may be due to greater hair loss resulting in a more straightforward distinction between balding patterns, or possibly just a greater self-awareness of degree of balding amongst those in the older age group. The results with regard to education were confusing with men who had only senior high school education seeming to be best at assessing their balding patterns. This may have just been due to small numbers in the groups. Other demographic characteristics not included in our questionnaire may have been of interest in determining factors that influence accuracy of men's self assessment of their balding pattern. These include factors such as marital status, occupation, and personality sub-types, and we would encourage any further research into the area to investigate the possible relationship between these aspects and the accuracy of men's self-assessment. Any extrapolation of our data needs to take into account the differing methods of data collection between our study and other studies in which the man may obtain advice from partners or friends in the home environment, as well as have access to mirrors and photographs to assist in their assessment of current and past balding patterns. These factors do not negatively affect our results however, as the use of such help would ultimately increase accuracy of balding assessment from the already acceptable level shown in our results, rather than detract from it. The study of the accuracy of previous balding was limited by small sample size. Participants had to be approached prior to the study to provide a photograph of them at age 35 years. This limited numbers of eligible participants, and also meant that the men may have viewed the photograph of themselves before completing the questionnaire, which may have increased their accuracy of retrospective assessment. Difficulties in assessing vertex balding from the photographs by the observer also became apparent, as often photographs did not provide an adequate view of the top and back section of a participant's head. It is unlikely that these limitations can be overcome, as it would be difficult to devise another method for the observer to retrospectively assess the participant's balding pattern at age 35. Previous research has demonstrated an intra-observer rate of consistency in assessment of 98–99% using the Hamilton-Norwood scale[ 2 , 10 ]. Hamilton himself classified 200 balding patterns and then repeated the process three months later without reference to the original classifications. All but one of the classifications were identical. The scale was modified in 1975 by Norwood. The Hamilton scale has been found to correlate with local hair density[ 14 ]. A more simple classification of balding patterns was recently been described for use in hair restoration surgery[ 16 ] but this scale has not been used in the epidemiological context. Conclusions From this study, we suggest that any further work requiring assessment of male balding patterns consider the use of trained observers as the gold standard of assessment. Where this is unattainable, we have shown that men's self evaluation is accurate enough to ensure reliability and validity of results. In addition, we believe this study demonstrates that if links are found between male balding patterns and health effects, that men can reliably determine their own balding pattern and assess their own risk. Competing interests The author(s) declare that they have no competing interests. Authors contributions RT and JM planned the study, collected the data, analysed the data under supervision, and wrote the study report for a student project. JL assisted in planning the study and drafted the manuscript LF assisted in planning the study, supervised data collection and analysis and edited the manuscript. All authors read and approved the final manuscript Pre-publication history The pre-publication history for this paper can be accessed here:

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539338

Genome Sequencing: Using Models to Predict Who's Next

It's hard to believe it was just ten years ago that scientists reported the first complete genome sequence of an organism, the bacterial pathogen Haemophilus influenzae . The list has grown considerably since then: add over 160 bacterial species (and counting), most major model organisms, and an ever-growing list of mammals—including, of course, humans. With 99% of our genome now fully sequenced, the Human Genome Project's next major goal is to identify all the functional elements contained in our 2.85 billion nucleotides. Such an effort is hardly trivial: producing the sequence of a mammalian-size genome can run from $10 to $50 million, the estimated price tag of the Cow Genome Project. In an ideal world, any organism would be fair game for sequencing, but in the real world, sequencing resources are scarce. Comparing genome sequences turns out to be a great way to identify regions that have important functions, but comparative genomics studies would be far more efficient if scientists could figure out in advance which genomes would reveal the most information about a particular question. Taking up that challenge, computational biologist Sean Eddy reports a statistical model that predicts how many genomes, and at what evolutionary distance, are needed for effective comparative genomic analyses. In addition to confirming some working principles of comparative genomics, the model also reveals a surprisingly simple guideline for future studies. Comparative genomics works by aligning sequences of different organisms to identify patterns that operate over both large and small distances. Aligning mouse chromosomes with human chromosomes, for example, shows that 99% of our protein-coding genes align with homologous sequences in mice. Underlying such analyses is the principle that DNA sequences that are highly conserved are likely to be functionally important. A common assumption is that adding more comparative genomes to the alignment helps distinguish functionally significant from irrelevant conserved sequences. How do you go about creating an abstract model that captures what Eddy calls the “essential flavor of comparative genomic analysis”? His model puts aside the specific characteristics of individual organisms, genomic features, and analysis programs in favor of identifying higher-level patterns and scaling relationships, specifically between the number of genomes, evolutionary distance, and feature size (features include genetic elements like exons and transcription factors). The model shows that the number of genomes required to identify conserved regions—that is, regions evolving under selection—scales inversely with the size of the feature being sought. Thus, to look for conserved sequences half as long, you need twice as many genomes, assuming a constant evolutionary distance and statistical power. For example, to identify a conserved human feature the size of a coding exon (about 50 nucleotides), it is sufficient to compare just the human and mouse genomes. But to identify conserved single nucleotides, you would need 55 comparative genomes at “mouse-like” evolutionary distances (roughly 75 million years). Things get a little trickier when varying evolutionary distance. We can see a substitution only at a given point in time: we can't tell how many times a site has changed, for example, or whether it changed at some point and then changed back. But at short evolutionary distances—where it's safer to assume no sites have changed more than once—the evolutionary distance is roughly the same as the fraction of sites identified as changed, and evolutionary distance and the number of genomes needed scale inversely. Therefore, the closer the evolutionary distance, the more genomes needed: one would need seven times as many comparative genomes using human/baboon distances, for example, compared to human/mouse distances. So when it comes to using primate sequences to study the human genome, our most distant relatives (such as lemurs) offer far more comparative analysis power than our next of kin (chimps and bonobos). While this model confirms the intuitive assumption that identifying smaller features requires more genomes, it reveals an inverse scaling relationship far more direct, and precise, than previously imagined. With the next phase of the Human Genome Project under way, Eddy's model offers valuable guidelines for identifying which genomes and how many might best meet this ambitious goal.

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543443

The use of bootstrap methods for analysing health-related quality of life outcomes (particularly the SF-36)

Health-Related Quality of Life (HRQoL) measures are becoming increasingly used in clinical trials as primary outcome measures. Investigators are now asking statisticians for advice on how to analyse studies that have used HRQoL outcomes. HRQoL outcomes, like the SF-36, are usually measured on an ordinal scale. However, most investigators assume that there exists an underlying continuous latent variable that measures HRQoL, and that the actual measured outcomes (the ordered categories), reflect contiguous intervals along this continuum. The ordinal scaling of HRQoL measures means they tend to generate data that have discrete, bounded and skewed distributions. Thus, standard methods of analysis such as the t -test and linear regression that assume Normality and constant variance may not be appropriate. For this reason, conventional statistical advice would suggest that non-parametric methods be used to analyse HRQoL data. The bootstrap is one such computer intensive non-parametric method for analysing data. We used the bootstrap for hypothesis testing and the estimation of standard errors and confidence intervals for parameters, in four datasets (which illustrate the different aspects of study design). We then compared and contrasted the bootstrap with standard methods of analysing HRQoL outcomes. The standard methods included t -tests, linear regression, summary measures and General Linear Models. Overall, in the datasets we studied, using the SF-36 outcome, bootstrap methods produce results similar to conventional statistical methods. This is likely because the t -test and linear regression are robust to the violations of assumptions that HRQoL data are likely to cause (i.e. non-Normality). While particular to our datasets, these findings are likely to generalise to other HRQoL outcomes, which have discrete, bounded and skewed distributions. Future research with other HRQoL outcome measures, interventions and populations, is required to confirm this conclusion.

1. Introduction Health Related Quality of Life (HRQoL) measures are now frequently used in clinical trials and health services research, both as primary and secondary endpoints [ 1 ]. Investigators are now asking statisticians for advice on how to plan and analyse studies that have used HRQoL measures. HRQoL measures such as the Short Form (SF)-36, Nottingham Health Profile (NHP) and European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 are described in Fayers and Machin [ 1 ] and are usually measured on an ordered categorical (ordinal) scale. This means that responses to individual questions are usually classified into a small number of ordered response categories, e.g. poor, moderate and good. The responses are often analysed by assigning equally spaced numerical scores to the ordinal categories (e.g. 0 = 'poor', 1 = 'moderate' and 2 = 'good') and the scores across similar questions are then summed to generate a HRQoL score. These 'summated scores' are usually treated as if they were from a continuous distribution and were Normally distributed. We will also assume that there exists an underlying continuous latent variable, Z, that measures HRQoL (although not necessarily Normally distributed), and that the actual measured outcomes, X, are ordered categories that reflect contiguous intervals along this continuum. This ordinal scaling of HRQoL measures means they generate data with discrete, bounded and non-standard distributions, which may lead to several problems in determining sample size and analysing the data [ 2 , 3 ]. Since HRQoL outcome measures may not meet the distributional requirements (usually that the data have a Normal distribution) for parametric methods of sample size estimation and analysis, conventional statistical advice would suggest that non-parametric methods be used to analyse HRQoL data. The bootstrap [ 4 , 5 ] is a data based simulation method for estimating sample size [ 6 ] and analysing data: including hypothesis testing (p-values), standard error (SE) and confidence interval (CI) estimation; which involves repeatedly drawing random samples from the original data, with replacement. So, in theory, computer intensive methods such as the bootstrap that make no distributional assumptions may be more appropriate for estimating sample size and analysing HRQoL data than conventional statistical methods. Conventional methods of analysis of HRQoL outcomes are extensively described in Fayers and Machin [ 1 ] and Fairclough [ 7 ]. They did not use the bootstrap to analyse HRQoL outcomes. As a consequence of this omission, the aim of this paper is to compare bootstrap computer simulation methods with standard methods of analysis of HRQoL measures (particularly the SF-36). We used the bootstrap for hypothesis testing, estimation of SEs and CIs for parameters, in four datasets (which illustrate the different aspects of study design). We then compared the bootstrap with standard methods of analysing HRQoL outcomes. These standard methods included: t -tests; multiple regression/analysis of covariance (ANCOVA) models fitted via ordinary least squares (OLS); response feature analysis or summary measures such as the Area Under the Curve (AUC) [ 8 ] and Generalised Linear regression Models (GLMs) [Pages 21–44, [ 9 ]] fitted using Generalised Estimating Equations (GEE) [ 10 ]. The remainder of this paper is structured into the following sections. The SF-36 HRQoL outcome is briefly described in Section 2. Section 2 also describes how the bootstrap can be used for hypothesis testing and confidence interval estimation. Section 2 ends with a description of the four example datasets. The results of conventional methods of analysis and bootstrap methods are compared in Section 3. Other issues, such as withdrawals and study sizes are discussed in Section 4. The final section (5) ends with a summary and conclusions. 2. Methods The bootstrap The term bootstrap derives from the phrase " to pull oneself up by one's bootstraps ". The phrase is thought to be based on one of the eighteenth century Adventures of Baron Munchausen by Rudolph Erich Raspe. The Baron had fallen to the bottom of a deep lake. Just when it looked like all was lost, he thought to pick himself up by his own bootstraps [Page 5, [ 4 ]]! The basic idea of the bootstrap involves repeated random sampling with replacement from the original data, to produce random samples of the same size of the original sample, each of which is known as a bootstrap sample , and each provides an estimate of the parameter of interest, e.g. mean. The "with replacement" means that any observation can be sampled more than once in each bootstrap sample. It is important because sampling without replacement would simply give a random permutation of the original data, with many statistics such as the mean being exactly the same [Page 115, [ 11 ]]. Repeating the process a larger number of times provides the required information on the variability of the estimator, since the standard error is estimated from the standard deviation of the statistics derived from the bootstrap samples. Bootstrap observed value of the test statistic The bootstrap is mainly used as a method for assessing statistical accuracy i.e. SE, biases and CIs. Throughout this paper we shall use the observed value of the test statistic or parameter estimate as our best guess at the true value of the unknown parameter or statistic. For example, if we are interested in estimating the population mean (from a random sample) it may seem that the best estimator of the mean of the population is the mean of all the bootstrap estimates. This is turns out not to be the case as the mean of the all the bootstrap means is biased. The observed sample mean, from the original data, is always the best estimate of the population mean. The same result applies for other statistics such as the median and regression coefficients. Confidence Interval estimation Suppose we wish to calculate a 95% confidence interval for a mean HRQoL from a sample. We take a random sample, with replacement from this data, of the same size as the original sample, and calculate the mean HRQoL of the data, in this bootstrap random sample. We do this repeatedly, a large number of times, say 1000. So we now have 1000 bootstrap samples, and 1000 estimates of the sample mean, one from each bootstrap sample. If these 1000 bootstrap sample means are ordered in increasing value, a bootstrap 95% confidence interval for the mean would be from the 25 th to the 975 th largest values. This is known as the percentile method and although it is an obvious choice, it is not the best method for bootstrapping confidence intervals, because it can have a bias, which one can estimate and correct for. This leads to methods such as the preferred bias corrected and accelerated (BC a ) method [ 4 , 5 ]. Using the bootstrap method, valid bootstrap confidence intervals can be constructed for all common estimators such as the sample mean, median, proportion, difference in means, and difference in proportions. We estimated BC a bootstrap confidence intervals using the bootstrap procedure in STATA v8 [ 12 ]. According to Efron and Tibshirani [Page 180, [ 4 ]] each interval , where and are the lower and upper bounds of the interval respectively, can be described by its length and shape , 'Shape' measures the symmetry of the interval about the point estimate . The standard Normal based intervals are symmetrical about , and hence have shape = 1.00. Shape is a measure of skewness of the CI about the point estimate. A shape > 1.00, implies the CI is positively skewed, with a long tail to the right, whereas shape < 1.00 implies the CI is negatively skewed. Hypothesis testing with the bootstrap Bootstrap methods can also be used for hypothesis testing. The two quantities that we must choose when carrying out a bootstrap hypothesis test are a test statistic and a null distribution for the data under the null hypothesis . Given these, we generate bootstrap values of the test statistic under the null distribution for the data and estimate the achieved significance level (ASL) by calculating the proportion of the bootstrap values of the test statistics, which are greater than or equal to the observed value of the test statistic from the original data. Several bootstrap test statistics are available for comparing the distribution of sample data in two independent groups. In considering a bootstrap hypothesis for comparing the two means, there is no compelling reason to assume equal variances and so we do not make this assumption. We used a bootstrap test statistic for comparing two means that use only the assumption of a common mean, under the null hypothesis [Page 224, [ 4 ]]. Linear regression: Model (residual) and case resampling Standard errors and CIs for regression coefficients can also be obtained using bootstrap methods. Two different approaches are possible, case and model (residual) resampling. For example with the simple linear model, y = a + bx , where y is the outcome variable and x is a predictor or explanatory variable, a is the intercept and b is the slope or gradient of the line, with n (x, y) pairs of HRQoL observations. Then case-based resampling involves drawing a bootstrap sample of size n , with replacement from these n pairs. Ordinary least squares (OLS) are then used to estimate the regression coefficients for this bootstrap sample of paired cases. Again we do this repeatedly, say 1000 times, so we now have 1000 bootstrap samples and 1000 estimates of the regression coefficients, one from each bootstrap sample. The standard error of these estimated coefficients is simply the standard deviation of these 1000 estimates. As before we can calculate BC a confidence intervals for these estimated regression coefficients. Case-based resampling may be entirely natural for situations where it is plausible that the ( x , y ) pairs have been drawn by random sampling from a population. However, case based resampling is less appealing if the x values were controlled for in some way, perhaps by the design of the study. In this situation the alternative model or residual based procedures could be used. For model based resampling the conventional fitted values and residuals are first obtained from the observed data. A bootstrap sample of the residuals is then drawn. These residuals are then added to the original regression equation (and x values) to generate new bootstrap values for the outcome variable. Ordinary least squares are then used to estimate the new bootstrap regression coefficients, for this bootstrap sample. This process (resampling of the residuals, adding them to the fitted values and estimating the regression coefficients) is repeated lots of times to estimate standard errors and confidence intervals for the regression coefficients from the bootstrap samples. Thus model based resampling is an example of the " parametric bootstrap " when the residuals from a parametric model are bootstrapped to give estimates of the standard error of the parameters. There is considerable debate about which form of resampling is more appropriate. Both forms of resampling can easily be implemented in STATA [ 12 ] and S-PLUS [ 13 ]. We now briefly describe the SF-36 outcome and the four example datasets. SF-36 Health Survey The SF-36 is one of the most commonly used HRQoL measures in the world today. It contains 36 questions measuring health across eight different dimensions – physical functioning (PF), role limitation because of physical health (RP), social functioning (SF), vitality (VT), bodily pain (BP), mental health (MH), role limitation because of emotional problems (RE) and general health (GH). Responses to each question within a dimension are combined to generate a score from 0 to 100, where 100 indicates "good health" [ 14 ]. Thus, the SF-36 generates a profile of HRQoL outcomes, on eight dimensions, with discrete, bounded and skewed distributions (see Figures 1 and 2 ) which makes statistical analysis and interpretation difficult [ 1 ]. Figure 1 Distribution SF-36 dimensions from CPSW data by group Figure 2 Distribution SF-36 dimensions from CPSW data by group The four datasets There now follows a brief description of the four datasets which are used throughout the rest of this paper. These datasets illustrate the use of HRQoL outcomes across a variety of study designs. There are three types of study: observational (both cross-sectional and with baseline and a single follow-up assessment), two group randomised controlled trial (RCT) and longitudinal RCT (with several follow-ups). CPSW Data: Costs & effectiveness of community postnatal support workers (CPSW): RCT [ 15 ] This RCT aimed to establish the relative cost-effectiveness of postnatal support in the community compared to the usual care provided by community midwives. Six hundred and twenty-three postnatal women were allocated at random to Intervention (n = 311) or Control (n = 312) groups. The intervention consisted of up to 10 home visits in the first postnatal month of up to three hours duration by a community postnatal support worker (CPSW). The main outcomes were HRQoL as measured by the SF-36 at six weeks postnatally. This study is unusual since no baseline HRQoL assessment was made. It was felt that it was inappropriate to assess HRQoL just prior to or immediately after childbirth. Our analysis is based on the 495 responders to the six-week postnatal questionnaire who completed all 36 items of the SF-36. This sample consisted of 241 women in the Control group and 254 women in the Intervention group. We will use this data to illustrate methods for simple two group cross-sectional comparisons of HRQoL scores using conventional (e.g. t -test and Mann-Whitney tests) and bootstrap hypothesis tests. We will also compare standard Normal theory ( t -test) based CIs with their bootstrap BC a equivalent. OA Knee Data [ 16 ] The aim of this longitudinal observational study was to evaluate two condition specific and two generic health status questionnaires for measuring HRQoL in patients with Osteoarthritis (OA) of the Knee, and offer guidance to clinicians and researchers in choosing between them. Patients were recruited from two settings, knee surgery waiting listings and rheumatology clinics. Four self-completion questionnaires including the SF-36 were sent to the subjects on two occasions 6 months apart. Two hundred and thirty patients returned the questionnaire at initial assessment, consisting of 118 patients awaiting total knee replacement (TKR) Surgery and 112 patients attending Rheumatology outpatient clinics. At the six-month follow-up assessment, 211 patients returned the questionnaire (109 and 102 in the Surgery and Rheumatology groups respectively). The data used here are based on the 211 patients returning both assessments. Since there was a difference in the baseline HRQoL and sociodemographic characteristics (age and gender) of the Clinic and Surgery groups, we use this dataset to illustrate multiple regression/ANCOVA methods with follow-up HRQoL as the outcome variable and baseline HRQoL, age, gender and group as covariates. We compare the conventional ordinary least squares (OLS) estimates of standard error (SE) and Confidence Interval (CI) for the group regression coefficient with their bootstrap counterparts. Leg Ulcer RCT data [ 17 ] The aim of this RCT, with one year of follow-up, was to establish the relative cost-effectiveness of community leg ulcer clinics that use four layer compression bandaging versus usual care provided by district nurses. Two hundred and thirty-three patients with venous leg ulcers were allocated at random to intervention (120) or control group (113). The intervention consisted of weekly treatment with four layer bandaging in leg ulcer clinic ( Clinic group) or usual care at home by the district nursing service ( Home group). The primary outcome was time to complete ulcer healing over the one-year follow-up. Secondary outcomes included HRQoL as measured by the SF-36 at baseline, three months and 12 months follow-up. We use these data to illustrate the use of summary measures such as the AUC for analysing longitudinal data, using conventional and bootstrap hypothesis tests. We will also compare standard Normal theory ( t -test) based CIs with their bootstrap BC a equivalent. Early Rheumatoid Arthritis RCT data [ 18 ] The Early Rheumatoid Arthritis or NAMEIT trial was a 48-week, randomised, double blind study to compare Neoral with methotrexate ( Neoral ) versus placebo plus methotrexate ( Placebo ) in patients with early severe rheumatoid arthritis (RA). The primary efficacy variable in this study was the attainment of American College of Rheumatology (ACR) criteria for improvement of rheumatoid arthritis. Secondary efficacy variables included patient assessment of health related quality of life (HRQoL). In order to assess the impact of the treatments on patients' health related quality of life, the SF-36 was completed by subjects at seven time-points, Week 0 (baseline), Weeks 8, 16, 24, 32, 40, and Week 48 at the end of the study or at the time of premature withdrawal from the trial. Three hundred and six subjects at 48 centres were actually entered into the study. One hundred and fifty-two subjects receiving methotrexate were randomised to the Neoral treatment group and 154 subjects receiving methotrexate were randomised to the Placebo group. Of the 306 subjects randomised, 227 completed the study. Seventy-nine randomised subjects discontinued from the study prior to completion. We use these data to illustrate more complex statistical models for analysing longitudinal data e.g. a marginal GLM fitted with GEEs and compare bootstrap SEs and CIs for the parameters with their conventionally estimated counterparts. 3. Results Dataset 1 CPSW Study: simple cross-sectional comparison of 6 week HRQoL for the Control vs. Intervention Groups Figures 1 and 2 show the histograms of the SF-36 dimension scores at six weeks post-natally for Intervention and Control groups. The graphs clearly show the bounded, skewed and discrete nature of the data for the SF-36 from this study. Table 1 shows the two sample t -test (with equal variances) and Mann-Whitney (MW) comparisons of the eight SF-36 dimension scores. If we assume a cut-off of p ≤ 0.05 for statistical significance, then the t -test suggests significant differences on two dimensions of the SF-36: RP and SF. On two other dimensions PF (p = 0.060) and BP (p = 0.065) the p -values are close to the arbitrary cut-off of 0.05, suggesting some differences although these may not be statistically reliable. The results of the MW tests suggest significant differences on four dimensions (PF, RP, BP and SF) of the SF-36. The only major contrast between the interpretation of the results of the MW and t -tests is on the BP and PF dimensions, where the former test suggests a difference and later not. Table 1 CPSW Study Simple cross-sectional comparison of 6 week HRQoL for Control vs. Intervention Groups SF-36 Dimension Group n mean sd Mean Diff t-test equal σ's P-value MW test P-value Bootstrap P-value Physical Control 241 89.9 14.5 2.6 0.060 0.015 0.057 Function Intervention 254 87.3 15.8 Role Control 241 74.3 38.1 9.1 0.009 0.004 0.010 Physical Intervention 254 65.2 39.5 Bodily Control 241 75.6 23.7 4 0.065 0.040 0.062 Pain Intervention 254 71.6 23.8 General Control 241 77.7 17.7 2.4 0.139 0.147 0.131 Health Intervention 254 75.3 18.5 Vitality Control 241 51.1 20.7 1.3 0.498 0.596 0.494 Intervention 254 49.8 21.7 Social Control 241 81.6 22.7 4.7 0.025 0.015 0.024 Function Intervention 254 76.9 24.2 Role Control 241 77.9 36.4 1.1 0.734 0.503 0.737 Emotional Intervention 254 76.8 35.5 Mental Control 241 72.9 17.2 -0.2 0.902 0.972 0.904 Health Intervention 254 73.1 16.7 The bootstrap p-value is based on 5000 bootstrap replications. The last column of Table 1 also shows the results of a bootstrap hypothesis test for comparing two means. It compares and contrasts the results of the p -values from a bootstrap hypothesis tests with the p -values from the standard two sample t -test with equal variances, and the MW test. Although they report quantitatively different p -values, the magnitudes are similar, and if we use a cut-off of p < 0.05 for statistical significance then the qualitative interpretation of the tests is the same. So in this example dataset there appears to be little advantage in using the bootstrap hypothesis tests compared to conventional hypothesis tests, such as the t- test, for testing equality of means. A major limitation of non-parametric methods, such as the MW test, is that they do not allow for the estimation of confidence intervals for parameters or allow for the adjustment of confounding variables such as baseline covariates. One way to estimate non-parametric CIs is via the bootstrap method. Table 2 compares and contrasts the Normal/ t -test (equal variances) based confidence intervals with the bootstrap BC a ones. Table 2 Comparisons of parametric and bootstrap estimates of confidence intervals for the eight dimensions of the SF-36 from the CPSW Study for Control vs. Intervention Groups SF-36 CIs Interval Dimension Mean Difference Lower Upper Length Shape Physical Normal ( t -test) -2.6 -5.2 0.1 5.4 1.00 Function Bootstrap BC A -5.2 0.0 5.2 0.98 Role Normal ( t -test) -9.1 -16.0 -2.3 13.7 1.00 Physical Bootstrap BC A -15.8 -2.3 13.5 1.02 Bodily Normal ( t -test) -4.0 -8.2 0.2 8.4 1.00 Pain Bootstrap BC A -8.1 0.3 8.4 1.03 General Normal ( t -test) -2.4 -5.6 0.8 6.4 1.00 Health Bootstrap BC A -5.6 0.8 6.4 0.99 Vitality Normal ( t -test) -1.3 -5.0 2.5 7.5 1.00 Bootstrap BC A -5.1 2.4 7.5 0.98 Social Normal ( t -test) -4.7 -8.9 -0.6 8.3 1.00 Function Bootstrap BC A -8.7 -0.6 8.1 1.03 Role Normal ( t -test) -1.1 -7.5 5.3 12.7 1.00 Emotional Bootstrap BC A -7.1 5.6 12.7 1.11 Mental Normal ( t -test) 0.2 -2.8 3.2 6.0 1.00 Health Bootstrap BC A -2.8 3.2 6.0 0.98 Mean difference = Intervention mean - Control mean BCa confidence intervals based 5000 bootstrap replications . The estimates and lengths of the CIs are almost identical. Table 2 also shows that the shape of the BC a CIs is almost symmetric about the point estimate of the mean difference except for the RE dimension, where there is some evidence of asymmetry. So again in this example dataset there appears little advantage in using the bootstrap BC a confidence intervals compared to conventional methods of confidence interval estimation. The bootstrap (and Normal) confidence intervals are calculated for a characteristic of the distributions (for example mean difference). The groups may have differences in distributions but similar characteristics e.g. mean [ 15 ]. For example, the MW tests suggests a significant difference (in distributions) for the PF, RP, BP and SF dimensions, but the bootstrap and Normal confidence limits for two out of four of these dimensions (PF and BP) includes zero; suggesting no differences in the mean HRQoL between the groups. When a hypothesis is tested using the bootstrap, the resampling is carried out assuming the null hypothesis H 0 is true. Whereas when confidence intervals for mean differences between two groups are estimated the resampling is carried out separately for each group. A useful analogy is with the comparison of proportions in two independent groups. Here the standard error for the hypothesis test is different to the standard error of the difference between the observed proportions used for estimating a confidence interval [Page 45, [ 19 ]]. Dataset 2 OA Knee: comparison of OLS multiple regression, bootstrap case and model based resampling SE and CI estimates for the group (surgery vs. clinic) parameter Table 3 shows the baseline socio-demographic and HRQoL characteristics of the two groups of OA patients those awaiting total knee replacement surgery (Surgical) and those having pharmacological treatment (Rheumatology). The group of patients awaiting surgery is significantly older and has significantly more men than the Rheumatology group. The Surgical group has significantly lower levels of PF prior to total knee replacement surgery than the Rheumatology group. Conversely the Surgical group has significantly higher levels of GH, V and MH compared to the Rheumatology clinic patients. For the other four dimensions of the SF-36 (RP, BP, SF and RE) there was no evidence of any difference in HRQoL between the two groups. Table 3 Baseline characteristics of the TKR Surgery and Rheumatology Clinic patients from the OA Knee study. Rheumatology Surgical 95% CI N Mean SD N Mean SD Mean Diff Lower Upper P-value Age (years) 102 64.2 (11.3) 109 71.1 (8.5) -6.9 -9.6 -4.2 0.001 SF-36 Dimensions Physical Function 97 28.2 (22.4) 95 21.2 (18.2) 7.0 1.2 12.8 0.019 Role Physical 96 11.5 (22.0) 99 12.9 (26.3) -1.4 -8.3 5.4 0.684 Bodily Pain 100 32.0 (19.5) 104 36.3 (23.4) -4.3 -10.3 1.6 0.154 General Health 94 43.9 (22.9) 96 57.3 (23.8) -13.3 -20.0 -6.6 0.001 Vitality 98 36.9 (19.0) 99 42.3 (19.3) -5.4 -10.8 0.0 0.050 Social Function 100 53.1 (30.6) 101 53.6 (27.6) -0.5 -8.6 7.6 0.910 Role Emotional 95 41.1 (44.2) 99 44.1 (44.6) -3.1 -15.6 9.5 0.632 Mental Health 99 62.7 (20.9) 100 68.2 (18.8) -5.5 -11.0 0.1 0.054 Gender Female 71 (69.6%) 59 (54.1%) (15.5%) (2.4%) (27.8%) 0.021† Male 31 (30.4%) 50 (45.9%) Total 102 (100%) 109 (100%) P-values from two independent samples t-test except (†) χ 2 test . We were interested in seeing whether or not there was a difference in HRQoL in OA patients after TKR surgery compared with pharmacologically treated patients. From previous studies using the SF-36 we know that HRQoL varies with age and gender [ 14 , 20 ]. Since there was a difference in the baseline HRQoL and socio-demographic characteristics (age and gender) of the Rheumatology clinic and TKR surgery groups, we use this dataset to illustrate multiple regression/ANCOVA methods with follow-up HRQoL as the outcome variable and baseline HRQoL, age, gender and group (TKR surgery or Rheumatology clinic) as covariates. The analysis involved using OLS to fit the multiple regression model with six month follow-up HRQoL as the outcome variable and age in years at baseline; gender of the patient (coded 0 for males and 1 for females); baseline HRQoL and treatment group variable (coded 0 = Clinic, 1 = Surgery) as explanatory covariates. The group regression coefficient estimate represents the difference in six-month follow-up HRQoL between the Rheumatology Clinic and TKR Surgery groups after adjustment for the patient's age, gender and baseline HRQoL. A positive value for the regression coefficient indicates the Surgery group has a better mean HRQoL at six months follow-up than the Clinic group after adjustment for the other covariates. Table 4 compares the OLS and bootstrap standard errors and confidence interval estimates for the group coefficient from the OA Knee data. All models include age, baseline HRQoL and gender as covariates in the regression. For the bootstrap methods the standard errors are the standard deviations of the coefficients from the 5000 bootstrap re-samples. For ease of interpretation and comparison only the estimates for the group coefficient are shown. Table 4 Comparison of multiple regression, bootstrap case and model based resampling SE and CI estimates from the OA Knee data Dependent GROUP coefficient 95% CI Interval Variable Model N SE / SE p Lower Upper Length Shape Physical Function OLS 165 13.3 3.07 4.31 0.001 7.19 19.32 12.14 1.00 Case 3.02 4.39 7.64 19.69 12.05 1.15 Model 3.05 4.35 7.49 19.49 12.00 1.08 Role Physical OLS 177 -0.5 4.89 -0.11 0.915 -10.16 9.12 19.29 1.00 Case 4.39 -0.12 -8.60 8.51 17.11 1.12 Model 4.86 -0.11 -10.11 8.93 19.04 0.99 Bodily Pain OLS 200 14.7 3.39 4.34 0.000 8.01 21.38 13.37 1.00 Case 3.41 4.30 7.81 21.41 13.60 0.98 Model 3.36 4.38 8.07 21.25 13.18 0.99 General Health OLS 173 4.7 2.01 2.32 0.021 0.71 8.65 7.95 1.00 Case 2.03 7.26 0.69 8.69 8.00 1.01 Model 1.98 2.37 0.70 8.48 7.78 0.95 Energy OLS 185 6.5 2.46 2.64 0.009 1.65 11.36 9.72 1.00 Case 2.50 2.60 1.75 11.63 9.88 1.08 Model 2.46 2.65 1.49 11.04 9.54 0.90 Social Function OLS 194 9.1 3.70 2.46 0.015 1.82 16.41 14.59 1.00 Case 3.52 2.59 2.14 16.06 13.92 1.00 Model 3.65 2.50 1.72 16.09 14.37 0.94 Role Emotional OLS 184 9.4 6.10 1.55 0.124 -2.60 21.48 24.08 1.00 Case 5.89 1.60 -1.90 20.85 22.75 1.01 Model 6.03 1.57 -2.37 20.90 23.27 0.97 Mental Health OLS 191 1.1 2.15 0.51 0.613 -3.15 5.33 8.48 1.00 Case 2.32 0.47 -3.54 5.42 8.96 0.94 Model 2.14 0.51 -3.17 5.12 8.28 0.95 Bootstrap BCa confidence intervals based 5000 bootstrap replications . The regression analysis suggests that at six month follow-up TKR surgical patients have significantly better HRQoL than Rheumatology treated clinic patients on five dimensions of the SF-36 (PF, BP, GH, V and SF) after adjustment for age, gender and baseline HRQoL. As can be seen from Table 4 the standard error estimates are almost identical for the three methods. Similarly the length of the confidence intervals is virtually the same for all three methods. Although the bootstrap CIs tend to be asymmetric about the point-estimate of the regression coefficient. Qualitatively all of the intervals from the three methods either include or exclude zero so the interpretation of the group regression coefficient is the same. Therefore, again in this example dataset, there appears to be little advantage in using bootstrap case or model based re-sampling to estimate standard errors and confidence intervals compared to conventional methods of confidence interval estimation from the OLS multiple regression model. Dataset 3 Leg ulcer: simple cross-sectional comparison of AUC for Home vs. Clinic Groups We are interested in comparing the HRQoL over the one-year follow-up between the Home and Clinic treated groups. The two groups were well matched at baseline for age, gender and HRQoL, except for the RE dimension of the SF-36, where there was some reliable statistical evidence of a difference (p = 0.052). The overall HRQoL of the leg ulcer patients over the 12-month study period (and three HRQoL assessments) can be summarised by the AUC. If we set the time units for the AUC calculation as a fraction of a year, then an AUC value of 100 implies the leg ulcer patient has been in "good health" for the entire 12-month follow-up period. Conversely an AUC value of 0 implies the leg ulcer patient has been in "poor health" for the entire 12-month follow-up period. Table 7 [See additional file 1 ] gives the results of simple comparisons of differences in mean AUC between the groups using the two independent samples t -test, the MW test and the bootstrap hypothesis test. The p -values from the t -test and the ASL from the bootstrap hypothesis tests are very similar. None of the p -values for the eight SF-36 dimensions are less than 0.05. Therefore there is no reliable statistical evidence to suggest a difference in mean AUC between the Clinic and Home treated leg-ulcer patients. Only the results of the MW test on the RE dimension of the SF-36 provide (p = 0.071) any evidence of a difference in AUC distributions between the groups, although even this p -value is not statistically significant using the conventional cut-off of 0.05. The table also contrasts the Normal theory based CI estimates from the t -test with the bootstrap BC a limits. The lengths of the intervals are very similar, although the bootstrap BC a intervals tend to have a non-symmetric shape. All the estimated CIs include zero, again suggesting no evidence of a difference in mean AUC (HRQoL) between the Clinic and Home group patients in the Leg Ulcer study. Dataset 4 Early RA: Comparison of robust and bootstrap SE's and CI's for the time and group coefficients with a GEE marginal model and exchangeable autocorrelation In the Early RA study, HRQoL assessment was carried out at 0, 8, 16, 24, 32, 40 and 48 weeks. With seven repeated HRQoL measurements, such as this, the best approach is to model the longitudinal data using GLMs. The modelling of longitudinal data takes into account the fact that successive HRQoL assessments by a particular subject are likely to be correlated. We used a marginal model with the Early RA data and used GEEs to estimate the regression coefficients. Marginal models are appropriate when inferences about the population average are the focus. For example, in a clinical trial the average difference between control and treatment is most important, not the difference for any one individual. In a marginal model, the regression of the response on explanatory variables is modelled separately from the within-person correlation. The marginal model is an extension of the linear regression model used with the OA Knee data. Longitudinal models require the specification of the auto- or serial correlation , which is the strength of the association between successive longitudinal measurements of a single HRQoL variable on the same patient. Several underlying patterns of the auto-correlation matrix are used in the modelling of HRQoL data. The error structure is independent (sometimes termed random ) if the off diagonal terms of the auto-correlation matrix are zero. The repeated HRQoL observations on the same subject are then independent of each other, and can be regarded as though they were observations from different individuals. On the other hand, if all the correlations are approximately equal or uniform then the matrix of correlation coefficients is termed exchangeable , or compound symmetric . This means that we can re-order (exchange) the successive observations in any way we choose in our data file without affecting the pattern in the correlation matrix. As the time or lag between successive observations increases, the auto-correlation between the observations decreases. A correlation matrix of this form is said to have an autoregressive structure (sometimes called multiplicative or time series ). Table 5 summarises the resulting 21 auto-correlation pairs for the assessments until week 48. The pattern of the observed auto-correlation matrix, gives a guide to the so-called error structure associated with the successive HRQoL measurements. Table 5 shows that the autocorrelation coefficients range between 0.19 and 0.85. For three dimensions of the SF-36, PF, GH and MH, the autocorrelation coefficients are moderately large (between 0.5 and 0.85). The pattern of values suggests that the assumption of compound symmetry is not unreasonable. Table 5 Auto-correlation matrices for the eight dimensions of the SF-36 from RA patients in the Early RA study assessed at seven time points a) Physical Function (n = 218) e) Vitality (n = 216) Week 0 8 16 24 32 40 48 Week 0 8 16 24 32 40 48 0 1.00 0 1.00 8 0.61 1.00 8 0.55 1.00 16 0.63 0.74 1.00 16 0.48 0.58 1.00 24 0.57 0.69 0.75 1.00 24 0.47 0.54 0.71 1.00 32 0.56 0.68 0.80 0.79 1.00 32 0.50 0.59 0.68 0.71 1.00 40 0.55 0.67 0.77 0.81 0.86 1.00 40 0.42 0.49 0.67 0.68 0.77 1.00 48 0.53 0.64 0.74 0.81 0.81 0.85 1.00 48 0.47 0.53 0.66 0.72 0.72 0.76 1.00 b) Role Physical (n = 212) f) Social Function (n = 219) Week 0 8 16 24 32 40 48 Week 0 8 16 24 32 40 48 0 1.00 0 1.00 8 0.40 1.00 8 0.44 1.00 16 0.35 0.53 1.00 16 0.43 0.53 1.00 24 0.29 0.39 0.57 1.00 24 0.39 0.55 0.63 1.00 32 0.19 0.30 0.56 0.67 1.00 32 0.36 0.46 0.63 0.70 1.00 40 0.34 0.42 0.52 0.60 0.61 1.00 40 0.38 0.51 0.58 0.64 0.71 1.00 48 0.27 0.40 0.59 0.67 0.64 0.71 1.00 48 0.34 0.45 0.58 0.64 0.71 0.71 1.00 c) Bodily Pain (n = 219) g) Role Emotional (n = 206) Week 0 8 16 24 32 40 48 Week 0 8 16 24 32 40 48 0 1.00 0 1.00 8 0.43 1.00 8 0.46 1.00 16 0.45 0.55 1.00 16 0.35 0.47 1.00 24 0.44 0.47 0.61 1.00 24 0.34 0.40 0.59 1.00 32 0.37 0.46 0.51 0.68 1.00 32 0.31 0.32 0.56 0.62 1.00 40 0.40 0.42 0.57 0.60 0.69 1.00 40 0.34 0.46 0.53 0.56 0.54 1.00 48 0.42 0.46 0.59 0.63 0.68 0.76 1.00 48 0.31 0.37 0.49 0.58 0.54 0.69 1.00 d) General Health (n = 209) h) Mental Health (n = 218) Week 0 8 16 24 32 40 48 Week 0 8 16 24 32 40 48 0 1.00 0 1.00 8 0.55 1.00 8 0.57 1.00 16 0.56 0.68 1.00 16 0.57 0.62 1.00 24 0.60 0.67 0.80 1.00 24 0.55 0.59 0.72 1.00 32 0.58 0.67 0.77 0.83 1.00 32 0.52 0.55 0.65 0.69 1.00 40 0.59 0.65 0.72 0.79 0.84 1.00 40 0.50 0.54 0.70 0.70 0.74 1.00 48 0.58 0.65 0.75 0.84 0.82 0.85 1.00 48 0.56 0.55 0.68 0.72 0.73 0.77 1.00 Correlations are Pearson's product moment coefficient . The process of fitting marginal models using GEE begins by assuming the simple independence form for the autocorrelation matrix, and fitting the model as if each assessment were from a different patient. Once this model is obtained the corresponding residuals are calculated and these are then used to estimate the autocorrelation matrix assuming it is of the exchangeable (or autoregressive) type. This matrix is then used to fit the model again, the residuals are once more calculated, and the autocorrelation matrix obtained. The iteration process is repeated until the corresponding regression coefficients that are obtained in the successive models converge or differ little on successive occasions [ 1 ]. Fayers and Machin [Pages 183–202, [ 1 ]] and Diggle et al [ 10 ] emphasise the importance of graphical presentation of longitudinal data prior to modelling. Figure 3 shows the mean levels of HRQoL in patients with RA, before and during treatment, for the eight dimensions of the SF-36. The curves for some dimensions of the SF-36 overlap (e.g. PF, GH, RE, and MH dimensions) suggesting that it may be unrealistic to assume that the mean difference in HRQoL values on these dimensions remains constant over time. For other dimensions such as BP, V and SF there is some evidence to suggest that for later HRQoL measurements the curves are parallel and that the mean difference between treatments is now fairly constant. Figure 3 Profile of mean SF-36 scores over time by treatment group EARLY RA data (Patients who completed all seven HRQoL assessments) The overlapping lines on some of the graphs in Figure 3 imply there may be a 'Treatment × Time' interaction. It is therefore important to test for any such interaction in any regression model. Fortunately, with the marginal model approach this is relatively easy to do and simply involves the addition of an extra regression coefficient to the model. If treatment is coded as a 0/1 variable (i.e. 0 = Placebo and 1 = Neoral) and assessment time as a continuous variable, then the additional interaction term is simply the product of these two variables (which will be 0 for all the Placebo group patients and equal to the HRQoL assessment time in the Neoral Group patients). Early RA marginal model analysis The marginal model we used for the Early RA data for analysing the seven HRQoL assessments over time was, Y ij = β 1 + β Base x Base_i + β Age x Age_i + β Sex x Sex_i + β Time t ij + β Group x Group_i + ε ij ,     (2) where Y ij is the HRQoL at time t ij post-baseline; t ij is the time of the QoL assessment, in weeks post baseline, of patient i at visit j ; x Base_i is the baseline HRQoL assessment for subject i ; x Age_i is the age (in years) of subject i at time 0 (baseline); x Sex_i is the gender of subject i ; x Group_i is the treatment group (0 = Placebo, 1 = Neoral) for subject i ; β 1 is a constant and ε ij is the residual error. The marginal regression models were fitted in STATA [ 12 ] using the xtgee command with an identity link function (link (iden)) and the robust standard errors option. The observed correlation matrices in Table 5 clearly show the off-diagonal terms are non-zero and that the assumption of an independent auto-correlation matrix for the marginal model is unrealistic. We will not consider models with an independent auto-correlation structure and will concentrate on reporting the results of models with an exchangeable correlation. None of the interaction term coefficients for the eight SF-36 dimensions were statistically significant (from zero). Thus there was no reliable evidence of a 'Treatment × Time' interaction on any dimension of the SF-36 (p > 0.05), irrespective of the autocorrelation structure. Therefore we will only report the results of the simpler model (2), without the interaction term. The beauty of the marginal model and the GEE methodology is that it is very flexible and can in principle deal with all the observed data from a HRQoL study. The subjects are not required to have exactly the same numbers of assessments, and even the assessments can be made at variable times. The latter allows the modelling to proceed even if a subject misses a HRQoL assessment. So it seems unrealistic and unreasonable to use bootstrap resampling methods for marginal models that can only utilise a balanced data set, with equally spaced QoL assessments. Since we are interested in fitting a marginal model and we are likely to have an unbalanced dataset with unequal observations per subject we used simple bootstrap case-resampling. Figure 4 shows the estimated within subject correlation matrices for the eight dimensions of the SF-36 if we fit the longitudinal model and assume a compound symmetric structure. The lower diagonal gives the observed matrix before the model fitting. The fitted autocorrelations ranged from 0.43 for the RE dimension to 0.63 for the PF and GH dimensions. On the whole, the model correlation estimates tend to be lower than the actual observed autocorrelations, for HRQoL assessments that are close together. Conversely the model correlation estimates tend to be larger than the observed correlations for HRQoL observations further apart in time. It will usually be the case that after model fitting the autocorrelations will appear to have been reduced [ 1 ]. The observed deviations between the fitted model and observed autocorrelations are not too great, suggesting that the assumption of compound symmetry is not unreasonable (Figure 4 ). Figure 4 Observed and estimated within-patient auto-correlation matrices (exchangeable model) from RA patients in the EARLY RA study. The lower diagonal gives the observed matrix before model fitting whilst the upper gives the exchangeable form after model-fitting a Table 6 shows the estimated regression coefficients for the group and time variables. There is some evidence that HRQoL increases over time for three dimensions of the SF-36, PF, BP and V. However, we are interested in the effect of treatment and comparing HRQoL over time across the Placebo and Neoral treated groups. Since there is no reliable evidence of a 'Group × Time' interaction the interpretation of the treatment group coefficient is relatively straightforward. The p -values for the treatment group regression coefficients in Table 6 suggest significant differences in HRQoL between the Neoral and Placebo groups on three dimensions of the SF-36 (RP, GH and BP). Table 6 Comparison of robust and bootstrap SE's and CI's from the EARLY RA data with a Marginal Model and exchangeable autocorrelation Dependent Coefficients 95% CI Interval Variable SE / SE p Lower Upper Length Shape Physical Function (n = 222) time 0.11 0.03 3.63 0.001 0.05 0.18 0.12 1.00 0.03 3.72 0.05 0.18 0.12 0.97 group 2.82 2.25 1.25 0.211 -1.60 7.24 8.84 1.00 1.72 1.64 -0.51 6.13 6.64 0.99 Role Physical (n = 221) time -0.06 0.07 -0.90 0.366 -0.19 0.07 0.26 1.00 0.07 -0.94 -0.19 0.06 0.25 0.91 group 9.49 3.93 2.42 0.016 1.79 17.19 15.40 1.00 3.22 2.95 3.63 16.62 12.99 1.22 Bodily Pain (n = 222) time 0.16 0.03 4.69 0.001 0.10 0.23 0.14 1.00 0.03 4.88 0.10 0.23 0.13 1.05 group 4.23 1.97 2.14 0.032 0.36 8.10 7.74 1.00 1.50 2.82 1.44 7.25 5.81 1.08 General Health (n = 221) time 0.04 0.03 1.67 0.095 -0.01 0.09 0.10 1.00 0.03 1.68 -0.01 0.09 0.10 0.95 group -4.61 1.96 -2.35 0.019 -8.46 -0.76 7.69 1.00 1.51 -3.04 -7.36 -1.28 6.07 1.21 Vitality (n = 220) time 0.09 0.03 3.09 0.002 0.03 0.14 0.11 1.00 0.03 3.05 0.04 0.15 0.11 1.24 group 2.67 1.80 1.48 0.14 -0.87 6.20 7.07 1.00 1.41 1.89 -0.19 5.42 5.61 0.96 Social Function (n = 222) time 0.03 0.03 0.77 0.442 -0.04 0.09 0.13 1.00 0.03 0.79 -0.04 0.09 0.13 0.97 group 2.40 2.11 1.14 0.255 -1.73 6.54 8.27 1.00 1.65 1.46 -0.72 5.88 6.61 1.11 Role Emotional (n = 221) time -0.02 0.07 -0.32 0.752 -0.17 0.12 0.29 1.00 0.08 -0.31 -0.18 0.12 0.30 0.93 group 4.14 3.91 1.06 0.29 -3.52 11.81 15.33 1.00 2.93 1.41 -1.54 10.11 11.64 1.05 Mental Health (n = 221) time 0.02 0.03 0.69 0.489 -0.03 0.07 0.10 1.00 0.03 0.68 -0.03 0.07 0.10 1.15 group 1.53 1.67 0.92 0.359 -1.74 4.79 6.53 1.00 1.34 1.14 -0.93 4.42 5.35 1.18 Note: The bootstrap estimates of SE and BC a Confidence Intervals are shown in italics below the model based estimates and are based on 1000 resamples . The bootstrap and robust standard errors for the time and group coefficients are different, although the bootstrap SE estimate tends to be the same size or somewhat smaller than its robust counterpart. However both bootstrap and robust SE estimates are of a similar order of magnitude. More importantly, the ratios of the estimated coefficient to its standard error are of similar size. A crude test of statistical significance is to examine this ratio, if it is bigger than 2.0 then the estimated regression coefficient is likely to be significantly different from zero. Table 6 shows that for all the models where the original (group or time) regression estimates are significant (i.e. ratios of estimate/SE > 2) then so too is the ratio of the estimate to its bootstrap standard error. When we compare the bootstrap BC a confidence intervals with the model- based estimates in Table 6 then the length of the bootstrap intervals tend to be the same size or slightly narrower than its robust counterpart. As before the bootstrap estimates are not constrained to be symmetric about the point-estimate of the regression coefficient. Qualitatively both the bootstrap and model based intervals include zero when the estimated regression coefficient is non-significant and exclude zero when the estimated coefficient is significant. Therefore, the actual practical interpretation of the confidence interval estimates is the same. That is for the RP, BP, and GH dimensions there is some evidence that the Neoral group has a better HRQoL than the Placebo group patients over time, after allowing for baseline HRQoL, age and gender. The use of the bootstrap to estimate SEs and CIs for marginal longitudinal models appears to offer little advantage (in the Early RA data) compared to the conventional robust estimates. 4. Discussion In the datasets and outcomes studied, and for the specific conventional analyses we used, we have shown that use of the bootstrap does not lead to different p-values, SE and CI estimates compared to conventional methods. On this basis, we cannot conclude the use of the bootstrap is more appropriate than conventional methods. The explanation for this conclusion and the extent of its generalisability deserve discussion. Ordinality of HRQoL outcomes One of the fundamental assumptions we have made, is that there exists an underlying continuous latent variable that measures HRQoL, and that the actual measured outcomes are ordered categories that reflect contiguous intervals along this continuum. If the goal of the analysis is to assess the magnitude of the treatment effect on this ordered outcome, then an appealing approach is to assign numeric scores to the ordered categories and then to compare means between groups using conventional linear regression methods. If interest lies elsewhere, for example in comparing the relative frequencies of cumulative probabilities in the ordered categories between treatments, then other techniques such as the proportional odds model would be more appropriate [ 9 , 2 , 21 ]. Heeren and D'Agostino [ 26 ] have demonstrated the robustness of the two independent samples t -test when applied to three-, four- and five point ordinal scaled data using assigned scores, in sample sizes as small as 20 subjects per group. Sullivan and D'Agostino [ 27 ] have expanded this work to account for a covariate when the outcome is ordinal in nature. They again assign numeric scores to the distinct response categories and compare means between treatment groups adjusting for a covariate reflecting a baseline assessment measured on the same scale. Their simulation study shows that in the presence of three-, four- and five point ordinal data and small sample sizes (as low as 20 per group) that both ANCOVA and the two independent sample t -test on difference scores are robust and produce actual significance levels close to the nominal significance levels. Generalisability The generalisability of the results could be called into question as they only apply to the limited number of datasets studied (four) and the SF-36 outcome. The SF-36 outcome is the most widely used generic HRQoL measure in the world today, so that is one obvious reason to use it [ 22 ]. Secondly, we had easy access to a variety of datasets that had previously used the SF-36 outcome. The four studies (CPSW, OA Knee, Leg Ulcer and Early RA), and datasets were well known to us. They illustrate the use of HRQoL outcomes across a variety of studies including cross-sectional surveys, RCTs, non-randomised before and after studies and longitudinal designs. So on practical and pragmatic grounds, we felt it was appropriate to use such datasets because of their familiar nature and the analysis was easy to understand and interpret. The SF-36 is a multi-dimensional outcome with eight dimensions. As described in the Introduction the eight dimensions have a variety of distributions. We believe these distributions are not atypical of other generic HRQoL measures such as the NHP and EORTC QLQ-C30. The distributions we considered were chosen based on our experiences with HRQoL data in a variety of settings. So we believe that our results about the bootstrap may have generalisability to other HRQoL outcomes (besides the SF-36) used in other studies and populations, although strictly speaking our results only apply to the SF-36 outcome and the observed datasets. Hence, we cannot make sweeping generalisations about the impact of the bootstrap on other HRQoL outcomes, used in other studies. Therefore, these results need to be replicated with other HRQoL measures in other datasets and populations. Missing values It should be noted that in the all four example datasets there is missing data. We assumed that any missing HRQoL values in these datasets were Missing Completely at Random (MCAR). This means that the probability of the HRQoL response being missing is independent of the scores on the previous observed questionnaires and independent of the current and future scores had they been observed. We have assumed that the reduced dataset represents a randomly drawn sub-sample of the full dataset and the inferences drawn can be considered reasonable. This is a strong assumption and unlikely to hold for missing HRQoL data [ 1 , 7 , 23 - 25 ]. Sample sizes of the example datasets The various datasets used in this study all had a sample size in excess of 100 patients. Some caution should be used in applying the results to smaller sample sizes. However the robustness of the conventional two-sample t -test and ANCOVA, for three-, four- and five point ordinal scale data using assigned scores has been demonstrated for sample sizes as small as 20 [ 26 , 27 ]. Simple bootstrapping may not be very successful in small samples anyway (say < 9 observations), since the observations themselves are less likely to be representative of the study population. As Campbell [Page 118, [ 11 ]] states, "In very small samples even a badly fitting parametric analysis may outperform a non-parametric analysis, by providing less variable results at the expense of a tolerable amount of bias." The bootstrap Bootstrap case resampling vs. model based resampling The results with the OA Knee data show that there is little to choose from between the case and model based resampling for the multiple linear regression model for estimating SEs and CIs. Since there was very little difference in the SE and CI estimates from the datasets used, for simplicity one would tend to favour a case based resampling approach. Indeed this was the resampling method for the longitudinal marginal model for the Early RA data. Bootstrap model based resampling for marginal model In the longitudinal Early RA for simplicity we used only a simple case based resampling for the marginal model and effectively carried out a stratified random resampling with replacement. That is we sampled with replacement blocks or clusters of each patients' repeated HRQoL responses. In theory, one should be able to use model or residual based resampling for the marginal model. The resampling procedure would be rather complex particularly for autoregressive autocorrelation structures and for unbalanced datasets, with HRQoL assessments at unequally spaced time points. One would have to take into account that the residuals were not independent and uncorrelated, and for the autoregressive correlation structure, that the correlation between residuals within a patient declined over time. This is a very interesting avenue and requires further exploration with other longitudinal datasets. Are the results surprising or unexpected? Finally, are the results all that surprising or unexpected? We have shown that the use of bootstrap methods for analysis (calculation of p-values, SE and CIs) appears to offer little advantage compared to standard methods in the four datasets studied. If we assume that there exists an underlying continuous latent variable that quantifies the HRQoL response of interest and that the goal of the analysis is to assess the magnitude of a treatment effect on the HRQoL outcome, by comparing means between groups. Then statistical theory says that if the distribution of the HRQoL data is Normal, so will be the distribution of the sample mean. Much more importantly, even if the distribution of HRQoL data is not Normal, as is frequently the case, that of the sample mean will become closer to a Normal distribution as the sample size gets larger. This is a consequence of the Central Limit Theorem (CLT) [Pages 304-7, [ 28 ]]. The Normal distribution is strictly only the limiting form of the sampling distribution as the sample size increases to infinity, but it provides a remarkable good approximation to the sampling distribution even when the sample size is small and the distribution of the data is far from Normal [Page 94, [ 29 ]]. This implies, for example, that the distribution of the sample means for the SF-36 HRQoL data shown in Figures 1 and 2 will be approximately Normal. Thus, if the investigator is planning a large study and the sample mean is an appropriate summary measure of the HRQoL outcome, then pragmatically there is no need to worry about the distribution of the HRQoL outcome and we can use standard methods to estimate sample sizes and analyse the data. Since dramatic effects are unlikely in HRQoL studies using the SF-36 as an outcome, large samples sizes are likely to be required [ 2 , 3 , 6 ]. So perhaps unsurprisingly, the results reflect the robustness of conventional methods with large sample sizes and the application of the CLT to sample means even for HRQoL data with such bounded, discrete and skewed distributions as shown in Figures 1 and 2 . So our research using the SF-36 HRQoL outcome and the four datasets has shown that bootstrap methods appear to produce p-values, SEs and CIs similar to conventional methods. When the standard and the bootstrap methods agree, we can be more confident about the inference we are making and this is an important use of the bootstrap [Page 118, [ 11 ]]. When they disagree more caution is needed, but the relatively simple assumptions required by the bootstrap method for validity mean that in general it is to be preferred. Thus, there appears to be little advantage in using the bootstrap for the analysis of SF-36 data, particularly if one is interested in comparing mean HRQoL between treatment groups. 5. Conclusions In the datasets we studied, using the SF-36 as an outcome measure, bootstrap methods produce results similar to conventional statistical methods. This is likely because the t -test and OLS multiple regression are robust to the violations of assumptions that HRQoL data are likely to cause (i.e. non-Normality). While particular to our datasets, these findings are likely to generalise to other HRQoL outcomes, which have discrete, bounded and skewed distributions. They may not generalise to HRQoL studies with smaller sample sizes of less than 100 subjects. Future research with other HRQoL outcome measures, interventions and populations, is required to confirm this conclusion. Supplementary Material Additional File 1 Table 7 – Leg Ulcer study simple cross-sectional comparison of AUC for Home vs. Clinic Groups Click here for file

