We investigated the relationship between cutaneous malignant melanoma and multiple sunburns in the Queensland population. Interview data were gathered from 236 case-control pairs concerning their lifetime experience of severe sunburns, their occupational and recreational sun exposure, and their skin type. Excluding the lentigo maligna melanoma subtype, an association between multiple sunburns and melanoma was evident. After controlling for other major risk factors there was a significant dose-response relationship (P less than 0.05): the estimated relative risk associated with 2-5 sunburns in life was 1.5, and with 6 or more was 2.4. This observation extends the hitherto circumstantial evidence of a causal relationship between exposure to solar ultraviolet radiation and melanoma, and suggests that precautionary measures could prevent the development of this disease in a proportion of cases in fair-skinned populations.


Studies of morphological integration provide valuable information on the correlated evolution of traits and its relationship to long-term patterns of morphological evolution. Thus far, studies of morphological integration in mammals have focused on placentals and have demonstrated that similarity in integration is broadly correlated with phylogenetic distance and dietary similarity. Detailed studies have also demonstrated a significant correlation between developmental relationships among structures and adult morphological integration. However, these studies have not yet been applied to marsupial taxa, which differ greatly from placentals in reproductive strategy and cranial development and could provide the diversity necessary to assess the relationships among phylogeny, ecology, development, and cranial integration. This study presents analyses of morphological integration in 20 species of australodelphian marsupials, and shows that phylogeny is significantly correlated with similarity of morphological integration in most clades. Size-related correlations have a significant affect on results, particularly in Peramelia, which shows a striking decrease in similarity of integration among species when size is removed. Diet is not significantly correlated with similarity of integration in any marsupial clade. These results show that marsupials differ markedly from placental mammals in the relationships of cranial integration, phylogeny, and diet, which may be related to the accelerated development of the masticatory apparatus in marsupials. The correlated evolution of traits is a fundamental issue in evolutionary biology, with great importance for understanding morphological evolution and the generation of morphological diversity Most studies of morphological integration focus on microevolutionary hypotheses , documenting the relationships among development, genetics and phenotypic integration, usually in single species . The few comparative studies conducted have focused on placental mammals Because placentals and marsupials differ greatly in the timing of cranial bone ossification A plausible null hypothesis is that evolutionary history (phylogeny) is correlated with similarity in patterns of morphological integration. Of the placental clades studied, however, only a few support this hypothesis These results from previous studies demonstrate that a complex relationship exists between phylogenetic relatedness, integration, and ecology across placental mammals. In addition, as suggested by Steppan As noted above, examination of morphological integration in marsupials is particularly important, because of the striking differences in the timing of cranial bone development between marsupials and placentals. Ossification of the anterior masticatory apparatus  is accelerated in marsupials relative to placentals. This heterochronic shift is related to the unique marsupial reproductive pattern in which neonates are birthed after a short gestation period and complete their early development attached to the teat Cranial landmarks were captured using an Immersion Microscribe G2×3-D digitizer. Fifty-seven landmarks were collected across the skull, emphasizing points of certain homology across taxa, such as tripartite sutures. In addition, landmarks corresponding to those in earlier studies also were used, to permit direct comparison with previous results. Landmarks are listed in Twenty species of australodelphian marsupials were included in this analysis, spanning Dasyuromorphia, Peramelia, and Diprotodontia . Taxa weData were collected from 13 to 16 adult specimens per species, for a total of 327 specimens from 20 species, and male and female specimens are as equally represented as possible . While hAnalytical methods follow previous studies Matrix correlation analysis was employed to assess similarity in patterns of morphological integration To test the relationship between MSI and phylogenetic relatedness, multiple phylogenetic similarity matrices were constructed for all of the taxa examined, using recently published phylogenetic hypotheses Perameles, Isoodon, and Macrotis) and Peroryctidae . Szalay Macrotis as the nearest outgroup to the rest of the peramelians included in this study, while Westerman et al. Peroryctes outside the remaining peramelians in this study. Each of these competing phylogenetic hypotheses for Marsupialia and for Peramelia was analysed separately to test the relationship between phylogeny and similarity of morphological integration +1, such that the most distantly-related species have a value of one and sister taxa have the maximum value. Matrix correlation analysis was used to test the correlation of various phylogenetic distance matrices with MSI. Mantel's test is used to determine the significance of the matrix correlation. Mantel's test randomly reorders the rows and columns of one of the two correlation matrices being compared and recalculates the matrix correlation between the two matrices An alternative analysis of phylogenetic relationship also was employed. Pairwise similarity of morphological-integration values were averaged for taxa related at various taxonomic levels , basal Dasyuromorphia , and Dasyuromorphia+Peramelia . Because diet is significantly correlated with phylogeny, the dietary-similarity matrix was regressed against the phylogenetic-distance matrix to isolate diet from phylogeny. The dietary similarity residual matrix (hereafter DSRM) was compared to the original MSI, using matrix correlation analysis with Mantel's test for significance.Across all australodelphian marsupials, there was a significant correlation with phylogeny using all topologies , Table 2Single pairs analysis was also conducted for each clade . When siNeither DSM nor DSRM were significantly correlated with similarity in morphological integration in any of the clades examined in this study , Table 3Within placental mammals, morphological integration has been analysed comparatively in Primates Matrix correlation analysis and single pairs analysis produced consistent results in most analyses. Both support a significant relationship between phylogeny and similarity of integration across australodelphian marsupials. These three orders are quite morphologically distinct and diverged 40–50 million years ago The three orders examined, however, display three different patterns with respect to phylogeny, size, and cranial integration. While Dasyuromorphia and Dasyuridae show significant correlations between phylogenetic distance and similarity of integration, both including and excluding size , DiprotoPeroryctes) that show particularly low similarity of integration with other taxa, whether including or excluding size. However, Perameles nasuta, which shows comparatively high similarity of integration with other peramelians when size is included, displays the lowest similarity of integration values when size is removed, most notably with the congeneric species Perameles gunnii (0.22). As the congeneric peramelid species reflects only a single comparison, between Perameles nasuta and Perameles gunnii, greater sampling of congeneric species is necessary to determine if that low similarity of integration among species is a general characteristic of Peramelia. However, these results suggest that size-related correlations are a more significant factor within Peramelia than in the other marsupial orders considered in this study, even though they occupy a smaller range of size than either Dasyuromorphia or Diprotodontia Peramelia shows an intermediate pattern between Dasyuromorphia and Diprotodontia, with marginally significant correlations in matrix correlation analysis when size is included, but not when it is removed. All three phylogenetic hypotheses for Peramelia produced similar results. Interestingly, the differences in correlation values between analyses with and without size are greater in Peramelia than in other clades. Likewise, in single pairs analysis, Peramelia shows increased similarity of integration with phylogenetic relationship when size is included, but a negative correlation when size is removed. This result seems to be primarily influenced by a few taxa , invertivorous (Myrmecobius), and folivorous  taxa, to species with a variety of mixed diets , frugivore (Fr), folivore (Fo), and carnivore (C). *The diet of Vombatus is primarily grasses and roots.(0.06 MB DOC)Click here for additional data file.Appendix S2Matrix of similarity of morphological integration. The lower triangle is the original MSI. The upper triangle is MSI without size.(0.15 MB DOC)Click here for additional data file.