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC543443.xml

548502

Phenotypic and genetic heterogeneity in a genome-wide linkage study of asthma families

Background Asthma is a complex genetic disease with more than 20 genome-wide scans conducted so far. Regions on almost every chromosome have been linked to asthma and several genes have been associated. However, most of these associations are weak and are still awaiting replication. Methods In this study, we conducted a second-stage genome-wide scan with 408 microsatellite markers on 201 asthma-affected sib pair families and defined clinical subgroups to identify phenotype-genotype relations. Results The lowest P value for asthma in the total sample was 0.003 on chromosome 11, while several of the clinical subsets reached lower significance levels than in the overall sample. Suggestive evidence for linkage (p = 0.0007) was found for total IgE on chromosomes 1, 7 and again on chromosome 11, as well as for HDM asthma on chromosome 12. Weaker linkage signals could be found on chromosomes 4 and 5 for early onset and HDM, and, newly described, on chromosome 2 for severe asthma and on chromosome 9 for hay fever. Conclusions This phenotypic dissection underlines the importance of detailed clinical characterisations and the extreme genetic heterogeneity of asthma.

Background Many chromosomal regions have been shown to be linked or associated to asthma and asthma-associated traits in humans [ 1 ]. More recently, asthma genes have been identified on chromosomes 2 [ 2 ], 13 [ 3 ], 14 [ 4 ], and 20 [ 5 ]. The investigation of the genetic aetiology aims at the improvement of preventive strategies, diagnostic tools and therapeutic alternatives [ 6 ]. These final steps have not yet been reached or are even in sight, while the reasons for this delay are unclear. The mainstay of all genetic studies has been genome-wide linkage scans in families with at least two asthma-affected siblings. Based on a previous analysis of a genome-wide scan of asthma [ 7 , 8 ] with inconsistent chromosomal findings to earlier studies, we decided to expand the initial sample with additional families by the same core protocol for clinical examination and using the same set of microsatellite markers [ 7 , 8 ]. The increased the number of identically pheno- and genotyped families could be used to define sub-phenotypes, which may be a promising strategy to explain the aetiological heterogeneity observed so far. Relevant clinical subsets may be defined by different age of onset, different disease course by degree of severity, extrinsic (allergic sensitization detectable) and intrinsic (no allergic sensitization detectable, symptoms often during infections of the upper respiratory tract) asthma type, and house dust mite allergy (HDM), as well as genetic background as judged by geographical origin of parents (table 1 ). We hypothesized that the restriction to a smaller well-defined sample would reduce heterogeneity and improve the power of detecting linkage. Linkage regions should show higher lod scores than in the total sample and lead from phenotype subgroups to a genotypic dissection. Table 1 Phenotypes of the 201 families included. Phenotype Definition No. of families Early onset asthma occurrence of asthmatic symptoms for one child before the age of 2 years and for his or her sib pair at least before the age of 4 years 67 Extrinsic asthma all asthmatic children are positive for at least one SPT or specific IgE 134 HDM SPT positive at least 3 family members with a positive skin prick test (SPT) for HDM 33 HDM RAST positive at least 3 family members with a positive result of serum specific IgE for HDM 42 Severe asthma asthma severity index (see table 3) for one asthmatic child is 4 or 5 and for his or her sib pair 3, 4 or 5 56 Seasonal variation: "Winter-Type" one affected child suffers from asthma attacks in the winter half-year and his or her sib pair at least not solely in the summer half-year 39 Seasonal variation: "Summer-Type" one affected child suffers from asthma attacks in the winter half-year and his or her sib pair at least not solely in the summer half-year 35 German nationality both parents are of German descent 170 Methods Clinical evaluation 97 families consisting of at least two children with confirmed clinical asthma were collected during the first stage of the German genome scan [ 7 ]. We now recruited another set of families during a period of 18 months. Trained staff from 3 university hospitals as well as 6 pediatric pulmonary practices carried out an identical phenotyping procedure as described previously [ 7 ]. This procedure contained detailed interviews of every family member, skin prick tests (SPT) of frequent allergens, blood samples (for IgE and allergen-specific IgE (RAST) measurements, eosinophil count), peak flow tests for a period of 10 days and dust collection at patients' homes. SPT and RAST assays included several pollens, animal furs, mould, and house dust mite allergens (ALK-SCHERAX, Hamburg, Germany). The ethics commission of "Nordrhein-Westfalen" approved all study methods and informed consent was obtained from all parents and children. Children with premature birth, low birth weight and any severe pulmonary disease other than asthma were excluded as prematurity (and or associated low birth weight) may be a major non-genetic risk factor for the development of pulmonary symptoms. Another 4 families were excluded after genotyping because of Mendelian errors. The additional 104 families comprised 452 individuals (table 2 ). Their 244 children had a mean age of 10.8 years. 75 families contributed 2 children, 22 families 3 children and 7 families 4 children). In total, the 201 families had 465 children; 255 were male, 210 female, and 413 had physician-diagnosed asthma. Table 2 Clinical characteristics of the 201 families (867 participants) that also include non-affected siblings of the core families. Descriptors of categorical variables include n/total sample in the first row and percent in the second row. Descriptors of continuous variables include mean values in the first row and standard deviation in the second row. Parents Children Stage 1 Stage 2 Stage 1 Stage 2 sex females 97/194 50.0% 104/208 50.0% 96/221 43.4% 114/244 46.7% Asthma diagnosis 33/194 17.0% 60/208 28.8% 200/221 90.5% 213/244 87.3% D.pter skin > = 3 mm 68/193 35.2% 58/206 28.2% 111/217 51.2% 107/242 44.2% D.far skin > = 3 mm 57/193 29.5% 48/207 23.2% 101/217 46.5% 88/242 36.4% D.pter. CAP class >1 51/183 27.9% 43/147 29.3% 124/217 57.1% 102/198 51.5% D.far. CAP class >1 46/183 25.1% 39/147 26.5% 122/217 56.2% 100/198 50.5% age (years) 39.8 +/-5.7 40.9 +/-5.5 11.0 +/-4.2 10.8 +/-3.4 onset asthma (years) not reliable not reliable 3.8 +/-2.8 4.3 +/-3.1 height (cm) 172.3 +/-9.0 172.3 +/- 9.9 146.9 +/-19.2 156.3 +/-18.1 weight (kg) 76.0 +/-14.9 78.0 +/-16.6 41.5 +/-17.0 47.4 +/-16.6 ln(Ige) (kU/L) 4.1 +/-1.7 4.1 +/-1.6 5.0 +/-2.0 5.0 +/-1.8 ln(eosinophil) (count / mm3) 0.3 +/-1.7 0.7 +/-1.5 1.1 +/-1.6 1.1 +/-1.6 FEV1 (ml) 3.594 +/-804 3.464 +/- 957 2.312 +/-883 2.861 +/-1.061 The trait "asthma" was based on the clinical diagnosis as described earlier [ 7 ]. "lnIgE" was used for analysis as a quantitative variable and categorized by cut-off point of 100 kU/l. Furthermore, the following phenotype subsets (see also table 1 ) were selected from the total study sample: Early onset asthma We created a subset of 67 families with one child having asthma symptoms before the age of 2 years and another child having asthma symptoms before the age of 4 years. In about half of the families within this subtype, both affected children had asthmatic symptoms before the age of 2 years. We were of course dependent on the recollection of the parents – but keeping in mind that the state of health of the children is of high priority in those families who joined our study and the period of time is quite clear, the data seemed valuable to us. Extrinsic/intrinsic asthma sample The group of families with atopic ("extrinsic") asthma was large, consisting of 134 families all with at least one positive skin prick test reaction or one increased specific IgE. Positive Dermatophagoides farinae skin prick test and increased specific IgE in serum Almost all family members participated in a skin prick test (SPT). We selected an "HDM SPT positive" group of 33 families with at least 3 family members showing a skin prick test reaction ≥ 3 mm. House dust mite allergy is quite common in asthmatic patients, and a known trigger factor for asthma attacks. Some studies have successfully used the combination with asthma as lead trait [ 9 , 10 ]. As the functional relevance might be slightly different in study participants with serum antibodies, another subgroup was based on the measurement of HDM-positive specific IgE at concentrations ≥ 0.35 kU/l, which resulted in a group of 42 families. Asthma severity Several interview questions related to asthma symptoms, diagnostic findings and therapy. We used information about asthma attack frequency, actual medication and emergency hospital visits for a severity index with 5 levels. This index is based on subjective patient information, current/previous therapy and influence on quality of life (table 3 ). "Severe Asthma" was seen in 56 families where one sib had at least grade 4 and another grade 3, 4 or 5. "Moderate Asthma" with one sib of maximum grade 2 and another with maximum grade 3 was defined in the same way. Table 3 Asthma severity grade definition. Severity grade Frequency and percentage in asthmatic children Clinical criteria Attacks (last 12 months) Asthma medication (last 12 months) At least 1 overnight hospital stay 1 29 (7.2%) none none none 2 118 (29.3%) none none yes none yes none 3 164 (40.8%) none yes yes once/month yes none 4 72 (17.9%) once/month yes yes at least once/month yes none 5 19 (4.7%) at least once/month yes yes Seasonal variation of asthma attacks All participants were asked to specify the months when asthma attacks occurred. 39 "winter-type" families had one affected child with symptoms only in the cold months and his or her sibs not a fully "summer-type". In contrast 35 "summer-type" families were found with one affected child with summer attacks and his or her sibs not purely "winter-type". Attacks year-round usually were seen with severe disease only. Sporadic attacks usually did not have any seasonal preferences while both the intrinsic, usually infection-related "winter-type", as well as the extrinsic, hay fever related "summer-type", appeared to be concordant within families. This observation may already support the hypothesis of different genes in different subtypes. German sample In 170 of 201 families, both parents were of German descent ("German"). In this subgroup, we tried to reduce the genetic heterogeneity by leaving out the remaining 31 families, of which 5 were from Sweden and 5 from Turkey. DNA analysis DNA was isolated from peripheral white blood cells using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's recommendation. For genotyping we used almost the same microsatellite marker as in the first scan [ 7 , 8 ]. The final analysis included 408 markers of which 364 were autosomal and 7 were based on the X-chromosome, all included in the previous scan as well as a set of 37 previous fine mapping markers. For the baseline marker set, the mean distance was 10 cM, with an average marker-information content of 0.87 and a mean heterozygosity of 0.79. Marker amplification was performed in microtiter plates, either in 96- (Peltier Thermal Cycler PT-225, MJ Research, Waltham, MA) or in 384-well format (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA). Fragment analysis of PCR pools was conducted on an ABI 3700 DNA sequencer and genotypes were scored using GENESCAN and GENOTYPER (ABI) software. In the second scan, 98% of all possible genotypes could be unequivocally determined. Statistical analysis Genotyping data were transferred to a SQL 2000 database, pre-checked with previously developed routines and exported in ASCII format for multipoint linkage analysis with MERLIN 0.9.3 [ 11 ]. Allele frequencies were estimated from the (unrelated) parental alleles. As different primers were used in some assays between the first scan in 1997 and the second scan in 2002, we adjusted the original allele size before replacing the allele size with its respective order. This procedure led to comparable allele frequency distributions in both scans. For ordering of all markers we used the Marshfield comprehensive human genetic linkage map that was slightly modified according to the marker order given by Golden Path 13 and expressed intermarker distances to Haldane cM. Error detection as implemented in MERLIN was then applied to discard unlikely genotypes from the analysis. Finally, P-values for qualitative traits were derived from the Kong and Cox method based on the score statistic S_all [ 12 ]. For the quantitative trait ln(IgE) a variance component was applied including only age and sex as covariates. Results Figure 1 shows the linkage results for asthma, total IgE and all other subgroups. Chromosomes are arranged by number from p-ter to q-ter with distance in centimorgans on a linear scale. Table 4 reports all loci with p-values below 0.01 in at least two adjacent markers for one phenotypic subgroup. Figure 1 Multipoint linkage results for all traits. Chromosomes are arranged with increasing numbers with orientation from p-ter to q-ter. Distance is given in centimorgans (cM) on a linear scale. A = asthma all, B = asthma German families, C = extrinsic asthma, D = HDM RAST positive, E = HDM SPT positive, F = severe asthma, G = early onset asthma, H = winter-type, I = summer-type, J = LnIgE continuous, K = LnLgE categorical. Table 4 Linkage results in 201 core families with at least 2 children having asthma. Weak linkage (p < 0.01) and suggestive linkage (p = 0.0007) is indicated in bold. GDB PIC Chr. cM (Marshfield) Asthma All Asthma German Extrinsic Asthma HDM RAST +ve HDM SPT +ve Severe Asthma Early Onset Winter-Type Summer-Type Ln(IgE) continuous Ln(IgE) categorical D1S478 0.8404 1 48.53 0.03 0.03 0.04 0.5 0.9 0.12 0.3 0.13 0.8 0.002 0.0011 D1S234 0.8559 1 55.1 0.07 0.08 0.04 0.3 0.9 0.12 0.2 0.2 0.8 0.0002 0.00009 D1S255 0.7710 1 65.47 0.005 0.04 0.0015 0.04 0.4 0.03 0.15 0.7 0.2 0.002 0.005 D1S197 0.8285 1 76.27 0.007 0.06 0.002 0.12 0.4 0.09 0.07 0.5 0.11 0.08 0.2 D1S484 0.8050 1 169.68 0.08 0.12 0.012 0.004 0.003 0.3 0.3 0.08 0.3 0.007 0.013 D1S431 0.9008 1 182.35 0.12 0.13 0.02 0.03 0.04 0.5 0.3 0.3 0.2 0.005 0.005 D1S2815 0.9201 1 188.85 0.06 0.09 0.012 0.06 0.07 0.3 0.3 0.2 0.03 0.0008 0.0007 D1S238 0.8722 1 202.73 0.4 0.7 0.4 0.2 0.1 0.3 0.8 0.2 0.6 0.004 0.003 D1S2655 0.8976 1 216.82 0.7 0.9 0.6 0.2 0.2 0.2 0.9 0.7 0.7 0.05 0.007 D2S2374 0.9039 2 54.96 0.03 0.07 0.04 0.011 0.008 0.04 0.02 0.8 0.4 0.3 0.5 D2S2328 0.9545 2 61 0.02 0.04 0.014 0.007 0.007 0.008 0.04 0.8 0.06 0.14 0.4 D2S2294 0.9617 2 64.84 0.02 0.03 0.02 0.003 0.02 0.003 0.5 0.8 0.02 0.13 0.4 D2S2298 0.9585 2 65.94 0.02 0.02 0.02 0.003 0.02 0.002 0.06 0.8 0.014 0.11 0.3 D2S2113 0.9331 2 88.15 0.3 0.5 0.3 0.2 0.4 0.008 0.13 0.7 0.3 0.5 0.5 D3S1597 0.8199 3 29.92 0.7 0.7 0.4 0.6 0.9 0.4 0.4 0.6 0.8 0.009 0.006 D3S1286 0.9021 3 41.56 0.4 0.3 0.3 0.2 0.3 0.13 0.2 1 0.4 0.02 0.007 D3S1300 0.8748 3 80.32 0.12 0.3 0.2 0.09 0.0014 0.3 0.5 0.3 0.8 0.4 0.09 D3S1285 0.7673 3 91.18 0.07 0.08 0.2 0.07 0.0008 0.2 0.2 0.13 0.6 0.5 0.5 D4S1607 0.8843 4 183.63 0.3 0.3 0.3 0.07 0.05 0.2 0.005 0.07 0.6 0.4 0.3 D4S1535 0.8938 4 195.06 0.05 0.07 0.013 0.009 0.03 0.03 0.006 0.007 0.11 0.07 0.09 D4S2924 0.8934 4 199.93 0.06 0.09 0.007 0.002 0.03 0.02 0.009 0.02 0.2 0.08 0.15 D5S426 0.8990 5 51.99 0.006 0.006 0.03 0.008 0.14 0.04 0.002 0.007 0.2 0.09 0.4 D5S418 0.9101 5 58.55 0.015 0.03 0.06 0.004 0.2 0.012 0.007 0.012 0.4 0.02 0.07 D7S484 0.9204 7 53.5 0.5 0.7 0.2 0.4 0.5 0.5 0.4 0.5 0.9 0.02 0.009 D7S528 0.9481 7 57.79 0.2 0.4 0.014 0.14 0.3 0.3 0.5 0.5 0.6 0.0012 0.0007 D7S510 0.9783 7 59.93 0.14 0.4 0.02 0.3 0.3 0.2 0.6 0.6 0.4 0.0009 0.0013 D7S485 0.9688 7 60.68 0.1 0.3 0.01 0.3 0.3 0.13 0.6 0.7 0.4 0.0007 0.0004 D7S2548 0.9596 7 62.28 0.2 0.4 0.05 0.4 0.5 0.12 0.7 0.7 0.3 0.003 0.002 D7S691 0.9403 7 63.67 0.08 0.2 0.02 0.4 0.5 0.08 0.6 0.6 0.3 0.003 0.007 D7S2506 0.9313 7 69.56 0.2 0.4 0.13 0.9 0.8 0.14 0.7 0.7 0.3 0.007 0.011 D7S663 0.9049 7 78.65 0.06 0.05 0.08 0.3 0.4 0.2 0.5 0.3 0.14 0.0007 0.001 D7S669 0.8351 7 90.42 0.08 0.05 0.04 0.4 0.2 0.13 0.5 0.5 0.02 0.0014 0.0012 D9S257 0.9630 9 91.87 0.09 0.06 0.3 0.3 0.4 0.4 0.4 0.4 0.004 0.5 0.5 D9S283 0.9681 9 94.85 0.015 0.01 0.1 0.2 0.3 0.2 0.11 0.1 0.002 0.2 0.3 D9S1796 0.9745 9 97.53 0.02 0.014 0.07 0.3 0.2 0.05 0.07 0.14 0.006 0.07 0.13 D9S1781 0.9565 9 99.4 0.015 0.007 0.05 0.14 0.2 0.11 0.03 0.03 0.003 0.07 0.15 D9S1851 0.9708 9 103.42 0.02 0.008 0.04 0.06 0.2 0.2 0.05 0.02 0.003 0.3 0.5 D9S1786 0.9825 9 104.48 0.013 0.006 0.03 0.04 0.11 0.13 0.05 0.02 0.002 0.4 0.5 D9S176 0.9798 9 105.02 0.02 0.009 0.05 0.09 0.1 0.2 0.14 0.03 0.005 0.5 0.5 D9S1690 0.9709 9 106.63 0.06 0.04 0.2 0.2 0.14 0.4 0.4 0.13 0.007 0.5 0.5 D9S1784 0.9667 9 111.99 0.11 0.04 0.2 0.07 0.2 0.3 0.6 0.4 0.008 0.5 0.5 D10S547 0.8313 10 29.15 0.14 0.2 0.4 0.8 0.8 0.6 0.3 0.2 0.9 0.007 0.005 D10S191 0.8726 10 37.9 0.4 0.5 0.2 0.3 0.5 0.8 0.5 0.3 0.8 0.008 0.012 D11S922 0.9045 11 2.11 0.2 0.4 0.4 0.3 0.02 0.13 0.3 0.9 0.06 0.0005 0.0006 D11S902 0.8133 11 21.47 0.3 0.3 0.5 0.9 0.3 0.3 0.5 0.6 0.5 0.006 0.003 D11S935 0.7997 11 45.94 0.5 0.4 0.4 0.4 0.4 0.4 0.6 0.9 0.05 0.003 0.04 D11S968 0.8094 11 147.77 0.003 0.007 0.05 0.11 0.3 0.04 0.12 0.6 0.2 0.0005 0.0002 D12S355 0.8343 12 74.58 0.07 0.08 0.06 0.003 0.04 0.4 0.2 0.012 0.6 0.3 0.4 D12S1684 0.9339 12 86.4 0.02 0.015 0.05 0.002 0.05 0.9 0.15 0.2 0.4 0.08 0.4 D12S1667 0.9727 12 92.89 0.05 0.08 0.2 0.003 0.04 0.8 0.15 0.08 0.4 0.03 0.2 D12S81 0.9707 12 94.44 0.08 0.12 0.2 0.005 0.02 0.8 0.3 0.09 0.3 0.013 0.2 D12S351 0.9822 12 95.56 0.02 0.013 0.05 0.002 0.0003 0.5 0.3 0.05 0.3 0.0009 0.02 D12S95 0.9785 12 96.09 0.012 0.007 0.04 0.002 0.0002 0.5 0.2 0.06 0.4 0.0012 0.02 D12S327 0.9757 12 97.78 0.02 0.008 0.07 0.008 0.0009 0.4 0.05 0.13 0.2 0.04 0.2 D12S1716 0.9590 12 101.45 0.04 0.005 0.2 0.02 0.006 0.4 0.06 0.2 0.2 0.011 0.06 D12S1706 0.9696 12 104.12 0.12 0.015 0.3 0.07 0.06 0.6 0.3 0.4 0.2 0.007 0.2 On chromosome 1 , two linkage areas for total IgE could be found at about 55 cM (p = 0.00009) and at 188 cM (p = 0.0007), which reached the threshold for suggestive linkage (p = 0.0007) [ 13 ]. On chromosome 2 at about 64 cM (p = 0.002) evidence was found for a locus for severe asthma and for house dust mite sensitive asthma. Early onset, together with HDM, is represented both on chromosome 4 at about 195 cM (p = 0.002) and on chromosome 5 at about 55 cM (p = 0.002). Three loci for total IgE regulation were found on chromosome 7 (65 cM, p = 0.0004) and chromosome 11 (2 cM, p = 0.0005; 147 cM, p = 0.0002), all with suggestive linkage according Kruglyak-Lander. On chromosome 9 at about 104 cM (p = 0.002) there is evidence for a locus linked to allergic rhinitis plus asthma ("summer-type"), having an effect mainly in families of German descent. Lastly, a locus showing suggestive linkage could be identified on chromosome 12 (95 cM, p= 0.0002) for house dust mite sensitised asthmatic patients. Discussion Several linkage regions in asthma families could be found in this extended sample, although again no linkage with P < 0.003 could be found with any marker for asthma. Asthma is apparently of such a heterogeneity that even investigating affected sib pairs from more than 200 families failed to yield significant results. Nevertheless, the quantitative trait IgE in these families, both as a discrete and continuous variable, led to three suggestive linkage findings on chromosomes 1, 7, and 11, which have all been discussed in prior publications (table 5 ). Table 5 Comparison of asthma loci Chr. Position This study (2004) Bradley (2002) [14] Cookson (2001)[15] Daniels (1996) [16] Dizier (2000) [17] Laitinen (2001) [18] Malerba (1999) [19] Ober (1998. 1999, 2000) [20-22] Xu J (2000, 2001) [23, 24] Xu X (2001) [25] Yokouchi (2000) [9] CSGA (1997) [26] 1 55cM IgE Asthma Asthma, Atopy Asthma Hispanic population 1 188cM IgE AD Slope IgE 2 64cM severe asthma HDM 3 50cM IgE HDM AD Loose Asthma 4 195cM early onset HDM Slope HDM and Asthma 5 55cM early onset HDM Slope BHR Asthma 7 65cM IgE IgE, Slope, Eosinophils SPT Asthma, IgE 9 104cM German summer type Asthma symptoms, Atopy 11 2cM IgE IgE SPT Asthma Hispanic population Asthma 11 147cM IgE IgE 12 95cM HDM Eosino-phils, IgE Asthma Asthma IgE, Asthma HDM and Asthma Asthma Phenotypic dissection Moreover, the strategy of phenotypic dissection proved to be quite successful, though the sample size of subsets seems to be very small. The size of the sample in a linkage study is known to be one of the most important factors for a significant finding [ 1 ], making the artificial limitation of subsets a double-edged decision. Clearly, there is a remarkable phenotypic heterogeneity of asthmatic diseases but – as these families represent different genetic influences-limitation of the sample size did not affect statistical properties as significance values were smaller compared to the whole study sample. In particular, the subgroups segregated by high disease severity, sensitisation by HDM, early onset and population origin attained better lower significance levels than the main trait (table 4 ). So far, results were not adjusted with the Bonferroni correction for multiple testing as this would have been too conservative for this explorative investigation. Subgroups are often closely related and highly overlapping. Nevertheless, there might be a problem with multiple testing and the criteria for suggestive linkage in a genome scan on a single phenotype [ 13 ]. Sample recruitment with restricted phenotypic requirements proved to be successful for some other traits as well. Early onset definitions have been used for cancer studies and metabolic disorders to increase the underlying genetic component [ 27 , 28 ]. Separation in sub-samples of ethnically diverse populations has also been conducted [ 26 ], but higher disease activity scores [ 29 ] seemed to give an advantage in other fields also. Other Asthma Genome Scans Recently, evidence was shown for asthma genes on chromosomes 2 [ 2 ], 13 [ 3 ], 14 [ 4 ], and 20 [ 5 ]. Our study does not support linkage loci in those regions. As the region on chromosome 13 was responsible for only 10% of the observed IgE serum level variability, this small effect may be easily overlooked in another study. A lack of replication does not necessarily mean an error in the primary study. Most likely, genetic heterogeneity is the cause, where the proportion of patients with a particular gene variant is different between studies. A polygenic model of the inheritance mode of asthma with minor effects by single genes could explain the highly diverse association and linkage results thus far. Frequent replication, however, may point toward lead genes. Most regions listed in table 4 have already been described and some of the traits are supported by the phenotypes proposed in our analysis. Table 5 summarizes previously published asthma/atopy loci in approx. 20 cM distance that would be consistent with our findings. Both loci on chromosomes 4 and 5 are supported by the phenotypes of early onset, HDM and "winter-type", which might relate to the clinical picture of chronic obstructive bronchitis. Phenotypes described by other studies include lung function variation and HDM, which is supported by our data. The "atopy" locus (defined by SPT and RAST) on chromosome 3 confirms linkage studies of atopic dermatitis (AD) [ 14 ] and might be the genetic link between both diseases. The locus on chromosome 12 with suggestive linkage for HDM in our study is known from many other reports. Also our first scan showed weak evidence for linkage in this region at D12S351 (p = 0.01) [ 7 ], whereas the best result for the HDM subset lies now about 0.5 cM apart at D12S95 with p = 0.0002. These consistent findings are probably based on frequent alleles playing an important role in extrinsic asthma. Linkage results for SPT and RAST for HDM were quite similar, but the HDM SPT positive group – which is a smaller group-, reached the suggestive linkage level on chromosome 12. SPT, even more than RAST, might represent the individual's allergic disposition. On chromosome 2, we found a locus in families with severe asthma that has not yet been described in other studies. A p-value of 0.007 has already been found in our first genome-wide scan [ 7 ] for D2S2298 and our main trait asthma. The same marker for severe asthma in both samples improved to a p-value of 0.002. Severe asthma is rather rare, and a disease-aggravating allele on chromosome 2 could be easily overlooked in a heterogeneous asthma sample. Severe asthma may be a lethal disease, while a severe-asthma gene would be of high importance for intensified treatment. The German "summer-type" locus on chromosome 9 is also not frequent in other studies. This might be due to the fact that many, but not all, genes are necessary for a common trait. Our first asthma scan has already shown linkage in this region with a p-value of 0.007 for D9S1784 [ 7 ], which now improves to p = 0.002 for the "summer-type" subset at D9S1786, about 7.5 cM apart. Conclusions We conclude that this phenotypic dissection is a useful tool to detect linkage in a heterogeneous disease like asthma because some of the sub phenotypes reached even better significance values than the main trait. We show that the precision of the phenotype can be more effective than expanding the sample size only. Unfortunately, large sample sizes are needed to assure at least moderate sample size in subsets. Grouping by early onset or disease severity could be applied to almost every complex disease, but for a more specific dissection, clinical expert knowledge is required. A prior analysis of clinical data can help to identify symptom clustering, which -if consistent within families- can reduce genetic heterogeneity. Competing interests The author(s) delcare that they have no competing interests. Authors' contributions MW, JA, and CS were involved in the study design, JA organised the sample collection, conducted the genetic analysis, and prepared the manuscript, MW organized funding and supervised the study. YAL and PN organised and helped with genotyping and revised the article critically for important intellectual content. SL, FR and MW carried out the statistical analysis and made substantial contributions to its conception and design, CS, DB, FF, HJ, JK, AK, AS, MS, WW, PW assisted in the recruitment of families and examined patients and therefore made substantial contributions to the acquisition of data, asthma severity grades are part of the thesis of CS. GS was responsible for the IgE analysis. Pre-publication history The pre-publication history for this paper can be accessed here:

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EvDTree: structure-dependent substitution profiles based on decision tree classification of 3D environments

Background Structure-dependent substitution matrices increase the accuracy of sequence alignments when the 3D structure of one sequence is known, and are successful e.g. in fold recognition. We propose a new automated method, EvDTree, based on a decision tree algorithm, for automatic derivation of amino acid substitution probabilities from a set of sequence-structure alignments. The main advantage over other approaches is an unbiased automatic selection of the most informative structural descriptors and associated values or thresholds. This feature allows automatic derivation of structure-dependent substitution scores for any specific set of structures, without the need to empirically determine best descriptors and parameters. Results Decision trees for residue substitutions were constructed for each residue type from sequence-structure alignments extracted from the HOMSTRAD database. For each tree cluster, environment-dependent substitution profiles were derived. The resulting structure-dependent substitution scores were assessed using a criterion based on the mean ranking of observed substitution among all possible substitutions and in sequence-structure alignments. The automatically built EvDTree substitution scores provide significantly better results than conventional matrices and similar or slightly better results than other structure-dependent matrices. EvDTree has been applied to small disulfide-rich proteins as a test case to automatically derive specific substitutions scores providing better results than non-specific substitution scores. Analyses of the decision tree classifications provide useful information on the relative importance of different structural descriptors. Conclusions We propose a fully automatic method for the classification of structural environments and inference of structure-dependent substitution profiles. We show that this approach is more accurate than existing methods for various applications. The easy adaptation of EvDTree to any specific data set opens the way for class-specific structure-dependent substitution scores which can be used in threading-based remote homology searches.

Background With the sequencing of entire genomes and the exponential growth of sequence databases on the one hand, and the significant number of known folds compared to the putative number of possible folds in the fold space on the other hand, sequence-structure comparison is currently one main challenge of the post-genomic era. To this goal, 3D-environments were used by Eisenberg and coll. in the early 90s to build statistical potentials indicating the probability of finding each amino acid in a given structural environment as described by the secondary structure, the solvent accessibility and the polarity of neighboring atoms [ 1 ]. Such statistical potentials were successfully applied to protein fold recognition [ 1 - 4 ] or protein model evaluation [ 5 , 6 ], and were shown to improve the quality of sequence-structure alignments [ 7 ]. Statistical potentials describing the propensity of a residue pair to be at a given spatial distance have proved successful as well [ 8 - 14 ], but are more difficult to use as information to guide sequence-structure alignments using dynamic programming. On the contrary, residue preferences for position-dependent structural environments are easily implemented in alignment programs [ 7 , 15 ]. Recent improvements in this field were achieved by (i) optimizing definition and classification of the 3D-environments, and (ii) by constructing substitution matrices instead of residue preferences, i.e. taking into account the native residue type [ 15 - 17 ]. Indeed, it has been shown that amino acid substitutions are constrained by the structural environment, each environment displaying a distinct substitution pattern [ 18 , 19 ]. The use of 64 distinct substitution matrices corresponding to different 3D environments based on secondary structure, solvent accessibility and hydrogen bonding, combined with structure-dependent gap penalty and with global or local alignment algorithms, provides good performance to the FUGUE software in fold recognition approaches [ 15 ]. In this paper we investigate the use of decision tree algorithms to automate and improve the classification of structural environments. The automation will allow easy adaptation to any particular selected data set, opening a way for the construction of various specific substitution matrices. Indeed, it appears that one problem in the use of statistical potentials for structure prediction is their lack of universality [ 13 ]. It may thus be worthwhile to derive potentials specific to prediction problems or to protein classes. The automated derivation proposed here will facilitate such developments. In the first part of the work we focus on automatically building and evaluating structure-dependent substitution scores. The emphasis is given to the development of a method for automatic selection of the most informative classifications of 3D environments in order to set up a versatile method allowing easy compilation of structure-dependent substitution scores for any given set of proteins. In a second part, the method is applied to a specific protein class, the small disulfide-rich proteins. Decision trees have attracted our attention for several reasons. Knowledge acquisition and description language are optimized automatically and do not require human expertise during the learning process. Thanks to the hierarchical organization of the inferred trees, classifications are obtained quickly and the chain of decisions leading to the prediction is explicit and can be easily interpreted (in contrast to artificial neural networks for example). Decision tree learning algorithms are also robust since they partition the data recursively. The first dichotomies near the tree root are then based on many examples and are therefore statistically reliable. To handle noise and uncertainty, the trees can be pruned during a post-processing step to remove possible misleading clusters at the bottom of the tree. The research field on this topic is well-established and the number of applications of decision trees to real world is huge. Methods Structure-dependent substitution profiles Standard substitution matrices are deduced from multiple sequence alignments of similar sequences [ 20 - 22 ]. To derive structure-dependent substitution matrices, multiple sequence alignments are also needed as well as a description of the 3D structure at each position in the alignment [ 15 , 17 ]. A schematic overview of the EvDTree method is displayed in Figure 1 . Since we want to make sure that all residues at the same position in the alignment do share similar 3D structures, we will only use multiple alignments obtained from structural superimpositions (step 1 in Figure 1 ). From this, we extract all observed "substitutions" for each residue type and the corresponding structural environment (step 2 in Figure 1 ). The word "substitution" here is used to describe the residue replacement observed at an equivalent position in two structurally similar proteins. Then, for each residue type, the structural environments and associated substitutions are classified using a decision tree algorithm and substitution scores are computed from residue distributions observed in each cluster of the classification tree (step 3 in Figure 1 ). Standard structure-dependent substitution matrices report the probability of 20 × 20 or 21 × 21 possible substitutions in a given structural environment [ 15 ]. In this work we classify 3D-environments and associated substitutions derived from alignments separately for each of the 21 residues (the 20 standard residues plus the half-cystine) using a decision tree algorithm (Figure 1 , step 3 and Figure 2 ). As a result, we get several structure-dependent substitution profiles for each type of residue, that each indicates the relative probabilities of all 21 possible substitutions of one residue type in a given structural environment. Since the selected structural environments differ between residue types, the substitution profiles cannot be gathered into structure-dependent substitution matrices. As an example of how structural environments may differ between residues, a solvent-exposed environment might refer to a solvent accessibility > 6% if the residue is a leucine, but to a solvent accessibility > 33% if the residue is a glutamine. Learning and test data sets Several data sets of structure-structure superimpositions and the corresponding alignments are available [ 23 - 25 ]. We have selected the database of homologous structure alignments, HOMSTRAD [ 23 ] for constructing both the learning and the test data sets of sequence-structure alignments. Main HOMSTRAD strengths are (i) a selection of accurate protein structures, (ii) a combination of automatic procedures and of manual inspections that guarantee low data noise, and (iii) its successful use in the derivation of the structure-dependent substitution matrices used in FUGUE [ 15 ]. Moreover, to facilitate comparison between our method and FUGUE, we selected the very same learning set previously used by Mizuguchi et al. This subset consists of 177 families extracted from the HOMSTRAD database and does not contain membrane proteins. From this HOMSTRAD subset, a set of 2.5 million of observed substitutions were extracted, one substitution corresponding to a residue in a reference structure, and the corresponding residue observed at the same position in a structurally similar protein. Moreover, to remove non-structural constraints from the sequence-structure alignments, the following filters were applied: - Residues involved in a domain-domain or chain/chain interface, were excluded. Residues are considered to be involved in an interface when their solvent accessibility varies by more than 7 % when comparing the protein environment in the complex and in the isolated chain/domain. The cut-off value was taken from Mizuguchi et al [ 15 ], who used a similar filter to remove residues at a chain/chain interface. - Residues that are not correctly superimposed in the structural superimposition were also excluded. The superimposition was considered good enough when the deviation between the two corresponding alpha carbons is below 3.5 Å. We assume that larger deviations may correspond to incorrect structural superimposition for the particular residue even though other residues are correctly aligned. Large deviations may also imply significant modifications in the 3D-environment. Although this 3.5 Å criterion is sometimes too restrictive, it actually leaves enough data for robust statistical estimations while removing most of aligned amino acid pairs whose respective structural contexts are not superposable. Application of the two above filters excluded about 20% of the initial substitutions leaving about 2 million substitutions for the learning process. This data set was split into (i) a learning data set containing 950 000 substitutions similar to the learning set used by Mizuguchi et al. (ii) a pruning data set containing 325 000 substitutions, and (iii) a test data set containing 355 000 substitutions. The learning data set has been in some cases filtered further based on the percentage of sequence identity between superimposed proteins, resulting in smaller sets of 500 000 (0–40% id) or 700 000 (0–60% id) substitutions, respectively. - Since we only work with three-dimensional structures, the oxidation state of any cysteine (free or disulfide bridged) is known. The symbol 'C' refers to disulfide bridged cysteines (half-cystines), whereas the symbol 'J' was used for free cysteines. Structural descriptors Since the decision tree algorithm is able to automatically select the most discriminating structural descriptor at each classification step (see below) we do not need to empirically determine the 'best' descriptors. In this work, twenty-three structural descriptors were provided to the classification algorithm. The secondary structure (ss1) was assigned to each residue according to STRIDE [ 26 ] into seven categories. Values are as follows: ss1 = 1, 2, 3, 4, 5, 6 and 7 for α-helices (H), 3 10 helices (G), π-helices (I), isolated bridges (B), extended conformations (E), turns (T) and coils (C), respectively. We also used a simpler 3-state description (ss2) deduced from the STRIDE assignment: ss2 = 1, 2 or 3 for helices (H or G), sheets (B or E) and coils (I, T, or C), respectively. Hydrogen bonds were determined using the Hbond software [ 27 ]. Four different descriptors were used for different type of interactions: side-chain...main-chain O atom (hb1), side-chain...main-chain N atom (hb2), side-chain acceptor...side-chain donor (hb3) and side-chain donor...side-chain acceptor (hb4). For each interaction type, the number of interactions was used as the descriptor value. Here again a simpler description (lh) was also implemented that takes value of 0, 1, 2, or 3 if the side-chain of the residue makes no hydrogen bond, makes hydrogen bond(s) with side-chain atom(s), makes hydrogen bond(s) with main-chain atom(s) or makes hydrogen bonds with both side-chain and main-chain atoms, respectively. Other structural parameters were obtained using the local program compilPDB [J.G.]. Beside the secondary structure, the local structure was also described by the Phi and Psi dihedral angles, and by Cα-Cα distances: d3 = Cα i - Cα i+3 , d4 = Cα i - Cα i+4 , d5 = Cα i - Cα i+5 , d6 = Cα i - Cα i+6 , d7 = Cα i - Cα i+7 . Other descriptors were the buried surface area (bur), percent of accessibility (pac), contact area with carbon atoms (C), nitrogen atoms (N), oxygen atoms (O), sulfur atoms (S), positively charged atoms (pp), negatively charged atoms (nn), or polar atoms (pol). For simplicity, these structural descriptors will now be called s 1 to s 23 . It should be noted that some structural descriptors are correlated (e.g., the Phi and Psi dihedral angles versus the d3 and d4 alpha carbon distances). However, this descriptive redundancy is not a problem since it is eliminated during the tree construction where the most informative descriptors only are selected, as explained below. Automated classification of structural environments using a decision tree algorithm The native structural environments observed in the learning data set were classified for each of the twenty amino acids, plus the half-cystine, resulting in twenty-one independent decision trees (Figures 1 and 2 ). The use of these decision trees is as follows: let ( a ( k ), s (k)) the position k in a protein for which we want to score substitutions, a ( k ) the residue type and s ( k ) = ( s 1 ( k ),..., s 23 ( k )) the structural environment description at this position. After the learning phase explained below, each tree node will be associated to particular structural descriptor s j and threshold S and will be linked by edges to two subnodes whose structural environments will be constrained respectively by the tests s j ≤ S and s j > S . The classification of ( a ( k ), s (k)) will be obtained by selecting the decision tree corresponding to residue type a ( k ) and then by running through the tree from its root node to an appropriate leaf following at each node the edge whose test, s j ≤ S or s j > S , is compatible with the value of the corresponding structural descriptor s j ( k ) (Figure 2 ). Contextual substitutions scores associated to the selected tree leaf, as explained in a further paragraph, will then evaluate each possible substitution of the amino acid a ( k ). According to the standard data mining terminology, the predictive variables are therefore the native amino acid type and its associated structural descriptors and the dependent variable to be predicted is the substituted residue at this position. During the learning phase which we will now describe, the goal of the decision tree construction is to optimize the predictive power of the structural descriptor test chosen at each node and therefore to maximize the bias of the statistical distributions of the substituted residues associated to each subnode towards a few types of amino acids. Ideally, tree leaves should be associated to only one type of substituted amino acid, but this never happens in practice because of the tree depth limitation and the data set noise. Let ( a ( i ), s ( i ), b ( i )) be the i -th example of the whole learning data set where a ( i ) is a native residue, s ( i ) = ( s 1 ( i ),..., s 23 ( i )) is its structural environment description and b ( i ) is the substituted residue as observed in a structurally similar protein at the same position as a ( i ). The main steps of the decision tree construction from the learning data set are as follows (Figure 2 ): 1. The decision tree for a given residue type A is initiated to a unique root node with an associated cluster c 0 = { i / a ( i ) = A } grouping all examples with native residue type A . 2. For each tree cluster c do : a. Test in turn each descriptor s j and each associated threshold S that creates possible dichotomies of c into two subclusters c 1 and c 2 . If s j has continuous values, 9 possible thresholds S are chosen to create dichotomies c 1 = { i ∈ c / s j ( i )≤ S } and c 2 = { i ∈ c / s j ( i )> S } corresponding to the 10 th , 20 th , ..., and 90 th percentiles of the statistical distribution of the considered descriptor. If s j is restricted to a few discrete categories, all possible dichotomies c 1 = { i ∈ c / s j ( i )== S } and c 2 = { i ∈ c / s j ( i )! = S } are created, where S is one of each possible value of s j . b. Select the optimal dichotomy from previous step which satisfies the tree constraints (see section (i) below) and minimizes the chosen splitting criterion (see section (ii) below). c. Insert the new clusters c 1 and c 2 as nodes in the tree by linking them to cluster c with respective edges labeled { s j ( i )≤ S } and { s j ( i )> S } or { s j ( i ) == S } and { s j ( i )! = S }. The structural environment associated to a particular cluster will be defined by all edge labels from the tree root to the considered tree node or leaf (see figure 2 ). 3. Finally, prune the tree according to the selected pruning method and pruning data set (see section (iii) below). It should be noted the choice of the optimal descriptor at a given tree level will depend on both the amino acid identity of the native residue and each structural descriptor previously chosen as splitting criteria along the tree path that leads to the considered node. Main parameters in the classification are (i) the tree constraints, (ii) the splitting criterion, and (iii) the tree pruning method. (i) Tree constraints - Tree depth: as the learning process goes deeper in the tree, more and more specific clusters are created. Beyond a certain depth, the chance that the corresponding rules can be applied to new examples outside the learning set drops significantly, resulting in an overfitting of the available data since deep clusters won't have enough associated examples to derive statistically significant distributions. Therefore, to avoid wasting time to partition the data into smaller and smaller clusters, maximum tree depths of 2 to 6 were tested. - Cluster cardinal: For the same reason as above, a minimum cardinal of examples was required for each cluster. We tested values between 200 and 1200 with increments of 200. - Tree balancing: A restriction on uneven distributions of samples among two clusters from the same parent was applied to prevent the creation of unbalanced trees which would require higher depth to fully partition the data. This restriction is achieved by the parameter sim cc measuring the cluster cardinal similarity between two subclusters obtained by splitting : where, n 1 is the cardinal of the subcluster 1 and n 2 the cardinal of the subcluster 2. (ii) Three different splitting criteria were tested - The Gini criterion evaluates the probability that two randomly selected elements in a cluster correspond to two different types of residues [ 28 ]: where P ( a | c ) is the relative frequency of residue type a in cluster c . To evaluate the quality of a given segmentation into several clusters, the splitting criterion is given by where n c is the number of elements in the cluster c and n is the total number of elements in all clusters. - The Shannon entropy [ 29 ] tries to limit the distribution of elements of the same class among several clusters. where P ( a | c ) is the relative frequency of residue type a in cluster c . - We also used a specifically developed splitting criterion called the "mean rank" MR . Each class (residue type) in a cluster is ranked according to the number of elements of this class in the cluster (rank 1 is assigned to the most frequent residue type and rank 21 to the least frequent one). The mean rank MR evaluates the mean rank for a randomly selected element in the cluster. Low MR indicates clusters with only few well represented classes. Such clusters would correspond to structural environments that induce significant bias in the sequence and therefore strong structural constraints. where R ( a | c ) and P ( a | c ) are the frequency rank and probability of the residue type a in the cluster c . (iii) Three different pruning methods were considered - The Pessimistic Error Pruning (PEP) [ 30 ] consists in recursively checking each cluster starting from the tree root and in cutting its corresponding subtree if this removal reduces the mean error rate estimated on the independent pruning test set by : where N ( c ) is the number of examples assigned to the cluster c and n ( c ) is the number of occurrences of the most frequent amino acid in the cluster c . Let C be the father cluster from which c is derived in the tree, then c and its subtree will be removed if E( c )≥E( C ). - The Mean Rank Pruning (MRP) has a principle similar to PEP, except that c will be removed if R ( c )< R ( C ) where R ( c ) and R ( C ) are respectively the mean ranks of the current cluster c and of its father cluster C averaged over the pruning test set. - The pessimistic Mean Rank Pruning (PRP) is a more stringent version of MRP using a confidence margin to prevent statistically biased clusters to be kept in the tree. The current cluster c will now be removed if R ( c )+ σ t 80 < R ( C ), where σ is the mean rank standard deviation over the pruning test set and the scaling factor t 80 = 1.82 corresponds to a 80% confidence level for a Gaussian distribution. Few other parameters were further optimized including - A mutation weight α = 1/ N f inversely proportional to the total number of residues N f in each protein family f of the learning data set. This insures that all structural families have similar importance in the derivation of the substitution probabilities. - A mutation weight β = 25/ ide inversely proportional to the percentage of identity ide between the two considered proteins. If ide <25%, then the mutation weight is decreased to 1. This reduces the importance of substitutions observed in similar sequences and could be used later to specialize EvDTree on different kinds of applications involving different sequence similarities. Residue specific environment-dependent substitution profiles Once trees have been constructed, statistical distributions of observed substitutions in each cluster are used to compute cluster-specific environment-dependent substitution profiles. As explained previously, the structural environment associated to a particular cluster will be defined by all edge labels from the tree root to the considered tree node or leaf. The probability for the amino acid a in the 3D environment s to be substituted by amino acid b is where is the number of observed substitutions of amino acid a by amino acid x in the 3D environment s . Smoothed probabilities Q ( b | a , s ) are then calculated as where A ( b | a, s ) is the a priori distribution of Topham et al. [ 19 ]. Relative weights are calculated as where is the total number of occurrences of amino acid a in 3D environment s , n is the number of classes (21 in this case), and σ is a normalization constant. We used the value of 5 previously used by Topham et al. [ 19 ]. It should be noted that the weight of this "a priori" distribution is inversely proportional to the number of available examples and is therefore maximum for undersampled substitutions. Then the log odds scores are calculated as where P ( b ) is the background probability of occurrence of amino acid b in the whole database. These log-odds are calculated for each node cluster of each native amino acid tree. Application and evaluation of environment-dependent and standard substitution scoring functions To evaluate the EvDTree scoring function, each example of associated native residue, structural environment, substituted residue ( a ( k ), s ( k ), b ( k )) from the test data set is classified by the tree corresponding to residue type a ( k ). Then the tree leaf corresponding to the structural environment s ( k ) is searched and its associated log-odds substitution scores are finally used to score the substituted residue b ( k ). To compare the EvDTree substitution scores with other scoring methods, we have used the mean rank ( MR ) as the criterion to evaluate the quality of scoring functions. For each example in the test data set, the 20 possible substitutions are scored as indicated above, and the observed (real) substitution b ( k ) is ranked according to its score among all other possible substitutions. The mean rank over all examples in the test data set is indicative of how well the scoring function is able to recognize as probable the "real" substitutions. A MR of 1 would mean that the scoring function always gave the better score to the observed substitution. At the opposite, a MR of 10.5 would indicate that observed substitutions are scored randomly. The main advantage of the mean rank criterion is that it is fast to calculate and it is independent from the absolute values of the scores, therefore allowing comparisons between very different scoring functions. Similar criteria based on ranking were previously used to evaluate 1D-3D scoring functions [ 31 ]. The evaluation of environment-dependent substitution matrices requires computing the 3D environments the very same way they were computed when deriving the scores. Thanks to Dr K. Mizuguchi who provided us with all the necessary tools, we could include the FUGUE environment-dependent substitution matrices into our evaluation process. To complement the MR evaluation, we also compared the performance of EvDTree with other scoring functions in sequence-structure alignments. To do this, 1000 sequence-structure alignments were selected from our test data set derived from the HOMSTRAD database. Each alignment was recalculated using a Smith and Watermann algorithm with several different substitution scoring functions. For each scoring function, the percentage of correctly aligned positions, according to the real alignments in the test data set, was compiled and used for comparisons. For each method, the gap opening ( Go ) and gap extension ( Ge ) penalties were optimized by comparing the alignments for several penalty combinations ( Go = 2, 5, 10, 15, 20; Ge = 2, 5, 10, 15, 20). Results and discussion Decision tree classifications Several learning data sets were compiled by filtering out observed protein substitutions (Figure 1 ) with sequence identity between superimposed proteins above thresholds of 40%, 60% or 80%. For each learning set, several combinations of parameters were tested for the construction of the EvDTree classifications and the calculation of the resulting structure-dependent substitution scores (Figures 1 and 2 ). For each run, a set of 21 decision trees was built and the corresponding scoring function was evaluated on the test data set using the mean rank ( MR ) criterion as explained in Methods. Due to the amount of data, CPU time limitations did not allow systematic examination of all parameter combinations, and the best parameters were determined through a limited trial and error protocol. The variation of the mean rank over sixteen different runs remained limited (6.74 < MR < 6.89) showing that the method is robust and is not critically affected by slight modifications of the parameters. The lowest MR (6.74) was obtained with the following protocol: - Minimum cardinal of any cluster = 600; - The minimal cluster cardinal similarity between two subclusters obtained by splitting of the parent cluster is sim cc = 0.1. - Segmentation criterion: mean rank - The pruning method is MRP. - Examples in the learning set are weighted according to the number of residues in the protein family: α = 1/ N f - No weighting is done in relation to the sequence identity (β = 25/ ide ) - Maximal sequence identity between superimposed proteins in the learning set = 60% The values of the obtained mean ranks could, at first sight, appear rather high. However, it is worth noting that only substitutions were ranked in the evaluations, i.e. protein positions occupied by the same amino acid in the two structurally superimposed proteins were not considered in the evaluation process. They are included, however, in the statistics during the calculations of the substitution scores. The evolution of the MR criterion along the learning process for alanine is shown in Figure 3 . As expected, the MR decreases regularly when calculated on the learning set. On the other hand, when calculated on the test data set, the MR decreases in the first few learning steps, then increases in the following steps. The difference between the two curves in the last steps is due to overfitting, i.e. learning specific rules from the learning set that cannot be generalized, thus reducing the predictive power on the test data set. Pruning the tree using an independent pruning data set removes clusters with reduced predictive power resulting in a flat curve in the last steps. Analysis of the EvDTree classifications For each residue type, the maximal tree depth used was 6, leading to a maximal number of leaves 21 × 64 = 1344, each of them potentially leading to a substitution profile that corresponds to one line in a classical substitution matrix. In other words, the total amount of data corresponds to 64 distinct substitution matrices, although it is not possible to associate substitution profiles to matrices since each profile corresponds to a different structural environment. The overall amount of data is nevertheless, in principle, comparable to the 64 environment-dependent substitution matrices used in the FUGUE system. However, the tree pruning step removed a significant number of clusters that do not afford improved information, leaving only 111 environments each associated to a specific substitution profile (data available from ). Structural descriptors, thresholds and values used in the first dichotomies of the root clusters for each residue type are displayed in Table 1 . Our main interest in using a decision tree classification is that, in principle, optimal splitting parameters and associated thresholds or values are automatically selected for each residue. As an example, it has been suggested that different boundaries on the fraction of area buried should be used for different residue classes when determining if a residue is exposed or buried [ 17 ]. Several examples of this can indeed be found in the EvDTree classifications: the selected structural descriptor for splitting the root cluster of serine and alanine substitutions is the percent of accessible area (pac), but the threshold is 3% for serine, whereas it is 10% for alanine (Table 1 ). The pac is also used as structural descriptor in the second dichotomy in the glutamic acid tree classification, with a threshold of 25%. The use of different accessibility thresholds by the decision tree algorithm fully supports previous observations by Rice and Eisenberg [ 17 ]. This observation also highlights the nice feature of decision trees that can be easily interpreted. Analysis of the most discriminating structural descriptor selected in the learning process, i.e. the descriptor selected for the first dichotomy of the root cluster for each residue type (denoted c 0 in Figure 2 ), shows that the secondary structure is the most discriminating parameter for aspartic and glutamic acids, lysine, arginine and asparagine (Table 1 ). Although contact polarity or solvent accessibility have been selected in subsequent dichotomies in most cases, it is clear that the substitution profile of charged residues primarily depends on the local structure. This result appears to be consistent with previous work by Gilis & Rooman [ 32 ] on the relative importance of local and non-local interactions in mutant stabilities. These authors showed that for solvent-exposed residues, the local structure is the most important factor, whereas distance potentials (i.e. 3D interactions) appear more suited to prediction of mutations in the protein core [ 32 ]. Here we show that substitution profiles for charged residues (which are largely solvent-exposed) mainly depend on the local structure. Another observation leads to a similar conclusion: for alanine and serine, the first selected structural parameter for splitting is the percent of accessibility (pac) and the most exposed resulting cluster is then split using secondary structure, whereas the most buried resulting cluster is split using the polarity of the protein environment (pol). These results confirm that, for solvent-exposed protein positions, the local structure is one main parameter that determines which amino acid can occupy this position. Four substitution profiles (i.e. log-odds substitution scores for one residue type into one structural environment) are displayed in Figure 4 . Comparison of the substitution profiles for alanine and aspartic acid in similar environments (exposed α-helix) reveals significant differences (Figure 4A ). This observation is not trivial since it could be postulated that, except for functional residues, the probability that a residue b occurs in a structural environment s only depends on s but is independent of the observed residue a in structurally similar proteins. The fact that, for similar structural environment, substitution profiles vary with the native residue probably indicates that purely structural descriptions probably lack some essential information, possibly related to the evolution process. This observation also illustrates the limits of environment-dependent statistical potentials in which the native amino acid is not taken into account. As an example, using data in Figure 4A , substitutions to Met in exposed α-helices appear more likely than substitutions to Leu when the native residue is Ala but the reverse is true when the native residue is Asp. Such differences cannot appear in environment-dependent statistical potentials such as 3D-1D scores that only describe the relative preference of residues for particular structural environments [ 1 ]. On the other hand, the substitution profiles for leucines in different structural contexts also display significant differences (Figure 4B ). Thus, substitutions Leu → Met are favored in exposed α-helical positions whereas substitutions Leu → Thr are favored in exposed non α-helical positions (Figure 4B ). This observation is not unexpected since it is well-known that β-substituted residues do not like to be in α-helices. Nevertheless it shows that EvDTree was able to extract consistent knowledge on sequence-structure relationships and it confirms previous observations that substitution scores are indeed structure-dependent [ 18 , 33 ], explaining why structure-dependent substitution matrices perform better than standard evolutionary matrices in fold recognition processes [ 1 , 15 , 17 , 34 ]. Structural information improves prediction of substitution probabilities The detailed impact of structural information for correct prediction of substitution probabilities can be approached by comparing the EvDTree substitution profiles with the evolutionary substitutions matrices GONNET [ 20 ] and BLOSUM62 [ 35 ]. To this goal, the "Mean Rank" criterion has been used (the lower the Mean Rank, the better the scoring function; see Methods and Data). Results by residue type and averaged over all residues are shown in Table 2 . Comparison of EvDTree with the Gonnet and BLOSUM62 matrices shows that EvDTree performs clearly better on average, and individually for most residues. Therefore, the use of the structural information in EvDTree does improve the predictive power of substitution profiles versus structure-independent substitution matrices. Moreover, a substitution matrix was built from the tree clusters of EvDTree, i.e. before any structural information is taken into account. This matrix, referred to as EvDTree0, is simply derived from the structural superimpositions in the learning data set and is thus similar to other structure-derived substitution matrices [ 36 ]. Interestingly, EvDTree0 performs better than evolutionary matrices suggesting that, despite a lower amount of data, structure-derived alignments can provide data of higher quality than sequence alignments for derivation of substitution matrices. The results in Table 2 show that EvDTree provides poorer evaluation than the evolutionary matrices for two residues, histidine and lysine, possibly due to an insufficient amount of data. It is also worth noting that histidine often participates in active sites or coordination sites, and the substitution probabilities may have been biased by this peculiarity. Filtering out the learning data set for coordination sites was performed by Shi et al [ 15 ], but such a filter was not implemented here. More surprisingly, five residues (Gly, Met, Pro, Gln, and Thr) and the free cysteine (J) do not display evaluation improvement by using structural information (compare EvDTree0 and EvDTree). The latter remark means that for these five residues no structural descriptor permitted efficient splitting of the data. It is likely that for these residues new descriptors or descriptor combinations remain to be discovered. Nevertheless, on average, the structural information significantly improves the performance and EvDTree appears as a clearly better scoring function than evolutionary matrices in evaluation of sequence-structure alignments. The EvDTree substitution profiles provide slightly better substitutions predictions Comparison with structure-dependent substitution matrices obtained by other groups is not as simple as for standard substitution matrices, because we must make sure that we compute the structural environment exactly the same way that was used for generating the matrices. We were able to compare EvDTree with FUGUE, thanks to the FUGUE accessory programs kindly provided by Dr K. Mizuguchi. As shown in Table 2 , the overall performance of EvDTree and FUGUE appear very similar. A comparison between these two scoring functions at different levels of sequence identity is displayed in Figure 5 . EvDTree provides slightly better performances at sequence identity above 30% but similar results below 30% (Figure 5 ). The reason for this remains unclear, but might be a result of our filtering of the learning set that removed all positions were Cα deviates from more than 3.5 A. Further optimization of the EvDTree learning set would probably be necessary for use in fold recognition programs at low sequence identity. The observation that EvDTree performs at least as well as the scoring function of FUGUE but is computed in a fully automated manner opens the way for future potential applications. For example, we show below that EvDTree can easily optimize fold-specific scoring matrices specific of small disulfide rich proteins leading to improved substitution scores for this particular class of proteins. Evaluation of EvDTree as scoring function in sequence-structure alignment Although the Mean Rank test determines the ability of a scoring function to correctly evaluate structure-compatible sequences, it does not determine how well a scoring function would actually perform in a particular application, e.g. fold recognition. In this paper, we do not focus on a particular application, but rather on the method to automatically derivate structure-dependent substitution profiles, and the Mean Rank appears as a simple and efficient criterion to rapidly compare different learning parameterizations or scoring functions. To verify that the theoretical evaluations using the Mean Rank have some significance for future real applications, we compared EvDTree with other methods used as scoring functions in sequence-structure alignments. The percent of correctly aligned positions for different methods and for different sequence identity ranges is displayed in Figure 6 . For each method, the best Gap opening and Gap extension penalties were roughly optimized by checking 25 combinations ( Go = 2, 5, 10, 15, 20; Ge = 2, 5, 10, 15, 20). The results shown in Figure 6 fully confirm previous analyses using the Mean Rank criterion: on average, better alignments are obtained using the structure-dependent scoring functions FUGUE and EvDTree, and EvDTree provides slightly better alignments than FUGUE above 30% of sequence identity but similar results at lower sequence identity. The slightly better accuracy of EvDTree for mid-range percentages of identities suggests that our approach could be particularly useful to improve sequence/structure alignments in homology modeling. The EvDTree learning algorithm could also be applied to fold recognition by optimizing scoring functions specific of particular protein families. Application of EvDTree to a specific class of proteins, the small disulfide-rich proteins The strongest potential of EvDTree is its ability to adapt itself to any particular set of structures. As a test case, we have applied EvDTree to small disulfide-rich proteins. Small disulfide-rich proteins display several peculiar structural features: (i) due to their small size, a larger number of residues than usual are solvent-exposed, (ii) regular secondary structures are limited and the content in turns and loops is high, (iii) the hydrophobic core is largely constituted by the disulfide bridges that are responsible for the high stability despite the small size, and (iv) glycine, proline, and, of course, cysteine residues are more frequent than usual. With all these peculiarities, small disulfide-rich proteins are not well-suited to standard prediction methods and it has been shown that these proteins score poorly using the standard PROCHECK database [ 37 ]. Evaluations of non-specific substitution scoring functions on small disulfide-rich proteins using the mean rank criterion are reported in Table 3 . Comparison of values in Table 3 for EvDTree, Gonnet, BLOSUM62 and FUGUE with those in Table 2 clearly support the idea that small disulfide-rich score poorly when using standard databases and methods. Thus, to test the ability of EvDTree to automatically adapt itself to a class of proteins with specific structural features, we have computed disulfide-rich specific substitution profiles (EvDTreeDS). For this, we have compiled a specific data set from the structural class "small disulfide" in the HOMSTRAD database. These data were complemented by data extracted from the KNOTTIN database [ 38 ]. The number of structural positions in the initial data set (about 40000) is clearly insufficient to divide this set into independent learning and test datasets. Therefore, a "leave-one-out" protocol was used. In this protocol, one protein family is excluded from the learning set and the resulting substitution scores are used to evaluate this family. This process is repeated for all protein families in the initial set. Also, due to the limited number of data, the tree pruning had to be performed using the learning data set, which is of course far less efficient than using an independent data set (Esposito, 1997), and resulted in a very limited pruning when compared to the general case. Mean rank evaluations of the new, specific, EvDTreeDS substitution scores on small disulfide-rich proteins are reported in Table 3 . Despite the limited set of data, comparison of the efficiency of the EvDTreeDS specific substitution profiles with standard EvDTree clearly shows that the automated classification method was able to extract, at least in part, the specific features of the small disulfide-rich proteins. Furthermore, the comparison of the structural descriptors used in the first partition of root clusters in EvDTree and EvDTreeDS classifications highlights interesting differences (Table 1 ). First, when the solvent accessibility percentage is the first used descriptor in both trees (half-cystines and alanines), the thresholds retained by the learning algorithm are different for the disulfide-rich proteins which are small and whose residues, except half-cystines, are more exposed to solvent on average. Accordingly, the selected threshold is lower for half-cystines but higher for alanines. Table 1 also reveals that most large hydrophobic residues (Leu, Phe, Tyr, His) switch their most discriminating descriptor from hydrophobic-hydrophilic measures in EvDTree (Pol, C) to secondary structure descriptors in EvDTreeDS (ss1 and ss2). This inversion can be interpreted by the reduction of the buried volume in the small disulfide-rich structures which cannot accommodate for large residues. This suggests that, in these proteins the stability is mainly due to disulfide bridges whereas the hydrophobic effect would be less crucial. Being more solvent exposed and less involved in hydrophobic packing, large hydrophobic residues might become more sensitive to local structure in small disulfide-rich proteins. More surprisingly, we also notice that charged and polar residues (Asn, Asp, Glu, Lys, Arg) display the opposite switch, i.e. a secondary structure descriptor is used in EvDTree but a solvent accessibility descriptor is used in EvDTreeDS. Analysis of the EvDTreeDS classification suggests that these residues, when in more buried positions, are far more conserved than when in more exposed positions. We think that beside the disulfide bridge core, additional elements of stability often occur through specific hydrogen bonding networks between charged or polar residues and the backbone, rather than through hydrophobic packing. Typical examples of this are the conserved glutamic acid in position 3 of cyclotides [ 39 ], or the conserved aspartic acid in position 15 of squash inhibitors [ 40 ] which both participate in multiple hydrogen bonding with the backbone. This might explain, at least in part, why the distinction between exposed and partially buried charged residues is more critical in small disulfide-bridged proteins. All these subtle modifications revealed by the EvDTree and EvDTreeDS classifications suggest that there is probably no universally optimal description language and that the choices and partitions of structural descriptors should be adapted to the class of proteins considered. Our new decision tree learning algorithm makes this fine tuning automatically from scratch whereas classical potentials are based on globally optimized description languages which may become suboptimal in specific contexts. Conclusions We have described a new method, EvDTree, based on decision tree classification of structural environments to automatically construct structure-dependent substitution profiles from a set of sequence-structure alignments. The EvDTree method was shown to perform similarly to the successful environment-dependent substitution matrices used in FUGUE (Shi et al. , 2001). Interestingly, the tree-pruning step removed a significant number of structural clusters yielding an average tree depth of 4 instead of the six allowed levels. This is an indication that clusters at higher levels corresponded to the learning of specific sequence-structure relationships that could not be generalized (Figure 3 ). It may be expected that as more high quality data will become available, this effect could be reduced and higher levels of the decision tree will gain better performances. In this work, we were interested in the development of a fully automatic method for the classification of structural environments and inference of structure-dependent substitution profiles. The evaluation of the intrinsic performance of the substitution profiles was primarily done on known sequence-structure alignments, using the mean rank of observed substitutions. We have shown that in this context, the EvDTree substitution profiles perform slightly better than other successful substitution matrices, and as such, the EvDTree matrices constitutes interesting elementary data for various applications. Moreover, comparison of the EvDTree substitution scores with other scoring functions for sequence-structure alignments led to results similar to the mean rank evaluation supporting the usefulness of the latter criterion. One specific strength of the EvDTree method is its easy automatic adaptation to any specific data set. Here we have shown that it is possible to obtain structure-dependent substitution profiles specific of small disulfide-rich proteins with better predictive power than standard substitution scores. This approach could be easily extended to other specific protein classes such as coil-coils, membrane proteins, etc. as soon as enough structures are available for learning. Fold-specific substitution matrices have recently been proposed for protein classification [ 41 ]. The EvDTree approach opens the way for class-specific or fold-specific structure-dependent substitution scores for use in threading-based remote homology searches. Decision trees based on different learning sets and with different depths could be optimized depending on the available protein structures and sequences of the fold family considered. The fact that, as stated above, the structural information did not yield better prediction for several residues in the EvDTree approach (Table 2 ) suggests that improvements are still possible. To this end, 3D environments from the decision trees yielding poor performances should be determined in order to design more appropriate structural descriptors. It is tempting to speculate that using combined structural descriptors, e.g. (Phi, Psi) angle pairs which can delineate particular regions of the Ramachandran plot or (dCi,i+j, dCi,i+k) distance pairs which can introduce some super-secondary structural constraints, could increase the accuracy of the decision trees. Alternatively, the use of linear combinations of descriptors in decision tree induction algorithms have been reported and could be used for structural classifications [42]. Authors' contributions After an initial program from JG, JCG coded a new version of the software to incorporate a novel algorithm. All methods were implemented and tested by JCG. The whole work was conceived by JCG, JG and LC and was supervised by LC and JG. All authors read and approved the final manuscript.

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524355

Metabolic aspects of low carbohydrate diets and exercise

Following a low carbohydrate diet, there is a shift towards more fat and less carbohydrate oxidation to provide energy to skeletal muscle, both at rest and during exercise. This review summarizes recent work on human skeletal muscle carbohydrate and fat metabolic adaptations to a low carbohydrate diet, focusing mainly on pyruvate dehydrogenase and pyruvate dehydrogenase kinase, and how these changes relate to the capacity for carbohydrate oxidation during exercise.