We examined the expression of pancreatic secretory trypsin inhibitor (PSTI) in colorectal cancer by immunohistochemical staining using an anti-PSTI antiserum, an in situ hybridisation technique utilising sulphonated PSTI cDNA probe, and a Northern blot hybridisation method, using a 32P-labelled PSTI cDNA probe. Immunohistochemically, PSTI was detected in 80 of 95 (84%) colorectal cancer cases. Analyses with in situ hybridisation as well as Northern blot hybridisation demonstrated PSTI mRNAs in immunohistochemically positive cases, showing PSTI could be produced in colorectal cancerous cells. Histologically well or moderately differentiated adenocarcinoma showed higher incidence of PSTI immunoreactivity than the other types. Furthermore, the intensity of the immunohistochemical staining for PSTI increased the more cases advanced, particularly in regard to depth of invasion and tumour size. Thus, PSTI expression is widespread in colorectal cancer, and occurs more commonly in advanced cases. Considering the suggestion that PSTI is a growth-stimulating factor as an well as inhibitor to proteolytic proteinase, the present findings may indicate that PSTI expressed in colorectal cancerous cells may play a role possibly closely associated with tumour development.


Plasmodium species and its vectors may result in adaptive changes in genes that are crucial components of the vector's defense against the pathogen. By analyzing which genes show evidence of positive selection in malaria vectors, but not in closely related non-vectors, we can identify genes that are crucial for the mosquito's resistance against Plasmodium.Co-evolution between CEC1, GNBP-B1 and LRIM1, in both vector and non-vector species of the Anopheles gambiae complex. Whereas little protein differentiation was observed between species in CEC1 and GNBP-B1, McDonald-Kreitman and maximum likelihood tests of positive selection show that LRIM1 underwent adaptive evolution in a primary malaria vector; An. arabiensis. In particular, two adjacent codons show clear signs of adaptation by having accumulated three out of four replacement substitutions. Furthermore, our data indicate that this LRIM1 allele has introgressed from An. arabiensis into the other main malaria vector An. gambiae.We investigated genetic variation of three anti-malarial genes; LRIM1 to P. falciparum infection, an adaptive response of a known anti-malarial gene in a primary malaria vector is intriguing, and may suggest that this gene could play a role in Plasmodium resistance in An. arabiensis. If so, our data also predicts that LRIM1 alleles in An. gambiae vary in their level of resistance against P. falciparum.Although no evidence exists to link the adaptation of  Plasmodium falciparum transmission. The need to identify refractory genes for this effort has focused much attention on the immune system of malaria's main vector in Africa, An. gambiae. The completion of the An. gambiae genome Plasmodium infection Despite ongoing control efforts during the last decades, malaria remains one of the most deadly infectious diseases. The vast majority of its burden is carried by people on the African continent, where 1 to 2 million people die annually from this disease Plasmodium infection affects the mosquito's fitness, we may expect the accumulation of adaptive amino acid substitutions in those anti-malarial genes that are crucial in specifically limiting Plasmodium infection in vector species, whereas such changes should not be found in closely related species that do not transmit malaria.So far little attention has been devoted to examining polymorphism of immunity genes in natural malaria vector populations An. gambiae has in fact undergone an adaptive response to P. falciparum infection is suggested by several lines of evidence. First of all, P. falciparum goes through severe bottlenecks during its life cycle in this mosquito Plasmodium infection. Furthermore, P. berghei, which is not transmitted naturally by An. gambiae, produces a much higher oocyst number in An. gambiae than its natural pathogen P. falciparum. In fact, in a review of studies estimating the fitness effect of Plasmodium infection on Anopheles species, reduced fitness was observed in 10 combinations of Plasmodium and Anopheles species that do not occur naturally, whereas in 10 natural combinations, including An. gambiae and P. falciparum, no fitness effects were observed Anopheles species have evolved to limit infections of the Plasmodium species they come into contact with. This is corroborated by the fact that the immune response to P. falciparum is specific, i.e. An. gambiae up-regulates different genes in response to infection with P. falciparum vs. P. bergheiP. falciparum in An. gambiae and An. arabiensis are typically low, ranging between 3–9% P. falciparum infection. However, it should be kept in mind that the data summarized above indicate that the rate and intensity of P. falciparum infection is likely to have been much higher when Anopheles mosquitoes first came into contact with this pathogen.That An. gambiae belongs to a complex of closely related species that includes another primary African malaria vector, An. arabiensis. Additionally it contains several species, i.e. An. melas, An. merus and An. bwambae, that occasionally transmit malaria locally, but do not have wide enough distributions to be considered important vectors. More importantly for the purpose of the present study, the An. gambiae complex also contains the highly zoophilic An. quadriannulatus A and An. quadriannulatus B, which are never or rarely exposed to the human-limited P. falciparum.i.e. CEC1, GNBP-B1 and LRIM1, in six species of the An. gambiae complex. CEC1  is a cecropin gene whose expression in An. gambiae is induced by infection with bacteria and Plasmodium bergheiAn. gambiae that express CEC1 24 hours after a blood meal, showed a 60% reduction in the number of P. berghei oocysts GNBP-B1 (ENSANGG00000015205) is a pattern recognition receptor whose expression is strongly upregulated in response to infection with both P. bergheiP. falciparumLRIM1 (ENSANGG00000010552) is a leucine-rich repeat immune protein that is an important plasmodium antagonist. This protein is up-regulated in response to infection with P. berghei, and silencing of this gene increases oocyst load 3.6-fold An. gambiae and An. arabiensis, to examine if these genes show signs of an adaptive response that may implicate them in the co-evolution of the mosquito vector and Plasmodium pathogen. Whereas no evidence for positive selection was found in CEC1 and GNBP-B1, our results clearly indicate that LRIM1 underwent an adaptive response in the An. arabiensis lineage. Additionally our data also indicate that LRIM1 has introgressed from An. arabiensis into An. gambiae.In this study, we investigated patterns of polymorphism in three anti-malarial genes, CEC1 gene, consisting of 177 bp of coding sequence and two introns comprising a combined 90 bp, was amplified. A total of 186 alleles of this gene were obtained from six species of the An. gambiae complex, 66 of which were unique . Very few fixed differences were present between species, and in most comparisons no fixed non-synonymous differences were found  for the coding region. Very few fixed replacement substitutions were observed between species  the annotation of this gene was altered, and it is now thought that these 858 bp represent