Review Exercise, an acute bout of muscular activity, requires an expenditure of energy above resting levels. This required mechanical energy is provided through the conversion of metabolic fuels into ATP, the base currency of chemical energy. Once produced, ATP is the only direct form of energy that is transferred and utilized by the contractile apparatus within the muscle. Fats are the predominant fuel source of resting skeletal muscle and during exercise, there is a complex interaction between skeletal muscle fat and carbohydrate (CHO) metabolism (see [ 1 ] for review). When evaluating the effects of exercise on skeletal muscle fuel utilization, there are many facets that must be taken into consideration. These include intensity and duration of the bout of exercise and the training status of the subjects. During low intensity physical activity (25% maximal oxygen uptake (VO 2max )), fat supplies the majority of metabolic fuel to exercising skeletal muscle [ 2 ]. As physical activity increases to moderate levels (65–70% VO 2max ), there is a shift to more reliance on CHO, specifically muscle glycogen [ 2 ]. However, at this level of physical activity, fat oxidation becomes increasingly important as the duration of exercise increases [ 2 ] or as training status improves [ 3 ]. The studies presented in this review utilize moderately active subjects (maximal oxygen uptake, 50–60 ml·kg -1 ·min -1 ) exercising at a workload of 65–75% VO 2max for 30–48 min. The sources of chemical energy that fuel exercising skeletal muscle are available through endogenous depots (intramuscular glycogen and triglycerides) or exogenous sources (plasma glucose and free fatty acids). In turn, these exogenous and endogenous fuel sources are replenished through dietary intake. As a result, there is an important relationship between diet and fuel metabolism in skeletal muscle. Diets low in carbohydrate content have become increasingly popular as a method of weight loss. These diets that limit daily dietary carbohydrate intake are termed low-carbohydrate diets (LCD). When evaluating the effects of LCD, there are a couple of factors that must be considered, as they may influence the measured outcome. These include the composition of the diet (since a LCD may replace the missing CHOs with either protein or fat), and the duration of the dietary period. For the purpose of this review, LCD will refer primarily to high-fat low-carbohydrate isocaloric diets with <50 g of CHO per day, with a composition of 3–8% CHO, 22–46% protein, and 51–75% fat, and consumed for 3–6 days. The present paper will briefly review human skeletal muscle metabolism during exercise and the importance of dietary CHO for metabolic energy production. It has been well documented that diets low in carbohydrates result in several metabolic and hormonal adaptations that improve fat oxidation and promote glycogen sparing in exercising skeletal muscle (see [ 4 ] for review). However, the mechanism(s) responsible for these changes in exercising skeletal muscle are still debatable, but could be the result of up-regulated fat and/or down-regulated carbohydrate metabolism. The emphasis of the present paper is on adaptive skeletal muscle CHO and fat metabolism in humans, and will compare recent studies that examine the effects of altered diets on key enzymes and how fatty acid composition and re-feeding of carbohydrates following these altered diets affect these enzymes. Data from other mammals are cited where necessary. Regulation of carbohydrate oxidation by low-carbohydrate diet Role of pyruvate dehydrogenase In order to understand the regulation of carbohydrate oxidation, the regulation of the mitochondrial enzyme pyruvate dehydrogenase (PDH) must be carefully considered. PDH is a multi-enzyme complex which catalyzes the irreversible oxidative decarboxylation of glycolytically-derived pyruvate to acetyl-coenzyme A (acetyl-CoA; Fig. 1 ) Because it is highly regulated, it plays a pivotal role in determining the proportion of acetyl-CoA which is derived from carbohydrate sources, thereby regulating flux through carbohydrate oxidation and indirectly determining the rate of fat oxidation. The amount of PDH in its active form (PDHa) determines its activity and regulation is achieved through reversible phosphorylation, catalyzed by an intrinsic PDH phosphatase (PDP), which dephosphorylates and activates PDH, and PDH kinase (PDK), which phosphorylates and inhibits PDH [ 5 ]. The E1 subunit of PDH has three known phosphorylation sites, with the first site being necessary for inactivation of the complex, and the other two sites acting as barrier sites to hinder phosphatase activation [ 6 ]. Figure 1 Activation of pyruvate dehydrogenase enzyme complex control by a phosphorylation and dephosphorylation cycle. Each of the covalent regulatory enzymes of PDH is subject to allosteric regulation. Phosphorylation of the complex is catalyzed by a family of four PDK isoforms (PDK1-4) which differ in their responsiveness to allosteric inhibition by pyruvate or activation by energy charge (ATP/ADP ratio), redox (NADH/NAD + ratio), and acetyl-CoA-to-free CoA ratio (see [ 7 ] for review). In addition, the kinases differ in their specificity for the different phosphorylation sites [ 7 ]. Thus, the relative activities of the PDK isoform population will determine the response of the PDH complex in acute situations. An intrinsic pair of phosphatases (PDP1 and 2) catalyze the dephosphorylation and activation of PDH [ 8 ]. PDP1 is the isoform which is activated in the presence of increasing concentrations of Ca 2+ ions (as would be expected during exercise), while PDP2 is activated when insulin levels are increased during dietary manipulations [ 8 ]. At rest, PDH is mainly phosphorylated and inactive due to high energy charge, redox, and acetyl-CoA-to-free CoA ratio and low pyruvate concentration, which maintain a high PDK activity. Phosphatase activity is low at rest, due to low intramuscular Ca 2+ levels. During exercise, Ca 2+ release from the sarcoplasmic reticulum is the primary stimulus that coarsely activates PDH whereas changes to pyruvate concentration, energy charge, and possibly redox fine-tune this activation (see [ 9 ] for review), in order to match PDH activation to the demand for CHO oxidation [ 10 ]. In addition to the importance of intramitochondrial effectors to the acute regulation of PDH activation in the first few seconds or minutes, long-term or chronic alterations to the activation state of PDH can be accomplished through stable changes in the absolute levels of PDK and/or PDP. The rate of activation of PDH is dependent on the activity ratio of PDK and PDP, and changes in the expression of either covalent modifier would alter the rate of activation or inactivation of PDH. These chronic alterations occur over hours or days and are independent of acute changes in intramitochondrial effector concentrations. Effects of low-carbohydrate diet In 1993, Putman and co-workers undertook a study to examine the effects of a short term low-carbohydrate diet on activation of skeletal muscle PDHa activity during moderately intense exercise (75% VO 2max ) [ 11 ]. In this study, a 6 d LCD was compared to a high-carbohydrate diet, shifting reliance from the two extremes, either towards fat or towards carbohydrate oxidation. Subjects completed muscle glycogen depleting exercise and then consumed either a LCD (< 3% energy from carbohydrate) or a high-carbohydrate diet (86% carbohydrate) for 6 d. At the end of the dietary intervention, subjects exercised at 75% VO 2max . The subjects exhausted in ~47 min following the LCD, and exhaustion coincided with hypoglycemia (~2.4 mM) and low levels of muscle glycogen (~32 mmol glucosyl units/kg dry muscle), indicating that that skeletal muscle and liver glycogen stores were limiting under these conditions and at this intensity of exercise. Following the high-carbohydrate diet, exercise was terminated at the same time as the LCD trial, and their blood glucose concentrations were maintained at ~5 mM throughout the exercise duration. Skeletal muscle glycogen content decreased during exercise but was still ~250 mmol glucosyl units/kg dry muscle at the end of exercise. At the onset of exercise during the high-carbohydrate trial, PDHa activity increased maximally in the first 15 minutes of exercise, reflecting the increased energy demand for carbohydrate oxidation at this workload. However, following the LCD, PDHa activity was maximally activated in the first 15 minutes of exercise, but the activation did not achieve the same levels as during the high-carbohydrate trial, effectively impairing the capacity for carbohydrate oxidation and possibly promoting fat oxidation for the duration of exercise at this workload (Fig. 2 ). The authors were unable to adequately explain the difference in PDHa activity between the trials based on acute changes in the concentrations of intra-mitochondrial effectors, suggesting that chronic regulation of the complex could be playing a role. Figure 2 Skeletal muscle pyruvate dehydrogenase in its active form (PDHa) at rest and during exercise in low carbohydrate (LCD) and high carbohydrate (HCD) diets. * denotes significance from LCD. Adapted from Putman et al. [11]. Subsequent studies demonstrated adaptive alterations at the level of PDK with resultant changes in PDH activation. PDK activity was adaptively increased in human skeletal muscle following 6 days of a LCD [ 12 ] (Fig. 3 ). PDK activity increased in as little as 24 hr and continued to increase in a linear fashion throughout the 6 d diet [ 13 ]. The increased PDK activity in human skeletal muscle was associated with increased PDK4 mRNA and protein expression, which was maximally increased after 24 h [ 13 ]. These studies suggest a selective increase in PDK4 expression with LCD. The increase in PDK activity during the LCD was associated with impaired glucose clearance from the blood in response to an oral glucose load in health young men [ 14 ]. Following as little as 56 h on the LCD, the 90 min area under the blood glucose and plasma insulin concentration vs. time curves increased 2-fold and 1.25-fold, respectively, during an oral glucose tolerance test [ 14 ]. Figure 3 Pyruvate dehydrogenase kinase (PDK) activity during six days of a LCD. a Significantly different from day 0. b Significantly different from day 1. Adapted from Peters et al. [12,13]. These increased levels of PDK4 protein and PDK activity would be expected to render the complex resistant to activation during exercise, as observed by Putman et al. [ 11 ] for two reasons: 1) increased multi-site phosphorylation of the PDH complex, and/or 2) decreased sensitivity of the complex to regulation by pyruvate. Increased PDK activity would be expected to enhance multi-site phosphorylation of the E1 subunit and make the complex more resistant to dephosphorylation and activation by the phosphatase [ 6 ]. Under normal dietary conditions the predominant isoform in human skeletal muscle is PDK2, which has a greater affinity for phosphorylation of site 1 (the inactivating site) of the E1 subunit [ 15 - 17 ]. However, as the population of PDK4 isoform increased, there would be enhanced phosphorylation of the 2 nd (barrier) site, since this isoform has a greater affinity for both site 1 and site 2 [ 15 - 17 ]. As well, PDK2 has a greater sensitivity to inactivation by pyruvate than PDK4 [ 18 ]. Thus at the onset of exercise with increased glycolytic flux, the increased levels of PDK4 protein would render the complex more resistant to activation due to increased PDK4 kinase activity even in the face of elevated muscle pyruvate concentrations [ 19 ]. A confounding factor in the Putman study was that subjects had undergone intense glycogen depleting exercise protocols prior to both dietary interventions, so the initial levels of skeletal muscle glycogen and glycogen utilization was considerably lower following the LCD [ 11 ]. In a subsequent study, subjects were asked to refrain from intense exercise throughout the study, and a LCD (~3% carbohydrate) was compared to a mixed diet (~55–60% carbohydrate) instead of a high-carbohydrate diet [ 20 ]. Subjects followed each 6 d dietary intervention with 30 min exercise at a slightly lower workload (65% VO 2max ). The object of the study was to match as closely as possible the glycogen utilization during exercise between the two trials. Although the initial skeletal muscle glycogen concentration was still ~50% lower in the LCD compared to the mixed diet, skeletal muscle glycogen utilization and pyruvate accumulation were similar during the 30 min of exercise in both trials. Unlike the attenuated activation of PDHa at the onset of exercise which was observed in the Putman study [ 11 ], these authors observed that the activation during exercise was identical between the two conditions. Thus, in spite of the fact that PDK activity and PDK4 isoform would be expected to increase to a similar extent as previous studies [ 13 ], these effects were overridden when initial muscle glycogen levels were higher and glycolytic flux to pyruvate was maintained [ 20 ]. It is clear from these studies that the intensity and duration of the exercise play a role in the regulatory changes observed during exercise following a LCD. As exercise intensity increases, the demand for muscle and liver glycogenolysis and muscle carbohydrate oxidation increases. These stores are not fully replenished following a very low carbohydrate diet, and therefore during intense exercise glycogenolytic flux and PDH activation are decreased following a LCD. Effect of fatty acid composition of low-carbohydrate diet The studies presented in this review demonstrate that LCDs decrease the activation of PDH in skeletal muscle at rest and during exercise, mediated through increased PDK activity and isoform expression. However, not all LCDs are created equally, and there is increasing interest in the composition of the fatty acids consumed. Recently, it has been demonstrated that substituting only ~12% of the fat in a LCD (~3% carbohydrate; 75% fat) with fish oils, which are high in omega-3 unsaturated fatty acids, attenuated the diet-induced increase in PDK activity in human skeletal muscle [ 21 ] (Fig. 4 ). These results are similar to earlier work in rodents, with the key difference being that the diet-induced increase in rat skeletal muscle PDK activity was completely abolished with the addition of fish oil [ 22 ]. In fact, the increase in PDK activity following a 28 d LCD diet could be completely reversed in as little as 24 h when fish oils were introduced into the high-fat diet [ 22 ]. Surprisingly, in both rat and human skeletal muscle in the resting and basal state, PDHa activity was not affected by the inclusion of fish oils suggesting that the total fat content of the diets was more important in determining the conversion of the complex in the basal state [ 22 ]. However, there is evidence from animal studies that a LCD which is rich in fish oils enhances muscle carbohydrate oxidation and glucose disposal in response to a challenge such as an insulinemic/euglycemic clamp. Jucker et al. [ 23 ] fed rats one of three experimental diets to study the effects on muscle metabolism: a LCD diet rich in safflower oil; a LCD rich in fish oil; or a high-carbohydrate (control) diet. They found that the safflower-fed rats were insulin resistant compared to control or the fish oil-fed rats. The increased whole body glucose disposal in the fish oil-fed rats correlated with increased insulin-stimulated muscle disposal of glucose through oxidation as determined with stable isotope tracer technology [ 23 ]. Thus, it would appear that the deleterious effects of high-fat feeding on carbohydrate oxidation and glucose disposal may be ameliorated when the dietary composition of fatty acids are considered carefully. The effect of altered fat composition on skeletal muscle metabolism during exercise has yet to be examined. Figure 4 Pyruvate dehydrogenase kinase (PDK) activity before and after three days of a LCD with and without n3 fatty acids. a Significantly different from pre diet. b Significantly different from post LCD diet. Adapted from Turvey et al. [21]. Effect of re-feeding of carbohydrate following low-carbohydrate diet In humans, there is little information on how rapidly the LCD-adapted increase in PDK activity and PDK4 protein may be reversed with carbohydrate re-feeding. Most re-feeding studies have used prolonged fasting as a perturbation, and very little work has been done in human skeletal muscle. In rodents, early studies in cardiac muscle indicated that re-feeding following 6 h starvation recovered PDHa activity to ~75% of normal levels in as little as 1–2 h. However, as the duration of the starvation period increased, the time course of the response to re-feeding was longer, in such that after 48 h of starvation, PDHa activity recovered to only ~25% of control values after 4 h [ 24 ]. In later rodent studies, this increasing resistance to PDH complex activation was accompanied by increased PDK activity, which correlated with the duration of the fast or high-fat diet [ 25 , 26 ]. Following 48 h starvation and re-feeding, PDK activity and PDK4 protein in skeletal muscle decreased ~50–60% in approximately 4 h of re-feeding [ 27 ]. However, little is known about the time course reversion of PDK activity and PDK isoform expression following a LCD in human skeletal muscle. In human skeletal muscle, Pilegaard et al. [ 28 ] recently examined changes in PDK4 mRNA concentrations in human skeletal muscle following fasting and re-feeding. Subjects fasted for 20 h and then were given either a high-carbohydrate meal or a high-fat meal. In muscle biopsies taken 1 h after the re-feeding meal, these authors found increased transcription rate and mRNA concentration of the PDK4 isoform regardless of the composition of the meal. Based on rodent studies of PDK activity, these data were unexpected, since it would be expected that the high-carbohydrate meal would suppress PDK4 gene expression. These data suggested that the skeletal muscle PDK 4 gene was very sensitive to metabolic disturbances. However, without measurement of PDK or PDH activity, the study gave little information regarding how quickly the fasting-induced increase in PDK activity was reversed with carbohydrate re-feeding. A recent study examining carbohydrate re-feeding following a 6 d LCD indicates that PDK activity is rapidly reversed and PDHa activity has fully recovered in as little as 3 h in resting human skeletal muscle [ 29 ]. Thus, the adaptive change in PDK activity observed in human skeletal muscle is rapidly reversed with re-feeding of carbohydrates, regardless of potential changes in PDK4 mRNA expression [ 28 ]. Regulation of fat oxidation by low-carbohydrate diet There is little information regarding the skeletal muscle adaptation on "the fat side" to a LCD. In human skeletal muscle, most studies restrict their measurements to gene expression or mRNA concentrations of the pertinent enzymes involved in fat oxidation, and very few have measured the more physiologically relevant concentrations of enzyme activity or protein concentration. Still, there is evidence in human skeletal muscle for increased activities of several regulatory enzymes and proteins in skeletal muscle fatty acid uptake and oxidation following high fat diets or LCD. Key steps include delivery of fatty acids to the muscle through muscle lipoprotein lipase (LPL), sarcolemmal fatty acid transporters and plasma membrane fatty acid binding proteins (FAT/CD26 and FABP pm respectively), mitochondrial uptake and oxidation through carnitine palmitoyl transferase I (CPT I), fatty acid beta-oxidation (marker enzyme β-hydroxy acyl CoA dehydrogenase (β-HAD)), and general oxidative capacity (marker enzyme citrate synthase (CS)). In response to a 4 week adaptation to a high fat (~62% fat) moderate LCD (~20% CHO), skeletal muscle LPL activity increased almost 2-fold, increasing fatty acid availability to the muscle and increasing intramuscular triglyceride content significantly [ 30 ]. In terms of muscle fatty acid uptake, there is evidence that the FAT/CD36 protein and mRNA were increased modestly (1.25-fold) after only 5 d on a moderate LCD (20% CHO), while FABP pm gene expression and protein content were unaffected by the diet [ 31 ]. In general, muscle uptake of fatty acids and very low density lipoprotein triglycerides, as well as plasma fatty acid oxidation were higher during exercise following a fat-rich LCD (21% CHO) when exercise training was combined with the diet perturbation [ 32 ]. In human studies, skeletal muscle CPT I is unaffected by LCD. This was demonstrated at the level of maximal enzyme activity following a 6 d LCD (~3% CHO) [ 12 ], and mRNA levels following a 5 d LCD (19% CHO) [ 31 ]. However, in skeletal muscle of rats fed a high-fat diet, CPT I enzyme activity capacity was increased up to 1.3 to 2-fold at 10 weeks, depending on the fatty acid composition of the diet [ 33 ]. In another rat study, increased gene expression of CPT I mRNA appears to be restricted to type I slow oxidative muscle fibers, since a significant increase was only documented in the soleus muscle, and not the extensor digitorum longus following 8 weeks of a high-fat LCD (0% CHO) [ 34 ]. Taken together, these studies suggest that the short term 5–6 d LCD perturbation may not be prolonged enough to evoke a significant change in activity or gene expression of this enzyme which regulates transport of fatty acids into the mitochondria for oxidation. This is further supported by the fact that in well trained human subjects, maximal CPT activity was modestly increased following a prolonged (4 week) very low LCD (<20 g CHO), although it is not clear from this data whether this measurements included CPT I and CPT II activity together [ 35 ]. Increased activity of a key marker enzyme for fatty acid beta-oxidation has been observed in human skeletal muscle during prolonged LCD perturbations as well. Although a 6 d LCD (3% CHO) did not alter β-HAD activity [ 12 ], Helge et al. [ 36 ] observed increased β-HAD activity following a 4 week LCD (20% CHO) perturbation in untrained subjects. However, they found no increase in either whole body VO 2max or CS activity, suggesting that the increase in beta-oxidation was specific rather than a generalized increase in oxidative capacity. Similarly, a more carbohydrate restricted diet (3% CHO) for 6 d did not alter CS activity in human skeletal muscle [ 12 ]. In contrast, results from some rat studies have demonstrated modest increases in CS activity of approximately 20% [ 37 - 39 ], with the largest increases demonstrated in type IIb fibers [ 40 ]. Although the increase in β-HAD activity in human skeletal muscle and possibly CS in rat muscle could potentially suggest an increase in oxidative capacity, recent research has demonstrated that there was no difference in human skeletal muscle mitochondrial density (as determined by electron microscopy), even though there was an increase in fat oxidation at rest and during incremental exercise following a 5 week high fat LCD (25–30% CHO) [ 41 ]. Conclusions In summary, following a 6 d LCD in human subjects, PDHa activation is attenuated during intense exercise and this is due at least in part to increased PDK activity and PDK4 gene expression. This decreased activation of PDHa decreases carbohydrate and increases fat oxidation during exercise. PDK activity increases in as little as 24 h on a LCD, and PDK activity increases linearly over the 6 d. Impaired glucose clearance in response to an oral glucose tolerance test was observed in healthy subjects following only 56 h of LCD, but this may be dependent on the fatty acid composition of the diet. With re-feeding of carbohydrates, PDK activity drops to pre-diet levels in 3 h, although this does not appear to correlate with mRNA concentration. If intense exercise is restricted and muscle glycogen stores and utilization rates are preserved during the LCD, the activation of the PDH complex is similar to that following a mixed diet. The up-regulation of enzymes involved in muscle fatty acid uptake and fat oxidation appears to be slower to response to a LCD perturbation. In addition, these adaptations appear to be of a smaller magnitude. In human studies there is evidence that muscle uptake of fatty acids is up-regulated by LCD through increased maximal activity of LPL and increased FAT/CD36. However, the maximal rate of mitochondrial transport of fatty acids through CPT I appears to be resistant to adaptive changes in response to the diet. In addition, although increased maximal β-HAD activity has been documented in response to LCD, there is no evidence that the overall oxidative capacity is elevated following a LCD in human skeletal muscle.

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538754

Gait unsteadiness and fall risk in two affective disorders: a preliminary study

Background In older adults, depression has been associated with increased fall risk, but the reasons for this link are not fully clear. Given parallels between major depression and Parkinson's disease, we hypothesized that major depression and related affective disorders would be associated with impairment in the ability to regulate the stride-to-stride fluctuations in gait cycle timing. Methods We measured stride-to-stride fluctuations of patients with two forms of mood disorders, unipolar major depressive disorder (MDD) and bipolar disorder, and compared their gait to that of a healthy control group. The primary outcomes were two measures of gait unsteadiness that have been associated with fall risk: stride time variability and swing time variability. Results Compared to the control group, the two patient groups tended to walk more slowly and with decreased swing time and increased stride time. However, none of these differences was statistically significant. Compared to the control group, swing time variability was significantly larger in the subjects with bipolar disorder (p < 0.0001) and in the subjects with MDD (p < 0.0004). Conclusions Patients with MDD and patients with bipolar disorder display gait unsteadiness. This perturbation in gait may provide a mechanistic link connecting depression and falls. The present findings also suggest the possibility that measurement of variability of gait may provide a readily quantifiable objective approach to monitoring depression and related affective disorders.

Background In older adults, depression has been associated with increased fall risk [ 1 , 2 ]. Epidemiological studies indicate that older adults who are more depressed have an increased risk of falls and fractures compared to their aged-matched peers and that this association persists even after adjusting for potential confounding effects such as medication usage [ 3 - 5 ]. The reasons for this link between depression and falls are not fully clear [ 3 ]. People with major depression have been shown to walk more slowly, with decreased push-off and more time spent with both feet on the ground [ 6 - 8 ]. These gait changes, however, do not necessarily explain the increased risk of falls associated with depression [ 9 , 10 ]. One possibility is that these two syndromes simply share common risk factors [ 11 ]. Alternatively, there may be some cause and effect influences mediating the association between depression and falls. For example, depression may not only lead to a slowed gait, but it could induce gait unsteadiness and a reduction in the ability to maintain a stable walking pattern. Cross-sectional studies suggest that individuals who report more signs of depression tend to be less steady on their feet [ 12 , 13 ]. To date, however, no studies have directly examined the dynamics of gait unsteadiness of patients with major depression or related affective disorders. Parallels between Parkinson's disease and depression also support the possibility of gait unsteadiness in the latter. Psychomotor studies have revealed deficits in the motor performance of persons with major depression similar to some of the motor disturbances observed in Parkinson's disease [ 14 , 15 ]. In both groups of patients, slowed motor performance can be seen in speech, simple reaction time tests, and gait [ 14 - 17 ]. Furthermore, in both groups, imaging studies reveal shrunken basal ganglia, the motor area primarily affected in Parkinson's disease, and changes in other dopamine dependent neural circuits [ 15 ]. In Parkinson's disease, basal ganglia dysfunction not only causes slowed movements, but also contributes to gait unsteadiness and increased stride-to-stride variability [ 18 - 20 ], a marker of fall risk [ 10 , 12 , 20 , 21 ]. Given the associations between fall risk and depression and the parallels between major depression and Parkinson's disease, we hypothesized that major depression and related affective disorders would be associated with gait unsteadiness and impairment in the ability to regulate the dynamics of gait stability. To this end, we measured the stride-to-stride fluctuations in the gait cycle timing of patients with two forms of mood disorders, unipolar major depressive disorder (MDD) and bipolar disorder, and compared their gait to that of a healthy control group. The primary outcomes were two measures of gait unsteadiness: stride time variability and swing time variability [ 12 , 19 , 20 ]. We anticipated that these two variability measures would be increased in the two patient groups compared to the control group. In secondary analyses, we examined the potential confounding influences of age, medications and other subject characteristics on these two measures of variability. Methods Subjects Patients with a clinically defined major affective disorder were recruited from the clinics of McLean Hospital, a psychiatric teaching center in Belmont, MA. Specifically, patients were invited to participate if they were being treated for either bipolar disorder or unipolar MDD, as defined by DSM-IV criteria [ 22 ]. Patients were excluded if they had clinically significant co-morbidities likely to affect gait including orthopedic disease, dementia, neurological disease, podiatry complaints or any known gait disturbances. To quantify disease severity, we used the Clinical Global Impression Scale (0: healthy; 7 most severe) [ 23 ]. Healthy subjects without depression, other affective disorders, or any of the above-mentioned co-morbidities likely to affect gait were recruited from among hospital employees and the community to serve as a healthy control group. The Human Studies committee of McLean Hospital approved the study. All subjects provided informed written consent prior to participating in the study. Assessment of gait unsteadiness Subjects were instructed to walk at their normal pace on level ground for 35 meters, to turn and to walk the same route back, and to continue walking for a total of six minutes. Study subjects were not aware of the specific questions addressed in this investigation. Previously described methods were used to evaluate gait dynamics and analyze the stride-to-stride fluctuations of gait timing [ 12 , 19 , 20 ]. Briefly, to measure the gait rhythm and the timing of the gait cycle (i.e., the stride time and the % swing time), force sensitive insoles were placed in the subject's shoes. These inserts produce a measure of the force applied to the ground during ambulation. A small, lightweight (5.5 × 2 × 9 cm; 0.1 kg.) recorder was worn on the ankle and held in place using an ankle wallet. An on board A/D converter (12 bit) sampled the output of the footswitches at 300 Hz and stored the data. Subsequently, the digitized data were transferred to a computer workstation for analysis using software that extracts the initial and end contact time of each stride (i.e., heel-strike and toe-off). With this information, the stride time or duration of the gait cycle (time from initial contact of one foot to subsequent contact of the same foot) and the % swing time were determined for each stride during the walk. The stride time is a measure of the gait cycle duration and the inverse of the cadence. The time spent with one foot in the air, relative to the gait cycle duration, defines the % swing time. Typically, the % swing time becomes smaller in diseases that affect gait (e.g., Parkinson's disease). To focus on the assessment of the intrinsic dynamics of continuous, usual walking and gait unsteadiness and to insure that the analysis was not influenced by outlier data points, previously described analysis methods were used [ 12 , 19 , 20 ]. Briefly, the first thirty seconds of each subject's walking time series were excluded to minimize any start-up effects and the last ten seconds were removed as well [ 12 , 19 , 20 ]. A median filter was then applied to each subject's time series to remove data points that were three standard deviations greater than or less than the median value of each time series [ 12 , 13 , 19 ]. The mean stride time and mean % swing time were computed. Two measures of stride-to-stride variability [ 12 , 19 , 20 ] were also assessed. 1) Stride time variability, the magnitude of the stride-to-stride fluctuations in the gait cycle duration, was calculated by determining the coefficient of variation (CV) of each subject's stride time. 2) Swing time variability was determined by calculating the % swing time CV. Values reported are for the left foot. Similar results were obtained if we analyzed each subject's right foot or the "best" foot (i.e., the one with the lowest variability). These two measures of stride-to-stride variability reflect gait unsteadiness and have been shown to be related to fall risk, i.e., larger variability has been associated with increased risk of falls [ 12 , 20 ]. Mean gait speed was evaluated by determining the time required to walk 6 laps (i.e., 210 meters). Statistical analysis Results are reported as means ± standard deviation. For continuous data, the Wilcoxon Rank Sum test (the non-parametric equivalent of the two-sample Student's t-test) was used to compare each patient group to the control group (We note that for all of the primary outcomes the conclusions were unchanged if parametric analyses were applied instead). Subgroup analyses were performed to evaluate the effects of subject characteristics such as age and use of certain drugs. A p-value less than or equal to 0.01 was considered statistically significant (Bonferroni-like adjustment for the five measures of gait). Statistical analysis was performed using SPSS for Windows (version 10.1). Results Subject characteristics Fifty subjects (17 women) were studied. Subjects were 36.3 ± 14.7 years old. Twenty-three of the subjects had bipolar disorder (mean age: 43.4 ± 14.2 yrs), nine subjects had MDD (mean age: 40.9 ± 16.9 yrs), and eighteen of the subjects were healthy controls (mean age: 24.9 ± 3.5 yrs). Scores on the Clinical Global Impression (CGI) scale were 0.2 ± 0.4, 3.8 ± 0.9, and 3.2 ± 0.7 for the control, bipolar and MDD groups, respectively. As expected, the mean CGI scores were significantly higher in both patient groups compared to the control group (p < 0.001 for both groups). Gait changes in bipolar disorder and MDD Table 1 summarizes the gait measurements of the three groups. Compared to the control group, the two patient groups tended to walk more slowly, with decreased swing time % and increased stride time. However, none of these differences was statistically significant (e.g., for gait speed, p = 0.32 and 0.10 in patients with bipolar disorder and MDD compared to controls, respectively). As hypothesized, stride time variability tended to be larger in both patient groups compared to the control group, but the differences were not significant (p > 0.29). Compared to the control group, swing time variability was significantly larger in the subjects with bipolar disorder (p < 0.0001) and in the subjects with MDD (p < 0.0004). These measures of gait were not significantly different between the two patient groups. Examples of time series showing the increased stride-to-stride fluctuations in swing time % in a subject with bipolar disorder and a subject with MDD are shown in Figure 1 . Table 1 Gait dynamics in the three subject groups Gait Measure Healthy Controls (n = 18) Bipolar Disorder (n = 23) Unipolar Major Depressive Disorder (n = 9) Mean Values Stride time (msec) 1120 ± 69 1161 ± 106 1200 ± 94 Swing Time (%) 37.0 ± 1.6 35.4 ± 2.1 35.8 ± 1.3 Gait speed (m/sec) 1.21 ± 0.16 1.14 ± 0.20 1.11 ± 0.14 Variability Measures of Unsteadiness Stride Time CV (%) 2.4 ± 0.6 2.9 ± 1.3 2.6 ± 0.9 Swing Time CV (%) 1.9 ± 0.3 3.0 ± 1.4* 3.6 ± 1.9* CV: coefficient of variation * p < 0.0005 compared to the control group Figure 1 Representative swing time series from two patients with affective disorders and a control subject. Data shown are from a healthy control subject (25 yr old male; swing time CV = 1.6 %), a subject with major depressive disorder (38 yr old male; swing time CV = 3.2 %), and a subject with bipolar disorder (20 yr old male; swing time CV = 3.7 %). Note the relatively large stride-to-stride fluctuations (increased variability) in the swing time of the two patients compared with the control subject. All but one of the subjects with MDD was being treated for acute illness, as were nineteen of the subjects with bipolar disorder. Among the subjects with bipolar disorder, the subjects whose disease was stable tended to walk more normally (closer to control values) compared to those subjects with bipolar disorder being treated acutely. Swing time variability tended to be larger (p = 0.044) in the subjects with bipolar disorder who were being treated acutely (3.2 ± 1.5 %) compared to those who were more clinically stable (2.2 ± 0.3 %). Among the subjects with MDD, swing time variability tended to be higher in subjects on antidepressants (n = 5) compared to subjects not taking antidepressants (p = 0.027). Other measures of gait and the CGI were similar in the MDD subjects who were on or off antidepressants (p > 0.41). One patient with MDD was taking a neuroleptic and none was taking mood stabilizers. Among bipolar patients, there were no significant differences in any of the gait measures in patients who were taking antidepressants (n = 15), neuroleptics (n = 14) or mood stabilizers (n = 12) compared to those who were not. Compared to controls, swing time CV was significantly increased among bipolar patients who were not taking antidepressants (p < 0.001), not taking neuroleptics (p < 0.002) or not taking mood stabilizers (p < 0.001). Stride time CV tended (p = 0.068) to be increased among bipolar patients who were taking antidepressants, neuroleptics, or mood stabilizers (n = 17), compared to those who were not (n = 6). Compared to the controls, swing time CV was significantly increased among the six bipolar patients who were not taking antidepressants, neuroleptics or mood stabilizers (p < 0.007). Subjects in the two patient groups were generally older than the subjects in the control groups. To evaluate if the group effects were only a result of these age differences, we analyzed the subset of all the subjects who were less than forty years of age. In this subgroup (n = 32), mean ages of all three groups were similar (p > 0.32) and CGI scores remained significantly higher in the two patient groups compared to the control group (p < 0.0001). The results of this subset analysis are consistent with those shown in Table 1 . For example, gait speed was similar among the younger subjects with bipolar disorder compared to the younger control subjects (p = 0.185) and among the younger subjects with MDD compared to the younger control subjects (p = 0.075). Among the subjects who were less than forty years old, swing time variability was significantly higher in the subjects with bipolar disorder compared to the control subjects (p = 0.010) and in the subjects with MDD compared to the control subjects (p = 0.002). Discussion Previous studies reported that patients with clinical depression walk more slowly and with less time spent in the swing phase of the gait cycle [ 6 , 7 ]. The present findings lend support to those reports. Although the differences were not statistically significant in this study, subjects with MDD and subjects with bipolar disorder generally tended to walk more slowly and with a shortened swing phase compared to the control group. Here we report, for the first time, changes in the stride-to-stride variability of these two patient groups. In both MDD and bipolar disorder, significant increases in swing time variability were observed. This finding raises the intriguing possibility that dynamic instabilities in the walking pattern may be a heretofore unrecognized feature of gait in severe clinical depression and related affective disorders, including subjects with MDD and bipolar disorder. To our knowledge, this is the first quantitative investigation of gait in bipolar disorder patients. Although not necessarily apparent to the clinical observer, the present quantitative study suggests that severe clinical depression and related major affective disorders may be associated with an unsteady gait, with certain features reminiscent of impairments in Parkinson's disease. There are, however, subtle but important differences. In advanced Parkinson's disease [ 18 , 19 ], gait speed and swing time are typically reduced to a much greater extent than that seen in the present study (e.g., one study reported a mean gait speed of 1.0 m/sec and a mean % swing time of 33.5 % in subjects with Parkinson's disease [ 19 ]; compare with Table 1 ). In Parkinson's disease, increased stride-to-stride variability is seen in the swing time (as in the present study), but the magnitude of the increase in swing time variability is larger than that seen in MDD and bipolar disorder [ 19 ]. For example, a previous study found that the mean stride time variability and swing time variability were 4.4 % and 5.7 %, respectively, among patients with Parkinson's disease [ 19 ], values that are much higher than those observed in the present study (compare with Table 1 ). In addition, whereas in Parkinson's disease increased variability is observed in the stride time as well as in swing time, in MDD and bipolar disorder the gait impairment appears in swing time regulation and the alterations are smaller than those observed in Parkinson's disease. In some previous studies that have examined both stride time variability and swing time variability, an increase in one measure was accompanied by an increase in the other measure [ 12 , 19 ]. This observation is not surprising since swing time is a key component of gait cycle timing and a sub-phase of the stride time. An interesting question is why increased swing time variability was observed among the patients with MDD and bipolar disease in the present study, while stride time variability was not significantly increased? A number of explanations may be possible. Our findings suggest that in some circumstances swing time variability may be a more sensitive marker of subtle underlying changes in neural control and in the locomotor system's ability to repeatedly and precisely generate and execute neural commands. While stride time variability evaluates consistency of the loading response, as reflected in the variability in the time between consecutive heel-strikes, swing time variability reflects the stride-to-stride consistency of both the loading and unloading of body weight. Thus, when the regulation of gait is largely intact and stride-to-stride variability is relatively small, as in the present study, changes in swing time variability may be present, while stride time variability remains largely unchanged. Another related explanation for the discrepancy been stride time variability and swing time variability has been suggested by Gabell and Nayack [ 24 ]. They proposed that stride time variability reflects gait patterning mechanisms and the rhythmicity of locomotor pacing while variability of stance and swing time more closely mirror dynamic equilibrium and postural control mechanisms. A complete explanation for the difference between swing time variability and stride time variability requires further investigation. We note, however, that such discrepant behavior among different measures of gait variability has also been observed in other studies [ 25 - 28 ]. For example in a study of patients with very mild Parkinson's disease [ 27 ], swing time variability was significantly increased while stride time variability was only slightly larger than control values. In older adults, depression is associated with an increase in the risk of falls and hip fractures [ 1 , 3 ]. The mechanisms responsible for this increase are not clear. The present findings suggest that depression and bipolar disorder may be associated with an increase in stride-to-stride variability which may, in turn, predispose to falls [ 10 , 12 , 20 , 21 ]. Young and middle-aged adults who are depressed generally do not have an increased fall risk, probably because depression by itself does not increase stride variability enough to cause falls and because other physiologic systems that help maintain gait and balance stability are generally intact. In older adults, falls are often multifactorial [ 1 ]. If age-associated changes occur in multiple systems (e.g., vision, balance), depression may further contribute to fall risk by increasing stride variability and exacerbating instability. One possibility is that the changes in basal ganglia function in depression [ 15 ] increase stride variability, as occurs in Parkinson's disease; however, the mechanisms whereby depression and other affective disorders influence gait and impair the regulation of stride variability remain to be determined. A previous study of gait in depression did not find any significant differences in the gait of subjects who were taking medications compared to those who were not [ 6 ]. Specifically, gait speed, stride time and swing time were apparently not related to medication usage. For these specific features of gait, our results support the previous findings. Gait speed and stride time were not related to medication usage in the subjects with bipolar disorder or MDD in the present study. Conversely, while swing time variability was increased relative to control values even in the subgroup of bipolar patients who were not taking any medications, among the patients with bipolar disease and the patients with MDD taking certain medications, variability of gait measures tended to be further increased. Although Lemke et al. did not find a medication effect on gait in patients with depression [ 6 ], the suggested relationship between gait and medication usage in the present study in the subjects with MDD is consistent with a recent study which reported that certain antidepressants may impair psychomotor abilities and integrative central nervous system function [ 29 ]. However, it is not apparent why such an effect was not observed among patients with bipolar disease and why antidepressants might affect the swing time variability of patients with MDD, but not of patients with bipolar disease. This preliminary study has a number of limitations. As noted, subjects in the control group were generally younger than the two patient groups (mean differences of 4 and 7 years). However, a number of considerations suggest that the observed increase in swing time variability in the patient groups was not simply a function of aging. 1) Subgroup analysis demonstrates that the differences between the groups persist even when only the relatively young subjects (< 40 years of age) are studied. 2) Like the variability of gait speed and stride length, previous work has shown that the variability of stride time and swing time is similar in healthy young and healthy older adults, and that increases in older adults are a sign of pathology [ 24 - 26 , 30 - 32 ]. For example, one study of the effects of physiologic aging on gait found stride time variability was essentially identical in a group of healthy young adults (mean age 24.6 years) and healthy older adults (mean age 75.7 yrs) [ 31 ], with values similar to those observed in the present study. The present study has other limitations and raises additional questions that should be addressed in future studies. Among subjects with MDD, we could not evaluate if there were differences between subjects treated for acute illness and those with more chronic symptoms. Further, among patient with bipolar disorder, we did not control for the patients' acute mood status. The present study may have also been underpowered to detect small differences between the patients with bipolar disorder and MDD. A prospective study of a larger number of patients, perhaps in different age groups before and after they undergo different types of therapy, including pharmacologic interventions, should be helpful in leading to a more complete understanding of the relationships among stride variability, fall risk, and medication usage. Such studies may also be helpful in determining the mechanisms whereby depression and related mood disorders influence stride variability, in assessing how this relationship changes over time and with respect to disease state (i.e., acute or stable symptoms), and in evaluating if stride variability measures may prove clinically useful as an objective measure of major affective disorders. Stride variability is less affected by a conscious effort to alter gait and more closely reflects intrinsic underlying impairment [ 10 ]. Additional biomechanical studies may also be helpful in elaborating the mechanisms underlying gait changes in affective disorders and possible causal links between swing time variability, gait instability, and the risk of falls. Conclusions Despite the limitations of this study, the present findings in a relatively small sample size indicate that patients with MDD and patients with bipolar disorder display gait unsteadiness evidenced by increased swing time variability, even while other measures of gait are not markedly changed. This apparent disparity between measures of stride variability, on the one hand, and gait speed and other features of gait, on the other, raises the possibility that stride-to-stride variability in major mood disorders may be more sensitive to certain subtle neuropsychiatric influences than measures based only on the mean value of a given parameter. Our findings also demonstrate that measurement of variability of gait may provide a readily quantifiable approach to monitoring depression and other affective disorders. Moreover, these results suggest that this perturbation in gait dynamics may provide a mechanistic link connecting depression and falls in older adults. List of abbreviations CV: coefficient of variation Competing interests The author(s) declare that they have no competing interests. Authors' contributions JMH, ALG and ALS conceived of the study, and participated in its design and coordination. JMH drafted the manuscript. JMH and CKP carried out the time series and statistical analyses. All authors helped to revise the manuscript and read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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555586

Retrovirology highlights a quarter century of HTLV-I research

In 1977, Takatsuki and co-workers described in Japan a human malignant disease termed adult T-cell leukemia (ATL). Three years later, in 1980, Gallo and colleagues reported the identification of the first human retrovirus, human T-cell leukemia virus type I (HTLV-I), in a patient with cutaneous T-cell lymphoma. This month, Retrovirology commemorates these two land mark findings by publishing separate personal recollections by Takatsuki and Gallo respectively on the discovery of ATL and HTLV.

Retrovirology as a medical study first emerged in the early 1900s. In 1908, Ellermann and Bang reported on the transmissibility of avian leucosis by cell-free filtrates, suggesting the involvement of a virus [ 1 ]. Shortly afterward, in 1910, Rous demonstrated that chicken sarcomas were infectious and when inoculated into healthy birds induced tumors [ 2 ]. Today, a plethora of oncogenic animal retroviruses including bovine leukemia virus, feline leukemia virus, gibbon ape leukemia virus, Jaagsiektse sheep retrovirus, murine leukemia virus, mouse mammary tumor virus, reticuloendotheliosis virus, simian T-cell lymphotropic virus, and Walleye dermal sarcoma virus has been described. Understanding how retroviruses cause cancer took a major step forward with the development of the cellular oncogene hypothesis in 1976. Thus Varmus, Bishop and colleagues [ 3 ] demonstrated that the viral oncogenes ( v-onc ) encoded by many retroviruses were captured originally from cellular sequences (i.e. c-onc ). To date, three general models of retroviral transformation are accepted: a) over-expression of v-onc ; b) cis -oncogenic effect from promoter insertion; and c) cis -oncogenic effect from enhancer insertion (Fig. 1A, B, C ). Figure 1 Panels A, B, and C show the three accepted ways by which a retrovirus may transform cells: capture of a c-onc and over-expression of v-onc by the provirus (A); promoter insertion upstream of a growth controlling cellular gene (B); and enhancer insertions either upstream or downstream of growth controlling cellular genes (C). Panel D shows the stepwise ways in which HTLV-I Tax oncoprotein may transform cells by i) inactivating checkpoints to induce tolerance of damaged DNA, and ii) permitting the accumulation of unrepaired DNA lesions which ultimately convert a normal cell to a transformed cell. Although not yet fully understood, HTLV-I is believed to transform human T-cells neither through the acquisition of a c-onc nor by cis -insertion effects on the cellular genome. Pioneering molecular biology studies by Mitsuaki Yoshida and colleagues led to the delineation of the HTLV-I transforming gene, Tax [ 4 ]. Tax has no cellular homologue; and it works in trans to disrupt cellular checkpoints and destabilize genome integrity [ 5 ] leading to transformation (Fig. 1D ). A more extensive discussion of the molecular biology of HTLV-I and its transforming function will be in an upcoming comprehensive review by Masao Matsuoka to be published in Retrovirology . Two articles in this month's Retrovirology describe respectively the discovery of adult T-cell leukemia [ 6 ] and HTLV-I [ 7 ].

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526218

Probiotic prophylaxis in patients with predicted severe acute pancreatitis (PROPATRIA): design and rationale of a double-blind, placebo-controlled randomised multicenter trial [ISRCTN38327949]

Background Infectious complications are the major cause of death in acute pancreatitis. Small bowel bacterial overgrowth and subsequent bacterial translocation are held responsible for the vast majority of these infections. Goal of this study is to determine whether selected probiotics are capable of preventing infectious complications without the disadvantages of antibiotic prophylaxis; antibiotic resistance and fungal overgrowth. Methods/design PROPATRIA is a double-blind, placebo-controlled randomised multicenter trial in which 200 patients will be randomly allocated to a multispecies probiotic preparation (Ecologic 641) or placebo. The study is performed in all 8 Dutch University Hospitals and 7 non-University hospitals. The study-product is administered twice daily through a nasojejunal tube for 28 days or until discharge. Patients eligible for randomisation are adult patients with a first onset of predicted severe acute pancreatitis: Imrie criteria 3 or more, CRP 150 mg/L or more, APACHE II score 8 or more. Exclusion criteria are post-ERCP pancreatitis, malignancy, infection/sepsis caused by a second disease, intra-operative diagnosis of pancreatitis and use of probiotics during the study. Administration of the study product is started within 72 hours after onset of abdominal pain. The primary endpoint is the total number of infectious complications. Secondary endpoints are mortality, necrosectomy, antibiotic resistance, hospital stay and adverse events. To demonstrate that probiotic prophylaxis reduces the proportion of patients with infectious complications from 50% to 30%, with alpha 0,05 and power 80%, a total sample size of 200 patients was calculated. Conclusion The PROPATRIA study is aimed to show a reduction in infectious complications due to early enteral use of multispecies probiotics in severe acute pancreatitis.

Background Infection of pancreatic necrosis is the major cause of death in acute pancreatitis [ 1 - 4 ] Small bowel bacterial overgrowth and subsequent bacterial translocation are held responsible for the majority of these infections [ 5 - 9 ] Antibiotic prophylaxis has been studied in several trials [ 10 - 13 ]. Recently, a well-designed placebo-controlled trial failed to show a reduction of infectious complications [ 14 ]. A multicenter trial from the Netherlands, using topical and enteral antibiotics to reduce bacterial overgrowth (selective bowel decontamination, SBD) showed a reduction in infected necrosis [ 15 ]. Despite favourable results, SBD has not been widely implemented due to the workload associated with it and the reluctance to use antibiotics for a long period of time with risks of bacterial resistance and fungal infection [ 16 , 17 ]. Microbial antibiotic resistance has become a worldwide problem due to excessive use. The World Health Organisation has advocated the use of microbial interference therapy: non-pathogens (probiotics) to restrain pathogens [ 18 ]. It is the goal of the present study to investigate the use of prophylactic probiotics as an alternative strategy. Several trials with enteral probiotics have shown a significant reduction of infectious complications both in acute pancreatitis and in patients undergoing major abdominal surgery [ 19 - 21 ] A well-designed placebo-controlled trial with Lactobacillus plantarum in patients with acute pancreatitis showed very interesting results: a significant reduction of infected pancreatic necrosis (1/22 versus 7/23 infected necrosis)[ 19 ]. However, some criticised this study because of the exclusion of biliary pancreatitis patients and some statistical flaws [ 22 , 23 ]. In these and other trials a single probiotic strain was used. It has been suggested that multispecies probiotics are more effective, because effects are strain specific. Combinations of probiotics can be designed so that strain-specific properties are additive or synergistic. Based on in vitro data, a selection of 6 out of 75 probiotic strains was made by Winclove Bio Industries (Amsterdam, the Netherlands) in co-operation with the Departments of Paediatric Immunology and Surgery, of the UMC Utrecht (Utrecht, The Netherlands). This paper describes the rationale of this product and the design of the study. Rationale for the efficacy of multispecies probiotics in acute pancreatitis The bacteria responsible for infection of (peri-)pancreatic necrosis most often originate from the gut [ 2 , 5 , 27 ] The pathophysiology of infection of peri-pancreatic necrosis and the steps amenable to therapeutic intervention are essentially unknown. Upper gastrointestinal dysmotility (UGID) has been observed in acute pancreatitis (AP) as well as in cholestasis and sepsis [ 6 - 8 ] UGID may lead to small bowel bacterial overgrowth (SBBO)[ 6 ]. In immunocompromised patients this bacterial overgrowth may lead to bacterial translocation (BT) [ 5 , 6 ] BT is not only held responsible for infectious complications, but it also contributes to the overproduction of pro-inflammatory cytokines during acute necrotising pancreatitis (ANP) [ 28 ]. These cytokines are key factors in the pathogenesis of multi-organ failure and sepsis. Prevention of UGID, SBBO and BT may lead to prevention of infected pancreatic necrosis and the resulting systemic complications. Intravenous antibiotic prophylaxis is considered an option to prevent pancreatic infection, but results from randomised clinical trials are conflicting [ 10 - 14 ]. Probiotics are living micro-organisms that upon oral delivery exert a range of health promoting properties. For a growing number of inflammatory diseases (gastrointestinal, airway or skin) probiotics are being used, with variable clinical outcome. It is hypothesised that probiotics have an effect on different levels. We developed a multispecies probiotic preparation that aims to prevent SBBO and BT in ANP through (1) stimulation of the production of anti-inflammatory cytokines, especially interleukin-10, at the level of the intestinal mucosa[ 29 ], (2) stimulation of gastrointestinal motility[ 30 ] and (3) competitive inhibition of opportunistic pathogens[ 31 ]. The individual strains each have their own capacity to inhibit growth of specific potential pathogenic micro-organisms (PMO's), as for instance Escherichia Coli or Enterococcus species . The combination of these probiotics, in vitro , inhibits the growth of all relevant PMO's known to infect pancreatic necrosis [ 31 ]. Methods / design Study objectives The study objective is to show that probiotics are effective in reducing the number of infectious complications during the course of acute pancreatitis. Primary endpoint The primary endpoint is the total numbers of infectious complications during the hospital stay for acute pancreatitis, see table 1. Secondary endpoints Secondary endpoints are mortality, necrosectomy, use of antibiotics, total hospital stay, intensive care stay, side effects, abdominal complaints by a patient visual analogue scale questionnaire, sequential organ failure assessment (SOFA) scores, bacterial resistance and total costs. Design PROPATRIA is a double-blind, placebo-controlled randomised multicenter trial. The randomisation is stratified according to the aetiology of the acute pancreatitis (ie. biliary versus non-biliary), also block-randomisation per hospital is used. Its design and timing of the investigations are presented in Figure 1 and Table 2 , respectively. Figure 1 PROPATRIA according to CONSORT. Table 1 Infectious complications Complication Definition Bacterial infection body temperature > 38 degrees and increased number of neutrophils and CRP in peripheral blood and one of the below: Infected pancreatic necrosis Positive fine needle aspiration culture or air bubbles in the pancreatic necrosis on CT-scan. Pneumonia Coughing, dyspnoea, radiography with infiltrative abnormalities, lowered arterial bloodgass. On the intensive care unit a positive endotracheal culture is mandatory. Urinary tract infection Dysuria with bacteraemia (>10.000 CFU/mL) Setting Patients will be enrolled from all 8 Dutch University Hospitals and 7 non-University hospitals. Patients A total of 200 adult patients with a first episode of predicted severe acute pancreatitis will be randomised. Eligibility criteria Inclusion Criteria • age equal to or above 18 years • first episode of acute pancreatitis • written and oral informed consent Exclusion criteria • post-ERCP pancreatitis • malignancy • infection/sepsis caused by a second disease • intra-operative diagnosis • immunocompromised patients • use of probiotics during admission Randomisation criteria After inclusion in the study, patients with predicted severe acute pancreatitis, represented by at least one of the following scores: 3 Imrie criteria, CRP 150 mg/L, APACHE II score 8, are randomised within the first 72 hours after the onset of abdominal pain. Patients with a predicted mild attack of acute pancreatitis do not receive the study product. They do give informed consent and are monitored. Ethics, informed consent This study is conducted in accordance with the principles of the Declaration of Helsinki and 'good clinical practice' guidelines. The independent ethics committee of all 15 participating hospitals approved the final protocol. Oral and written informed consent in form is obtained from the patient before inclusion in the trial. Safety All the probiotics used in this study have a long history of use in the food industry. Probiotics have been studied in many critical ill and immunocompromised patients without any serious adverse events being noted. There is one trial that studied probiotics in acute pancreatitis patients and no serious advents were noted. If an infection with one of the administered probiotics might occur, this could be treated with antibiotics. During administration of the study-product both the patient and the nursing staff are asked to register any potential side effect or adverse event. An independent monitoring committees will discuss all reported (serious) adverse events. Statistical analysis Intention- to-treat The analysis will be performed on the basis of an intention-to-treat (ITT) population and with respect to ITT principles. Also a per-protocol analysis and an analysis for necrotising versus non-necrotising pancreatitis will be performed. Interim-analysis For ethical reasons it is desirable to end a therapeutic experiment once a statistical significant difference in treatment results has been reached. This study uses the stopping-rules according to Snapinn [ 32 ]. An interim-analysis will be performed after the data of the first 100 patients (50% fraction) is obtained. According to Snappin, the trial will be ended at this interim-analysis at p < 0,0081. The study will also be ended in case of adverse events without possibility of positive outcome, p > 0,382. The monitoring committee will discuss the results of the interim-analysis and advice the steering committee. The steering committee decides on the continuation of the trial. Sample size It is anticipated that probiotics will lead to a reduction of infectious complications from 50% (% of patients) to 30%. The sample size calculation is based on α = 0.05, and a power of 80% This leads to a required sample size of 188 patients. Taking into account a 5% loss-to-follow up, a total of 2 × 100 patients will be randomised. Based on hospital data of 2002 about 500 patients have to be included in order to randomise 200 patients with predicted severe acute pancreatitis. There is one post-discharge follow-up after three months. The expected study end is in 2006 (2 years inclusion period). Randomisation The randomisation list was generated by using the website Randomization.com . According to this list a stratified random allocation of probiotics and placebo was performed. Each participating hospital received a series of subsequently numbered identical containers with probiotics or placebo. Patients with biliary cause of the pancreatitis are allocated to the lowest possible number available whereas patients with non-biliary cause are allocated to the highest possible number available (stratification). Blinding Both the probiotics and placebo are packed in identical, numbered sachets. These sachets are packed in identical, numbered containers. The probiotics and placebo are identical in weight, colour, smell and taste. All doctors, nurses, research staff and patients involved are unaware of the treatment administered to the patient. Treatment program Patients eligible for inclusion are followed during 72 hours after onset of the abdominal pain. When a patient meets a randomisation criteria (preferably within 24 hours), a nasojejunal feeding tube is passed and administration of the study product (Ecologic ® 641, Winclove Bio Industries, Amsterdam, The Netherlands) and fibre enriched tube feeding (Multifibre ® , Nutricia, Zoetermeer, The Netherlands) is started. Ecologic ® 641 consists of 6 strains of viable and freeze-dried bacteria, namely 4 lactobacilli: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus salivarius, Lactococcus lactis, and 2 bifidobacteria: Bifidobacterium bifidum and Bifidobacterium lactis in a total daily dose of 10 10 bacteria. The study-product is administered twice daily through a nasojejunal tube for a maximum of 28 days. The treatment is stopped when a patient is diagnosed with infected pancreatic necrosis, is discharged or dies. A standard protocol for the treatment of acute pancreatitis is followed. During ERC with sphincterotomy in case of biliary pancreatitis antibiotic prophylaxis is allowed. Prophylactic use of proton pump inhibitors is only allowed in case of a clinical history of peptic diseases like peptic ulcer disease and reflux esophagitis. Prophylactic use of antibiotics is not allowed. At 7–10 days after admission, a routine CT scan is performed to detect pancreatic necrosis. Fine needle aspiration in (peri)pancreatic collections is performed only in case of clinical suspicion of infected necrosis. Further culturing, imaging and treatment are all based on clinical findings. Monitoring A research nurse monitors the participating centres and patients. Every 6 months all centres are visited by a second independent research nurse who checks, at least, 10% of every patient's data. Follow-up Patients are followed during their hospital stay. There is one follow-up visit, 3 months after discharge, including an abdominal ultrasound and a short questionnaire regarding abdominal pain and daily activities. Discussion Only patients with predicted severe acute pancreatitis are randomised. Patients with mild acute pancreatitis are considered not eligible for randomisation because of their low risk to develop infectious complications. If such patients would be included, a very large number of patients would be needed to demonstrate a significant effect between the treated and the non-treated patients. To properly introduce the concept and potential of probiotic prophylaxis, the use of prophylactic antibiotics was discussed during the preparations of the study. About 30% of the hospitals participating in the study commonly used prophylactic antibiotics, once a patient was diagnosed with pancreatic necrosis. Because of the lack of evidence it was decided, even before the results of the most recent German trial[ 14 ] were presented, not to administer prophylactic antibiotics in case of pancreatic necrosis without clinical suspicion of infected necrosis. When patients do receive antibiotics, for instance because of an urinary tract infection, effort is made to administer the study product with a 4-hour interval in order to minimise interference of the antibiotics with the probiotics. The maximum of 72 hours between onset of symptoms and start of treatment was decided upon, based on the consideration that probiotics are expected to prevent infection of pancreatic necrosis. Therefore the probiotics should be administered prior to the stage that bacterial overgrowth starts and intraluminal bacteria migrate across the mucosal barrier. Experimental studies have shown that bacterial overgrowth occurs very early, within 24 hours after onset, in the course of acute pancreatitis and reduction of the bacterial load in the proximal small bowel by intraluminal antibiotics reduces the risk of infection of pancreatic necrosis [ 8 , 33 , 34 ]. The presence of pancreatic necrosis can only be detected reliably 5–7 days after onset [ 35 ]. This is too late to effectively prevent bacterial overgrowth and translocation and therefore "predictive laboratory scores" are used as randomisation criteria and not diagnosis of pancreatic necrosis on CT scan. The scoring systems used are also simple and generally available. A major disadvantage though, is the limited positive predictive value and the high number of false positives [ 36 , 37 ]. During interim-analysis the number of false positives will be calculated and the sample size may be adjusted. All randomised patients will receive early enteral feeding by a jejunal feeding tube. Since patients with predicted severe pancreatitis would develop severe pancreatitis in only 50% of the cases, the fraction with a mild course would normally not receive a nasojejunal feeding tube. It is unavoidable to prevent this over treatment for patients with a mild course because the current scoring systems fail to select all patients with a severe course and early intervention is warranted. The primary outcome parameter 'total of infectious complications', was chosen because it was shown in previous trials that also the number of pulmonary and urinary tract infections can be reduced by probiotics [ 19 - 21 ] This fits in with the concept that such complications are secondary to bacterial translocation. All of the infectious complications are documented in the study period until the patient reaches one of the study-endpoints: infected necrosis, discharge or in-hospital death. Conclusion PROPATRIA is a double-blind, placebo-controlled randomised multicenter trial that aims to show a reduction in infectious complications by the enteral use of a multispecies probiotics preparation in patients with predicted severe acute pancreatitis. Authors' contributions MB drafted the manuscript HT, EB, VN, LA and HG edited the manuscript All authors participated in the design of the study MB and EB performed the statistical analysis. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. PROPATRIA committee members Steering Committee -HG Gooszen (chairman), MGH Besselink (principal investigator), HM Timmerman, LMA Akkermans, VB Nieuwenhuijs, E Buskens, UMC Utrecht; H van Goor, GT Bongaerts, UMC St. Radboud Nijmegen. Monitoring Committee -IHM Borel Rinkes (chairman), B Oldenburg, Y van der Graaf, W Renooij, UMC Utrecht; E Stobberingh, University Hospital Maastricht. Key staff at coördinating centre MGH Besselink (principal investigator), VJM Zeguers (trial research nurse), J Oors (auditor), HG Rijnhart (data manager), HM Timmerman, LMA Akkermans, HG Gooszen, UMC Utrecht. Clinical centres and investigators The last investigator per hospital is the local principal investigator. University Hospital Groningen: RJ Ploeg, MR Kruijt Spanjer, HS Hofker; University Medical Center St. Radboud Nijmegen: H Buscher, A Nooteboom, H van Goor; University Hospital Maastricht: JP Rutten; CHC De Jong; St. Elisabeth Hospital Tilburg: T Drixler; C van Laarhoven; Erasmus Medical Center Rotterdam: G van ’t Hof, EJ Kuipers CHJ van Eijck; Canisius Wilhelmina Hospital Nijmegen: B Houben, L Ootes, A Tan, C Rosman; Medical Center Rijnmond Zuid Rotterdam: N Wijffels, L van Walraven, J Lange; Leiden University Medical Center: A Haasnoot, S Schaapherder; Gelderse Vallei Hospital Ede: B Witteman; St. Antonius Hospital Nieuwegein: TL Bollen, B van Ramshorst; University Medical Center Utrecht: KJ van Erpecum; Meander Medical Center Amerfoort: R Frankhuisen, MA Brink; Vrije Universiteit Medical Center Amsterdam: CJ Mulder, MA Cuesta; Rijnstate Hospital Arnhem: E Spillenaar Bilgen, P Wahab; Academic Medical Center Amsterdam: DJ Gouma, O van Ruler, MA Boermeester. List of abbreviations CRP c-reactive protein APACHE II Acute Physiology and Chronic Health Evaluation II ERC endoscopic retrograde cholangiography CT scan computer tomographic scan Table 2 Study Flowchart Visit Admission Day 2 Day 5 Day 10 Day 14 Day 21 Day 28 Day 35 Discharge 3 months Informed consent X Clinical scores X X X X X X X X X US# X X CT* X Laboratory X X X X X X X X Study product X § X § X X X X X # US = abdominal ultrasound to detect gallstones (at admission) or pseudocyste (at follow-up) * CT = abdominal computer tomographic scan to detect (peri-)pancreatic necrosis § = Within 72 hours after onset of pain the administration of the study product is started. Pre-publication history The pre-publication history for this paper can be accessed here:

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524369

Impact of seasonal variation, age and smoking status on human semen parameters: The Massachusetts General Hospital experience

Background To investigate the relationship of human semen parameters with season, age and smoking status. Methods The present study used data from subjects recruited into an ongoing cross-sectional study on the relationship between environmental agents and semen characteristics. Our population consisted of 306 patients who presented to the Vincent Memorial Andrology Laboratory of Massachusetts General Hospital for semen evaluation. Sperm concentration and motility were measured with computer aided sperm analysis (CASA). Sperm morphology was scored using Tygerberg Kruger strict criteria. Regression analyses were used to investigate the relationships between semen parameters and season, age and smoking status, adjusting for abstinence interval. Results Sperm concentration in the spring was significantly higher than in winter, fall and summer ( p < 0.05). There was suggestive evidence of higher sperm motility and percent of sperm with normal morphology in the spring than in the other seasons. There were no statistically significant relationships between semen parameters and smoking status, though current smokers tended to have lower sperm concentration. We also did not find a statistically significant relationship between age and semen parameters. Conclusions We found seasonal variations in sperm concentration and suggestive evidence of seasonal variation in sperm motility and percent sperm with normal morphology. Although smoking status was not a significant predictor of semen parameters, this may have been due to the small number of current smokers in the study.

Background Male factor infertility has been identified as the main or secondary cause in >40% of infertile couples. The number of office visits each year for infertility in the United States has risen from 600,000 in 1968 to 2 million by the 1990's. Semen analysis is a central component in male infertility evaluation [ 1 , 2 ]. The present study was designed to evaluate the relationship of human semen parameters to season, age and smoking status. Seasonal variations in semen parameters have been reported in both fertile and infertile men [ 3 - 6 ]. Saint Pol et al. found a significant seasonal variation in sperm count, with highest sperm counts in late winter and early spring and lowest concentration in late summer [ 5 ]. There are several studies that suggest that an increase in age is associated with a decline in semen parameters [ 6 - 8 ]. Paulson and coworkers identified an inverse association between age and total sperm count, but did not find an age related decrease in fertilization rate or a decrease in live birth rate in the oocyte donation model [ 9 ]. There is a consistent association between cigarette smoking and fertility in women [ 10 - 13 ]. However, the data on cigarette smoking and measures of male fertility are less clear. Künzle et al. found an association between smoking and reduced semen quality [ 14 ] while others found no strong relationship [ 15 ]. The present study explores the relationship of human semen parameters with season, age and smoking status, while controlling for abstinence interval. In an earlier publication, we reported on the relationship between semen parameters, season, and age using data from a retrospective review of an existing laboratory database at the Massachusetts General Hospital Vincent Memorial Andrology Laboratory [ 16 ]. The present study uses data from subjects recruited into an ongoing study exploring the relationship between environmental agents and semen parameters. There is no overlap with subjects from our earlier study. Methods Study participants The Human Subject Internal Review Board committees of the Harvard School of Public Health and the Massachusetts General Hospital approved the study (IRB #96-7545). Informed consent was obtained from each participant before entering the study. The study population consisted of patients (n = 306) presenting to the Vincent Memorial Andrology Laboratory of Massachusetts General Hospital (MGH) between January 2000 and January 2002 for semen analysis as a component of infertility evaluation. Of the 306 subjects, age was not available for five study subjects despite several attempts at follow up after the initial study visit. Sperm morphology was not performed on two subjects with azoospermia. Men were approached only if the referring physician had previously received a brief description of the study. The Andrologist approached patients and asked if they were willing to meet the research nurse to learn about the research study, noting that it was optional and not part of their clinic appointment. All patients approached had the opportunity to decide for themselves if they wanted to participate in the research study. Inclusion criteria for this study were: 1) scheduled for a routine semen analysis as a patient at the Vincent Memorial Andrology Laboratory, 2) either English or Spanish speaking, 3) age 18–54 years, 4) had not had a vasectomy, and 5) was not currently receiving hormone therapy. All subjects received an explanation of the study in a private setting prior to obtaining informed consent. Information on lifestyle, such as smoking history and medical history was obtained by nurse interview. From the date that semen specimen was produced, samples were classified by season as follows: winter = December, January, February; Spring = March, April, May; Summer = June, July, August; and Fall = September, October, November. Collection of samples Semen was collected by masturbation in a private room in the hospital. The semen specimen was allowed to liquefy for at least 20 minutes in an incubator at 37°C and was analyzed within 60 minutes after collection. A routine semen analysis was performed which included the following parameters: semen volume, pH, sperm concentration, sperm motility, and sperm morphology. Laboratory evaluation Semen volume and pH Volume was determined and sample color and viscosity were recorded. Semen pH was measured within one hour of ejaculation (Color pHast; EM Science, Gibbstown, NJ, USA). Concentration and motility All fresh semen samples were analyzed for sperm concentration and motion parameters by the HTM-IVOS Semen analyzer (Hamilton-Thorn 10HTM-IVOS, Beverly, MA, USA). Setting parameters and the definition of measured sperm motion parameters for CASA were established by the manufacturer: (frames acquired: 30; frame rate: 60 Hz; straightness (STR) threshold: 80.0%; medium VAP cutoff: 25.0 um/s; and duration of tracking time: 0.38 sec). To measure both sperm concentration and motility, aliquots of semen samples (5 μl) were placed into a pre-warmed (37°C) Makler counting chamber (Sefi – Medical Instruments, Haifa, Israel). A minimum of 200 sperm from at least four different fields was analyzed from each specimen. Percent motile sperm was defined as World Health Organization (WHO) grade "a" sperm (rapidly progressive with velocity ≥ 25 μm/s at 37°C) plus "b" grade sperm (slow/sluggish progressive with velocity ≥ 5 μm/s but < 25 μm/s) [ 17 ]. Morphology Using the "feathering" method at least two slides were derived from each fresh semen sample [ 17 ]. The resulting thin-smear slide was allowed to air dry before staining which was carried out using a Diff-Quik staining kit, consisting of three solutions (Dade Behring AG, Düdingen, Switzerland). Slides were then mounted with a microscopy cover glass (Fisher Scientific, Pittsburgh, PA, USA). Morphological assessment was performed at a magnification of 100X with an oil immersion using a Nikon microscope (Nikon Company, Tokyo, Japan). As the slide was scored, the normal and abnormal spermatozoa (head defects, midpiece defects and tail defects) were noted. Sperm morphology was determined using the strict criteria by Kruger et al. [ 18 ]. From each sample at least 200 spermatozoa was counted from two slides. Results were expressed as percentage of normal spermatozoa, head defects, midpiece defects and tail defects. Statistical analysis Multiple regression analyses (SAS version 8.2, SAS Institute, Cary, NC, USA) was performed to investigate whether there were differences in semen parameters with respect to season, age, and smoking status. Winter was used as the reference season. We also investigated month-to-month variation in semen parameters. For each semen parameter, a separate multiple regression was performed. Semen analysis parameters were entered into the models both untransformed and after square root transformation because of their skewed distribution. Since the square root transformed results were similar to the untransformed results, and are simpler to interpret, only the untransformed results are presented. To explore whether the semen parameters and age relationships were linear, age was used as both a continuous and categorical variable (less than 30 years, 30 to 40 years, and greater than 40 years old). Abstinence time was modeled as an ordinal five-category variable (2 or fewer days, 3, 4, 5, and 6 or more days). Results Subject's ages ranged from 18.2 to 54.3 years. The mean (± SD) age was 35.9 (± 5.6) years. The majority of the subjects (67%) were between 30 and 40 years old; only 11% were <30 years and 21% were >40 years old. Mean sperm concentration was above the WHO reference values [ 17 ]. Average (± SD) sperm concentration was 103 (± 95.5) M/ml with range = 0 – 655.4 M/ml. Mean (± SD) percent motility and percent normal morphology were 47.6% (± 25.5) and 6.9% (± 4.7), respectively. Mean (± SD) semen volume was 3.0 ml (± 1.6), with a range = 0.1 – 11.0. The mean (± SD) semen pH was 8.4 (± 0.3), with range = 6.8 – 9.0. Mean (± SD) abstinence interval was 1.7 (± 1.4) days. Distributions of semen parameters for the study population are presented in Table 1 . Table 1 Distributon of semen parameters for study subjects referred for infertility evaluation at Massachusetts General Hospital (n = 306). parameter mean ± SD range age (yrs) a 35.9 5.6 18.2–54.3 volume (ml) 3.0 1.6 0.1–11.0 pH 8.4 0.3 6.8–9.0 concentration (M/ml) 103 95.5 0–655.4 motility (%) 47.6 25.5 0–89.0 morphology (%) b,c 6.9 4.7 0–24.0 Note: a age was not recorded for 5 patients b by Tygerberg-Kruger strict morphology c morphology was not recorded for 2 patients Seasonal variations in semen quality are shown in Table 2 . The number of semen samples obtained in the Spring, Summer, Fall, and Winter were 62, 81, 82, 81, respectively. Mean sperm concentration in the spring (137.2 million/ml) was significantly higher than in the winter (99.2 million/ml), summer (93.1 million /ml) and fall (90.6 million/ ml), (p-value < 0.05). These seasonal differences remained after adjusting for age and abstinence times. Figure 1 shows the month-to-month median sperm concentration across the study. Median sperm concentration by month was higher in March, April, May and June than all other months (Figure 1 ). Mean sperm motility also was higher in the spring (52.3%) than in the summer (47.7%), fall (47.1%) and winter (44.3%). This was higher when compared to winter ( p = 0.06). Table 2 Seasonal variation in semen parameters observed among study patients referred for infertility evaluation at Massachusetts General Hospital ( n = 306). Spring Summer Fall Winter Parameter mean ± SD mean ± SD mean ± SD mean ± SD volume (ml) 3.0 1.5 2.9 1.6 2.9 1.5 3.1 1.9 PH 8.4 0.2 8.4 0.2 8.4 0.3 8.4 0.3 concentration (M/ml) 137.2 a 122.1 93.1 80.1 90.6 81.5 99.2 95.3 motility (%) 52.3 b 22.4 47.7 26.9 47.1 25.1 44.3 26.3 morphology (%) c 7.5 4.9 6.7 4.2 7.0 5.1 6.4 4.8 Note : Spring = March/April/May ( n = 62); Summer = June/July/August ( n = 81); Fall = Sept/Oct/Nov ( n = 82), and Winter = Dec/Jan/Feb ( n = 81). a p < 0.05 vs. Summer, Fall, and Winter by multiple regression analysis method b p = 0.06 vs. Winter c by Tygerberg-Kruger strict morphology Figure 1 Median sperm concentration by month. There were also seasonal variations in sperm morphology parameters. The mean percent normal morphology in Spring (7.5%) was greater than in Summer (6.7%), Fall (7.0%) or Winter (6.4%), though not statistically significant. These seasonal differences remained after adjusting for age and abstinence interval. Mean semen volume and pH were similar across season. There were no statistically significant relationships between age with semen volume, sperm concentration, percent motility, and percent normal morphology. However, there was little variability in age since the majority of men (> 65%) were between 30 and 40 years old. In the present study, only 22 men were current smokers and 57 were ex-smokers. After controlling for age and abstinence time, there was no statistically significant relationship between semen quality and smoking status, though current smokers tended to have lower mean sperm concentration (83 million/ml) than never smokers (104 million/ml). Discussion Although there are numerous previously published studies investigating the relationship among semen parameters and season, age and smoking status, the data are not entirely consistent. To determine if these associations are robust, replication is required. It is the accumulation of consistent observations from epidemiological studies that provides confidence in the findings. Therefore, we believe the present study adds to the literature since it provides replication of seasonal trends in semen parameters. In addition, the present study was conducted in men residing in the Northeast region of the United States, an area with distinct seasons and one that has not been well represented in the literature on this topic. Our study has several strengths. The center selected as the site of this study (the Vincent Memorial Andrology Laboratory of Massachusetts General Hospital) has a large and readily accessible population of men seeking infertility evaluation. Based in a large tertiary care facility, the Andrology Laboratory draws patients from diverse backgrounds throughout the New England region of the United States, receiving referrals from physicians in the community and the medical center. Our study also had potential limitations. Since it is known that there is within-person variability in semen parameters, using a single sample to characterize an individual may introduce measurement error, likely to be random. Another potential limitation is that it is possible that if some men recently moved to the New England area this may introduce bias. However, of the men in the study, 96% of them lived in New England area for at least 3 months prior to their semen analysis, the period of sperm development. Therefore, the concern with recent immigration to the New England area would be minimal. In the present study, we found higher sperm concentrations, motility and percent normal morphology in the spring than in other seasons. This may partially explain seasonal patterns of births in United States, where there is a deficit of spring births [ 19 ], conceived in the summer. Our data are in agreement with previous reports of seasonal variation in sperm concentration with spring having the highest concentration. Gyllenborg et al. [ 20 ] found high sperm counts in the spring as compared to the summer. Two other retrospective studies found peak sperm concentrations in the spring and winter [ 4 , 21 ]. In our earlier publication, a retrospective review of semen analysis results from a different cohort of men, we found higher median sperm concentrations in the winter (111.6 million/mL) as compared to the fall (87.7 million/mL), with a median spring concentration of 90.0 million/mL [ 16 ]. In our earlier study we were unable to control for abstinence interval, thus if winter abstinence times were longer than spring this may partially account for the differences between studies. Seasonal variations in sperm morphology were recently explored by Centola and Eberly in a large sample of California men [ 6 ]. Although they did not find statistical significant seasonal variations in percent normal morphology, there were significant seasonal differences in percent tail defects, percent tapered forms, and percent immature sperm. In our earlier study we found higher median percentage of sperm with normal morphology in the winter (9.0%) as compared to spring (6.5%) [ 16 ]. It is unclear why these results differed from the present study results. The literature on seasonal variations in sperm motility is largely inconsistent [ 5 , 6 , 22 , 23 ]. Effects of temperature and hours of daylight may partially explain seasonal variations in semen quality. Sperm production in humans is known to decrease when testicular temperature is raised by experimental techniques [ 24 ]. Normal spermatogenesis requires a temperature 2–3°C below rectal temperature [ 25 ]. We think temperature and photoperiod may play a role in seasonal variations in semen quality. To explore this further, we are presently conducting a study collecting information on lifestyle factors, such as alcohol and drug use, environmental and occupational exposures, and personal factors such as stress. The information will allow us to further explore risk factors for altered semen quality. Spermatogenesis occurs over approximately three months and does not seem to vary in duration among men [ 25 ]. Chia et al. reported there were no significant month-to-month fluctuations in semen volume and sperm density among men who resided in the tropics [ 26 ], where there are minimal changes in temperature, unlike the seasonal variation of climate in the New England region. In our study, improved semen parameters in the spring may reflect spermatogenesis during the cold New England winter. While we did not find a relationship between age and semen parameters, there was little variability in age in our study population. In our earlier study we found inverse associations between age and sperm concentration, motility and morphology [ 16 ]. However, the age range was larger in this earlier study. In another study, Schwartz and coworkers found an improvement in semen characteristics up to 25 years of age [ 27 ], followed by a leveling off and a subsequent decrease. However, the age relationships were not statistically significant for sperm count, semen volume, and the total number of spermatozoa. Statistically significant decreases in sperm concentration with advanced age were found among 29 'older' fathers (mean age 50.3 years) compared with those from 35 'younger' fathers (mean age 32.2 years) [ 7 ]. A recent review of the literature by Kidd et al. suggested that increased age was associated with a decline in semen volume, sperm motility, and sperm morphology but not with sperm concentration [ 8 ]. Studies have shown associations between cigarette smoking and infertility in women, time to conception, and risk of spontaneous abortion, as well as reduction in fecundity [ 11 , 12 ]. Data on associations between cigarette smoking and male fertility are unclear [ 11 , 15 , 28 , 29 ]. Hughes and Brennan [ 11 ] reported that there were no consistent effects on semen quality among male smokers. Similarly, a survey of more than 4,000 European couples attempting to become pregnant failed to find an effect of male smoking on fecundity [ 15 ]. However, in contrast, in a cross-sectional study of men with proven fertility (n = 243), cigarette smoking was associated with significantly lower semen volume (but not other semen parameters) after adjusting for age and alcohol consumption [ 29 ]. Results from a meta-analysis (including >1000 men) indicated that smokers' sperm density is on average 13–17% lower than that of nonsmokers [ 28 ]. The available data suggest that cigarette smoking was associated with a significant decrease in sperm density, total sperm count, total motile sperm, and the percentage of normal forms [ 14 ]. In our study, only 22 men were current smokers and 57 were ex-smokers. After controlling for age and abstinence time, there was no significant relationship between semen quality and smoking status, though current smokers tended to have lower sperm concentration. The present study is ongoing and we will reexamine these relationships in a larger dataset. In addition, we are also collecting information on other lifestyle and personal factors, such as physical exercise, stress, and alcohol intake. Their relationship with semen parameters will be investigated in the future when we have a larger number of subjects. Conclusions We found seasonal variations in sperm concentration and suggestive evidence of seasonal variation in sperm motility and percent sperm with normal morphology. Although smoking status was not a significant predictor of semen parameters, this may have been due to the small number of current smokers in the study. Competeting interests The authors declare that they have no competing interests. Authors' contributions ZC, LG-B, IS, and RH all contributed equally to this work and reviewed the manuscript.

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479044

The Molecular Biology of Wound Healing

Anyone who's endured their share of childhood scrapes has probably heard some version of the motherly admonishment, “Don't pick that scab, you'll just make it worse!” It turns out, Mom was on to something. Tissue damage in humans triggers a well-characterized response marked by rapid blood clotting and a recruitment of epidermal cells to the injury. When you remove a scab, you're also removing some of the newly regenerated tissues growing underneath, thereby interfering with the healing process. Many different cell types and proteins have been linked to the repair process, but the complexity of the mammalian wound response has challenged efforts to determine their individual roles. Only the first step—the signaling cascade that promotes blood clotting—is understood at the molecular level. Since dissecting the wound response pathway at the molecular level requires an organism that lends itself to relatively easy genetic manipulations, Michael Galko and Mark Krasnow of Stanford University turned to the quintessential genetics organism, Drosophila melanogaster , to create a novel system for studying wound healing. After stabbing fruitfly larvae with a needle to create a nonfatal puncture wound, Galko and Krasnow then analyzed significant morphological, cellular, and molecular events at various stages of healing. Immediately after wounding, the larvae begin to bleed, and about 15 minutes after wounding, the wound darkens and a “plug” of cellular debris forms in the wound. The plug staunches the bleeding and provides a protective layer. Within two hours the plug's outer layer forms a dark, tough scab that presumably serves as an effective barrier to further blood loss. Two or three days later, the healing process is complete. Galko and Krasnow observed the activity of epidermal cells during healing by labeling their nuclei with a fluorescent protein and staining their membranes. Soon after wounding, cells along the wound margin elongated and aligned themselves toward the wound, then fused together to form a large, multinucleate (multiple nuclei) epidermal cell surrounding the scab. Over the next six hours, these cells also spread along and through the plug, re-establishing a continuous epidermis. Since this epidermal spreading resembles that seen during a developmental stage of the fruitfly, where the process depends on the JNK signaling pathway, the authors investigated JNK signaling to shed light on the genetics and cellular events of healing. Sure enough, they found that the JNK pathway was activated during the peak hours of wound healing. Inhibiting the pathway in fly mutants—the classic approach in fly genetics—had dramatic effects on the wound-healing process. The early stages of healing—including plug and scab formation—weren't affected, but epidermal spreading to regenerate the intact epidermis was either blocked or defective. In contrast, larvae with defects in a gene required for the generation of crystal cells—a type of blood cell implicated in processes linked to scab formation—could not properly form scabs. In these scabless wounds, the JNK pathway was hyperactive, epidermal cells at the wound's margin started to spread to close the wound but often failed, and the wound did not heal. Score one for Mom. But apparently Mom wasn't totally right: As Galko and Krasnow discovered, a scab isn't always necessary. When the authors pinched larvae without puncturing the overlying cuticle, the wounds did not form scabs. However, they still saw many of the same processes at work around this “pinch” wound that they saw around the puncture wound, and the pinch wounds healed efficiently. This indicates, the authors argue, that the scab functions primarily to provide stability to the wound site and help restore tissue integrity when both the epidermis and overlying cuticle are damaged during wounding. While the stages of wound healing outlined here occur at a particular time and place, these results suggest that each stage is controlled by distinct genetic programs and signaling pathways triggered by the wound. Since many aspects of the fly wound response resemble those in mammals, it's likely that the molecular components are also shared. That makes identifying the molecular underpinnings of wound healing a high research priority. And thanks to the powerful system presented here, this task should be all the easier.

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555592

Health care reform and job satisfaction of primary health care physicians in Lithuania

Background The aim of this research paper is to study job satisfaction of physicians and general practitioners at primary health care institutions during the health care reform in Lithuania. Methods Self-administrated anonymous questionnaires were distributed to all physicians and general practitioners (N = 243, response rate – 78.6%), working at Kaunas primary health care level establishments, in October – December 2003. Results 15 men (7.9%) and 176 women (92.1%) participated in the research, among which 133 (69.6%) were GPs and 58 (30.4%) physicians. Respondents claimed to have chosen to become doctors, as other professions were of no interest to them. Total job satisfaction of the respondents was 4.74 point (on a 7 point scale). Besides 75.5% of the respondents said they would not recommend their children to choose a PHC level doctor's profession. The survey also showed that the respondents were most satisfied with the level of autonomy they get at work – 5.28, relationship with colleagues – 5.06, and management quality – 5.04, while compensation (2.09), social status (3.36), and workload (3.93) turned to be causing the highest dissatisfaction among the respondents. The strongest correlation (Spearmen's ratio) was observed between total job satisfaction and such factors as the level of autonomy – 0.566, workload – 0.452, and GP's social status – 0.458. Conclusion Total job satisfaction of doctors working at primary health care establishments in Lithuania is relatively low, and compensation, social status, and workload are among the key factors that condition PHC doctors' dissatisfaction with their job.

Background In 1989, after regaining its independence, Lithuania inherited an extremely centralised health care system that mainly conditioned ineffective health care management and resource usage. The above situation was an outcome of the former Soviet health care policy, as it did not encourage people or the state to treasure or safeguard its citizens' health [ 1 ]. The Soviet model of medicine that existed in Lithuania was based exclusively on the exaggerated focus and development of the hospital level, whereas the need to develop the primary health care (PHC) level was practically ignored. The situation has changed a lot though, and the current Lithuanian health policy states that priority shall be given to the development of primary health care [ 3 , 2 ]. Speaking of the very concept of primary health care, it should be stated that it first was offered in 1978 in Alma- Ata, Kazakhstan. Later on the primary health care concept, policy and strategy were further developed in such documents as "Health to Everybody – 2000" and "Health in the 21 st Century" [ 3 ]. As regards the health care policy, the above documents are considered of utmost importance in Lithuania as well as other EU countries. During the health care reform, the Lithuanian government came to a decision that the greater part of the health care budget should be allocated to primary health care establishments – consolidation of their basis and improvement of respective personnel qualification, which was expected to lead to a rise in the latter's work efficiency. In 1992, the training programme of general practitioners (GPs) was renewed in Lithuanian higher medical schools, and in 1996 the GP Statute was ratified under Lithuanian Health Norm MN 14:1996. District physicians and paediatricians were retrained as GPs, which requires more thorough competence than being a physician, as GPs are supposed to be capable of consulting, diagnosing and providing primary level help in all area. They have to be competent in internal disease treatment and can send patients to secondary level specialists merely in exceptional cases. The number of general practitioners in Lithuania is increasing, and it amounted to 1,150 at the beginning of 2003 [ 4 ]. As stated in the announcements of the National Board of Health, the situation in the Lithuanian health system is changing; nevertheless, it faces a number of problems. 78.6 % (1,146 out of 1,458) of GP graduates in 1994–2001 were retrained from district physicians and paediatricians [ 5 ]. Another problematic issue is lack of experience in organising their activities. Furthermore, GPs are often delegated many additional functions, such as accounting, work organisation in their establishments, and co-ordination with other institutions. Considerable instability of the legal basis as well as environment makes a serious negative impact too. Besides a great part of those trained as GPs do not follow their professional career, as they are tempted by considerably higher salaries that are offered by pharmaceutical companies and possibilities of working abroad [ 5 ]. This tendency may heighten even more now after Lithuania has joined the European Union, as this offers doctors considerably more opportunities to leave the country. The "loss" of doctors has significant economic consequences as well, as training of qualified general practice physicians is rather costly (a two year internship of one GP costs the state about EUR 8,700); therefore, it is essential to retain qualified specialists in the country. General medicine practice in health care systems of industrialised countries is considered a remarkably well working system that seeks to assess health needs of individuals and to become familiar with their living conditions, taking into consideration their family and social environment [ 6 ]. Nevertheless, recent research shows that "the crisis of general medicine" in the West is being widely discussed [ 6 ]. In the contemporary era of specialised medicine, the nature of GP's work is a direct opposite of specialisation: in terms of pathology, it covers all areas; in terms of diagnostics, GPs do not have the required equipment to make a diagnosis; working time is unlimited (from 54.1 to 62.2 hours per week); GPs constantly have to negotiate with patients the medicine to be prescribed and are high dependability on patients (calls to homes are not planned). Furthermore, 30 – 60% of cases in GP's work make psychosomatic problems of patients, which requires more psychological rather than medical knowledge. A GP is assessed not only as a specialist, as the above-mentioned criteria tend to be predominant. One of the ways to look into the existing PHC situation and possible problems is to study job satisfaction of GPs and physicians, as the attitudes people hold toward their jobs are referred to as job satisfaction and this is one of the most widely studied work-related attitudes. Job satisfaction is closely related to the lower level employee turnover and intentions to quit their jobs [ 7 - 9 ]. Since there is a lack of general practice physicians in Lithuania, it is vital to build conditions that would motivate them to stay and work in the Lithuanian health care system, which may be accomplished through the enhancement of job satisfaction at the primary health care level. Job satisfaction is highly important in building up employee motivation and efficiency, as higher job satisfaction determines better employee performance and higher level of patient satisfaction [ 9 ]. The aim of this research is to investigate job satisfaction of physicians and general practitioners in primary health care institutions during the health care reform in Lithuania. In 2003, there were 1,608 GPs and physicians working in Lithuanian PHC establishments [ 4 ]. The largest number of primary health care institutions were in Kaunas, the second largest city of Lithuania, (27% of all Lithuanian PHC institutions), and GPs provided services to 42% of the city's population. This research studies GPs and physicians who working in Kaunas. Research tasks: 1. To determine motives for choosing a PHC doctor's profession. 2. To compare total job satisfaction of Lithuanian PHC physicians' and GPs' as well as their intentions to recommend their children to choose a PHC level doctor's profession. 3. To assess factors which determine job satisfaction of PHC physicians, GPs who chose their specialization at higher school, and GPs who were retrained as GPs later in their career. Review of research on job satisfaction in health care institutions Job satisfaction comprises positive and/or negative attitudes held by individuals in respect to their job [ 10 ]. Employees with higher job satisfaction believe that the organisation will continue satisfying them for a long time; they care about the quality of their work, are more committed to the organisation, stay in it longer, are more productive, feel responsible for the working environment, and strive to make it satisfying [ 6 - 8 ]. Job satisfaction reduces employee turnover, absenteeism, and the number of thefts at work, which in turn reduces organisational costs [ 10 ]. Job satisfaction is closely related to the nature of work, the quality of management and working environment. Not only does it influence good employee performance, it also maintains good employee health and longevity. As to the most valued aspects of satisfaction, they comprise such things as compensation, promotion opportunities, fringe benefits, bonuses, management, co-workers, working conditions, nature of work, communication, security [ 11 , 12 ]. Bronsky and Cook (1994) interviewed 334 Ohayo University graduate physicians [ 13 ]. For the interviews, they used Job Discriminative Index questionnaire, in which satisfaction is assessed according to five sub-scales ("work", "co-workers", "management", "current reward" and "promotion opportunities"). The research showed that job satisfaction is not directly related to practical aspects of physicians. In 1989 American Physical Therapeutics Association (APTA) carried out a survey of hiring and retention aspects [ 14 ]. This study showed that physicians were satisfied with their work. The highest job satisfaction was gained from "autonomy", whereas the smallest was given by "reward". Okerlund et all (1994) research revealed that Utah physicians were satisfied with their job [ 15 ]. Respondents indicated the followings factors as the most important and having the greatest effect on their job satisfaction: "working freedom", "assistance in skills development", and "salary and fringe benefits". The survey also showed that the key factors of doctors' willingness to leave their practice comprised such things as big clerical workload, dissatisfaction with reforms, high patient expectations and big clinical workload. While young doctors willing to leave practice mainly emphasised communication problems and big clinical workload, older doctors were mainly discontent with changes in the health care system and big clerical workload. Differences between genders were insignificant. Rozier' et al (1998) found out that intrinsic factors, such as ethical practice, improvement of patient health, and perception of satisfaction were more important than big salary and important title [ 16 ]. These groups of factors were significantly affected by the gender factor, i. e. how employees combine work and family responsibilities. Barnes (1998) conducted research of job satisfaction among rehabilitation physicians, occupational therapy specialists, and speech pathologists. Set apart, there are three factors that describe job satisfaction: external context factors and internal context factors, as well as internal content factors [ 17 ]. External factors that affect job satisfaction comprise a competitive salary and additional rewards such as release from work due to family matters, flexible work schedule, and child raising support. The stated internal context factors were "less substantial but inherent to work". These could be factors that are controlled by external forces and directly affect internal employee satisfaction, such as adequate working hours and workload, stable working environment, and support of administration. Barnes also pointed out internal content factors that, first of all, are controlled by the professional himself/herself and that may affect employee's competence and feeling of effectiveness in the organisation. The author concludes that internal factors have more influence on employee career satisfaction than external. Speakman et al (1996) conducted a study of 106 Texas (USA) doctors' job satisfaction [ 18 ]. Respondents stated that their work was "a challenge" in a positive sense: it enabled them to use their capabilities and was stimulating. The doctors also pointed out that they were given sufficient autonomy at work and independence in decision-making, and were able to learn and improve their work. The doctors, however, were dissatisfied with the clerical aspect of their work. Dowell et al (2000) conducted research, the purpose of which was to examine job satisfaction and stress in the work of GPs in New Zealand. Their job satisfaction level was rather high [ 19 ]. However, rural doctors were less satisfied with their job than those working in cities. Besides GPs who worked individually were less satisfied with their work than group practice GPs. 177 GPs (46%) stated that work affected their physical health, and 220 (57%) stated that they had been thinking about quitting general practice. The main causes of stress and dissatisfaction were as follows: too much bureaucracy and restrictions, health care reforms, long working hours, and work "by the telephone". Methods Sample Research was conducted in October – December 2003. Questionnaires were distributed to all Kaunas PHC physicians and GPs (N = 243) in person. The response rate amounted to 78.6 % (191 questionnaire returned). Questionnaire Development Total job satisfaction of the respondents was measured on a seven-point scale, where 7 stood for highly satisfied and 1 for highly dissatisfied. Intentions to recommend one's children to choose a PHC level doctor's profession were measured on a seven-point scale, where 7 stood for "strongly agree" and 1 for "strongly disagree". The level of satisfaction with different job characteristics was assessed using a job satisfaction questionnaire that embraced questions related to the PHC work specifics. Eight sections of questions were included in order to assess satisfaction with different job characteristics: ability utilisation at work, workload, colleagues, compensation, creativity, autonomy, management, and social status. All statements were measured on a seven-point scale, where 7 stood for highly satisfied and 1 for highly dissatisfied. The survey data was processed using SPSS version 11.00 statistical package for data analysis. The statistical data reliability was checked according to χ 2 criteria, degrees of freedom number (df) and statistical significance. Relationship between two independent variables was assessed relying on Spearmen's rank correlation, taking into consideration the value of the correlation ratio and statistical significance. Reliability notation: p < 0.05 – statistically significant, p < 0.01 – highly significant. Results Demographic characteristics of the respondents 15 men (7.9%) and 176 women (92.1%) participated in the research, among which 133 (69.6%) GPs and 58 (30.4%) physicians. The analysis of the distribution of the respondents according to their age and specialization (see Table 1 ) revealed that GP and physician distribution according to their age statistically varied: the percentage of GPs in younger age groups (25–34, 35–44, 45–54 years) is higher than that of physicians. The reason for this is that the specialisation of physicians is no longer included into higher school programmes; only GPs are trained there, and increasingly more young doctors retrain from internists to GPs [ 5 ]. Comparing the data of January 1998 and December 2002, we can see that the number of GPs increased by 6.6 times, whereas the number of physicians decreased by 42.6%. Table 1 Characteristics of the respondents Age group (years) Specialisation Total GPs* Physicians* n % n % n % 25–34 23 17.3 1 1.7 24 12.6 35–44 28 21.1 8 13.8 36 18.8 45–54 73 54.9 16 27.6 89 46.6 55–64 9 6.8 26 44.8 35 18.3 65 and more - - 7 12.1 7 3.7 Total 133 100.0 58 100.0 191 100.0 * - χ 2 = 63.360; df = 4; p = 0.000 Motives of choosing phc doctor's profession With the health care reform underway, it is of great importance to determine how GPs became who they are, and what motives made them chose this specialisation. During the survey, the respondents were divided into 3 groups: the first group comprised GPs who had requalified from other specialisations, the second – GPs who chose this specialisation while studying at higher school, and the third one comprised physicians. In all three groups, doctors admitted to have chosen their profession mostly because no other profession was of interest to them. This motive was indicated by 80% of GPs who chose their specialisation at higher school, 63.4% of GPs who changed their qualification, and 77.4% of physicians. The dominating motive of "no other speciality being of interest" can be explained by the fact that the work of a doctor is easiest to imagine and perceive. Total job satisfaction and intentions to recommend one's children to choose a phc level doctor's profession Total job satisfaction of the respondents was 4.74 point. Recommending a profession to one's own child makes an important factor that shows if an employee is satisfied with his/her work or not. Most respondents (75.5%) do not intend to recommend their children to choose a PHC level doctor's profession. The assessment of the respondents' general satisfaction with work and their intentions to recommend their children to choose a primary level doctor's profession did not reveal any statistically significant differences between the specialisations (p > 0.05). However, the research revealed a tendency among physicians to be generally more satisfied with their work and more willing to recommend their children to choose a PHC level doctor's profession: total job satisfaction of physicians was 5.03 and intentions to recommend their children to follow their career comprised 2.56, while those of GPs respectively made 4.64 and 2.19. Satisfaction with different job characteristics The study of the satisfaction level with different job characteristics shows that respondents were most satisfied with autonomy at work – 5.28, relationship with colleagues – 5.06, and management quality – 5.04 (see Table 2 ), while compensation (2.09), social status (3.36), and work load (3.93) turned to be causing the highest dissatisfaction among the respondents. Statistically significant differences of satisfaction between the three categories of respondents were determined with two job characteristics, i.e. compensation and social status. GPs who chose specialisation at higher school were more satisfied with compensation (2.68) and social status (4.03) than GPs who changed their qualification (respectively 2.00 and 3.09) and physicians (respectively 1.90 and 3.45) (see Table 2 ). Table 2 Means score of satisfaction with job characteristics according to specialization and its choice conditions Job characteristics Specialisation and its choice Mean score of satisfaction (standard deviation) GPs who have requalified (standard deviation) GPs who chose specialisation at higher school (standard deviation) Physicians (standard deviation) Ability utilisation at work 4.79 (1.50) 4.51 (1.60) 5.18 (1.56) 4.84 (1.54) Volume of work 3.69 (2.00) 4.29 (1.77) 4.24 (2.11) 3.93 (2.00) Co-workers 5.05 (1.66) 5.26 (1.75) 4.92 (1.68) 5.06 (1.68) Compensation* 2.00 (1.49) 2.68 (1.80) 1.90 (1.250) 2.09 (1.51) Creativity 3.96 (1.66) 4.74 (1.80) 4.30 (1.83) 4.19 (1.75) Autonomy 5.12 (1.65) 5.23 (1.62) 5.65 (1.25) 5.28 (1.57) Management 4.86 (1.78) 5.17 (1.74) 5.30 (1.84) 5.04 (1.79) Social status* 3.09 (1.76) 4.03 (2.01) 3.45 (1.85) 3.36 (1.85) * – p < 0.05 In order to determine the main factors that cause satisfaction and/or dissatisfaction with work, the relationship between total job satisfaction and job characteristics was analysed. Calculations of Spearmen's ratios revealed the strongest correlation between total job satisfaction and such characteristics as autonomy at work (0.566), workload (0.452) and GP's social status (0.458) (see Table 3 ). When studying the relationship between the intention to recommend one's children to choose a PHC level doctor's profession and averages of satisfaction with job characteristics, the strongest correlation was again found between the intention to recommend one's children to choose a PHC level doctor's profession and autonomy at work (0.371) as well as GP's social status (0.329). Table 3 Spearmen's ratio between job characteristics and total job satisfaction, and intention to recommend one's children to choose a GP's profession Job characteristics Total job satisfaction Intention to recommend one's own children to choose a GP's profession Ability utilisation at work 0.402** 0.241** Volume of work 0.452** 0.228** Co-workers 0.349** 0.239** Compensation 0.240** 0.255** Creativity 0.383** 0.151* Autonomy 0.566** 0.371** Management 0.358** 0.175* Social status 0.458** 0.329** ** – p < 0.01; * – p < 0.05 Discussions and conclusions Taking into consideration PHC level doctors' satisfaction with various aspects of work, a conclusion can be drawn that the health care reform that is being implemented meantime and that makes general practice physician the key figure and which should encourage expansion of GP institution is channelled in the right direction. Relying on the data of the conducted research, GPs who chose specialisation while studying at higher school are more satisfied with some aspects of work than physicians and GPs who requalied. However total job satisfaction of doctors who work in the primary health care is relatively low (4.74 on a 7 point scale). Doctors who have worked for 30 – 39 years are most satisfied with their job. Total job satisfaction of GPs and physicians does not have statistically significant difference. Most PHC level doctors (75.5%) do not intend to recommend their children to follow their career. Such characteristics as autonomy at work, social status, and workload have the biggest impact on total job satisfaction. Stamps and Piedmonte's (2000) research shows that the more autonomy activities possess and the less monotony exists at work, and the more employees are satisfied with their work [ 20 ]. The research revealed that compensation, social status, and workload are among the key factors causing PHC doctors' dissatisfaction with their jobs. Thus it can be assumed that the above factors condition low total job satisfaction. In conclusion it can be stated that the nature of a PHC doctor's work and rather low salaries condition relatively low job satisfaction among Lithuanian PHC doctors. Competing interests The author(s) declare that they have no competing interests. Authors' contributions These authors contributed equally to this work. Pre-publication history The pre-publication history for this paper can be accessed here:

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555592.xml

555590

Computational verification of protein-protein interactions by orthologous co-expression

Background High-throughput methods identify an overwhelming number of protein-protein interactions. However, the limited accuracy of these methods results in the false identification of many spurious interactions. Accordingly, the resulting interactions are regarded as hypothetical and computational methods are needed to increase their confidence. Several methods have recently been suggested for this purpose including co-expression as a confidence measure for interacting proteins, but their performance is still quite poor. Results We introduce a novel computational method for verification of protein-protein interactions based on the co-expression of orthologs of interacting partners. The performance of our method is analysed using known S. cerevisiae interactions, and is shown to overcome limitations of previous methods. We present specific examples of known and putative interactions that are detected by our method and not by previous methods, and suggest that they represent transient interactions that might have been conserved and stabilized in other species. Conclusion Co-expression of orthologous protein-pairs can be used to increase the confidence of hypothetical protein-protein interactions in S. cerevisiae as well as in other species. This approach may be especially useful for species with no available expression profiles and for transient interactions.