about half of the coding sequence of LRIM1. We obtained a total of 138 alleles from six species, of which 108 were unique (Dxy ranged from 1.03 to 3.06 (per 100 bp) between species. In contrast to CEC1 and GNBP-B1 however, McDonald-Kreitman tests of positive selection indicated a significant excess of fixed non-synonymous differences between An. arabiensis and An. quadriannulatus A, An. merus as well as An. bwambae . However, a branch test did show that it is significantly larger than ω along the background branches . Values of ω along the branches leading to An. melas, An. merus and An. bwambae/An. quadriannulatus A were estimated to be 0.299, 0.298 and 0.147 respectively  and is also found in one of the “arabiensis-like” An. gambiae alleles.A total of four non-synonymous substitutions occurred along the foreground branch . Three oAn. arabiensis is fixed for nucleotide A at sites 704, 706 and 707 (codons 235 and 236). With the exception of An. gambiae, all other species are fixed for nucleotides T, G and G at these respective positions. The few “arabiensis-like” An. gambiae alleles also have the (AAA) arrangement. Positions 416 through 718, a 302 bp stretch, cluster these An. gambiae (AAA) alleles with An. arabiensis (An. gambiae (AAA) alleles with the rest of An. gambiae. Additionally, no fixed differences were found between An. gambiae and An. arabiensis anywhere in this gene.abiensis . Only poLRIM1 is located inside the 2La inversion. Therefore we determined the karyotype of our An. gambiae samples with respect to the 2La arrangement. Out of 32 specimens from Cameroon, three were 2La/+ heterozygotes, all of which were also heterozygous for the (AAA)/(TGG) alleles. The karyotype of one (AAA)/(TGG) heterozygote was not clear, as it produced a second band of unexpected size. The remaining four (AAA)/(TGG) heterozygotes, as well as all (TGG) homozygotes, carried the 2L+/+ karyotype. These findings confirm that the (AAA) allele is at very high frequency or even fixed in the 2La inversion in Cameroon, and is present at very low frequency in the 2L+ arrangement (≈7%).LRIM1 in An. arabiensis showed signs of a recent selective sweep, a HKA test was performed by comparing the polymorphism/fixed differences ratio of LRIM1 to CEC1 and GNBP-B1. A selective sweep reduces the amount of standing genetic variation within a species, as indicated by a relatively low ratio. However, no significant differences were found between the genes, and in fact, this ratio was considerably higher for LRIM1 (20/20.2) as compared to CEC1 and GNBP-B1 (17/28.1).To examine if CEC1 and GNBP-B1 did not show any signs of positive selection, and in particular, showed little or no differentiation between malaria vectors and the non-vector species, indicating that these genes are largely subject to purifying selection. In two of the species comparisons GNBP-B1 showed a significant excess of non-synonymous polymorphisms. Some cloning error is expected to be present in the GNBP-B1 data set. Since a majority of possible mutations are non-synonymous, random errors will bias the observed number of non-synonymous polymorphisms upward. However, the number of PCR errors in the data is not nearly high enough to explain the difference. Therefore, most likely purifying selection is responsible, with numerous slightly deleterious substitutions present at low frequency in populations, but which are prevented from going to fixation. In contrast, LRIM1, a gene with no known homologue in other organisms, shows clear signs of positive selection in An. arabiensis.An. gambiae and An. arabiensisLRIM1 in An. arabiensis. First of all, a demographic explanation should affect all genes. As noted before, GNBP-B1 has a higher ratio of non-synonymous/synonymous polymorphisms than fixed differences in most or all populations, including An. arabiensis. This indicates the presence of a relatively large number of slightly deleterious alleles in this gene, few or none of which became fixed in ancestral populations. This is contrary to the demographic explanation. More importantly however, it is extremely unlikely that three out of four fixed amino acid changes would occur in two adjacent positions in a 285 amino acid protein, if the random process of genetic drift were responsible.As pointed out by MacDonald and Kreitman, an excess of non-synonymous fixed differences between species may result from a much smaller population size in the past LRIM1 provides strong evidence that the presence of “arabiensis-like” alleles in An. gambiae is caused by introgression and not by the retention of ancestral polymorphism. It is unlikely that recombination would not have broken down a linkage group of at least 352 bp, if it were maintained in the population for long time. Furthermore, the introgression hypothesis is supported by the complete absence of fixed differences between these species anywhere in the LRIM1 gene.Polymorphisms are shared between species in all three genes we examined, and there is no doubt some of this is due to the retention of ancestral polymorphism. However, the pattern of polymorphism observed in An. gambiae (AAA) and An. arabiensis , introgression has occurred multiple times, after which, according to the shared polymorphism between the An. gambiae (AAA) and (TGG) alleles (positions 324 and 327), these alleles recombined between position 327 and 416. The introgression of LRIM1 from An. arabiensis into An. gambiae is also consistent with previous studies that have shown that introgression between these two species has occurred in the past nd2 chromosome, on which LRIM1 is located, has been shown to transfer most readily between An. gambiae and An. arabiensis. Based on the shared polymorphisms between LRIM1 in An. arabiensis has evolved in direct response to malaria infection, but our observation that this anti-malarial gene shows distinct signs of positive selection in a primary malaria vector, but not in a species that does not transmit Plasmodium, is intriguing. This suggests that the observed adaptive evolution of LRIM1 may have been the result of the infection of An. arabiensis with Plasmodium, with at least two adjacent amino acids playing a crucial role in this adaptation. If this is true, variation for Plasmodium resistance should be present at LRIM1 in An. gambiae.Mosquitoes, like other organisms, encounter numerous pathogens during their life cycle, all of which could potentially exert selection pressure on the immune system. In fact, molecules that play a role in host-parasite interactions are one of the main groups of proteins on which positive selection has been demonstrated LRIM1 contains a leucine-rich repeat (LRR), which in other LRR genes is crucial for the three-dimensional structure by folding it into an arc LRIM1, but the two neighboring adaptive amino acids (i.e. 235 and 236) are located well outside the leucine-rich repeat region (positions 30–160), suggesting that they could have a more specific function.LRIM1 is known to play a role in suppressing P. berghei infection in An. gambiaeLRIM1 on P. falciparum infection in field-collected An. gambiaeP. berghei infection was confirmed. This could be because the action of LRIM1 in An. gambiae is specific against P. berghei. However, it is also possible that only some LRIM1 alleles suppress infection with P. falciparum, and these may even be specific for certain P. falciparum strains. Another study demonstrated the