Background Protein-protein interactions (PPIs) have a central role in most biological processes, and identifying these interactions is an important goal of biological research. PPIs are the subject of extensive experimental studies, but the majority of them remain unknown. In the last few years, high-throughput techniques were developed for the identification of PPIs on a genomic scale. Yeast two-hybrid [ 1 , 2 ] and mass spectrometric analysis of protein complexes [ 3 , 4 ] were used to produce large sets of PPIs. However, these techniques are known to suffer from many false positives and the resulting PPIs are typically regarded as putative [ 5 , 6 ]. Thus, the development of computational methods for assessment and verification of putative PPIs is crucial [ 7 - 10 ]. Two such methods were proposed, that are based on the co-expression [ 11 ] and conservation [ 9 ] of PPIs, respectively. Here we propose to extend these methods by considering co-expression of orthologous protein pairs. We demonstrate the predictive power of our approach and discuss its advantages. Results Verification by mRNA co-expression It was previously shown that interacting pairs of proteins are often correlated in their expression profiles [ 11 , 12 ]. The correlation of expression profiles was therefore proposed as a confidence measure for putative PPIs [ 7 , 10 , 13 ]. However, this approach has three major limitations. First, many pairs of non-interacting proteins are also co-expressed (false positives). Second, many pairs of interacting proteins are not co-expressed (false negatives). Third, to properly determine co-expression, mRNA expression profiles from a large and diverse set of conditions are needed, rendering this approach inapplicable for most organisms. Former studies that used co-expression to identify PPIs did not explicitly examine its predictive power, or did not use a random set of protein-pairs as control for evaluating its performance. We thus carried out an analysis to evaluate the predictive power of this approach for S. cerevisiae , in order to later compare it to our new method. High quality S. cerevisiae expression data is available for many conditions, making it an ideal organism for the use of co-expression for validation of PPIs. We extracted a reference set of 1656 known interaction from the MIPS database [ 14 ], and generated a random set by randomly choosing pairs of proteins. Cosine correlation over our entire set of S. cerevisiae conditions was used to compare the levels of co-expression between the reference set and the random set (see methods). The results of this analysis are summarized in Figure 1 . The cumulative distributions of expression correlations in both sets are compared, showing higher degrees of co-expression in the reference set than in the random set (Figure 1a ). The resulting predictive power is shown in Figure 1b , where each dot represents a possible correlation threshold for PPIs prediction. The percentages of protein-pairs passing each threshold from the random and reference sets are shown in the horizontal and vertical axes, respectively. For example, the threshold shown in Figure 1 (0.155) which leads to the correct verification of 30% of the reference set (497 true positives), results also in the false verification of approximately 9% of the random set (~149 false positives). Applying this to a set of putative PPIs with 50% false positives (as estimated for the S. cerevisiae yeast two hybrid sets [ 5 , 6 ]) results in a filtered subset with approximately 23% false positives (9% divided by 39%). We verified that the performance of this method is largely independent of the exact set of conditions that is used, and that filtering the conditions or choosing them specifically for each pair of proteins does not improve the performance (not shown). Conservation of PPIs Another approach that was proposed to verify or predict PPIs is based on conservation of interactions [ 9 , 15 , 16 ]. In this approach (termed "interologs"), pairs of proteins whose orthologs are known to interact in other species are assumed to interact. Such a method can potentially reveal many conserved PPIs, but it is currently limited by the availability and accuracy of interaction data. Without relying on putative interactions, the available set of S. cerevisiae PPIs only correspond to a small fraction of the biologically meaningful interactions, and the situation is much worse for other species. Consequently, this method has so far been based only on S. cerevisia e PPIs, including putative ones, to predict interactions in other organisms. Giot et al. used putative S. cerevisia e PPIs from mass spectrometric analysis to verify Drosophila PPIs found by yeast two-hybrid. Only 65 out of the ~2000 Drosophila putative interactions were identified as having an orthologous interaction in S. cerevisia e. This set was then used to train a statistical model for assignment of confidence scores to putative PPIs. Li et al. used putative S. cerevisia e PPIs gathered from several sources to predict C. elegans PPIs (rather than verify an existing set of putative PPIs). Out of the 5534 predicted C. elegans PPIs, only 949 were identified as having an orthologous interaction in S. cerevisia e [ 16 ]. The use of conserved interactions to verify a putative set of PPIs is therefore very limited, since only a small fraction of the putative set would have a known orthologous interaction. Furthermore, using putative PPIs in order to increase the coverage of this approach will decrease its accuracy and introduce many more false positives. Orthologous co-expression Motivation In order to overcome the limitations of the two methods described above, we propose to integrate them and detect PPIs by orthologous co-expression, i.e. co-expression of the orthologs of the interacting partners (Figure 2a ). A conserved interaction may be co-expressed only in a subset of the organisms in which it is present, so combining knowledge of co-expression from multiple organisms can be informative. The use of orthologous co-expression for verification of PPIs is also supported by three previous observations. First, in order to preserve their interaction and functionality, interacting partners should co-evolve [ 17 ]. Sequence analysis was previously used to uncover co-evolution at the sequence level [ 18 ], but it may also be present at the level of gene expression. Second, as shown in two recent papers, co-expression of functionally linked proteins is more likely to be conserved than the co-expression of random pairs of proteins [ 19 , 20 ]. Hence, orthologous co-expression can replace co-expression, and serve as a better measure to identify functional links in general and PPIs in particular. Third, interacting protein-pairs are more likely to have pairs of orthologs in other species than randomly selected protein-pairs. This observation was made previously for different ascomycota species [ 21 ], and can also be seen in our analysis of more distant organisms (Figure 2b ). Since orthologous co-expression can only be computed for conserved protein-pairs, the increased conservation of interacting protein-pairs will also increase the percentage of interacting pairs where orthologous co-expression can be computed, and lead to higher percentage of real PPIs out of the total predicted protein-pairs. Performance To examine whether orthologous co-expression can indeed be used to predict PPIs, we focused on S. cerevisia e orthologs from five species ( C. elegans, E. coli, A. thaliana, D. melanogaster , and H. sapiens ). Orthologous pairs of the protein-pairs in the reference and random sets were identified by BLAST [ 22 ], and their co-expression was measured using cosine correlation over the entire sets of mRNA expression data (see methods). Co-expression values of the random set orthologs in each organism were used to determine the 5% significance correlation thresholds. The percentage of interactions with significant orthologous co-expression in each organism (out of all the interactions where orthologous co-expression can be computed, i.e. interactions with both orthologs and expression profiles at that organism) is shown in figure 2c . Indeed, for all five organisms we found that orthologous-pairs of known PPIs are more likely to be co-expressed than that of random protein-pairs. Interestingly, the percentages of orthologous-pairs of PPIs with significant co-expression in E. coli and D. melanogaster are even higher than the percentage of PPIs with significant co-expression in S. cerevisiae (Figure 2c ). Note, however, that less than 3% of the reference set had orthologous-pairs in E. coli and orthologous co-expression was computed only for 38 PPIs, so the high E. coli value might be a result of insufficient statistics. The ability to predict PPIs by orthologous co-expression strongly depends on the percentage of interactions where orthologous co-expression can be computed (i.e. where both proteins are conserved and have expression profiles), so the percentages of PPIs that can be predicted by each organism is lower than 7% for all five organisms (Figure 2c ). To overcome the lower coverage of each organism we combined the information from all five organisms. We examined the predictive power of this approach by repeating the analysis shown in Figure 1 , when the yeast co-expression is replaced by the sum of the orthologous co-expression from the five other species (figure 2d ). To avoid over-fitting, we only considered simple summation of the co-expression in different species. Notably, although S. cerevisiae co-expression was omitted from the analysis, the predictive power of this approach was better than that of S. cerevisiae co-expression alone (Figure 2d ). Combining S. cerevisiae and orthologous co-expression The correlation between S. cerevisiae co-expression and orthologous co-expression of the true interactions in the test set is only 0.34. This means that the two methods are complementary, and that except for detecting interactions between co-expressed proteins, orthologous co-expression can also detect interactions between proteins that are not co-expressed in S. cerevisiae , but their corresponding orthologous are co-expressed in other species. Examples of known interactions from the test set with low co-expression in S. cerevisiae but high orthologous co-expression are shown in Table 1 . In these 30 cases, the co-expression in S. cerevisiae is very low or even negative, but the orthologous co-expression is high in at least two species, such that they are easily detected by our approach. Based on the complementarities of the two methods, namely S. cerevisiae and orthologous co-expression, we proceeded by adding the orthologous co-expression to S. cerevisiae co-expression (figure 2d ). The addition significantly improved the results of both methods. Using the same example as mentioned above, the percentage of protein-pairs identified from the random set is reduced from 9% to 5%, while the percentage of proteins-pairs identified from the reference set remained 30%. Transient interactions In a previous study relating gene expression to PPIs, Jansen et al. classified protein complexes as 'permanent' and 'transient' [ 12 ]. The subunits of permanent complexes were shown to be highly co-expressed, in contrast to transient complexes where co-expression was very low. Transient interactions are therefore harder to detect by co-expression as well as by experimental methods. To test the performance of our method on transient interactions we examined the nine protein complexes classified as transient: pre-replication complex, replication complex, anaphase promoting complex (APC), TAFIIs, SAGA complex (Spt-Ada-Gcn5-acetyltransferase), CCR4 complex, RSC complex, SRB complex (kornberg's mediator) and SWI/SNF complex. Assuming all pair-wise interactions in these complexes, we compared the percentage of protein-pairs with significant S. cerevisiae or orthologous co-expression for each complex and for the combined set (Figure 3a ). Orthologous co-expression is slightly better than S. cerevisiae co-expression at identifying interactions in the reference set, but the differences in performance increase considerably when transient complexes are examined. In the combined set of 764 transient interactions, orthologous co-expression identifies almost three times (2.68) more interactions than S. cerevisiae co-expression. Moreover, for five out of the nine transient complexes, orthologous co-expression identifies at least three times more interactions than S. cerevisiae co-expression, while the opposite occurs only in one complex – RSC, which is also the smallest complex examined. These results suggest that orthologous co-expression is especially useful for detection of transient interactions. Specialization of interacting proteins can lead to high orthologous co-expression Why are there interacting protein-pairs which are not co-expressed in S. cerevisiae , while their corresponding orthologs are co-expressed in other species (Table 1 ; Figure 3a )? The observation that interacting protein-pairs are co-expressed is believed to be a result of their need to be present in similar amounts at different conditions. However, for transient interactions occurring only in specific processes, this requirement might affect only a small number of conditions, and hence might have a slight influence on the global levels of co-expression. In contrast, the orthologs of such interacting proteins might have adopted a stable interaction, resulting in co-expression at many conditions. Such transient interactions will not be detected by co-expression, and might also be hard to find using experimental methods, but orthologous co-expression may help to identify them. Moreover, one of the interacting proteins may be multifunctional, interacting with several proteins depending on context. The expression of such pleiotropic proteins is likely to be constitutive, and will not show correlation to that of its interacting partners. However, the pleiotropic protein might have several specialized orthologs in other species, each performing distinct functions, and co-expressed with the corresponding orthologs (Figure 3b ). Note that in such cases the specialized ortholog may not be the closest one in sequence. However, allowing each protein to have multiple orthologs and choosing the maximal correlation can also increase the orthologous co-expression of false interactions. Consequently, such an approach only reduced the performance of our method (not shown). Specific examples To examine if specialization of interacting proteins can account for the high orthologous co-expression of protein pairs in Table 1 and in the transient complexes, we looked in more details at specific examples. Here we provide three examples supporting this notion. 1. CDC28 is the only cyclin-dependent kinase (CDK) in S. cerevisiae involved in cell cycle transitions [ 23 ]. CDC28 interacts with different proteins at different stages of the cell cycle, including G1 and B-type cyclins ( CLNs and CLBs , respectively) and CDC6 . Indeed, no detectable co-expression is found between CDC28 and its interacting partners (Table 1 ; not shown for CLNs ). In contrast, CDC28 has several orthologs in higher eukaryotes (up to five distinct CDKs in mammals), each devoted to specific processes or tissues [ 23 ], and the orthologs that were found by our analysis in H. sapiens , D. melanogaster and C. elegans (CDK2, CDC2 and CDK-1 , respectively) are highly co-expressed with the corresponding orthologs of CDC6 and the B-type cyclins (Table 1 ). 2. Yeast TAF5 is a component of at least two transient complexes, the general transcription factor TFIID and the SAGA complex [ 24 ]. However, its human ortholog ( TAF5 ) is only known to be a part of the TFIID complex, while a second ortholog ( TAF5L ) is known to be in both TFIID, and the human equivalent of SAGA [ 25 ]. As expected, the co-expression of human TAF5 and the other proteins in human TFIID is higher than that of yeast TAF5 and the other proteins in yeast TFIID (not shown). 3. The opposite case of two S. cerevisiae paralogs with only one ortholog in higher eukaryotes, though less common, may also help to identify PPIs. The nascent polypeptide associated complex (NAC), consists of an alpha subunit ( EGD2 ) and a beta subunit (either EGD1 or BTT1 ) [ 26 ]. BTT1 is not co-expressed with EGD2 , presumably since EGD1 and BTT1 are alternating beta subunits that bind both the ribosome and the alpha subunit ( EGD2 ). In contrast, D. melanogaster and C. elegans have only one known orthologous beta subunit, which are co-expressed with the corresponding orthologs of EGD2 (Table 1 ). Predictions Table 2 shows examples of low confidence putative interactions with low co-expression but high orthologous co-expression. These interactions were found by high-throughput yeast two-hybrid [ 1 ], and considered low confidence (they had less than 3 interaction sequence tags and were not included in the core data; also not supported by co-expression). However, in light of the high orthologous co-expression from at least two species, we predict that they represent true interactions. In support of that, both proteins in all these examples are localized to the same cellular compartment (according to the MIPS database [ 14 ]). Some of these proposed interactions might also fit the model in Figure 3 . For example, SMT3 is the only SUMO gene in S. cerevisiae , which is known to modify TOP2 (DNA Topoisomerase II) and other proteins [ 27 ]. However, in vertebrates there are three known SUMO genes: SUMO1 , SUMO2 , and SUMO3 . As suggested by the model in Figure 3 , SMT3 is not co-expressed with TOP2 , but one of its human orthologs ( SUMO1 ), is highly co-expressed with the human ortholog of TOP2 ( TOP2A ; see Table 2 ). Discussion We presented here a new computational method for verification of PPIs that is based on the co-expression of orthologous protein-pairs, and demonstrated its predictive power using PPIs identified in S. cerevisiae . This method extends two of the former methods, namely co-expression of interacting proteins and conservation of interactions (interologs). The first method can only be applied to organisms with expression data and its performance depends on the amount and quality of that data. Our method overcomes this limitation by integrating sequence and expression data from other organisms. It can thus be applied to any sequenced organism, particularly for those without available expression data, thereby replacing the missing data. Moreover, it performs better than the former method even for S. cerevisiae , where many high quality expression data is available, and is especially better in identifying transient interactions. It is difficult to evaluate our approach for other species, since we do not have large representative sets of known interactions, but the success in yeast is promising. The proposed method also overcomes the limitation of the interologs approach, namely the small fraction of interactions that is known to date. Our method uses expression rather than interaction data, which makes it capable of giving evidence for a much larger number of interactions. mRNA expression profiles are being generated by many different labs for a wide range of organisms. The improved quality of existing expression profiles as well as the addition of profiles for other organisms will improve the performance of our method. Further improvements can be achieved by giving different weights to the co-expression from different organisms (not shown). A weight can be given to each organism according to the reliability of its expression profiles, or according to its evolutionary distance from the studied organism. During the writing of this manuscript, a related approach was suggested [ 28 ]. Based on the codon adaptation index (CAI) as an estimator for average expression levels, Fraser et al. examined co-evolution of expression levels from four fungi closely related to S. cerevisiae , and used that to predict PPIs in S. cerevisiae . This approach is complementary to the one that we have proposed. Thus, mRNA expression should be used directly when possible, even from relatively distant species (such as D. melanogaster ), and CAI should be used from closely related species without available expression data. Finally, the methods described here are still not accurate enough to verify specific PPIs, but they provide additional evidences and are useful for assessment and filtering of high-throughput PPIs data sets, in order to produce smaller sets of higher confidence, and direct further investigations. Complementary methods should be combined to create a general scheme for verification of putative PPIs, for example by considering only those interactions that are verified by at least two or three methods [ 7 ] or using supervised machine learning approaches [ 29 ], thus improving the performance of each method alone. Conclusion We have shown that expression data from multiple organisms can be used to increase the confidence of hypothetical PPIs by considering co-expression of orthologs of the presumed interacting partners. For organisms such as S. cerevisiae , with highly characterized expression profiles, orthologous co-expression may be combined with co-expression of the actual proteins, whereas for other, less studied organisms, it may replace the missing expression profiles. Notably, this method is especially useful for detection of transient interactions which presents a known weakness of most prediction methods. The success of this method also implies that PPIs tend to be conserved in different organisms, even as distant as yeast and human, further supporting the use of comparative approaches in proteomics. Methods Interactions sets – a reference set of S. cerevisiae interactions was extracted from the MIPS (Munich Information Center for Protein Sequences) PPI database [ 14 ] at 22/01/04. We excluded genetic interactions, self-interaction, interactions found by high-throughput experiments, interactions without expression data, and redundancies, resulting in a set of 1656 interactions. We did not use larger databases such as the one compiled by von Mering et al. [ 7 ] since they are more likely to contain false interactions and are also biased towards co-expression since this information was used in their construction. Randomly generated set of the same size was used as control, and averaged over ten trials. Self-interactions were excluded from the random set. The random set may include real interaction, but their expected frequency is much less than 1%. Transient complexes were taken from Jansen et al. [ 12 ]. The transient set was constructed by combining the pair-wise interactions from each transient complexes and removing redundancies (some protein pairs were present in more than one complex). mRNA expression data – datasets for six organisms were collected from different sources, as described in [ 19 ], and can be downloaded from our home page [ 30 ]. All datasets were normalized to have a mean of 0 and standard deviation of 1 for each condition. Expression correlation – cosine correlation over the entire expression data of each organism was used as a measure of co-expression. Former analysis suggested that cosine correlation is the optimal measure of co-expression for the purpose of detecting PPIs [ 13 ]. Many genes in all six organisms have missing values in the expression data, so the expression correlations of many orthologous pairs cannot be calculated. To decrease the dependency of our approach in the availability of expression data and to improve its performance, we replace the missing correlations by estimated values. We used the corresponding yeast co-expression when the yeast and orthologous co-expression are combined (green curve in figure 2d ). In contrast, when orthologous co-expression is used alone (red curve in figure 2d ), the yeast expression data is assumed to be unavailable (in order to show the applicability of the method to organisms without expression data) and an expected correlation is calculated for each species, based on the union of the reference and random sets (average expression correlation of orthologous pairs in a specific species, over the reference and random sets combined with equal weights). The expected correlations are greater than zero for all five species; so putative PPIs are actually given positive scores for the existence of an orthologous pair, corresponding to the notion that PPIs are more likely to have pairs of orthologs [ 21 ]. Orthologous proteins – orthologs were found using blastp [ 22 ] with a P-value threshold of 10 -7 , and alignment length threshold of 0.3. The ortholog with the most significant p-value that had available expression data was used to measure co-expression. Other studies had used a reciprocal best-hit BLAST search for finding orthologous; we use a less strict criterion in order to apply the orthologous co-expression method to more protein-pairs. P-values and Significance – by sampling 100,000 protein pairs we determined p-values for S. cerevisiae and orthologous co-expression as the fraction of pairs with equal or greater correlation of expression profiles; P-values of 0.05 (not corrected for multiple testing) were used as thresholds for significance.

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517951

Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification

Sequence-based typing (SBT) is one of the most comprehensive methods utilized for HLA typing. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations which occur when two or more different allele combinations produce identical sequences. The purpose of this study is to investigate the probability of this occurrence. We performed HLA-A,-B SBT for Exons 2 and 3 on 676 donors. Samples were analyzed with a capillary sequencer. The racial distribution of the donors was as follows: 615-Caucasian, 13-Asian, 23-African American, 17-Hispanic and 8-Unknown. 672 donors were analyzed for HLA-A locus ambiguities and 666 donors were analyzed for HLA-B locus ambiguities. At the HLA-A locus a total of 548 total ambiguous allele combinations were identified (548/1344 = 41%). Most (278/548 = 51%) of these ambiguities were due to the fact that Exon 4 analysis was not performed. At the HLA-B locus 322 total ambiguous allele combinations were found (322/1332 = 24%). The HLA-B*07/08/15/27/35/44 antigens, common in Caucasians, produced a large portion of the ambiguities (279/322 = 87%). A large portion of HLA-A and B ambiguous allele combinations can be addressed by utilizing a group-specific primary amplification approach to produce an unambiguous homozygous sequence. Therefore, although the prevalence of ambiguous allele combinations is high, if the resolution of these ambiguities is clinically warranted, methods exist to compensate for this problem.

Introduction The precise identification of HLA Class I and Class II alleles is critical for successful hematopoietic progenitor transplants, the development of peptide based viral and cancer vaccines, and investigating immune response [ 1 , 2 ]. DNA sequencing is one of the most comprehensive methods available for HLA typing. Sequence-based typing (SBT) involves PCR amplification of specific coding regions of HLA genes and sequencing of the amplicons [ 3 , 4 ]. SBT allows for a detailed interpretation of HLA alleles by comparing nucleotide sequences of the polymorphic, and sometimes conserved, regions of the HLA gene to a database of possible allelic combinations. While SBT permits the highest resolution of genotypes, like all typing methods, it has limitations. One of the inherent problems when using SBT is the interpretation of ambiguous allele combinations which can occur for several reasons [ 5 - 7 ]. There exist two main types of ambiguous typing results obtained with SBT. The first is when a heterozygous sequence can be explained by more than one possible pair of alleles within the region analyzed. The second exists when alleles are defined by a polymorphism outside the region analyzed. In addition to these two situations, a third type of ambiguity arises when an allele has an incomplete sequence in the region analyzed. The prevalence of ambiguities in HLA typing relates to the nature of polymorphisms which exists in the sequence of the major histocompatibility complex (MHC) Class I and Class II genes. The majority of polymorphisms that distinguish one MHC allele from another are oftentimes due to gene conversion, recombination and exon shuffling events. Due to this, polymorphic motifs at given positions are generally shared among several alleles. Sequence-based typing involves PCR amplification and sequencing of specific HLA exons, which are known to be polymorphic, from genomic DNA. For each HLA locus both alleles are amplified and sequenced; therefore, it is not always possible to determine exactly which two alleles were responsible for sequence results. For example two or more different allele combinations can combine to produce identical sequences due to the heterozygous base pair combinations, the first type of ambiguity. More specifically, in the Class I region, HLA-B*070201, 3503 would have the same nucleotide sequence as HLA-B*0724, 3533 in positions 559 and 560 (Figure 1 ). In this example, the SBT produces a heterozygous base pair combination at positions 559 and 560 with an international union of biochemists (IUB) designation of K(G+C)W(A+T). Therefore, the interpretation to the high resolution level can not be made because it is not known which allele combination is correct. Figure 1 Two different HLA-B allele combinations that yield identical sequence based-typing results. The amplification and sequencing of exon 3 of HLA Class I from a HLA-B*070201/B*0724 subject and a HLA-B*3503/3533 subject produce the same results. Both G and C are detected at nucleotide 559 and both A and T at nucleotide 560. The second type of ambiguity relates to defining a polymorphism outside the region analyzed. For example, many HLA-A alleles are defined by a polymorphism located in Exon 4 (Table 3 ). Traditionally, for Class I typing most laboratories only sequence Exon 2 and Exon 3; for Class II typing most laboratories only sequence Exon 2. This approach has been the standard due to the functional relevance of this region which defines the peptide groove of Class I and Class II molecules, respectively. However, some Class I alleles have identical sequences across Exons 2 and 3. To resolve these alleles it is necessary to analyze the gene at the region where they differ. As DNA sequencing has become easier and more widely applied to defining HLA alleles, additional polymorphisms have been found in other exons, and also in the introns. Table 3 Specific A locus sequence-based typing ambiguities discovered during this study and the reason that the ambiguity occurs. 1 A*010101/04N 0104N is resolved as a C insertion at bp 628 in exon 4. 2 A*2402/09N/11N 24020101 is unresolvable from 24020102L. 2409N is resolved in exon 4 at bp 742 by a T substitution. The base change responsible for 2409N creates a premature termination on exon 4. 2411N is the result of the same insertion at 0104N. 3 A*010101/04N, 0201/09/43N or 0236, 3604 0209 is resolved as an A substitution in exon 4 at bp 779. 0243N is resolved as a C insertion at bp 780. 0236 and 3604 are not defined in exon 4. 4 A*020101/43N, 030101 or 0226, 0307 or 0234, 0308 0226 and 0308 are not defined in exon 4. As a result resolution can not be determined. 5 A*0201/09/43N, 2301/07N or 0236, 2304 The 02 alleles are resolved in exon 4 as described above. 2307 N is resolved by a C insert at bp 628 in exon 4. Neither 0236 nor 2304 are defined in exon 4. 6 A*020101/09/43N, 240201/09N/11N or 0212, 2413 or 0236, 24031/33 The 02 and 24 alleles are resolved in exon 4 as described above. Alleles 2413 and 0236 are not defined in exon 4. 7 A*0201/09, 2501 or 0206, 2503 The 02 alleles are resolved in exon 4 as described above. 2503 is not defined in exon 2. 8 A*030101, 240201/09N/11N or 0308, 2407 The 24 alleles are resolved in exon 4 as described above. 0308 is not defined in exon 4. 9 A*030101/2501 or 0308/2502 or 3204/6601 0308, 2502, and 3204 are not defined in exon 4. 10 A*A*030101, 2601 or 0308, 2613 0308 and 2613 are not defined in exon 4. 11 A*240201/09N/11N, 2601 or 2406, 2608 The 24 alleles are resolved in exon 4 as described above. 2406 is undefined in exon 4. 12 A*240201/09N/11N, 2902 or 24031, 2903 The 24 alleles are resolved in exon 4 as described above. 24020101, 2902 and 240301, 2903 are unresolved. 13 A*020101/09/20/43N, 310102/3102 3102 is not defined in exon 4. 14 A*240201/09N/11N, 3201 or 2432, 3203 Both 2432 and 3203 are not defined in exon 4. 15 A*240201/09N/11N, 680102/11N or 2406, 6809 or 2407, 680301 680102 and 6811N are not resolvable. 2406 and 6809 are not defined in exon 4. Exon 4 would resolve 24020101 from 2409N and 2411N. 24020101, 680102/11N is unresolvable from 2407, 680301. 16 A*2502, 7401 or 2502, 7402 or 3201, 6601 7402 is not defined in exon 4. 2502, 7401 is unresolvable from 3201, 6601. Finally, an ambiguity may be due to incomplete sequence information, because not all alleles have been sequenced for the same exons. For some alleles the entire sequence is not known in the region that is amplified. For example, A*010101 has been sequenced from Exon 1 through Exon 8, but A*010102 has been sequenced only in Exon 2 and Exon 3. Numerous ambiguities arise due to an incomplete sequence in Exon 4. The minimum requirements for submission of new sequences into reference databases of HLA sequences are the sequencing of Exon 2 and Exon 3 for Class I and Exon 2 for Class II. The relevance of completely identifying the polymorphisms found by SBT needs consideration. In clinical respects, it may not always be necessary to resolve ambiguities that involve a silent non-coding polymorphism and/or an intron polymorphism. Exceptions will exist to this situation where the polymorphism negates or impairs expression (e.g. A*24020102L or B*15010102N – both are due to an intron polymorphism). However, for investigations of genetic inheritance or disease association, the definition of all polymorphisms may be significant. The purpose of this study was to summarize the incidence, nature, and cause of ambiguous HLA SBT results. This represents an important step toward developing strategies to reduce or eliminate this problem. Methods DNA Isolation Genomic DNA was isolated from peripheral blood using the Gentra PUREGENE ® isolation kit (Gentra Systems, Minneapolis, MN, U.S.A). The DNA was resuspended in Tris HCl buffer (pH 8.5) and the concentration was measured using a Pharmacia Gene Quant II Spectrophotometer. The DNA was then stored at -70°C until testing. Sequence-Based Typing The primary PCR amplification reaction consists of a 1.5 kb reaction encompassing exon 1 through intron 3 of the HLA region. All reagents necessary for primary amplification and sequencing are supplied in the HLA-A or HLA-B AlleleSEQR Sequenced Based Typing Kits (Forensic Analytical, Hayward, CA, U.S.A.). The primary amplification PCR products were purified from excess primers, dNTPs, and genomic DNA using ExoSAP-IT (Amersham Life Science, Cleveland, OH, U.S.A.) Each template was sequenced in the forward and reverse sequence orientation for exon 2 and Exon 3 according to protocols supplied with the SBT kit. Excess dye terminators were removed from the sequencing products utilizing an ethanol precipitation method with absolute ethanol. The reaction products were reconstituted with 15 μl of Hi-Di™ Formamide (PE Applied Biosystems/Perkin-Elmer, Foster City, CA, U.S.A.) and analyzed on the ABI Prism ® 3700 DNA Analyzer with Dye Set file: Z and mobility file: DT3700POP6 {ET}. Results Sequence based typing analysis of HLA-A and B alleles was performed on a population of 676 normal donors. The racial distribution of the subjects studied was: 615 Caucasian, 13 Asian, 23 African American, 17 Hispanic and 8 Unknown. 672 of the 676 subjects were analyzed for the presence of HLA-A locus ambiguities and 666 were analyzed for HLA-B locus ambiguities. Each allele was counted separately in this analysis in order to determine the total percentage of ambiguous allele combinations. Four new potential alleles were found. At the HLA-A locus a total of 548 ambiguous allele combinations were found. This represented 41% of all HLA-A alleles (548 of 1344) (Table 1 ). Approximately half, 51% (278 of 548) of these ambiguities were due to the fact that Exon 4 analysis was not performed (Table 2 and Table 3 ). HLA-A*01 and HLA-A*24 are very prevalent alleles and most ambiguities involving these alleles could be resolved by performing Exon 4 analysis. For example the ambiguity most prevalent for HLA-A*01 in this study was HLA-A*0101/0104N. The sequences of these two alleles are identical across Exons 2 and 3; the difference between these two alleles occurs at position 627insC, which is located in Exon 4. Table 1 Prevalence of ambiguous sequence-based typing allele combinations among 672 people analyzed at the HLA-A locus and 666 analyzed at the HLA-B locus Allele Occurrence Allele Occurrence A*01 174 B*07 51 A*02 162 B*08 56 A*03 43 B*13 2 A*23 2 B*14 3 A*24 104 B*15 26 A*25 5 B*18 17 A*26 26 B*27 37 A*29 3 B*35 34 A*31 4 B*39 3 A*32 5 B*40 31 A*66 1 B*44 44 A*68 16 B*51 14 A*74 3 B*52 1 B*55 2 B*58 1 TOTAL 548 TOTAL 322 Table 2 Nature and Resolution of HLA-A and -B Allele Ambiguities HLA-A Locus HLA-B Locus Portion of alleles with ambiguities 41% 24% Most frequently involved alleles A*01, 02, and 24 B*7, 8, 15, 35, and 44 Methods used to resolve ambiguities Exon 4 sequencing (51%) Many different methods A*02 subtyping (30%) A large portion of HLA-A locus sequence-based typing ambiguities involved HLA-A*02, 30% or 162 of 548. Some of the HLA-A*02 ambiguities can also be resolved via Exon 4 sequencing and most of the other HLA-A*02 ambiguities can be resolved with traditional A*02 molecular subtyping methodologies using sequence specific primers or sequence specific probes. Not all HLA-A ambiguous allele combinations can be resolved as simply as those involving A*01, A*024 and A*02. Most of the remaining 19% (108 of 548) of the HLA-A ambiguities cannot be resolved with Exon 4 analysis (Table 3 ). Review of the HLA-B locus results revealed 322 ambiguous allele combinations among the 1332 total HLA-B alleles (24%). Antigens HLA-B*07/08/15/27/35/44, common in Caucasians, produced the largest portion of the ambiguities (279 of 322 or 87%). Each of these ambiguities had an independent reason for occurring. Table 4 lists some of the more common B locus ambiguities seen in this study. The reason for each ambiguity is variable; however a large portion of the ambiguities are related to cis/trans allele combinations. Table 4 Comparison of B locus ambiguities seen with the standard single tube amplification sequence-based typing method utilized in this study and those expected to be seen when utilizing a new two tube sequence-based typing method. Ambiguities using a single tube amplification B*270502 or 270502/13 or 2713 B*070201, 0801 or 0705/06, 0807 B*070201, 1402 or 0726, 1403 B*0702, 150101 or 0707, 1507 or 0709, 1563 B*070201, 180101/17N or 0707, 1814 or 0726, 1813 B*07021, 400101/0102 or 0705/06, 4033 B*070201, 440201/19N/27 or 0720, 4416 or 0724, 4421 B*0801, 180101/17N or 0804, 1807 or 0812, 1814 B*0801, 400101/0102 or 0804, 4007 B*0801, 440201/19N/27 or 0802, 4409 B*150101/15, 3503/13 B*1503, 3501 or 1529, 3528 B*3501, 400101/0102 or 3520, 4007 B*350101/40N/42, 4402/19N/27 or 3510, 4412 B*400101, 4402 or 400102, 4402 or 4042, 4414 B*4001, 510101 or 4007, 5107 Ambiguities reduced by 56% if Two Tube Group Amplification is Used Remaining ambiguities: B*070201, 180101/17N or 0707, 1814 or 0726, 1813 B*070201, 440201/19N/27 or 0720, 4416 or 0724, 4421 B*0801, 180101/17N or 0804, 1807 or 0812, 1814 B*0801, 440201/19N/27 or 0802, 4409 B*150101/15, 3503/13 B*1503, 3501 or 1529, 3528 B*3501, 400101/0102 or 3520, 4007 B*400101, 4402 or 400102, 4402 or 4042, 4414 B*4001, 510101 or 4007, 5107 Discussion While SBT provides the best available typing of HLA-A and B antigens, it is limited by sequence results that don't allow the precise identification of alleles. We found that 41% of HLA-A alleles and 24% of HLA-B alleles were ambiguously typed. The ambiguities involve some of the most frequent HLA-A and HLA-B antigens: A*01, A*02, A*24, B*07, B*08, B*15, B*27, B*35, and B*44. However, ambiguous allele combinations occur in all loci tested in HLA. The IMGT/HLA Sequence Database maintains an updated listing of all ambiguous possibilities [ 5 , 8 ]. The need to initiate additional testing to clarify ambiguous allele combinations must consider whether it is practical to obtain the information and if the information is useful and valuable. The clinical need for the highest resolution HLA typing possible is an important variable that must be considered. When typing is performed for cancer and viral vaccine development studies, high resolution allele data may be necessary to determine if a subject has an HLA type that is appropriate for a study. Utilization of high resolution data may also have implications for hematopoietic progenitor cell transplantation. Transplants involving partially mismatched or unrelated donor-recipient pairs require a higher resolution typing, but those involving HLA identical siblings may not. If it is necessary to resolve an ambiguous typing, a variety of different methods can be used. If the ambiguity is due to an allele that has not been completely sequenced or because the ambiguity is outside the region amplified by the SBT assay, the resolution is dependent on the nature and complexity of the ambiguity. Traditionally, for Class I sequencing purposes most laboratories have performed Exon 2 and Exon 3 analysis alone and for Class II sequencing only Exon 2 analysis. Many of the ambiguities can be resolved by sequencing Exon 4. In fact, in this study the largest portion of the typing ambiguities can be resolved by sequencing exon 4. However, many polymorphisms in exon 4 have no functional significance, so it may not be worthwhile resolving most ambiguities involving exon 4. The requirement for the analysis of Exon 4 to reduce the incidence of typing ambiguity has now been realized by commercial kit manufacturers. Both Celera Diagnostics (Alameda, CA) and Forensic Analytical/Atria Genetics (South San Francisco, CA) now include reagents for analysis of exon 4 in the HLA-A and -B kits. As the use of SBT increases, more data may become available from non-traditional exons in addition to those that have been traditionally sequenced and the number of ambiguities due to unknown sequences will decrease. If the ambiguity is due to an identical heterozygote sequence, as shown in figure 1 , the ambiguous allele combinations can sometimes be addressed by utilizing a group-specific primary amplification approach. In this approach each allele is amplified separately by using group specific primers for the alleles in question. A homologous sequence for each separate allele can then be obtained by sequencing the product of the group specific amplifications. Currently, there are commercially available kits (Forensic Analytical, Hayward, CA) for group specific amplification of the B locus. These kits allow the primary amplification of a specific group. Upon discovery of a particular ambiguity, a group specific amplification is done to separate out the allele pair. The resultant sequence will be homozygous for each allele in question. Another method, which will reduce the number of ambiguities in the B locus, is the utilization of a two tube group amplification approach (DYNAL Biotech, Brown Deer, WI). This method allows for resolution of ambiguities by taking into account the cis/trans allele combinations which result from simultaneous nucleotide incorporation for DNA templates being sequenced. This method allows for separation of ambiguities when the ambiguity has arisen due to a cis/trans situation. Ambiguities utilizing this method are reduced by 56%. (Table 4 ) Another method for separation of alleles is Haploprep™. (Genovision, Philadelphia, PA). Haploprep™ physically separates a diploid sample into its haploid components. Once the haplotypes are separated, routine HLA typing methods can be performed to determine the alleles. This laboratory is currently conducting studies to determine the efficacy of this product. Several of the remaining HLA loci ambiguities can be managed utilizing in-house custom group specific primary amplification mixes. Other methods which have been utilized to produce a homologous sequence include cloning, reference strand conformational analysis (RSCA), Pyrosequencing™ and denaturing high-performance liquid chromatography (DHPLC) [ 9 - 11 ]. Pyrosequencing™ is being explored by this laboratory and results at this time are preliminary. This method relies on the identification of a correct dispensation order of nucleotides during the Pyrosequencing™ process. Each ambiguity would require a separate dispensation order to be determined due to the unique nature of each ambiguity. The initial setup of this technology may be cumbersome; however, once established it may become very streamlined due to the availability of data on different ambiguous allele combinations. Each one of these methods has advantages and concerns which must be thoroughly investigated by the laboratory. Some, not all, ambiguous allele combinations produced by having identical heterozygote combinations can be resolved utilizing traditional sequence specific primers (SSP) or sequence specific oligonucleotide probes (SSOP). This may be a more viable approach for laboratories if they are already performing one of these technologies. In conclusion, although the prevalence of ambiguous allele combinations is high, methods exist to compensate for this problem. As the HLA field continues the discovery of new alleles, alternative approaches to discerning ambiguous allele combinations will need to be investigated in order to reduce the ever-growing number of ambiguities.