existence of such genotype by genotype interactions between P. falciparum and An. gambiae, by showing that no single strain of P. falciparum was best at infecting all of a set of iso-female An.gambiae lines LRIM1 is located inside the 2La inversion. While An. arabiensis is fixed for this 2La arrangement, An. gambiae is 2La/+ polymorphic. Since LRIM1 alleles introgressed from An. arabiensis into An. gambiae, we may expect that these An. gambiae (AAA) alleles are mostly found in the 2La arrangement. Interestingly, the mosquitoes that failed to show an effect of LRIM1 knockdown on P. falciparum infection 2L+). Our molecular karyotyping of the 2La inversion in our An. gambiae specimens shows that the (AAA) allele is indeed found at very high frequency in 2La inversions, whereas it is present in very low frequency in 2L+ (≈7%).Although LRIM1 vs GNBP-B1/CEC1 did not show a relatively low level of genetic variation in LRIM1 in An. arabiensis. In fact, the relative level of polymorphism was higher in this gene than in CEC1 and GNBP-B1. Therefore we have no indication that LRIM1 in An. arabiensis is currently involved in an evolutionary arms race. This also implies that possible selective sweeps occurred long enough ago to allow mutation to regenerate polymorphism.Pathogen-host co-evolution has mainly been considered in terms of an evolutionary arms race LRIM1 has undergone adaptive evolution in a primary malaria vector. This could be because this gene has evolved in response to P. falciparum infection in this species. If so, LRIM1 is expected to play a role in the resistance of An. arabiensis against P. falciparum. So far the immune system of this mosquito species has not yet been investigated, and our data suggest the possibility that a knockdown of LRIM1 will enhance infections of P. falciparum in An. arabiensis. If LRIM1 did indeed evolve in response to P. falciparum infection in An. arabiensis, this gene also deserves further study in An. gambiae, in particular with respect to potential variation in resistance between the two major alleles found in this species.The data presented here indicate that the anti-malarial gene An. gambiae were collected from the villages of Mbebé and Nyabessan, Cameroon in Dec. 2005. An. gambiae from Mali were collected from Banambani in 2000. Adult An. arabiensis females from Cameroon were collected from Kousseri in Dec 2005. Adult An. melas were collected in Ipono, Cameroon, Dec. 2005. Larvae of An. gambiae, An. arabiensis and An. bwambae from Bwamba county, Uganda (2004) were kindly provided by Ralph Harbach. DNA extractions of An. merus from Furvela, Mozambique (2001 and 2003) were kindly provided by David O'Brochta. An. quadriannulatus A from Kruger National Park, South Africa, were kindly provided by Anton Cornel. Sample sizes for each gene and species are represented in Adult females of et al.et al.An. gambiae specimens belonged to the S molecular form. Molecular identification of 2La karyotypes was performed following White et al.CEC1, GNBP-B1 and LRIM1 were designed using Primer3 An. gambiae genome and anneal to the flanking or non-transcribed regions of the genes CEC-A and LRIM1 was performed using Amplitaq Gold polymerase (Perkin Elmer) using respectively the following primer pairs CECin1 (GTTAGCAGAGCCGTCGTCTT)/CECin12 (ACAGTCGGTTCAAAGCGTTC) and LRIM1in6 (AGGTAACGGACAGCAGCCTA)/LRIM1in9 (GTCCGGTACTGCTCCTTGAG). The following program was used for PCR amplification of CEC1 and LRIM1; 2 min at 94°, 35 cycles of 30 sec at 94°, 30 sec at 52° and 45 sec at 72°, followed by 20 min at 72°. PCR products were excised from an agarose gel and purified using the Gel Purification Kit (Qiagen) and submitted for direct sequencing. A subset of the sequences from individuals heterozygous for two or more positions were amplified again and cloned using the TOPO-TA cloning kit (Invitrogen). Individuals were selected for cloning such that all observed polymorphic sites were represented in the final data set. From each individual a single colony was sequenced. PCR of GNBP-B1 was performed using Platinum High Fidelity Taq (Invitrogen) with the primer pair GNBPin1 (GTTTGGTAGGGGACGAATGA) /GNBPIN20 (GCGCTTTCAGTGGTTTGTTT) using the following program: 2 min at 94°, 35 cycles of 30 sec at 94°, 30 sec at 52° and 90 sec at 72°, followed by 20 min at 72°. Direct sequencing of the PCR product of GNBP-B1 was not possible in many cases because of the presence of indels. Therefore, PCR products of this gene were cloned and sequenced as outlined above. However, nine sequences were produced through direct sequencing, allowing for an estimation of the PCR/cloning error. Sequencing was performed on an ABI 3730 Genetic Analyzer using Big Dye v 3.1 (Applied Biosystems )DNA was extracted using the DNeasy tissue kit (Qiagen). Species and molecular form diagnostics were performed following Fanello LRIM1 and CEC1 samples that were cloned were also sequenced directly, we were able to derive both alleles from each individual while removing PCR/cloning errors. The PCR error in the GNBP-B1 sequences amplified using the proof-reading polymerase was estimated to be 0.625 per 1000 bp. Therefore, each 1188 bp GNBP-B1 allele for which no direct sequence was available is expected to have an average of 0.74 errors.Based on a comparison between direct sequencing and plasmid sequencing, the PCR/cloning error using Amplitaq Gold was estimated to be approximately 1.5 per 1000 bp. However, because all Dxy values were calculated using DnaSP 4.0 All sequences were aligned using MEGA3.1 CEC1 and GNBP-B1, subsequent analyses were limited to LRIM1. Aimed at reducing the computational effort, a reduced LRIM1 data set, containing 70 sequences, was used for phylogenetic analyses and maximum likelihood tests of positive selection. This data set was compiled in such a way that at every observed polymorphism and fixed difference, i.e. the relevant information for tests for maximum likelihood tests of positive selection, was retained. This reduced data set was used to construct 50% majority-rule consensus trees with MrBayes 3.1.2 Since few or no fixed differences were observed in LRIM1 alleles from An. gambiae clustered within An. arabiensis  along the major branches of the tree. Under the neutral model the relative number of synonymous and non-synonymous substitutions is expected to be 1. Under positive selection, amino acid substitutions are favored and ω>1, whereas under purifying selection amino acid substitutions are prevented and ω<1. The An. arabiensis lineage was designated as the foreground branch, i.e. the branch of interest, and model 2 free ω was compared to model 0 to test if ω along the foreground branch was significantly larger compared to the ω along the background branches, i.e. all other branches. Model 2 ω = 1, with the ω value fixed at 1 along foreground branch, was compared to model 2 free ω to test if ω along the foreground branch was significantly larger than 1. Model 1 was used to estimate ω along the central branches of the tree  of (1.19 MB TIF)Click here for additional data file.Figure S2LRIM1 in five species of the An.gambiae complex. Posterior probabilities are indicated along branches. An. arabiensis samples from Uganda, Madagascar and Mali are indicated by UG, MAD, and MAL respectively, with all remaining An. arabiensis samples originating from Cameroon.Bayesian tree (unrooted) of (0.72 MB TIF)Click here for additional data file.