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545783

Sparse graphical Gaussian modeling of the isoprenoid gene network in Arabidopsis thaliana

A novel approach for modeling gene-regulatory networks, based on graphical Gaussian modeling, is used to create a network for the isoprenoid biosynthesis pathway in Arabidopsis .

Background The analysis of genetic regulatory networks has received a major impetus from the huge amounts of data made available by high-throughput technologies such as DNA microarrays. The genome-wide, massively parallel monitoring of gene activity will increase the understanding of the molecular basis of disease and facilitate the identification of therapeutic targets. To fully uncover regulatory structures, different analysis tools for transcriptomic and other high-throughput data will have to be used in an integrative or iterative fashion. In simple eukaryotes or prokaryotes, gene-expression data has been combined with two-hybrid data [ 1 ] and phenotypic data [ 2 ] to successfully predict protein-protein interaction and transcriptional regulation on a large scale. If the principal organization of a gene network has been established, differential equations may be used to study its quantitative behavior [ 3 , 4 ]. In higher organisms, however, little is known about regulatory control mechanisms. As a first step in reverse engineering of genetic regulatory networks, structural relationships between genes can be explored on the basis of their expression profiles. Here, we focus on graphical models [ 5 , 6 ] as a probabilistic tool to analyze and visualize conditional dependencies between genes. Genes are represented by the vertices of a graph and conditional dependencies between their expression profiles are encoded by edges. Graphical modeling can be carried out with directed and undirected edges, with discretized and continuous data. Over the past few years, graphical models, in particular Bayesian networks, have become increasingly popular in reverse engineering of genetic regulatory networks [ 7 - 10 ]. Graphical models are powerful for a small number of genes. As the number of genes increases, however, reliable estimates of conditional dependencies require many more observations than are usually available from gene-expression profiling. Furthermore, because the number of models grows super-exponentially with the number of genes, only a small subset of models can be tested [ 10 ]. Most important, a large number of genes often entails a large number of spurious edges in the model [ 11 ]. The interpretation of the graph within a conditional-independence framework is then rendered difficult [ 12 ]. Even a search for local dependence structures and subnetworks with high statistical support [ 7 ] provides no guarantee against the detection of numerous spurious features. Some of these problems may be circumvented by restricting the number of possible models or edges [ 10 , 13 ] or by exploiting prior knowledge on the network structure. So far, however, this prior knowledge is difficult to obtain. As an alternative approach to modeling genetic networks with many genes, we propose not to condition on all genes at a time. Instead, we apply graphical modeling to small subnetworks of three genes to explore the dependence between two of the genes conditional on the third. These subnetworks are then combined for making inferences on the complete network. This modified graphical modeling approach makes it possible to include many genes in the network while studying dependence patterns in a more complex and exhaustive way than with only pairwise correlation-based relationships. For an independent validation of our method, we compare our modified graphical Gaussian modeling (GGM) approach with conventional graphical modeling in a simulation study. We show at the end of the Results section that our approach outperforms the standard method in simulation settings with many genes and few observations. For a further evaluation with real data, we apply our approach to the galactose-utilization data from [ 14 ] to detect galactose-regulated genes in Saccharomyces cerevisiae . The main aim of this methodological work, however, was to elucidate the regulatory network of the two isoprenoid biosynthesis pathways in Arabidopsis thaliana (reviewed in [ 15 ]). The greater part of this paper is therefore devoted to the inference and biological interpretation of a genetic regulatory network for these two pathways. To motivate our novel modeling strategy, we first describe the problems that we encountered with standard GGMs before presenting the results of our modified GGM approach. Results Isoprenoids serve numerous biochemical functions in plants: for example, as components of membranes (sterols), as photosynthetic pigments (carotenoids and chlorophylls) and as hormones (gibberellins). Isoprenoids are synthesized through condensation of the five-carbon intermediates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In higher plants, two distinct pathways for the formation of IPP and DMAPP exist, one in the cytosol and the other in the chloroplast. The cytosolic pathway, often described as the mevalonate or MVA pathway, starts from acetyl-CoA to form IPP via several steps, including the intermediate mevalonate (MVA). In contrast, the plastidial (non-mevalonate or MEP) pathway involves condensation of pyruvate and glyceraldehyde 3-phosphate via several intermediates to form IPP and DMAPP. Whereas the MVA pathway is responsible for the synthesis of sterols, sesquiterpenes and the side chain of ubiquinone, the MEP pathway is used for the synthesis of isoprenes, carotenoids and the side chains of chlorophyll and plastoquinone. Although both pathways operate independently under normal conditions, interaction between them has been repeatedly reported [ 16 , 17 ]. Reduced flux through the MVA pathway after treatment with lovastatin can be partially compensated for by the MEP pathway. However, inhibition of the MEP pathway in seedlings leads to reduced levels in carotenoids and chlorophylls, indicating a predominantly unidirectional transport of isoprenoid intermediates from the chloroplast to the cytosol [ 16 , 18 ], although some reports indicate that an import of isoprenoid intermediates into the chloroplast also takes place [ 19 - 21 ]. Application of standard GGM to isoprenoid pathways in Arabidopsis thaliana To gain more insight into the cross-talk between both pathways at the transcriptional level, gene-expression patterns were monitored under various experimental conditions using 118 GeneChip (Affymetrix) microarrays (see Additional data files 1 and 2). To construct the genetic regulatory network, we focused on 40 genes, 16 of which were assigned to the cytosolic pathway, 19 to the plastidal pathway and five encode proteins located in the mitochondrion. These 40 genes comprise not only genes of known function but also genes whose encoded proteins displayed considerable homology to proteins of known function. For reference, we adopt the notation from [ 22 ] (see Table 1 ). The genetic-interaction network among these genes was first constructed using GGM with backward selection under the Bayesian information criterion (BIC) [ 23 ]. This was carried out with the program MIM 3.1 [ 24 ] (see Materials and methods for further details). The network obtained had 178 (out of 780) edges - too many to single out biologically relevant structures. Therefore, bootstrap resampling was applied to determine the statistical confidence of the edges in the model (Figure 1b ). For the bootstrap edge probabilities, only a cutoff level as high as 0.8 led to a reasonably low number of selected edges (31 edges, Figure 2 ). However, a comparison between bootstrap-edge probabilities and the pairwise correlation coefficients suggested that for such a high cutoff level, many true edges may be missed. For example, the gene AACT2 appears to be completely independent from all genes in the model although it is strongly correlated with MK , MPDC1 and FPPS2 (see Additional data file 4 for the correlation patterns). This phenomenon had already been observed in a simulation study by Friedman et al . [ 25 ] and may be related to the surprisingly frequent appearance of edges with a low absolute pairwise correlation coefficient but a high bootstrap estimate (Figure 1c ). Although there is no concise explanation for this pattern, one conjecture would be that the simultaneous conditioning on many variables introduces many spurious edges with little absolute pairwise correlation but high absolute partial correlation into the model. Our modification for GGMs is to improve upon this drawback. Application of our modified GGM approaches As described in more detail in Materials and methods, our approach aims at modeling dependencies between two genes by taking the effect of other genes separately into account. In the hope of identifying direct co-regulation between genes, an edge is drawn between two genes i and j when their pairwise correlation is not the effect of a third gene. Each edge has therefore a clear interpretation. We have developed two versions of our method: a frequentist approach in which each edge is tested for presence or absence; and a likelihood approach with parameters θ ij , which describe the probability for an edge between i and j in a latent random graph. One main benefit of the second version over full graphical models is that one can easily test on a large scale how well additional genes can be incorporated into the network. This allows the selection of additional candidate genes for the network in a fast and efficient way. We have applied and tested our modified GGM approaches by constructing a regulatory network of the 40 genes in the isoprenoid pathways in A. thaliana and by attaching 795 additional genes from 56 other metabolic pathways to it. Figure 3 shows the network model obtained from the frequentist modified GGM approach. Because we find a module with strongly interconnected genes in each of the two pathways, we split the graph into two subgraphs, each displaying the subnetwork of one module and its neighbors. Our finding provides a further example that within a pathway many consecutive or closely positioned genes are potentially jointly regulated [ 26 ]. In the MEP pathway, the genes DXR , MCT , CMK and MECPS are nearly fully connected (upper panel of Figure 3 ). From this group of genes, there are a few edges to genes in the MVA pathway. Among these genes, AACT1 and HMGR1 form candidates for cross-talk between the MEP and the MVA pathway because they have no further connection to the MVA pathway. Their correlation to DXR , MCT , CMK and MECPS is always negative. Similarly, the genes AACT2 , HMGS , HMGR2 , MK , MPDC1 , FPPS1 and FPPS2 share many edges in the MVA pathway (lower panel of Figure 3 ). The subgroup AACT2 , MK , MPDC1 and FPPS2 is completely interconnected. From these genes, we find edges to IPPI1 and GGPPS12 in the MEP pathway. Whereas IPPI1 is positively correlated with AACT2 , MK , MPDC1 and FPPS2 , GGPPS12 displays negative correlation to the four genes. In contrast to the conventional graphical model, we could now identify the connection between AACT2 and MK , MPDC1 and FPPS2 . In general, we found a better agreement between the absolute pairwise correlation and the selected edges (frequentist approach) or the probability parameters θ (latent random graph approach). Figures 4a and 4b show the selected edges and θ -values as a function of the absolute pairwise correlation. Attaching additional pathway genes to the network Following construction of the isoprenoid genetic network, 795 additional genes from 56 metabolic pathways were incorporated. Among these were genes from pathways downstream of the two isoprenoid biosynthesis pathways, such as phytosterol biosynthesis, mono- and diterpene metabolism, porphyrin/chlorophyll metabolism, carotenoid biosynthesis, plastoquinone biosynthesis for example. Using the second version of our method, that is, the latent random graph approach, we compared θ -values for all gene pairs in the network with and without attaching these additional genes (Figure 4b and 4c ). As expected, the parameters θ for the edge probabilities decreased if additional genes were included in the isoprenoid network (see Materials and methods). After addition, if for a gene pair i , j , θ ij dropped by more than 0.3, it was assumed that the dependence between i and j could be 'explained' by some of the additional genes. To find these genes out of all additionally tested candidates k , GGMs with genes i , j and k were formed. A gene k was considered to explain the dependency between i and j when an edge between i and j was not supported in the GGM, that is, when the null hypothesis ρ ij | k = 0 was accepted in the corresponding likelihood ratio test. k was then taken to 'attach well' to the gene pair i , j . Thus, for each gene pair i , j whose parameter θ ij dropped by more than 0.3, we obtained a list of well-attaching genes. Genes appearing significantly frequently in these lists of well-attaching genes were assumed to connect well to the complete genetic network. We tested for significance by randomization: For each gene pair i , j , a randomized list of well-attaching genes was formed with the same size as the original gene list. To explore which pathways attach significantly well to the MVA and MEP pathways, the portion of genes from each of the 56 pathways was summed over all gene pairs i , j . These sums were then compared for the originally attached genes and the sums of randomly attached genes in 100 datasets. Table 2 shows the pathways whose genes were found to attach significantly frequently to the MVA pathway, the MEP pathway, or both pathways. Interestingly, from all 56 metabolic pathways considered, we predominantly find that genes from downstream pathways fit well into the isoprenoid network. These results suggest a close regulatory connection between isoprenoid biosynthesis genes and groups of downstream genes. On the one hand, we find strong connections between the MEP pathway and the plastoquinone, the carotenoid and the chlorophyll pathways (experimentally supported by [ 15 , 16 , 27 ]). On the other hand, the plastoquinone and phytosterol biosynthesis pathways appear to be closely related to the genetic network of the MVA pathway. On a metabolic level, our results are substantiated by earlier labeling experiments using [1- 13 C] glucose, which revealed that sterols were formed via the MVA pathway, while plastidic isoprenoids (β-carotene, lutein, phytol and plastoquinone-9) were synthesized using intermediates from the MEP pathway [ 27 ]. Moreover, incorporation of [1- 13 C]- and [2,3,4,5- 13 C 4 ]1-deoxy-D-xylulose into β-carotene, lutein and phytol indicated that the carotenoid and chlorophyll biosynthesis pathways proceed from intermediates obtained via the MEP pathway [ 28 ]. In contrast, a close connection between the MVA and the MEP pathways could not be detected. This suggests that cross-talk on the transcriptional level may be restricted to single genes in both pathways. In a further analysis step, we examined which gene pairs the four identified pathways (plastoquinone, carotenoid, chlorophyll, and phytosterols) attached to. Genes from the plastoquinone pathway were predominantly linked to the genes DXR , MCT , CMK , GGPPS11 , GGPPS12 , AACT1 , HMGR1 and FPPS1 , supporting the hypothesis that AACT1 and HMGR1 are involved in communication between the MEP and MVA pathways. Genes from the carotenoid pathway attached to DXPS2 , HDS , HDR , GGPPS11 , DPPS2 and PPDS2 , whereas the chlorophyll biosynthesis appears to be related to DXPS2 , DXPS3 , DXR , CMK , MCT , HDS , HDR , GGPPS11 and GGPPS12 . Genes from the phytosterol pathway attach to FPPS1 , HMGS , DPPS2 , PPDS1 and PPDS2 . Incorporating 795 additional genes into the isoprenoid genetic network would not have been feasible with standard GGMs as the graphical model would have had to be newly fitted for each additional gene. Also, hierarchical clustering would not have been an appropriate tool for detecting the similarities in the correlation patterns between the two isoprenoid metabolisms and their downstream pathways. Figure 5 shows the hierarchical clustering of the 40 isoprenoid genes and 795 additional pathway genes based on the distance measure 1 - | σ ij |, where σ ij denotes the pairwise correlation between genes i and j . The positions of the MVA pathway genes (labeled 'm') and the non-mevalonate pathway genes (labeled 'n'), respectively, are shown to the right of the figure. The symbol + represents the positions of genes from the downstream pathways identified in Table 2 , whereby the vertical line is drawn to distinguish between genes downstream of the mevalonate and the non-mevalonate pathway. From Figure 5 it can be easily seen that there is no clear pattern of (positional) association between genes of the isoprenoid biosynthesis and downstream pathways in the hierarchical clustering. Simulation study For an independent comparison between the modified and the conventional GGM approaches, we simulated gene-expression data with 40 genes and 100 observations. This simulation framework corresponds to the data for isoprenoid biosynthesis and is thought to be only exemplary at this point. An extensive simulation study is currently underway and will be presented elsewhere. Following recent findings on the topology of metabolic and protein networks [ 29 , 30 ], we simulated scale-free networks in which the fraction of nodes with k edges decays as a power law ∝ k - γ . For metabolic and protein networks, γ is usually estimated to range between 2 and 3, which would result in very sparse networks with fewer edges than nodes in our simulation settings. To allow for denser networks, we generated 100 graphs each for γ = 0.5, 1.5 and 2.5. With 40 nodes, these graphs then comprised 88.3, 49.7 and 30.5 edges on average. For each edge, the conditional dependence of the corresponding gene pairs was modeled with a latent random variable in a structural equation model as described in [ 31 ]. Further details are of technical nature and are omitted here. The use of latent random variables enabled us to model partial correlation coefficients according to the previously defined network structure while ensuring positive definiteness of the complete partial correlation matrix. This matrix was then transformed into a covariance matrix Σ, from which synthetic gene expression data with 100 observations were sampled according to a multivariate normal distribution N (0,Σ). The performance of the graphical modeling approaches was monitored using the rate of true and false positives in receiver operator characteristics (ROC) curves (see [ 11 ] for a short introduction). For the standard graphical model, bootstrapping would have been too time-consuming, so we ranked all edges according to their sequential removal in the backward selection process. Figure 6a shows the ROC curves for the graphical modeling with backward selection and the modified graphical modeling approaches (frequentist and latent random graph approach). We also included the ROC curve for network inference with pairwise correlation coefficients. It can be seen that the modified GGM approaches outperform the conventional graphical modeling. Both the frequentist and the latent random graph method show a similar performance. Also, it should be noted that a simple measure such as the pairwise correlation can be quite powerful in detecting conditional dependencies between genes. ROC curves depict the true-positive rate as a function of the false-negative rate. However, in our setting where the false-positive edges by far outnumber the true-positive ones, the proportion of true positives among the selected edges is also of interest (Figure 6b ). Note that this proportion is the complementary false-discovery rate 1-FDR [ 32 ]. Figure 6b provides further evidence that the modified GGM approaches have a better performance than standard GGM. Application to galactose utilization in Saccharomyces cerevisiae For further evaluation, we applied our approach to the galactose-utilization dataset from [ 14 ] to detect galactose-regulated genes in Saccharomyces cerevisiae . Ideker et al . [ 14 ] used self-organizing maps to cluster 997 genes with significant expression changes in 20 systematic perturbation experiments of the galactose pathway. From the nine galactose genes under investigation, two subgroups with three and four genes, respectively, were found in two of the 16 clusters. Nine of the 87 genes in these two clusters carried GAL4p-binding sites and are thus candidate genes for regulation by the transcription factor GAL4p. Among these candidate genes, GCY1 and PCL10 are known to be targets of GAL4p [ 33 ], and YMR318C has been implicated in another binding-site study [ 34 ]. After incorporating all yeast genes into our network of the nine galactose genes, 13 genes were found to attach significantly well. Among these, GCY1 and PCL10 were also detected. Furthermore, three out of the remaining 11 candidate genes ( MLF3 , YEL057C and YPL066W ) had GAL4p-binding sites. These genes were also identified in [ 14 ]. This result shows once more that with our approach we are not only able to model the dependence between genes but also find genes whose expression profiles fit well to the original genes in the model. In contrast to [ 14 ], we did not have to rely on gene clusters with a high occurrence of galactose genes to find these genes. Discussion Analysis of gene expression patterns, for example cluster analysis, often focuses on coexpression and pairwise correlation between genes. Graphical models are based on a more sophisticated measure of conditional dependence among genes. However, with this measure, modeling is restricted to a small number of genes. With a larger set of genes, it is rather difficult to interpret the model and to generate hypotheses on the regulation of genetic networks. In our approaches, in the search for significant co-regulation between two genes all other genes in the model are also taken into account. However, the effect of these genes is examined separately, one gene at a time. Because of this simplification, modeling can include a larger number of genes. Also, each edge has a clear interpretation, representing a pair of significantly correlated genes whose dependence cannot be explained by a third gene in the model. Our frequentist method has a resemblance to the first two steps in the SGS and PC algorithms [ 31 ]. By restricting the modeling to subnetworks with three genes, we avoid the statistically unreliable and computationally costly search for conditional independence in large subsets, as in the SGS algorithm. Also, we avoid having to remove edges in a stepwise fashion, as in the PC algorithm. Therefore, we do not run the risk of mistakenly removing an edge at an early stage, which leads to improved stability in the modeling process. By using a Gaussian model, we can only reveal linear dependencies between genes. For handling nonlinearities, gene-expression profiles should be discretized and analyzed in a multinomial framework. In principle, it should be straightforward to adopt our approach to a multinomial model. Because we focused on linear dependencies, we have not addressed this problem so far. For the isoprenoid biosynthesis pathways in A. thaliana , we constructed a genetic network and identified candidate genes for cross-talk between both pathways. Interestingly, both positive and negative correlations were found between the identified candidate genes and the corresponding pathways. AACT1 and HMGR1 , key genes of the MVA pathway, were found to be negatively correlated to the module of connected genes in the MEP pathway. This suggests that in the experimental conditions tested, AACT1 and HMGR1 may respond differently (than the MEP pathway genes) to environmental conditions, or that they possess a different organ-specific expression profile. In either case, expression within both groups seems to be mutually exclusive. On the other hand, a positive correlation was identified between IPPI1 and members of the MVA pathway, suggesting that this enzyme controls the steady-state levels of IPP and DMAPP in the plastid when a high level of transfer of intermediates between plastid and cytosol takes place. Although we have considered only metabolic genes in this analysis, the method can be extended to identify genes encoding other types of proteins belonging to the same transcription module. In fact, transcription factors and other regulator proteins, as well as structural proteins such as transporters, are often found in the same expression module [ 26 ]. Our results suggest that the expression of genes belonging to the chlorophyll and carotenoid biosynthesis pathways is controlled by a module that possibly includes genes from the MEP pathway. Similarly, the expression of genes in the phytosterol pathway appears to be influenced by genes from the MVA pathway. For the downstream regulation of plastoquinone biosynthesis, however, genes from both pathways seem to be involved. This finding is in agreement with the dual localization of enzymes from the plastoquinone pathway in either the plastid or the cytosol. The regulation of this pathway may therefore depend on processes happening on the metabolic and regulatory level in both compartments. We have shown in a simulation study that for gene-expression data with many genes and few observations, the modified GGM approaches have performed better in recovering conditional dependence structures than conventional GGM. However, a final evaluation of our inferred network for the isoprenoid biosynthesis pathways in A. thaliana can only be made on the basis of additional knowledge and biological experiments. At this stage, the use of domain knowledge has provided some means of network validation. As genes from the respective downstream pathways were significantly more often attached to the isoprenoid network than were candidate genes from other pathways, we are quite confident that our method can grasp the modularity in the dependence structure within groups of genes and also between groups of genes. Such modularity would have been difficult to detect by standard graphical modeling or clustering. Materials and methods Graphical Gaussian models (GGMs) Let q be the number of genes in the network, and n be the number of observations for each gene. The vector of log-scaled gene-expression values, Y = ( Y 1 ,..., Y q ) is assumed to follow a multivariate normal distribution N ( μ ,Σ) with mean μ = ( μ 1 ,..., μ q ) and covariance matrix Σ. The partial correlation coefficients ρ ij | rest , which measure the correlation between genes i and j conditional on all other genes in the model are calculated as where ω ij , 1 , j = 1,..., q are the elements of the precision matrix Ω = Σ -1 . Using likelihood methods, each partial correlation coefficients ρ ij | rest can be estimated and tested against the null hypothesis ρ ij | rest = 0 [ 5 ]. An edge between genes i and j is drawn if the null hypothesis is rejected. Since the estimation of the partial correlation coefficients involves matrix inversion, estimators are very sensitive to the rank of the matrix. If the model comprises many genes, estimates are only reliable for a large number of observations. Commonly, the modeling of the graph is carried out in a stepwise backward manner starting from the full model from which edges are removed consecutively. The process stops when no further improvement can be achieved by removal of an additional edge. The final model is usually evaluated by bootstrapping to exclude spurious edges in the model. Modified GGM approaches Let i , j be a pair of genes. The sample Pearson's correlation coefficient σ ij is the commonly used measure for coexpression. For examining possible effects of other genes k on σ ij , we consider GGMs for all triples of genes i , j , k with k ≠ i , j . For each k , the partial correlation coefficient ρ ij | k is computed and compared to σ ij . If the expression level of k is independent of i and j , the partial correlation coefficient would not differ from σ ij . If on the other hand, the correlation between i and j is caused by k since k co-regulates both genes, one would expect ρ ij | k to be close to 0. Here, we use the terminology, that k 'explains' the correlation between i and j . In order to combine the different ρ ij | k values in a biologically and statistically meaningful way, we define an edge between i and j if ρ ij | k ≠ 0 for all remaining genes k . In particular, if there is at least one k with ρ ij | k = 0, no edge between i and j is drawn since the correlation between i and j may be the effect of k . Our approach can be implemented as a frequentist approach in which each edge is tested for presence or absence or alternatively, as a likelihood approach with parameters θ ij , which describe the probability for an edge between i and j in a latent random graph. Frequentist approach For the gene pair i , j and all remaining genes k , p-values ρ ij | k are obtained from the likelihood ratio test of the null hypothesis ρ ij | k = 0. In order to combine the different p -values ρ ij | k , we simply test whether a third gene k exists that 'explains' the correlation between i and j . For this purpose, we apply the following procedure: (1) For each pair i , j form the maximum p -value p ij ,max = max{ p ij | k , k ≠ i, j }. (2) Adjust each p ij ,max according to standard multiple testing procedures such as FDR [ 32 ]. (3) If the adjusted p ij ,max value is smaller than 0.05, draw an edge between the genes i and j ; otherwise omit it. The correction for multiple testing in step 2 is carried out with respect to the possible number of edges ( q ( q - 1))/2 in the model. Implicitly, multiple testing over all genes k is also involved in step 1. However, because the maximum over all p ij | k is considered, a multiple testing correction is not necessary. Latent random graph approach The frequentist approach has the disadvantage that a connection between two genes i and j is either considered to be present or absent. Also, it is not taken into account whether an edge between i and k respectively j and k is truly present when we test for ρ ij | k = 0. In our second method, we introduce a parameter θ ij as the probability for an edge between two genes i and j in a latent random graph model. Let θ be the parameter vector of θ ij for all 1 ≤ i < j ≤ q and y = ( y 1 ,..., y n ) be a sample of n observations. For estimating θ , we maximize the log-likelihood L ( θ ) = log P θ ( y ) via the EM-algorithm [ 35 ]. Let θ t be a current estimate of θ . Further, let g be the unobserved graph encoded as an adjacency matrix with g ij ∈ {0,1} depending on whether there is an edge between genes i and j or not. In the E-step of the EM-algorithm, the conditional expectation of the complete data log-likelihood is determined with respect to the conditional distribution p ( g | y , θ t ), By assuming independence between edges, Equation (1) becomes and further, after replacing and summing out Equation 2 we find P ( g ij = 1| y , θ t ) and P ( g ij = 0| y , θ t ) at the right side of Equation (3) are approximated by the statistical evidence of edge i , j in GGMs with genes i , j and k . As we only want to estimate the effect of k on the correlation between i and j , we distinguish only the two cases whether k is a common neighbor of i and j , for example, g ik = 1 and g jk = 1 or not. When k is a common neighbor, we test ρ ij | k ≠ 0 versus ρ ij | k = 0. When k is not a common neighbor of i and j , we test σ ij ≠ 0 versus σ ij = 0 for the pairwise correlation coefficients instead. Thus, we obtain where and are p -values of the corresponding likelihood ratio tests. After replacing Equation (4) in Equation (3), the M-step of the EM-algorithm, that is the maximization of E θ (log P θ ( g )| y , θ t ) with respect to θ , leads to an iterative updating scheme θ t → θ t +1 with In summary, we determine the probability parameters θ as follows (1) For gene pairs i , j , compute P ( ρ ij | k ≠ 0) and P ( σ ij ≠ 0) for all genes k ≠ i , j . (2) Starting with θ 0 , apply iteratively Equation (5) until the error | θ t +1 - θ t | drops below a prespecified value, for example 10 -6 . Our latent random graph approach also enables us to fit a large number of additional genes into a constructed genetic network. In this case, for a gene pair i , j in step 1 of the analysis, the partial correlation coefficients ρ ij | k are not only computed and tested for genes k in the model but also for the additional candidate genes. However, the iteration in step 2 is not extended to these candidate genes. In other words, θ ij is only iteratively updated in Equation (5) if both genes i , j are in the original model. For candidate genes k , θ ik and θ jk are kept fixed at a prespecified value, for example 1, and are not re-estimated in the EM-iteration process. This outline introduces a second level into the modeling process. At the first level, the network between the original genes is constructed. At the second level, we test how additional candidate genes influence the parameters θ . If these candidates have an effect on the correlation between i and j , θ ij will decrease. Thus, by comparing the original network with the network inferred from allowing for additional genes in step 1, we can determine which candidate genes lower the θ -values and, accordingly, fit well into the network. Additional data files Additional data is available with the online version of this paper. Additional data files 1 and 2 contain the gene expression values of the isoprenoid genes (Additional data file 1 ) and the 795 genes from other pathways (Additional data file 2 ). Additional data file 3 contains a more detailed description of the microarray data (such as experimental conditions, hybridization and standardization). Additional data file 4 describes the correlation pattern of the 40 isoprenoid genes. Supplementary Material Additional data file 1 The gene expression values of the isoprenoid genes Click here for additional data file Additional data file 2 The gene expression values of the 795 genes from other pathways Click here for additional data file Additional data file 3 A more detailed description of the microarray data (such as experimental conditions, hybridization and standardization) Click here for additional data file Additional data file 4 The correlation pattern of the 40 isoprenoid genes. Click here for additional data file

/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545783.xml

545959

Microsatellite instability as prognostic marker in bladder tumors: a clinical significance

Background Carcinoma of urinary bladder is one of the leading causes of death in India. Successful treatment of bladder cancer depends on the early detection & specific diagnostic approaches. In the present study, microsatellite instability (MSI) has been evaluated as a prognostic marker in patients with superficial urinary bladder cancer in lower urinary tract for determining risk of recurrence. Methods A total of 44 patients with bladder tumors diagnosed with Transitional Cell Carcinomas [TCC] from lower urinary tract were selected for the study. Tumors were staged and graded according to AJCC-UICC (1997) classification and patients were followed with cystoscopy as per the protocol. Polymerase chain reaction (PCR) was done to amplify microsatellite sequences at mononucleotide BAT – 26, BAT – 40, TGFβ RII, IGFIIR, hMSH3, BAX and dinucleotide D2S123, D9S283, D9S1851 and D18S58 loci in blood (control) and tumor DNA. PCR products were separated on 8% denaturing polyacrylamide gel and visualized by autoradiography. Results MSI was observed in 72.7% of tumors at BAT – 26, BAT – 40, D2S123, D9S283, D9S1851 and D18S58 loci. Good association of MSI was seen with tumor stage and grade. MSI – High (instability at > 30% of loci) was frequently observed in high stage (40.6%) and high grade (59.4%) tumors. Of 24 tumors of Ta-T1 stage with different grades, 11 (9/18 high grade and 2/6 low grade tumors) recurred in the mean duration of 36 months. MSI positivity was significantly high in patients who had one or more recurrences (p = 0.02 for high grade and 0.04 for low grade tumors). Conclusions MSI may be an independent prognostic marker for assessing risk of recurrence in superficial tumors irrespective of the grade. Further studies on progression would help in stratifying the patients of T1G3 for early cystectomy vs bladder preservation protocol.

Background Amongst the genitourinary cancer, carcinoma of the urinary bladder is one of the leading causes of death in Indian population. Transitional cell carcinoma (TCC) is the commonest histopathological variant where stage and grade are the two important prognostic factors to know the clinical behavior of these tumors. Superficial tumors with different grades behave differently e.g. tumors with high grade recur early and progress to invasive bladder cancer very soon. This behavior of same stage of the tumor but with varied grades is attributed to genetic alterations. Bladder cancer manifesting from superficial to aggressive muscle invasive tumors undergoes a sequence of genetic alterations. Primary chromosomal aberrations are associated with tumor development while secondary chromosomal abnormalities lead to progression to a more advanced stage. A frequent loss of heterozygosity (LOH) on chromosomes 4, 5, 8, 9, 11 and 17 is considered a major event in the carcinogenesis of bladder cancer [ 1 , 2 ]. Defects in mismatch repair (MMR) genes result in replication errors and genetic instability. Faulty mismatch repair, generally observed as somatic variation in size of microsatellites (short tandem repeat sequences in genome) is referred as microsatellite instability (MSI) [ 3 ]. MSI and LOH in bladder cancer have been reported by several investigators [ 4 , 5 ]. A successful treatment of bladder cancer depends on early detection and more specific diagnostic approaches. Preneoplastic changes of the bladder epithelium or superficial tumors as an early event precede invasive bladder carcinomas. Though the higher grade and stage portends a worst prognosis, superficial tumors of same stage and grade have different outcome in different patients. Due to limited utility of these prognosticators in patients with superficial bladder tumor, there is a need to analyze new molecular parameters in predicting the prognosis and risk of recurrence. The following study is based on MSI analysis in tumor tissues to evaluate its utility as a marker for early detection of recurrent bladder carcinomas from lower urinary tract and thus help in deciding treatment modalities. Methods Patient selection Total of 44 patients with male & female ratio of (42:2) of TCC with a mean age of 62 years were included for the study after the approval from ethical committee. All the patients selected for the study were not having any familial cancer syndrome or had previous history of cancer to the best of our knowledge. All the tumors were resected transurethrally from the lower urinary tract. Part of superficial tissue specimen obtained after transurethral resection of bladder tumor (TURBT) was collected immediately in liquid nitrogen. Matched control sample (5 ml of peripheral blood) from all patients was collected in 200 μl of 0.5 M EDTA. The tumor stage and grade was assigned according to the TNM classification by American Joint Committee on Cancer (AJCC-UICC, 1997) [ 6 ]. Tumors of superficial nature classified as T1 or Ta while with deep muscular invasion were assigned as T2 or T3. Similarly tumor grading was done as G1 (low grade) and G2 or G3 (high grade). Patients were followed for recurrence (the number of times patient develops the tumor) every three months for 36 months with cytology and cystoscopy. The clinical and pathological characteristics of all the patients are summarized in Table 1 . Table 1 Clinical and pathological features of the patients diagnosed with bladder Carcinoma Case no. @ Age/ $ Gender Stage Grade #Recurrence MSI BC 1 45/M T2 High, G3 2 *High (BAT – 26, BAT – 40) BC 2 59/M T1 High, G2 1 High (BAT – 26, BAT – 40, D9S1851) BC 3 66/M Ta Low, G1 0 High (D9S283, D9S1851) BC 4 39/M T2 High, G3 0 High (BAT – 26, BAT – 40, D9S283) BC 5 72/M T2 High, G3 0 High (BAT – 40, D9S1851) BC 6 59/M T2 High, G2 1 Low (D9S1851) BC 7 78/M T2 High, G3 0 High (D9S283, D9S1851) BC 8 52/M Ta High, G2 0 High (BAT – 40, D9S283, D9S1851) BC 9 71/M Ta High, G2 0 **Low (BAT – 40) BC 10 84/M T1 High, G2 0 ***MSS BC 11 55/M T1 High, G1 0 MSS BC 12 53/M T2 High, G2 0 Low (D18S58) BC 13 52/M T3 High, G3 0 High (D9S283, D9S1851) BC 14 40/M T3 High, G3 0 MSS BC 15 55/M T2 High, G3 0 MSS BC 16 60/M T2 High, G3 0 High (BAT – 40, D9S283, D18S58) BC 17 66/M T1 High, G3 1 High (BAT – 40, D2S123, D9S283, D9S1851, D18S58) BC 18 80/M T1 High, G3 1 High (BAT – 26, D9S283, D18S58) BC 19 42/M T1 High, G2 0 Low (D9S283) BC 20 73/M T3a High, G3 0 MSS BC 21 55/M T2 High, G3 0 Low (D2S123) BC 22 58/M T2 High, G3 0 High (BAT – 26, D2S123) BC 23 70/M T1 High, G2 0 Low (BAT – 26) BC 24 53/M Ta High, G3 0 High (D9S1851, D18S58) BC 25 60/M T2 High, G3 0 High (D9S283, D9S1851) BC 26 54/M Ta Low, G1 0 MSS BC 27 72/M Ta High, G2 0 MSS BC 28 58/M T1 High, G3 0 Low (D9S283) BC 29 80/M T2 High, G3 0 High (D9S283, D9S1851) BC 30 64/M Ta High, G2 2 Low (BAT – 40) BC 31 74/M Ta High, G3 0 High (D9S283, D9S1851) BC 32 60/M T1 Low, G1 0 MSS BC 33 41/M T1 Low, G1 3 Low (BAT – 26) BC 34 66/ F T2 High, G3 0 High (D2S123, D9S283) BC 35 53/M T1 Low, G1 0 Low (D2S123) BC 36 66/M T1 High, G2 1 High (D2S123, D9S283, D18S58) BC 37 55/M T2 High, G3 0 High (BAT – 26, BAT – 40, D9S1851) BC 38 65/M T2 High, G3 0 Low (D18S58) BC 39 69/M T1 Low, G1 0 Low (D9S283) BC 40 74/M T1 High, G2 1 MSS BC 41 72/ F T2 Low, G1 0 MSS BC 42 71/M T1 High, G3 1 Low (D9S283) BC 43 64/M T2 High, G3 0 High (BAT – 40, D9S1851) BC 44 73/M T1 High, G2 1 Low (D9S283) @ Age = (years); $ Gender = (M: Male/F: Female); *MSI – H = MSI – High; **MSI – L = MSI – Low; ***MSS = microsatellite stable # Recurrence (0, 1, 2, 3) = Number of times the tumor recurred DNA isolation Superficial tumor tissue specimens of histologically confirmed bladder tumors and peripheral blood (frozen) of the same patient were processed for DNA isolation using phenol-chloroform extraction method [ 7 ]. MSI analysis Table 2 demonstrates the characteristic features of the microsatellite markers evaluated in the present study. The primer sequences for mono and dinucleotide microsatellite markers were searched from human genomic database. Polymerase chain reaction (PCR) amplification of DNA was done using primers of concentration of 6 pmol, 200 μM dNTPs, 10 mM Tris – Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.25 units of Taq polymerase (MSI, Fermentas), 100 ng DNA and 2 μCi [α- 32 P] dCTP (specific activity: 4000 Ci/mM) (BRIT, India) in a volume of 25 μl. PCR conditions involved an initial denaturation at 95°C for 3 min followed by 30 cycles (95°C for 1 min, 50°C to 60°C for 2 min and 72°C for 3 min) and a final extension at 72°C for 8 min. PCR products were mixed with equal volume of formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% Xylene cyanol), denatured for 5 min at 95°C and loaded onto 8% polyacrylamide gel containing 7 M urea. Gels were run at 55 W for 2 hours, transferred onto a Whatman sheet followed by an exposure to X-ray film (Kodak) for desired time and then developed. Table 2 Characteristic features of microsatellite markers examined in urinary bladder tumors Microsatellite marker Repeat pattern Chromosomal location ~ PCR product size BAT 26 (A) 26 5 th intron of hMSH2, 2p 117 – 130 bp BAT 40 (A) 40 2 nd intron of β hydroxy steroid dehydrogenase 94 – 112 bp BAX (38 – 41) (G) 8 19q13.3 – q13.4 94 bp TGFβ RII (665 – 737) (A) 10 3p22 73 bp IGFIIR (4030 – 4140) (G) 8 6q26 – 27 110 bp HMSH3 (381 – 383) (A) 8 5q 150 bp D2S123 (CA) 13 TA 2p16 197 – 227 bp (CA) 15 (T/GA) 7 D9S283 (CA)n 9q13 – q22 178 – 203 bp D9S1851 (CA)n 9q22.3 143 – 159 bp D18S58 (GC) 5 GA(CA) 17 18q22.3 144 – 160 bp The tumor was designated unstable if its PCR product had altered band pattern when compared to alleles in corresponding matched blood DNA [ 8 ]. Out of he motifs studied, BAT-26 & BAT-40, the mononucleotide poly A repetitive loci have been shown to exhibit polymorphism [ 9 ]. The change either borderline or major deletions/ insertions at this loci is compared in tumor tissue of the same patient with the normal tissue in colorectal tumors [ 8 ]. Tumors were called MSI-High (MSI-H) when they showed instability at > 30% of loci and MSI-Low (MSI-L) if they showed at or less than 30% of loci. Statistical analysis Statistical tests, including, 2 × 2 contingency table, Fisher's exact probability test (one or two tailed), Karl Pearson's correlation test were applied to assess the relation between the microsatellite instability in tumors and clinicopathological parameters. A student t test was applied to compare the number of genomic alterations between tumors of different grades and stages. Results A panel of ten microsatellite markers situated on chromosomes 2, 3, 5, 6, 9, 18 and 19 were screened to look for microsatellite instability in superficial tumor tissues and compared with blood DNA. Alterations were detected in 32 of 44 patients (72.7%). Out of the six mononucleotide microsatellite markers analyzed, only BAT – 26 and BAT – 40 in 17.7% and 24.4% of the cases could demonstrate changes respectively while TGFβ RII, IGFIIR, hMSH3 and BAX were microsatellite stable (MSS). BAT-26 & BAT-40 exhibited borderline changes in tumor DNA as compared to control DNA. We also sequenced the tumor & normal PCR product for these microsatellites to prove the change. The dinucleotide markers- D2S123, D9S283, D9S1851 and D18S58 exhibited altered electrophoretic migration pattern in 15.5%, 40%, 31.1% and 17.8% bladder tumors respectively. The most frequent microsatellite alteration was detected on the markers of chromosome 9 (D9S283 followed by D9S1851) (Fig 1 and 2 ). Figure 1 Changes in allelic pattern indicated by an arrow, observed in superficial tissue (Ts) as compared to blood (germline DNA, N) of patients with bladder carcinoma: (A) Deletion at BAT – 26; (B) Insertion at BAT – 40; (C) Insertion at D2S123; (D) Loss of heterozygosity at D9S283; (E) Biallelic alteration at D9S1851 and (F) Deletion at D18S58 Figure 2 Superficial tumor tissues and blood (control) of bladder tumor patients demonstrated no change at (A) TGFβ RII; (B) BAX; (C) hMSH3 and (D) IGFIIR A significant association of MSI – H with T2 or T3 stage tumors (2 × 2 contingency table, p = 0.05) and ≥ G2 grade tumors (Fisher's exact probability test, two tailed, p = 0.08) was observed (Table 1 ). Total genomic alterations were analyzed among different stages and grades. Changes were comparable among (24/44) T1-Ta and (20/44) T2-T3 tumors that included 35 (13 insertions, 6 deletions, 10 LOH and 6 BA) and 31 (10 insertions, 8 deletions & LOH and 5 BA) respectively (p > 0.05). High grade (G2-G3) (37/44) carcinomas encompassed 63 alterations (24 insertions, 14 deletions, 15 LOH and 10 BA) and low grade (G1) (7/44) bladder tumors had only 5 (1 each deletion & BA and 3 LOH) (p = 0.01) Recurrence of tumors (mean duration of 36 months) was correlated with MSI in 24 superficial (Ta-T1) tumors in patients who turned up for regular follow up (Table 1 ). This group comprised 18 high grade and six low grade tumors. In the high grade tumors, 13 were MSI+, and 8 of them (61.5%). showed recurrence while only one (1/5, 20%) MSI- recurred. Amongst low grade tumors (6), 2 recurrences were noted only in MSI+ group and none of the MSI- showed recurrence (Fisher's exact probability two tailed test) (p = 0.02 for high grade tumors) and (p = 0.04 for low grade tumors). Discussion This present work is a continuation of the previous published work where thirty bladder tumors were analyzed for the presence of MSI at BAT-26, BAT-40, TGFβ RII, IGFIIR, hMSH3 and BAX. The initial results encouraged examining the role of MSI/ LOH in more number of tumors with expanded panel of markers [ 7 ]. In this paper, 44 patients of bladder cancer are examined for MSI at BAT-26, BAT-40, TGFbRII, IGFIIR, BAX, & hMSH3 & D2S123, D9S283, D9S1851 & D18S58 dinucleotide repeat motifs. The MSI results are further analysed with clinicopathological features: stage and grade of the tumors & its recurrence in due course of time. The importance of MSI in the diagnosis of recurrence in superficial cases irrespective of grade & thus advocate the bladder cystectomy as a treatment modality in these cases. Genomic instability measured by changes in tumor tissues as compared to blood of the same patient at repetitive loci was detected in 72.7% cases of bladder carcinoma. It differs from previous observation, which shows infrequent occurrence of MSI in TCC using different microsatellite markers [ 10 ]. However, low frequency of MSI with alterations of dinucleotide repeats in TCC of the urinary tract was found as 21% and 16.6% in two independent studies [ 11 , 12 ]. MSI and allelic loss in a series of 26 upper urinary tract tumors using 5 informative microsatellite markers were examined & this study supports the presence of MSI in upper urinary tract which is rare event in bladder cancer [ 13 ]. Another study describes MSI and loss of respective MMR protein by immunostaining in a patient with a urothelial carcinoma of the ureter and a strongly positive history of cancer, who was subsequently found to have HNPCC [ 14 ]. In the present study a significant association of MSI with tumor stage and grade in sporadic bladder tumors suggested MSI as an early event in tumorigenesis. These results confirm the previous finding where MSI examined in TCC of bladder with low stage and grade using few microsatellite markers mostly confined to chromosome 9 [ 15 ]. Another study reports 100% tumor instability as determined by dinucleotide repeat analysis in 14 cases of urinary bladder of different stages and grades [ 16 ]. Many studies show relatively high proportion of tumors with mutations in di, tri, and tetra nucleotide repeat motifs, although each tumor exhibits only few such mutations [ 4 ]. Recently, a novel form of MSI, termed as EMAST (elevated microsatellite instability at selected tetranucleotide repeats) has been found to be significantly associated with mutations in p53 among the bladder cancer tumors, but no indication of elevated EMAST in tumors with abnormal p53 staining without mutation. EMAST likely reflects a particular pattern of somatic events that are interactive with p53 mutation, particularly common in skin cancer and limited to non-invasive disease in bladder cancer [ 17 ]. The difference between these studies and ours may be attributed to the number and identity of microsatellite motifs studied. Despite clear-cut prognostic differences, genetic alterations were comparable in superficial (Ta-T1) and invasive bladder carcinomas (T2-T3) suggesting the role of MSI in progression of bladder cancer as well. However, strong association of MSI – H with T2-T3 and G2-G3 was observed. MSI at ≥ 30% of loci has been found in 59.4% (19/32) of TCC bladder, which is not in accordance with reported earlier [ 11 , 12 ]. A good association of MSI – H with high grade superficial tumors may help in deciding radical surgery to begin with. Bladder cancer presents as superficial tumor in 75% of the patients, which can easily be removed by transurethral resection (TUR). Around 60–80 % of these treated patients develop recurrence in due course of time. Out of them, 15% progress to higher grade and stage. With so much potential for recurrence, patients need to be followed up with cystoscopy at regular intervals. Although many new tumor markers have been proposed but all have limitations with respect to execution and interpretation in predicting the recurrence of bladder tumors [ 18 ]. Among the molecular markers, alterations in p53, p21 WAF/C1P1 , Rb, c-erb B-2 are reported to be associated with tumor recurrence and progression but little is known to address MSI [ 19 ]. MSI analysis gives higher sensitivity and easy to execute among other molecular markers, thus making it a valuable marker for detection of recurrence. To the best of our knowledge, this is the first study reporting MSI as a good prognostic marker that correlates with risk of recurrence in superficial (Ta-T1) tumors irrespective of the grade. This may help in deciding radical treatment at an early stage. We could not study the genetic changes during the progression of tumor, which means the extension of superficial tumor confined to the mucosa and submucosa to deep musculature of the bladder. Limitation of this study is a small number of patients but initial trends show a strong correlation of MSI with recurrence irrespective of the grade of the tumor. Further multicentre trial is needed to prove this concept. Conclusions MSI has been observed to play important role in evolution, initiation and progression in bladder tumors. Patients with high grade superficial disease are reported to have higher incidence of MSI. Also high frequency of MSI in superficial tumors showing recurrence irrespective of grade may provide an indication for more radical approach to improve the survival. Competing interest The author(s) declare that they have no competing interests. Authors' contribution MV carried out the molecular genetic studies, participated in analyzing the data & drafted the manuscript. AM provided the clinical material & information, helped in analyzing the data & designing the manuscript. RDM helped in manuscript drafting. BM participated in its designing of the study & manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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Maternal plasma viral load and neutralizing/enhancing antibodies in vertical transmission of HIV: A non-randomized prospective study

Background We examined the association and interaction between maternal viral load and antibodies in vertical transmission of HIV in a non-randomized prospective study of 43 HIV-1 infected pregnant women who attended the San Juan City Hospital, Puerto Rico, and their 45 newborn infants. The women and infants received antiretroviral therapy. Methods A nested PCR assay of the HIV-1 envelope V3 region and infant PBMC culture were performed to determine HIV status of the infants. Maternal and infant plasma were tested for HIV neutralization or enhancement in monocyte-derived macrophages. Results Twelve (26.7%) infants were positive by the HIV V3 PCR assay and 3 of the 12 were also positive by culture. There was a trend of agreement between high maternal viral load and HIV transmission by multivariate analysis (OR = 2.5, CI = 0.92, p = 0.0681). Both maternal and infant plasma significantly (p = 0.001 for both) reduced HIV replication at 10 -1 dilution compared with HIV negative plasma. Infant plasma neutralized HIV (p = 0.001) at 10 -2 dilution but maternal plasma lost neutralizing effect at this dilution. At 10 -3 dilution both maternal and infant plasma increased virus replication above that obtained with HIV negative plasma but only the increase by maternal plasma was statistically significant (p = 0.005). There were good agreements in enhancing activity in plasma between mother-infant pairs, but there was no significant association between HIV enhancement by maternal plasma and vertical transmission. Conclusion Although not statistically significant, the trend of association between maternal viral load and maternal-infant transmission of HIV supports the finding that viral load is a predictor of maternal-infant transmission. Both maternal and infant plasma neutralized HIV at low dilution and enhanced virus replication at high dilution. The antiretroviral treatments that the women received and the small sample size may have contributed to the lack of association between HIV enhancement by maternal plasma and vertical transmission.