Five percent of the Swiss population attribute symptoms to electromagnetic fields (EMF). General practitioners (GPs) might play a key role in recognising an emerging health risk, since they are the first to observe and follow up persons who attribute symptoms to EMF. It is unclear to what extent EMFs have become an issue in general practice and which experiences GPs report from the consultations.We conducted telephone interviews in a random sample of GPs in Switzerland in order to assess the frequency of consultations in primary care due to EMF and the GPs' experience with these patients.342 general practitioners were interviewed, corresponding to a response rate of 28.2%. 69% of the GPs reported at least one consultation due to EMF, but GPs with a certificate in complementary medicine were much more likely to report EMF consultations. The median of EMF consultation numbers within one year was three. An overview of the most recent EMF-related consultation per GP yielded sleep disorders, headaches and fatigue as the most often reported symptoms and mobile phone base stations, power lines and the own use of mobile phones as the main EMF sources suspected to be associated to symptoms. GPs judged the association between EMF and the symptoms to be plausible in 54% of the cases. There was no combination of symptoms and EMF sources that was remarkably and consistently judged to be a plausible cause of the symptoms.In our survey, GPs often judged the association between the health problems and the suspected exposure to be plausible. This plausibility assessment seems to be based on grounds of preventive positions in a situation of scientific uncertainty. More research effort is needed to obtain more insight on a potential association between long term EMF exposure and unspecific symptoms. Everyone is exposed to a complex mixture of electric and magnetic fields at many different frequencies. In the scientific literature and in the media, increasing attention is paid to the potential adverse effects of electromagnetic fields (EMF) on human health, especially since the introduction of modern telecommunication technologies. Regarding radiofrequency EMF, such as those from base stations or mobile phones, the Stewart Report concluded in 2000 that "the balance of evidence to date suggests that exposures to RF radiation below guidelines issued by the National Radiological Protection Board (NRPB) and the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines do not cause adverse health effects to the general population" . In 2004So far, it is unclear to what extent EMFs have become an issue in general practice and which experiences GPs report from these consultations. In 2005, an estimate based on a representative survey yielded that more than half of the Swiss population perceived EMFs as potentially harmful and 5% attributed symptoms to EMF  reMost of the GPs reported little experience with EMF consultations in general practice: 71% of all GPs reported to have had fewer than five EMF consultations in the previous year. Of the GPs with few or no EMF consultations in the previous year, the majority still estimated an association between health complaints and EMF sources to be plausible in 52% of the cases. This makes it unlikely that GPs develop their plausibility assessment based on frequent and repeated experience with EMF-related consultations.There were some indications that GPs did not feel confident about counselling persons with symptoms attributed to EMF, e.g. 75% expressed to need more information on the topic for their work as a GP and less than half of the GPs reported to have a standard approach on how to deal with these patients. 53% would welcome the implementation of a national or regional interdisciplinary environmental medicine counselling centre.From the general practitioners' observations no obvious symptom-EMF source pattern could be extracted, which would link a specific EMF source to a specific health complaint and thus call for a new direction of investigation that is not yet pursued by researchers. However, the scientific uncertainty regarding long term effects of EMF on unspecific symptoms is mirrored in the GPs plausibility assessments: The majority of GPs believed that exposure to everyday EMF could cause symptoms, and the relation between EMF and the symptoms of their EMF-related consultations was judged to be "plausible" in more than half of the cases. Thus, we conclude that more research effort is needed to obtain more insight on a potential association between long term EMF exposure and unspecific symptoms.CATI – computer assisted telephone interviewEMF – electromagnetic fieldsGP – General PractitionerICNIRP – International Commission on Non-Ionizing Radiation ProtectionNRPB – National Radiological Protection BoardThe author(s) declare that they have no competing interests.AH coordinated the study and drafted the manuscript and MR conceived of the study. Both authors designed the study, developed the questionnaire used in the survey, analysed and interpreted the data, revised the draft and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Expression of alpha-1-antitrypsin (AAT) in tumour cells of 102 surgically resected lung adenocarcinomas was examined by immunohistochemical method using anti-AAT antiserum. While only 13 cases (13%) were negative for AAT expression, 89 cases (87%) contained AAT at varying degrees. The degree of AAT-positive tumour cells was significantly higher in advanced cases than in early cases. Clinical follow-up study of the patients, particularly in stage I, showed that strongly AAT-positive cases have poor prognosis than weak-to-moderately AAT-positive or AAT-negative cases. Thus, AAT expression status in tumour cells of lung adenocarcinoma may be a biological marker of prognostic significance in regard to tumour growth.


Individuals with severe Z α1-antitrypsin (AAT) deficiency have a considerably increased risk of developing chronic obstructive lung disease (COPD). It has been hypothesized that compensatory increases in levels of other protease inhibitors mitigate the effects of this AAT deficiency. We analysed plasma levels of AAT, α1-antichymotrypsin (ACT) and secretory leukocyte protease inhibitor (SLPI) in healthy (asymptomatic) and COPD subjects with and without AAT deficiency.Studied groups included: 71 asymptomatic AAT-deficient subjects  identified during Swedish neonatal screening for AAT deficiency between 1972 and 1974; age-matched controls ; older asymptomatic ZZ (n = 10); healthy MM ; and COPD patients . Plasma levels of SLPI, AAT and ACT were analysed using ELISA and immunoelectrophoresis.No significant difference was found in plasma ACT and SLPI levels between the healthy MM and the ZZ or SZ subjects in the studied groups. Independent of the genetic variant, subjects with COPD (n = 19) had elevated plasma levels of SLPI and ACT relative to controls (n = 153) .Our findings show that plasma levels of ACT and SLPI are not elevated in subjects with genetic AAT deficiency compared MM controls and do not appear to compensate for the deficiency of plasma AAT. Serine proteases play key roles in coagulation, fibrinolysis, and in kinin and complement activation. The activities of these enzymes are controlled at least in part by specific serine protease inhibitors, most of which are serpins. The relationship between serine protease and serpin inhibitor levels has been examined extensively, since an imbalance between them is linked to local tissue injury and to many pathologies, including cancer, autoimmune diseases, chronic obstructive lung disease (COPD), inflammation and infectious diseases -5.The serine protease inhibitor found in highest concentration in plasma is α1-antitrypsin (AAT). Severe AAT deficiency in the homozygous Z variant, which differs from the wild type M variant in the substitution of Glu-342 by Lys, was first recognized as a hereditary condition predisposing to COPD on the basis of low plasma levels  of AAT . This siIndividuals with severe AAT deficiency have at least a 20-fold increased risk of developing lung disease, especially if they smoke . AAT is It has been hypothesized that compensatory increases in other protease inhibitors and/or decreased leukocyte activity may reduce the severity of AAT deficiency by favourably affecting the overall protease/protease-inhibitor balance in AAT-deficient individuals -22. To f1 and FEV1/FVC%) was found among the studied subject groups with ZZ, SZ and MM variants of AAT.The first study group included 71 asymptomatic, 31 year old, AAT-deficient subjects: ZZ, n = 48, 24 females and 24 males, age 31.1 ± 0.52 years and SZ, n = 23, 12 females and 11 males, age 31 ± 0.58 years, identified in the Swedish neonatal screening study during 1972–1974 -22. ClinThe second study group consisted of 20 healthy MM AAT adults , 10 asymptomatic ZZ AAT adults  and 20 COPD patients, among them 10 patients with ZZ AAT  and 10 patients with MM AAT . COPD was diagnosed according to the NHLBI/WHO Workshop guidelines . The ZZ Blood was taken by venipuncture, plasma was directly separated by centrifugation and stored at -20°C or -80°C until assayed. AAT-phenotyping was performed by isoelectric focusing at the