Background The rate of HIV-1 infection has been increasing rapidly among women of childbearing age. At the end of 2003 women accounted for 50% of adults living with HIV/AIDS worldwide [ 1 ]. Consequently, the number of pediatric AIDS cases due primarily to perinatal (peripartum or intrapartum) transmission is rapidly increasing. Mother-to-child transmission accounts for more than 90% of all HIV infections in infants and children worldwide. In 2003 an estimated 2.1 million children under 15 years were living with HIV/AIDS [ 1 ]. Zidovudine (ZDV) given as either an intensive or short course regimen significantly reduces perinatal transmission [ 2 , 3 ]. However, because of its cost, ZDV is not always available in poorer countries of the world. Successful use of nevirapine therapy in preventing perinatal transmission offers hope for more affordable treatment for poor women worldwide [ 4 , 5 ]. However, in 2003, only one in ten pregnant women was offered services for preventing mother-to-child HIV transmission [ 1 ]. Further, whether treated with ZDV or nevirapine, a portion of HIV-positive women still transmit virus to their offspring vertically and the problem of maternal-infant transmission through breast milk remains unsolved. Therefore, there is need for continued studies of viral and immunological factors associated with maternal-infant transmission of HIV so that other effective and affordable strategies to prevent transmission may be developed. Although some studies show no association between the presence of HIV neutralizing antibodies in maternal sera and the risk of perinatal transmission [ 6 , 7 ], other studies report a reduction in the risk of vertical transmission in pregnant women whose sera contain neutralizing antibodies to HIV [ 8 ]. A number of studies have indicated lower transmission rates from infected pregnant women with high antibody titer or with high affinity/avidity antibody to conserved portion of HIV-1 glycoprotein 41 [ 9 ], to the CD4 binding site [ 10 ] or the V3 loop of glycoprotein 120 [ 11 , 12 ], and to the p24 Gag protein [ 13 ]. Other studies have reported that non-transmitting mothers more frequently have such antibodies to their own virus than do transmitting mothers and that transmitting mothers rarely have neutralizing antibody against their own children's isolates [ 14 , 15 ]. In contrast, a study by St. Louis, et al. [ 16 ] found no evidence that anti-V3 loop antibody protected against perinatal transmission. Further, a study by Lallemant, et al. [ 17 ] showed that mothers with higher antibody titers to peptides corresponding to the V3 region of gp120 and the immunodominant domain of gp41 had a higher risk of perinatal transmission. The authors hypothesized that women who display the broadest antibody response to V3 may be experiencing the greatest viral turnover [ 18 ] which could make them more at risk for transmitting virus to their offspring. In contrast to neutralizing antibodies, non-neutralizing antibodies may enhance HIV infection by binding to the virus and facilitating its uptake by cell types that carry immunoglobulin (Fc) or complement receptors. Antibodies that enhance HIV replication in vitro by either Fc gamma receptor- or complement receptor-mediated endocytosis (FcγR-ADE or C'-ADE) have been identified in sera from HIV-1-infected individuals [ 19 - 25 ] and from many gp120-vaccinated volunteers [ 19 , 20 ]. An in vitro study of antibody dependent enhancement (ADE) of HIV-1 infection in human term syncytiotrophoblast cell cultures suggested that both FcγR-ADE and C'-ADE may contribute to maternal-infant transmission of HIV-1 [ 26 ]. Pancino, et al. [ 27 ], reported that mother-infant transmission of HIV was associated with maternal antibodies to the envelope gp160 and to a highly conserved domain of the trans-membrane glycoprotein. Mann, et al. [ 28 ], observed that certain combinations of antibody subclasses occurred more frequently in mothers who transmitted HIV-1 to their offspring than in non-transmitters and suggested that ADE may occur in mother-infant transmission of HIV-1. However, C'-ADE was not found to be associated with maternal-infant transmission of HIV [ 29 ] and the role of FcγR-ADE in maternal-infant transmission has not been determined. Maternal plasma virus load has been shown to be strongly associated with perinatal transmission of HIV [ 27 , 30 , 31 ] and it was reported that there is no absolute threshold of maternal viral load below which HIV transmission does not occur [ 32 ]. Although transmission did not occur at a threshold below 2,000 copies/ml [ 18 , 33 ] or below 1000 copies/ml [ 34 ], more recent meta-analysis has demonstrated that occasional transmission does occur below a viral load threshold of 1,000 copies/ml [ 35 ]. Another study indicated that viral load correlated with vertical transmission in women at the clinical stage A1 (asymptomatic) of infection [ 36 ]. However, the association of both viral load and enhancing activity (presumably by FcγR-ADE) in maternal plasma and vertical transmission of HIV has not formerly been examined. Thus, we examined the association of these factors (independently and combined) in vertical transmission using samples from mother-infant pairs from San Juan, Puerto Rico, previously described by Melendez-Guerrero et al [ 37 ]. Neutralization/enhancement of a R5 tropic strain of HIV-1 subtype B by maternal plasma was examined in monocyte-derived macrophage cultures. Results and Discussion Study sample A cohort of 43 HIV-1 subtype B infected pregnant women attending antenatal clinic at the San Juan City Hospital, Puerto Rico, was enrolled into a prospective study from their first antenatal visit until delivery [ 37 ]. Eleven women were enrolled during their first trimester, 24 during their second trimester, and 8 during their third trimester of pregnancy. Approximately forty-five HIV-1 infected women gave birth at the San Juan City hospital during 1998. All of the women recruited into the study received some form of antiretroviral therapy as detailed and referenced [ 3 , 38 - 44 ] in Table 1 . All of the infants were enrolled into the study shortly after delivery and most (86%) were also enrolled in the antiviral protocols during their first six weeks of life (Table 1 ). One mother had triplets, therefore, a total of 43 mothers and 45 infants (regarded as 45 mother-infant pairs) were enrolled. Table 1 Number and percent of HIV positive pregnant women and infants assigned to the different AIDS Clinical Trails Group (ACTG) protocols or to zidovudine (ZDV) ACTG Protocol # Number of women (%) Dose and frequency 185 [38] 16 (37.2) Mother : ZDV according to 076 protocol [3] plus anti-HIV immune serum globulin (HIVIg) or immune globulin (Ig) (200 mg/kg) every 28 days followed by 1.0 mg/kg/hr continuous infusion during labor and delivery. Infant : HIVIg (200 mg/kg) or normal Ig within 12 hrs of birth plus oral ZDV syrup (2.0 mg/kg) every 6 hours after birth (beginning within 8–12 h) and continuing for 6 weeks. 249 [39] 4 (9.3) Mother : Didanosine (ddi) IV (1.6 mg/kg) on day one, during pregnancy, followed by an oral dose (200 mg) one week after the initial dose. Oral ddi is then administered every 12 hrs until labor starts and every 12 hrs after delivery until 6 weeks post-partum. During labor and delivery patients receive a loading dose followed by continuous infusion. Infant : Oral ZDV syrup (2.0 mg/kg) every 6 hours after birth and continuing to week 6. 250 [40] 5 (11.6) Mother : ZDV plus Nevirapine (200 mg/kg) single dose during labor. Infant : Oral ZDV syrup (2.0 mg/kg) every 6 hours after birth and continuing to week 6 plus single dose of Nevirapine (2 mg/kg) after birth. 296 [41] 3 (7.0) Mother: ZDV as in protocol 185. Infant : Oral ZDV syrup (2.0 mg/kg) every 6 hours after birth and continuing to week 6. 316 [42] 9 (20.9) Mother: Nevirapine (200 mg oral dose) or the corresponding placebo during delivery plus ZDV (as in 076). Infant: ZDV perinatal prophylaxis (2.0 mg/kg) plus single 2.0 mg/kg oral dose of Nevirapine or Nevirapine placebo administered between 48 and 72 hrs of life. 324 [43] 3 (7.0) Mother : ZDV before and after delivery as is usual but oral administration of ZDV (300 mg) every 3 hrs, 3 doses total during delivery. Infant : Oral ZDV syrup (2.0 mg/kg) every 6 hours after birth and continuing to week 6. 332 [44] 1 (2.3) Mother: Stravudine (d4T, 30–40 mg) during pregnancy and 0.05 mg/kg/hr during delivery in combination with 3TC 150 mg followed by 150 mg during delivery. Infant: Stravudine (d4T, 1 mg/kg) single dose on day 35–42, in combination with 3TC (2.0 mg/kg/dose). ZDV [3] 2 (4.7) Mother: ZDV (2.0 mg/kg) every 28 days followed by 1.0 mg/kg/hr continuous infusion during labor. Infant : Oral ZDV syrup (2.0 mg/kg) every 6 hours after birth and continuing to week 6. Demographic and clinical characteristics of the mothers and vertical transmission The mean age of the mothers was 24 years (range 14–38 years; Table 2 ). Majority of the women (88.4%) were infected through heterosexual contact. Based on the 1993 CDC revised classification system for HIV infection and disease progression, only 6 of the mothers (those in the A3 and B3 categories) were classified as AIDS cases [ 45 ]. Approximately two-thirds (67.4%) of the mothers had viral load levels below 10,000 RNA copies/ml and were classified as having low viral loads (LVL) based on the categorization by Contopoulos-Ioannidis & Ioannidis [ 46 ]. The remaining 32.6% of mothers had viral loads above 10,000 RNA copies/ml and were classified as having high viral loads (HVL). CD4+ T cell counts in the women ranged from 23 to 1165 cells/mm 3 of blood with a mean count of 425 cells/mm 3 . Twenty-six of the 43 women delivered their babies by normal vaginal delivery; the remaining women had cesarean sections (Table 2 ). Table 2 Age, source of infection, clinical status and transmission outcome for HIV-positive mothers Variable Number (%) Age of mothers (years) a < 25 14 (32.6) 25–30 12 (27.9) > 30 16 (37.2) Missing 1 (2.3) Source of infection Heterosexual contact 38 (88.4) IV drug use 3 (7.0) Unknown 2 (4.6) Clinical status (CDC classification 45) A1 11 (25.6) A2 14 (32.6) A3 1 (2.3) B1 2 (4.6) B2 10 (23.3) B3 5 (11.6) Viral load (copies/ml) b <10,000 (range 83–9,078) 29 (67.4) ≥ 10,000 (range 10,220–484,703) 14 (32.6) Mode of delivery Vaginal 26 (60.5) Cesarean 17 (39.5) Transmission outcome V3 PCR+ Infants 12 (26.7) V3 PCR- Infants 33 (73.3) Culture+ Infants 3 c (6.7) Culture- Infants 42 (93.3) a Mean age 24 years (range 14–28 years) b Mean viral load 28,112 copies/ml (range = 83 – 484,703 copies/ml) c HIV infection confirmed by PCR and culture Twelve (26.7%) of 45 infants were HIV V3 positive indicating that HIV transmission had occurred (Table 2 ). However, only three of these twelve infants were also HIV culture positive. This infection rate of approximately 7.0% observed is similar to the rate of 8.3% observed for ZDV treated mothers and infants in the 076 study [ 4 ]. Based on the V3 PCR results an equal number of mothers with low and high viral load (6 in each group) transmitted HIV to their infants. The mean log viral load of transmitting mothers 3.77 + 0.31 (median = 3.77) was higher than of the non-transmitting mothers 3.54 + 0.15 (median = 3.44) but the difference was not statistically significant (p = 0.474). This is probably due to the small sample size. The mothers of the three infants who were HIV culture positive all had high viral load levels (484,703, 11,642 and 10,220 RNA copies/ml) but the levels in two of the three were close to the 10,000 RNA copies/ml cut-off value used to distinguish LVL from HVL. Since perinatal transmission occurs mostly at or during delivery, the viral load in the genital tract (which may be similar to maternal plasma level) [ 47 ] may be an important determinant in maternal-infant transmission. The mothers of HIV V3 positive infants had a non-significantly higher mean CD4+ T count (526 cells/mm 3 , median = 508 cells/mm 3 ) compared to non-transmitting mothers (mean CD4+ T count of 413 cells/mm 3 ; median = 336 cells/mm 3 ). Maternal CD4 cell counts have been shown to be a less effective predictor of transmission of HIV than viral load [ 18 ]. Further, most of the women in the study (86%) had CD4+T cell counts above the 200 cells/mm 3 level which is used to define AIDS. Only one of the three women whose infants were HIV culture positive had AIDS (CD4 = 149 cells/mm 3 ). The other two women had CD4 counts of 510 and 538 cells/mm 3 . Neutralization/enhancement of HIV treated with maternal and infant plasma The effect of plasma (at 10 -1 to 10 -3 dilutions) from the mothers (transmitters and non-transmitters) and infants (HIV V3+ and HIV V3-) on HIV replication was examined in the neutralization/enhancement assay (Table 3 ). HIV p24 antigen in culture fluids was determined using the coulter p24 antigen assay kit (Coulter, Miami FL) according to the manufacturer's instructions. The mean value of HIV p24 antigen by dilution was calculated from seronegative samples. The percent change (increase or decrease) in p24 antigen of the maternal or infant plasma from the mean of the seronegative samples was calculated. Neutralization was defined as 70% or greater reduction in HIV p24 antigen in cultures treated with HIV positive plasma compared with cultures treated with HIV negative plasma. Enhancement was defined as 100% or greater increase in p24 antigen in cultures treated with HIV positive plasma compared to the p24 level in cultures treated with HIV negative plasma. Traditionally enhancement of HIV in PBMC cultures has been defined as a 1.5 to 2.5-fold or greater increase in virus replication as a result of treatment with immune sera [ 21 , 25 , 48 ]. Table 3 shows that at 10 -1 dilution, over 90% of plasma samples from both groups of mothers (transmitters/non-transmitters) and infants (HIV V3+/ HIV V3-) neutralized HIV. At 10 -2 dilution the percentages of plasma that neutralized HIV dropped to approximately 75% for both groups of mothers and to 58% and 70% for HIV V3+ and HIV V3- infants respectively. The percentages again dropped at 10 -3 dilution to ≤ 67% for mothers and ≤ 33% for infants (Table 3 ). Chi-square or Fisher's exact test were used to compare the proportion of plasma from transmitter versus non-transmitter mothers and between HIV V3+ and HIV V3- infants that neutralized, enhanced or resulted in no change in HIV replication. No significant differences were found. Table 3 Neutralization/enhancement of HIV infection by plasma from 12 transmitter and 33 non-transmitter mothers and 12 HIV V3 positive and 33 HIV V3 negative infants Plasma Dilution and effect Transmitter mothers Number (%) Non-transmitter mothers Number (%) HIV V3+ infants Number (%) HIV V3- infants Number (%) 10-1 Neutralized 11 (92) 31 (94) 11 (92) 30 (91) Enhanced 0 (0) 0 (0) 0 (0) 0 (0) No change 1 (8) 2 (6) 1 (8) 3 (9) 10-2 Neutralized 9 (75) 25 (76) 7 (58) 23 (70) Enhanced 1 (8) 2 (6) 2 (17) 1 (3) No change 2 (17) 6 (18) 3 (25) 9 (27) 10-3 Neutralized 8 (67) 19 (58) 4 (33) 9 (27) Enhanced 0 (0) 5 (15) 4 (33) 7 (21) No change 4 (33) 9 (27) 4 (33) 17 (52) The mean percent change in p24 antigen and standard errors for mothers and infants were plotted for each group by plasma dilution (Figure 1 ). The Wilcoxon signed-ranked test was used to determine whether the percent change was significantly different from zero. Both the maternal and infant plasma significantly (p = 0.001 for both groups) reduced HIV replication at low (10 -1 ) dilution when compared with HIV negative sera (Figure 1 ). At 10 -2 dilution the infant plasma still significantly (p = 0.001) reduced virus replication, but the maternal plasma lost neutralizing activity (Figure 1 ). At 10 -3 dilution, the maternal plasma significantly increased virus replication (p = 0.005) above seronegative plasma and the infant plasma showed a non-significant increase (88%) in HIV replication (Figure 1 ). These findings of neutralization by plasma of HIV positive individuals at low dilutions and enhancement at higher dilutions are similar to data published by Jolly and Weiss [ 49 ], which showed that neutralizing and enhancing antibodies can occur simultaneously in sera of HIV-infected individuals. If neutralizing antibody is present, enhancement is seen only at high dilutions, whereas, if only enhancing antibody is present, enhancement is observed without, or at low, plasma/serum dilutions. Figure 1 Neutralization/enhancement of HIV-1BaL by maternal and infant plasma diluted 10 -1 – 10 -3 . Virus replication (determined by p24 antigen (pg/ml) in culture fluids collected 2–4 days post-infection) is compared to replication of virus treated with plasma from HIV-1 seronegative (sn) women. The data represent the average of all maternal and infant samples. Two independent infections were conducted with each sample in duplicate (4 replicates) for each of 43 maternal and 45 infant plasma. Maternal and infant plasma significantly reduced HIV replication (p = 0.001 for both) at 10 -1 dilution compared to HIV negative sera. Infant plasma also significantly reduced HIV replication (p = 0.001) at 10 -2 dilution. At 10 -3 dilution maternal plasma significantly increased HIV replication (p = 0.005) above HIV negative sera and infant plasma showed a non-significant increase (88%) in HIV replication. Enhancing activity in plasma from mothers and infants Sixty-nine percent (31/45) of plasma from mother-infant pairs were also tested in the neutralization/enhancement assay at higher dilutions (10 -4 to 10 -6 ). Examination of enhancing activity in plasma of mothers and infants showed that there were good agreements (60% or greater) in enhancement status between mother-infant pairs. For example, at 10 -4 dilution, the plasma of 7 infants of 9 mothers (78%) whose plasma enhanced HIV replication also exhibited enhancement, and at 10 -6 dilution, the plasma of 8 infants of 10 mothers (80%) whose plasma enhanced HIV replication also exhibited enhancement (data not shown). Comparison of p24 antigen between transmitter and non-transmitter mothers or their infants at 10 -4 to 10 -6 dilutions showed no significant difference between the two groups of mothers or infants. The lack of association of enhancing activity and HIV transmission in this study is similar to the findings for C'-ADE by Gras, et al. [ 29 ]. We examined complement-independent (presumably FcγR-mediated antibody dependent) enhancement in primary human macrophages because we thought that this type of enhancement would be more relevant to HIV clinical disease and transmission. FcγR-mediated enhancement is characteristic of diseases such as dengue and feline infectious peritonitis for which ADE has been best demonstrated to occur [ 50 , 51 ]. In addition, macrophages are important target cells for infection and replication in vivo by most HIV-1 variants [ 52 - 54 ]. However, our results showed no association between enhancing activity in maternal or infant plasma and maternal-infant transmission of HIV. The negative results could in part be due to the various anti-retroviral protocols to which the women were assigned. Correlation between maternal and infant p24 antigen levels and maternal viral load with maternal and infant p24 antigen Using Spearman's correlation, we examined the association between maternal viral load and enhancing activity in vertical transmission of HIV by using mothers' characteristics (viral load and p24 antigen value) and the infants' p24 antigen values. A correlation matrix showed a positive association between mothers' p24 antigen values and those of their infants (Table 4 ). However, low positive or no associations were found between maternal viral load and maternal p24 antigen values at low dilutions (10 -1 – 10 -3 ) and low negative associations at higher dilutions (10 -4 – 10 -6 ). There were low positive associations between maternal viral load and the p24 antigen values of their infants at low dilutions (10 -1 – 10 -3 ) and low negative correlations at higher dilutions except at 10 -5 dilution (Table 4 ). However, none of the values was statistically significant. Table 4 Correlation (Spearman) of maternal and infant p24 antigen levels and maternal viral load with maternal and infant p24 antigen Plasma dilution p24 antigen (M vs I) Correlation a log (p-value) b MVL and Mp24 Correlation c log (p-value) b MVL and Ip24 Correlation d log (p-value) b 10 -1 0.412 (0.005) 0.12 (0.448) 0.09 (0.562) 10 -2 0.59 (0.0001) 0.18 (0.249) 0.11 (0.473) 10 -3 0.72 (0.0001) 0.14 (0.355) 0.04 (0.832) 10 -4 0.40 (0.028) -0.15 (0.423) -0.16 (0.394) 10 -5 0.75 (0.0001) -0.10 (0.581) 0.31 (0.091) 10 -6 0.69 (0.0001) -0.34 (0.064) -0.31 (0.089) M = mother; I = infant; MVL = maternal viral load; Mp24 = maternal p24 antigen; Ip24 = infant p24 antigen a Spearman correlation coefficient between log values of maternal and infant p24 antigen. b p-value to test for statistically significant correlation. c Spearman correlation coefficient between log values of maternal viral load and maternal p24 antigen. d Spearman correlation coefficient between log values of maternal viral load and infant p24 antigen. Maternal viral load and enhancing antibodies as predictors of vertical transmission of HIV Univariate analysis of type of treatment, stage of HIV disease, method of delivery and CD4+ T cell count of the mothers indicated no significant association with vertical transmission of HIV (p > 0.05). The combined effect of maternal viral load and enhancing antibodies as potential risk factors for the vertical transmission of HIV-1 was examined in a multivariable model. Log viral load was treated as a continuous variable in the model and enhancement was categorized as enhancement versus neutralization. There was a trend of association between maternal viral load and transmission of HIV-1 so that the higher the viral load, the more likely mothers were to transmit HIV-1 to their infants (OR= 2.5, CI= 0.93 – 6.67, p = 0.0681). This is in agreement with other studies discussed earlier that show viral load as a predictor of maternal-infant transmission [ 27 , 30 , 31 ]. The non-significant result in this study is probably due to the small sample size. Also, twice as many mothers (29) in this study had low plasma HIV RNA levels (<10,000 RNA copies/ml) compared to those with high viral load levels (14 mothers). The preponderance of women with low viral load levels may be the result of the effect of the different antiretroviral protocols that the women were given during pregnancy. Whereas Dickover, et al. [ 30 ] showed that women given zidovudine during gestation showed an eight-fold median decrease in plasma HIV RNA levels (p < 0.001), Sperling, et al., [ 32 ] have shown that ZDV treatment had only a minimal effect in decreasing maternal HIV RNA levels. In this study all of the women except one (given stravudine) received ZDV treatment and in most cases ZDV was given along with one other anti-retroviral drug or with HIVIg (Table 1 ). There was no significant association between enhancing activity (based on p24 antigen values) in the plasma of mothers who transmitted HIV-1 to their infants based on the HIV V3+ status of the infants (OR = 0.92, CI = 0.41 – 2.07, p = 0.8492). Conclusion In agreement with published data, multivariate analysis showed a trend of association between maternal viral load and maternal-infant transmission of HIV. The non-significant difference in the mean log viral load of transmitting and non-transmitting mothers is probably due to the small sample size. No significant associations were found between HIV antiretroviral treatment protocols, classification of HIV disease, method of infant delivery and CD4+ T cell count of the mothers and vertical transmission of HIV. Both maternal and infant plasma significantly neutralized HIV infection at low (10 -1 ) dilution and enhanced virus replication at higher dilution (10 -3 ). Neutralizing and enhancing antibodies can occur together in the blood of HIV positive individuals and the neutralizing effect can be lost at high plasma dilution. There were good agreements in the neutralizing or enhancing activity of the plasma from mothers-infant pairs. That is, when plasma of the mothers neutralized or enhanced HIV infection, their infants' plasma showed similar activity. However, there was no significant association between virus enhancement by maternal plasma and vertical transmission of HIV. Thus, enhancing activity in plasma of these HIV-infected mothers was not a dominant factor in vertical transmission of HIV. Methods Collection, processing and testing of maternal and infant blood samples for HIV Blood samples were taken from the mothers during each trimester of pregnancy and during delivery. Blood samples from the infants were collected from the umbilical cord at birth and at 1–2, 3–4 and 5–12 months. Blood was collected in vacutainer tubes containing ACD anticoagulant and centrifuged at 2500 rpm for 15 minutes. The plasma was stored frozen (-20°C) for use in maternal viral load and HIV neutralization/enhancement assays. The remaining blood was diluted in phosphate-buffered saline (PBS) pH 7.4 and processed by Ficoll-hypaque density gradient for cell isolation. For virus isolation, one million cells from each mother or infant were co-cultured with HIV seronegative donor cells previously stimulated by PHA as described in the ACTG virology manual [ 55 ]. The remaining cells (10 5 ) were stored frozen at -85°C. The HIV status of the infants was determined using a nested PCR assay of the HIV-1 envelope variable (V3) region as previously described [ 37 , 56 ]. This V3 PCR assay was conducted in duplicates and repeated on the infant samples at 1–2 months, 3–4 months and 5–11 months. Culture for HIV was repeated on infant samples collected at 3, 6 and 12 months and all except 3 infants were culture negative. Determination of maternal viral load HIV RNA copies in maternal plasma was determined by the amplicor HIV monitor test (Roche Diagnostics, Branchwater, NJ, USA) at the Puerto Rico ACTG-certified laboratory [ 37 ]. A 142 base-pair sequence in the HIV gag gene was amplified by RT-PCR for viral load determination. The mean log viral load was calculated and compared between transmitting and non-transmitting mothers to determine the effect of maternal viral load on HIV vertical transmission. Preparation and titration of HIV-1BaL for the neutralization/enhancement assay HIV-1BaL stock was prepared in primary macrophages as reported previously by Jolly [ 57 ]. Briefly, HIV-1BaL supernatant fluid (1 × 10 4.6 TCID 50 /ml) obtained from the NIH AIDS Research and Reference Reagent Program was used to inoculate fresh cultures of macrophages grown on 75 ml tissue culture flasks. The cultures were washed 24 hours later and incubated with fresh media. Supernatant fluids were harvested at 7 and 14 days post-infection, clarified by centrifugation at 1800 rpm for 10 minutes and used as stock virus for these studies. The stock contained 5 × 10 5 TCID 50 /ml. Neutralization/enhancement assay A neutralization/enhancement assay was conducted using maternal or infant samples and HIV-1BaL. Maternal plasma samples collected during the third trimester of pregnancy were used in these assays since most prenatal HIV infections occur in the third trimester [ 58 ]. Briefly, 10-fold dilutions of heat inactivated (56°C for 30 min) plasma samples were mixed 1:1 with 10 3 TCID 50 /ml of virus and pre-incubated at 37°C for 30 min. The mixtures were then inoculated into replicate cultures of monocyte-derived macrophages as prepared previously [ 57 ] in 8 well chamber slides and incubated at 37°C for 6 hours in a 5% CO 2 incubator. The inocula were removed and the cells washed, and incubated with fresh media for up to 8 days. Culture supernatant fluids were then collected on days 2, 4, 6, and 8 and tested for p24 antigen using the Coulter assay (Coulter, Miami, FL, USA). Cultures treated with HIV-negative sera were used as controls. All maternal and infant plasma samples were tested at three 10-fold dilutions; 69% of samples were tested up through six 10-fold dilutions. Two independent assays were conducted for each maternal or infant plasma sample and each sample was run in duplicate on each assay (4 replicates). Statistical analysis Descriptive statistics such as mean, median, range were calculated to summarize maternal characteristics such as viral load, CD4+ T cell count and p24 antigen levels. The number of HIV vertical transmission among infants was summarized and the univariate association of maternal characteristics, such as, treatment (type of antiretroviral therapy), stage of disease based on the 1993 CDC classification [ 45 ], method of delivery (C-section vs. vaginal), and CD4+ T cell count, with HIV transmission in infants was evaluated using the Fisher's exact test. The simultaneous effect of maternal viral load and p24 levels on infant transmission was evaluated using the logistic regression model. Odds ratio and 95% confidence intervals were calculated for the effect of both factors in the model. Correlation between maternal versus infant p24 antigen levels and between maternal viral load and maternal or infant p24 antigen levels was evaluated using the Spearman's correlation coefficient. Enhancement, decrease (neutralization), or no change in p24 levels compared to seronegative control was assessed for mother-infant pairs. The distribution of virus p24 antigen was compared between transmitting vs. non-transmitting mothers and between HIV V3+ infants and HIV V3- infants using Fisher's exact test. Competing interests The author(s) declare that they have no competing interests. Authors' contributions LMG and PEJ were involved in conception and design of the study, collection of the samples, interpretation of the data and drafting and critical review of the manuscript. PK and MA were involved in performing the laboratory experiments that resulted in the acquisition of the data, in data entry, and in drafting and revising the manuscript with LMG and PEJ. HLW was responsible for data analysis and interpretation along with PEJ, LMG and PK. All authors have read and approved the final manuscript.

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524357

Extreme conservation of noncoding DNA near HoxD complex of vertebrates

Background Homeotic gene complexes determine the anterior-posterior body axis in animals. The expression pattern and function of hox genes along this axis is colinear with the order in which they are organized in the complex. This 'chromosomal organization and functional correspondence' is conserved in all bilaterians investigated. Genomic sequences covering the HoxD complex from several vertebrate species are now available. This offers a comparative genomics approach to identify conserved regions linked to this complex. Although the molecular basis of 'colinearity' of Hox complexes is not yet understood, it is possible that there are control elements within or in the proximity of these complexes that establish and maintain the expression patterns of hox genes in a coordinated fashion. Results We have compared DNA sequence flanking the HoxD complex of several primate, rodent and fish species. This analysis revealed an unprecedented conservation of non-coding DNA sequences adjacent to the HoxD complex from fish to human. Stretches of hundreds of base pairs in a 7 kb region, upstream of HoxD complex, show 100% conservation across the vertebrate species. Using PCR primers from the human sequence, these conserved regions could be amplified from other vertebrate species, including other mammals, birds, reptiles, amphibians and fish. Our analysis of these sequences also indicates that starting from the conserved core regions, more sequences have been added on and maintained during evolution from fish to human. Conclusion Such a high degree of conservation in the core regions of this 7 kb DNA, where no variation occurred during ~500 million years of evolution, suggests critical function for these sequences. We suggest that such sequences are likely to provide molecular handle to gain insight into the evolution and mechanism of regulation of associated gene complexes.

Background Eukaryotic genome contains a large excess of non-coding sequences. Conservation of these sequences among species is a strong indication of their functional significance. With the availability of genome sequences it is possible to identify such sequences taking a comparative genomics approach [ 1 - 4 ]. The clusters of homeotic genes, which are expressed in a coordinated manner [ 5 ], are among the most conserved regions of the vertebrate genome. Clustering of genes that are regulated in a linked manner has been noticed in several other cases [ 6 , 7 ]. However, the molecular mechanism behind such coordination in regulation is not yet understood. Several mechanisms have been proposed that link the organization of homeotic genes and the spatio-temporally controlled expression [ 8 ]. Colinearity in hox complexes was first discovered in Drosophila [ 9 ] and later studies on the bithorax complex have demonstrated the role of chromatin organization in its regulation [ 10 ]. Recent studies on the HoxD complex suggest a role for higher order chromatin organization in the regulation of this complex involving up to 20 kb upstream region [ 11 ]. Results and discussion We compared genomic regions flanking hox complexes in order to identify conserved regions with potential regulatory function. Here we report that the upstream regions of HoxD complexes of human, mouse, rat, sacred baboon, horn shark, zebra fish and puffer fish contain long stretches of extremely conserved sequences. In the 25 kb region upstream of the HoxD complex from these organisms we found an extremely conserved region spread in three blocks located within 7 kb from the 3' end of the Evx-2 gene. These conserved regions, designated as C onserved R egion 1 , C onserved R egion 2 and C onserved R egion 3 (CR1, CR2 and CR3) (Fig. 1 ) show a degree of conservation not seen before among distant species. Detailed analysis of each region spanning to several hundred base pairs, in particular the CR2 shows several stretches of 100 % conservation, Fig. 2 . We also noticed longer stretches of conservation among mammals, which gradually shortens as we go towards lower vertebrates, defining the core of each conserved region, across the vertebrate classes, see Additional file 1 . This and the fact that in case of shark, as compared to mammals, the intervening sequence lengths between CR2 and CR3, and CR1 and Evx-2 is shorter by ~1300 bp and ~600 bp, respectively (Fig. 1 ) suggest that starting from the shorter conserved regions, additional unique sequences have progressively been acquired and conserved during the evolution of primates from lower vertebrates. This may reflect the molecular basis of conservation and elaboration of Hox gene regulation during evolution of these species [ 12 ]. Figure 1 Schematic representation of sequence conservation in the HoxD upstream region. Human sequence (AC009336; from position 56601 to 64095) was compared to the corresponding sequences of Papio hamadryas (AC116665), Heterodontus francisci (AF224263), Mus musculus (AC015584), Fugu rubripes (CAAB01000449) and Rattus norvegicus (NW_042732). Sequences that are conserved across vertebrates are shown as blocks. The conservation extends beyond these blocks within primates and rodents. ESTs found in the database corresponding to this region are also shown. ESTs mapping to CR3 are BB838602 from mouse 8 cell embryo and BU129154 from chicken 36 stage limb; and those mapping to CR1 are AA620964 from human testis; BB332383, BB335110, BB334358, BB333569 from 6 and10 days mouse neonate medulla oblongata and BU255316 from chicken 36 stage limb. Figure 2 Comparison of conserved regions from human, mouse and shark. Conserved bases of mouse and shark are shown as '.' and '-' indicates indels. Underlined sequences of human indicate primers that were used for amplification of the corresponding sequence from different vertebrates. Universal occurrence of these sequences in all vertebrate classes was confirmed by their amplification using primers from human HoxD complex (Figure 3 ) followed by Southern hybridization and sequencing (unpublished observation). Furthermore, using CR1, CR2 or CR3 as query we searched genomic sequences of variety of eukaryotes in available databases. This search indicated that these sequences are single copy and vertebrate specific. While these conserved regions appear to be a key component of the HoxD complex of all vertebrates looked at, we did not find such a degree of conservation in the flanking regions of other hox complexes ( HoxA , B and C ) of vertebrates. In order to trace back the evolutionary origin of such sequences, it will be of interest to investigate occurrence of these sequences at the corresponding region in the hox complexes of species of urochordata, cephalochordata or even agnatha. In the tunicate Oikopleura dioca , where hox genes are dispersed but the spatial pattern seen in other animals is still present [ 13 ], we did not find CR1, CR2 or CR3. Also, we did not find any significant conserved region corresponding to these CRs in the amphioxus genomic region that contains the cluster of hox genes. It appears, therefore, that these extremely conserved sequences have originated in the vertebrates where the hox complex has additional distinct features of tight clustering compared to the insect hox clusters and the temporal colinearity, not seen in invertebrates. Figure 3 Conservation of CR1, CR2 and CR3 in all vertebrate classes. PCR amplification of different vertebrate genomic DNA samples using primers designed based on the human sequence. Lanes: M – size marker indicated in bp, hu – human, mo – mouse, ch – chicken, co – cobra, fr – frog and zf – zebra fish. The arrows indicate the corresponding products that have been confirmed by direct sequencing as well as Southern hybridization using human CRs as probe. Several recent reports using comparative genomics approach have identified conserved non-coding regions among different vertebrates [ 14 - 16 ] but none to the degree that we report here. The mechanism that may require such a high degree of conservation is not known. It is not, therefore, immediately clear what precisely is the role of these sequences. EST database search revealed that part of CR1 and CR3 are transcribed without any significant ORF but no EST corresponding to CR2 or any other part of the 7 Kb region was found, Fig. 1 . A possible mechanism could involve RNA from this region that may function by base pairing to the genomic target sites. If that is the case, such high conservation could be expected. Role of transcription in the regulation of bithorax complex is emerging from recent studies [ 17 ]. Conclusions While such an extreme conservation of several hundred nucleotides over half a billion years in a region that does not code for any known proteins certainly implicates essential role for such sequences, probably in the regulation of HoxD complex, no known regulatory element requires such extreme conservation extending up to hundreds of base pairs. It is, therefore, likely that these elements could be components of a novel mechanism common to all vertebrates that regulates this gene complex. We are tempted to suggest that such a strongly conserved region from fish to human linked to a gene complex that is known to determine body axis formation may be the key determinant of molecular basis of early ontogeny. Early embryos of all vertebrates show striking similarity and we suggest that these elements may control the early expression pattern of HoxD which leads to similar pattern of the embryo shape. The gradient of conservation seen in this region from fish to human may further signify the evolutionary history of this locus and diversification of the morphological features along the anterior-posterior body axis of the vertebrate classes. Methods Sequence analysis The genomic sequences that contained Evx-2 and any of the Hoxd genes were downloaded and annotated using gene/ORF prediction tools. Similar approach was used for other hox complexes. Homology searches of the upstream sequences of HoxD region from human (AC009336; from nucleotide 56601 to 64095) was carried out using the BLAST program of NCBI. The sequences that showed significant homology were further used to analyze the extent of homology by BLAST 2 program. The conserved regions from each sequence was obtained and subjected to multiple sequence analysis using Clustal X. In order to identify the expressed sequences corresponding to the conserved sequence, the conserved sequences along with the unique sequences were BLASTed against EST databases (human, mouse and dbEST). The contigs that showed significant homology to the upstream sequences of human HoxD were annotated using the tBLASTx program and searching the translated amino acid sequence in the Swissprot database. Repeat masker program was used to look for repeat content. Genebank sequences used in this study are as follows: AC116665 Papio hamadryas , AF224263 Heterodontus francisci , AC015584 Mus musculus , AC009336 Homo sapiens , CAAB01000449 Fugu rubripes and NW_042732 Rattus norvegicus . DNA isolation, PCR amplification, sequencing and Southern hybridization For the isolation of genomic DNA blood samples of human, chick and cobra ( Naja naja ) were used while liver tissue of mouse and muscle tissue of frog ( Bufo melanostictus ) and zebra fish were used. Standard protocol of DNA isolation was followed which included lysis, RNase A and proteinase K digestions followed by phenol/chloroform extraction and precipitation. Concentration and quality of the genomic DNA was checked on 0.7% agarose gel and UV absorption spectrophotometry. Based on the sequence of conserved regions primers were designed to amplify the three regions CR1, CR2 and CR3. Primers used in this study to amplify conserved regions from different vertebrate species were:CR1 forward- GAGGCTGTTCACACTGGTGG,CR1 reverse- ATCATGCTCTCTGATGGACC,CR2 forward- GCATCGTAATCAGTTCGGTC,CR2 reverse- TGATACAAGCTGATACCGTC,CR3 forward- GCTATTCAAAATGTTATTTGAG and CR3 reverse- CTGTAATGAAGAAAAGATTTATG. The 25 μl reaction was performed using 100 ng template DNA and 5 pmol each of forward and reverse primers. PCR protocol was as follows: initial denaturation step of 94°C for 3 min was followed by 35 cycles of 94°C for 1 min, 57°C for 1 min and 72°C for 1.30 min and final extension step at 72°C for 7 min. Authenticity of the PCR products was confirmed by direct sequencing and Southern hybridization, using the corresponding human DNA as probe. Note An earlier version of this article was deposited in the 'Deposited Research' section of Genome Biology, , [ 18 ]. While this manuscript was in reviewing process, a report comparing human genome to several other mammalian sequences identified many highly conserved noncoding sequences [ 19 ]. Interestingly, this study also identified CR2 as uc.108 near HOXD and, in agreement to our observation, noted only a "core" conserved region in fish, suggesting that additional parts of the ultraconserved region were innovations after the common ancestor with fish. Authors' contributions CS carried out the sequence analysis, PCR amplification and Southern analysis. SS participated in sequence analysis and DNA isolation from several organisms. AT carried out the sequencing of PCR products and participated in the sequence alignments. RKM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Size and degree of conservation of CR1, CR2 and CR3 in different vertebrates. Core of conserved regions and extended conserved regions between indicated species is shown as length of sequence and degree of conservation. Non-overlapping blocks of vertebrate conservation is indicated based on human, baboon, rat, mouse and shark comparison. Click here for file

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