Department of Clinical Chemistry, University Hospital.Secretory leukocyte proteinase inhibitor (SLPI) was analysed using ELISA kits  according to the manufacturer's instructions. Absorbance was measured spectrophotometrically at 450 nm using a microplate reader (Labsystems). The minimum detectable level of SLPI was 0, 0625 ng/ml.The plasma concentration of ACT and AAT was determined by the rocket immunoelectrophoresis method based on a quantitative estimation of proteins by electrophoresis in 1% agarose gel containing monospecific antibodies against ACT or AAT at a concentration of 5.5 μg per square gel area . The ACTThe statistical package SPSS for Windows  was used for statistical calculations. Differences in the means were analysed for their statistical significance with the one-way ANOVA combined with a multiple-comparisons procedure (Scheffe multiple range test). The equality of means was analysed for statistical significance with an independent two sample t-test and Pearson correlation analysis. Tests showing p < 0.05 were considered to be significant. Data are expressed as mean ± SD.As expected, plasma levels of AAT in 31 year old subjects were ranked: MM>SZ>ZZ and are significantly different among the groups  , while ZZ COPD patients had 25% higher plasma AAT levels compared to asymptomatic ZZ cases, although this was not statistically significant. We also compared levels of ACT and SLPI in healthy/asymptomatic and COPD individuals independent of the genetic variant of AAT and age. As shown in Table To contain the potential injurious effects of serine proteases, the anti-proteases developed in a parallel network consisting of "alarm", locally produced such as SLPI and elafin/ESI/SKALP, and "systemic" inhibitors. The latter, such as α2-MG, ACT and AAT ,29, are To further evaluated this hypothesis, we analysed plasma levels of AAT, ACT and SLPI and compared in both healthy and COPD adult patients with and without AAT deficiency. We analysed these inhibitors in two different groups of individuals, the first group consisting of 31 year old asymptomatic AAT deficiency individuals from the prospective follow-up study , and theReports published from the prospective follow-up study of ZZ and SZ individuals up to age 26 years, focusing on clinical health, lung and liver function tests and plasma markers of the protease/protease inhibitor balance, have shown that ZZ and SZ subjects had significantly higher plasma concentrations of α2-MG, ACT and antithrombin III  at age 8The increased protease inhibitor plasma levels in young AAT deficiency subjects relative to normal subjects could be linked to age rather than to a ZZ or SZ phenotype. For instance, α2-MG levels in cord blood and in young children have been found to be very high and to fall fairly rapidly from age 15 to 20 years, and to continue to decline gradually until the age of 30 to 40 -35. SimiIt is also important to point out that, for example, when subjects were 26 years old, but not 18 and 31 years old, SLPI was found to be significantly higher in AAT-deficient subjects than in wild type controls. These inconsistent findings during the follow-up studies suggest that plasma may not be the relevant biological fluid to measure SLPI levels. Alternatively, because SLPI is produced locally in the airways and is regulated by various pro-inflammatory stimuli, its plasma levels when analysed in healthy (asymptomatic) subjects may not reflect the real situation. One can not exclude that under inflammatory conditions the compensatory increase in SLPI and/or other protease inhibitors may reduce the severity of AAT deficiency by favourably affecting the overall protease/protease-inhibitor balance in AAT-deficient individuals.Our data show small increases in ACT and SLPI, as well as AAT, concentrations in normal MM subjects with COPD relative to well subjects. These differences exceed those for the same parameters for ZZ subjects with and without COPD and suggest that the COPD disease state could confound conclusions drawn from comparative studies seeking to attribute phenotypes (elevated protease inhibitor levels) to genotypes .We conclude that there is no clear evidence for compensatory up-regulation of protease inhibitors, such as ACT and SLPI, in healthy (asymptomatic) subjects with severe AAT deficiency. Thus, the changes in circulating levels of protease inhibitors and their likely impact on individual susceptibility to lung disease remains to be confirmed through further studies.1, forced expiratory volume in 1 second; FVC, forced vital capacity;AAT, alpha-1-antitrypsin; PiZZ, homozygous AAT-deficiency variant; PiMM, wild type AAT variant; ACT, alpha-1-antichymotrypsin; COPD, chronic obstructive pulmonary disease; α2-MG, alpha2-macroglobulin; SLPI, Secretory leukocyte proteinase inhibitor; FEVThe author(s) declare that they have no competing interests.CH carried out the analysis, participated in the interpretation of data and helped to draft the manuscript. EP and TS collected patient material. UW and AW helped with the interpretation of data and the study design. SJ designed the study, carried out the final statistical analysis and wrote the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Asthma affects 1 in 8 school-aged children in industrialized countries, making it the most common chronic illness in this group. Now a meta-analysis of child asthma studies led by pharmaceutical scientist Fawziah Marra of the University of British Columbia shows that children diagnosed with asthma were twice as likely as nonasthmatics to have received antibiotics before age 1. The more courses of antibiotics a child received in the first year of life, the higher the risk for asthma.Chest, examined the link between antibiotic exposure in babies and subsequent development of asthma, as well as the dose–response relationship. Marra’s team analyzed four prospective studies and four retrospective studies conducted between 1999 and 2004. Each study involved between 263 and 21,120 children, including cases who had been diagnosed with asthma between the ages of 1 and 18 years. The number of antibiotic courses taken ranged from one to seven, and averaged three.The meta-analysis, reported in the March 2006 issue of Pooling the data from all eight studies revealed a twofold risk of developing asthma with at least one course of antibiotics. Each additional course raised asthma risk 1.16 times. Information about the antibiotics prescribed could not be obtained from the studies.The findings support the “hygiene hypothesis,” which proposes that an immune system that doesn’t get enough practice killing germs (due to either an excessively clean environment or overuse of antibiotics) will become overly sensitized and overreact to normally harmless environmental agents such as pollen and dust.Marra and her colleagues recently launched a community education campaign in British Columbia called “Do Bugs Need Drugs?” The program uses media ads, classroom visits, and educational materials to teach health professionals and the general public about the overuse of antibiotics. The campaign emphasizes the difference between bacterial and viral infections, useful preventive measures such as hand washing, and the need to use antibiotics wisely. “In children, antibiotics are commonly used to treat ear infections, upper respiratory tract infections, and bronchitis,” says Marra, even though many such infections are viral and don’t respond to antibiotics. Some parents may refuse to leave a doctor’s office without a prescription.The information gained from the meta-analysis is valuable for physicians who are striving to cut back on prescribing antibiotics, says W. Michael Alberts, president of the American College of Chest Physicians: “It can help to convince parents of young children to hold off on giving antibiotics unless absolutely necessary.”


Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation.The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of  Toxoplasma gondii is a member of the phylum Apicomplexa. Several apicomplexans cause severe disease in humans  while others cause economic losses to livestock . T. gondii in particular affects immunosuppressed individuals giving rise to life-threatening encephalitis in AIDS patients. In pregnant women toxoplasmosis can lead to congenital infection causing a variety of birth defects. Clinical disease is associated with the tachyzoite life stage causing tissue lesions by rapid proliferation Toxoplasma divides through a very distinct internal division mechanism termed endodyogeny, wherein two parasites are assembled within a mother parasite The obligate intracellular parasite Toxoplasma cell division, the basal complex acts in place of an actin-independent contractile ring in the late stages of cell division. The contractile force is probably generated by Centrin2, a calcium dependent contractile protein The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with various structures that have a role in cell division th but degenerated and partial MORN motif T. gondii and P. falciparum have provided evidence that MORN1 is associated with both the cytoskeleton and a membrane component, consistent with its localization at the IMC extremities MORN1 is composed of a short unique N-terminus, 14 membrane occupation and recognition nexus (MORN) repeats, a linker of 5 amino acids between MORN domains 6 and 7 and a C-terminus composed of a 15T. gondii and dissected its phenotype in tachyzoites. Our results show that MORN1 is critical for viability and that the lack of MORN1 results in defective organelle partitioning and basal complex assembly. The most distinctive effect of these defects is the appearance of double and multi-headed parasites as a result of incomplete daughter abscission and sequential rounds of budding. These results indicate the absence of checkpoints on daughter maturation before entering the next round of cell division. Furthermore, we exploited the conditional MORN1 knock-out parasites to identify functional domains within MORN1 by complementation with a series of MORN1 deletion constructs.The distinct sub-cellular localization of MORN1 in the Apicomplexa and the function of MORN motifs in other systems suggest that MORN1 may act as a scaffolding protein organizing protein complexes with a critical role in mitosis and cytokinesis. To test this hypothesis we generated a conditional MORN1 knock-out in T. gondii. We anticipated that MORN1 likely would be essential and therefore we engineered a conditional knock-out parasite line using the Tet-off system 2) under control of the Tet7sag1 promoter. A clone was selected wherein the expression levels of the Myc2-tagged transgene closely matched that of the endogenous locus  was used; no additional acylation sites were detected) The conditional knock-out MORN1 parasite provides a powerful tool to identify functional domains within MORN1 through complementation studies with mutated MORN1 constructs. For example this could address whether different sections of MORN1 are required for centrocone localization than for apical or basal complex localization. In addition, we also wished to assure that the MORN1 null phenotype is indeed due to lack of MORN1. To answer these questions we generated a full-length MORN1 complementation construct as well as a series of MORN1 deletion mutants and a point mutant. To guide the construction of the MORN deletion mutants we assessed MORN1 for any functional motifs. As reported before, the MORN1 protein consists of 14 complete MORN domains and 1 partial MORN domain (PM) at the C-terminus . The N- To dissect the highly modular MORN1 structure we designed a ‘sliding window’ of MORN domain deletion mutants and additionally generated a mutant construct wherein the linker was deleted . Growth restoration was not to full wild-type levels as judged by the size of the plaques, although no significant reduction in plaque number was observed . The cau2-tagged MORN1 in these parasites did not completely disappear. This suggests that MORN1.3–8 prevented Myc2-MORN1 degradation and indicates the presence of strong aggregates.Complementation with the deletion constructs missing one or more MORN domains never resulted in the establishment of stable parasite populations, with the exception of a construct consisting of MORN domains 3–8 (MORN1.3–8). As shown in Toxoplasma daughter budding is a highly complex process that requires the coordinated assembly of numerous elements. We have recently demonstrated that the cytoskeleton that organizes the IMC is of even greater complexity than previously anticipated Toxoplasma division, i.e. maturation of the pellicle coordinated with mother disassembly is akin to the late stages in mammalian cells completing abscission. Although in mammalian cells the initial stages of cytokineses are mediated by an actin-myosin mediated cleavage furrow ingression, which compares with the basal complex constriction in Toxoplasma, the final segregation is organized by a septin and membrane remodeling based machinery T. gondii harbors machinery analogous to the septin based machinery to complete cell division. An argument against such machinery is that even in normal parasite development abscission is not complete and parasites are often observed to share a cytoplasmic bridge at their basal end Toxoplasma breaks the small cytoplasmic bridge present after completion of cytokinesis either by mechanical stresses within the vacuole or alternatively, by pulling away in opposite directions upon egress. Such a mechanic abscission model has been shown for the related ciliates It is somewhat surprising there is no checkpoint for assembly or contraction of the basal complex before progressing toward disassembly of the mother's cytoskeleton, as this appears to be crucial for the formation of viable parasites. Therefore we questioned how this compares to well-studied model organisms. The late stages in While the effects of the loss of MORN1 on completion of cytokinesis are very strong, the formation and partitioning of other organelles and structures are largely intact with one prominent exception: the loss of apicoplast division . The apiUpon complementation of the MORN1-KO with a full-length MORN1 encoding plasmid complete restoration of the wild-type phenotype is accomplished . Effortsin vitro2-tagged MORN1 allele was lost and Myc2-MORN was constitutively expressed. This indicates a genomic rearrangement or other mutations accrued in this phenotypic selection process.A MORN1 null mutant recently isolated by Heaslip and colleagues shows a similar phenotype yet is viable In conclusion, we have shown that loss of MORN1 results in an early loss of apicoplast division and prevents the assembly of the basal complex. As a result parasites fail to fully separate while still re-initiating new rounds of division resulting in the formation of the striking multi-headed parasites. Finally, we showed that the linker region between MORN domains 6 and 7 potentially serves as hinge critical for flexibility in the MORN1 complex and is a critical region for correct functioning of MORN1. Future work will be needed to address a potential role of palmitoylation and/or other post-translational modifications in the function of MORN1.RH strain parasites and transgenic derivatives were used throughout this study. Parasites were maintained in human foreskin fibroblasts (HFF) as previously described 2-MORN1/DHFR, primers MycF-1, MycF-2, MycR-1 and MycR-2 were hybridized to form the tandem Myc tag and were cloned BglII/AvrII into the pmorn1-YFP-MORN1/sagCAT plasmid NotI and the ends filled end in with T4 DNA polymerase followed by BglII digestion releasing the Myc2-MORN1-3′dhfr segment. This segment was cloned into pDHFR-Tet7sag1-SpeI by SpeI digestion, filling in the ends and subsequent BclI digestion to generate pTet7sag1Myc2-MORN1/DHFR .All oligonucleotides used are provided in Supplementary The MORN1 open reading frame in cosmid PSBMG48 was replaced with a MORN1-flanking sequence flanked PCR amplified CAT cassette as previously described using primers MORN1_recomb-F and MORN1_recomb-R 2(MCS)/sagCAT plasmid PmeI/BglII), the first YFP (BglII/AvrII), the second YFP (AvrII/EcoRV), or the entire tandem YFP cassette (BglII/EcoRV). The sagCATsag cassette was replaced by the sagBLEsag cassette XhoI digest swapping. The megaprimer method was used to generate internal deletion or point mutations All MORN1 deletion plasmids were cloned based on the ptub-YFPPlasmids used that have been described previously are ptub-H2b-mRFP/sagCAT Immunofluorescence assays (IFA) were performed as described previously 2, with cells grown in MatTek glass bottom culture dishes. Images were processed to account for cell drifting.Live cell time-lapse movies were acquired using an I×71 inverted epifluorescence microscope (Olympus) with a 100X oil immersion lens (UPlanApo 1.35 NA). Images were recorded using a Photometrics Coolsnap HQ camera and brightness/contrast were processed using SoftWoRx software (Applied Precision). Time-lapse imaging was performed in a humidified chamber heated to 37°C with 5% CO4, dehydrated in ethanol, treated with propylene oxide and embedded in Spurr's epoxy resin. Sections were stained with uranyl acetate and lead citrate prior to examination in a JEOL 1200EX electron microscope For transmission electron microscopy (TEM), parasites were grown in HFF confluent T25 tissue culture flasks, released by trypsinization, and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2. The samples were post-fixed in OsO4 and dehydrated through an ethanol series. The coverslips were critical-point dried and coated with 20 nm gold. Samples were examined with a JEOL JSM-6340F field emission scanning electron microscope.For scanning electron microscopy (SEM), extracellular parasites were harvested following 48 hours of growth in the presence of ATc by filtration , washed twice in PBS and then fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 at 4°C overnight. Fixed cells were settled onto poly-L-lysine coated coverslips, washed twice in 0.1 M phosphate buffer, post-fixed with 1% OsOAs described previously Essentially as described previously 2 incubator at 37°C. After these incubation times, cells were washed with 1× PBS, and Ed1 medium without ATc was added. 7–10 days later, the monolayer was stained with crystal violet to visualize plaques A T25 flask confluent with HFF cells was inoculated with 50–200 freshly lysed MORN1-KO parasites in Ed1 medium and incubated for 0, 12, 18, 24, 48, 72, or 168 (1 week) hrs with 1 µg/ml ATc in a humidified 5% COTable S1Oligonucleotides used in this study. Restriction enzyme sites are underlined, point mutations are represented in underlined, bold font.(0.03 MB DOC)Click here for additional data file.Figure S1MORN1 knock-out strategy and identification of mutant clones. (A) MORN1 spanning cosmid PSBMG49 was recombineered to replace the MORN1 open reading frame with a gentamicin (for selection in E. coli) and CAT cassette (for selection in T. gondii). (B) The MORN1 replacement cosmid was transfected into a parasite clone expressing the Tet-transactivator and a Tet7sag1 promoter controlled Myc2-MORN1 construct . Twenty-(0.53 MB TIF)Click here for additional data file.Figure S2Viability of the MORN1-KO phenotype upon ATc withdrawal after various times of ATc induction. (A) MORN1-KO parasites were allowed to plaque by inducing the phenotype for the indicated duration with ATc, followed by 9 days of growth in absence of ATc. Plaques were counted and plotted relative to the uninduced MORN1-KO strain. Average of three replicate experiments is shown; error bars denote standard deviation. Only 1 data point was collected at 96 hrs induction. (B) Stained plaque assays of the time course of ATc induction followed by 7 days plaque growth (0 hrs) or 9 days plaque growth .(2.39 MB TIF)Click here for additional data file.Figure S3Single channel fluorescence panels corresponding with (10.05 MB TIF)Click here for additional data file.Figure S4Dissection of the MORN1 overexpression phenotype by MORN1 deletion mutants. (A) Schematic representation of MORN1 and the tested deletion mutants. The MORN domains are numbered and their location indicated by green rectangles. The partial MORN motif at the N-terminus is labeled “PM” and the 5 amino acid linker region between MORN domains 6 and 7 is indicated with an arrowhead. The names of the constructs that were re-cloned under their endogenous promoter and without a tag for functional complementation studies are highlighted with a black background. (B) Summary of the observed YFP locations as outlined in panel C for the tested deletion constructs. Viability was determined by selecting for stable transfectants . (C) Representative images of the phenotypes that could be discerned. In several panels the parasites are co-transfected with H2b-RFP to identify the nucleus. WT: wild type; AG: aggregation (inclusion bodies); Fib: Fibers; CC: centrocone; AC: apical complex; iBC: immature basal complex; mBC: mature basal complex; Cyt: cytoplasm. Arrows and arrowheads as indicated in the panel. All MORN1 constructs were driven by the α-tubulin promoter and at the C-terminus fused to YFP. All microscopy was performed on live parasites.(1.71 MB TIF)Click here for additional data file.Figure S5Single channels fluorescence images of (6.52 MB TIF)Click here for additional data file.Movie S1MORN1-KO parasites expressing YFP-IMC3 and FNR-RFP were induced for 12 hrs with ATc and then imaged for 14 hrs. Time points are indicated where every time point represents a 10 min lapse.(2.12 MB MOV)Click here for additional data file.


The reactivity of alpha-1-antitrypsin (AAT) with Lens culinaris agglutinin (LCA) was studied by crossed immuno-affinity electrophoresis of the sera of 246 subjects from 6 groups , carcinoma metastatic to the liver and normal controls). Two species of AAT (LCA-reactive and -nonreactive species) were detected on crossed immuno-affinity electrophoresis in a gel containing LCA. The percentages of LCA-reactive species of AAT in neoplastic diseases of the liver were significantly higher than those in benign liver diseases and normal controls. There was no correlation between the percentage of LCA-reactive species of AAT and serum AAT concentration in any group. Furthermore, in studying 15 pairs of serum samples before and after the subsequent development of HCC, the percentage of LCA-reactive species of AAT after HCC occurrence was significantly higher than that before, although there was no statistically significant difference between the serum AAT concentration before and after development of the disease. The latter 15 patients were all of the normal protease inhibitor phenotype (PiMM) and no change in phenotype was observed before and after the development of HCC. The results indicate that measurement of the reactivity of AAT with LCA can be a useful marker for the diagnosis of HCC and carcinoma metastatic to the liver, especially when serum concentrations of alpha-foetoprotein or other tumour markers are within the normal ranges.


Deficiency of Alpha-1-antitrypsin (AAT) can be a genetic condition that increases the risk of developing liver, lung and possibly gastrointestinal disease. Since many autistic children also have gastrointestinal disorders, this study was designed to measure serum concentration of AAT and establish AAT genotypes in autistic children, age and gender matched non-autistic siblings, parents and controls.We used an indirect ELISA with monoclonal IgG to AAT to measure AAT serum concentrations in 71 members from 16 families of individuals with autism and 18 controls (no family history of autism). We used a duplex polymerase chain reaction to detect M, S and Z alleles for alpha-1 antitrypsin expression in 52 members of 12 of the above families.A significantly high number of autistic family members had lower than normal serum levels of AAT when compared to controls. Autistic children with regressive onset had significantly lower levels of AAT compared to controls, and a significant number of autistic children with low serum AAT also had hyperbilirubinemia, gastrointestinal disease and respiratory problems. We also found that a significantly high number of these individuals had the PiMZ genotype and correspondingly low levels of serum alpha-1 antitrypsin.Knowing that low levels of alpha-1 antitrypsin may be inherited, and that low levels of AAT may be associated with GI disease in autistic children, genotyping autistic children may help identify individuals susceptible to developing digestive problems. Alpha-1-antitrypsin (AAT) is the most abundant circulating serine protease inhibitor with normal serum concentration of 85–250 mg/dL, but peak concentrations as great as fourfold that of normal may occur during inflammation.3AAT deficiency is a genetic condition that increases the risk of developing a variety of diseases including pulmonary emphysema, cirrhosis of the liver and possibly GI disease. It is caused by mutations in the AAT gene coding8The protease inhibitor or Pi system has been used to name the various mutations of the AAT gene.12Alpha-1 antitrypsin (AAT) is mostly secreted by hepatocytes and, to a lesser extent, by lung epithelial cells and phagocytes. It inhibits a variety of serine proteinases, but its preferred target is human neutrophil elastase (HNE), for which it demonstrates the highest affinity.13The major function of AAT in the lungs is to protect the connective tissue from HNE released from triggered neutrophils, as supported by the development of pulmonary emphysema early in life in subjects affected by severe inherited deficiency of AAT.17Alpha-1 antitrypsin production increases markedly after stresses such as surgery, injury, infection, or inflammation and with estrogen administration.18Toxic chemicals found in cigarette smoke, air pollutants, oxidizing chemicals, and oxidizing agents released by activated neutrophils, all inactivate a critical portion of the alpha-1-antitrypsin molecule.19If autistic children express low levels of alpha-1-antitrypsin.21Children with autism frequently have nongastro-intestinal symptoms suggestive of digestive diseased, such as reflux esophagitis.Since some autistic children have GI problems which might be associated with inflammation, we hypothesized that low AAT levels may be associated with these conditions.In this study, we determined AAT serum concentrations of 71 members from 16 families of individuals with autism, compared these to concentrations of AAT in 18 controls (parents with no family history of autism), and found that a significant number of family members had lower than normal AAT levels (less that 85 mg/dl). Using a duplex polymerase chain reaction, we detected M, S and Z alleles for alpha-1 antitrypsin expression in 52 family members from 12 of the above families. We found a significantly high number of family members (n= 37) with the MZ genotype. A significant number of these heterozygotes also had correspondingly low levels of serum alpha-1 antitrypsin.Since AAT concentration may be lower than that of the normal population, detection of low serum AAT and establishment of AAT genotype, might be useful tools for assessing prognosis in autism. Also, a better understanding of the incidence of AAT deficiency in autistic families and its potential relationship to gastrointestinal, liver and lung diseases, may lead to better therapy.We used an indirect ELISA and western blotting, as previously described29Purified Alpha-1 antitrypsin (Sigma) at concentrations of 1 μg, 0.1μg, 0.01 μg and 0.001 μg per 100 μl of bicarbonate buffer (pH 9.6), and 100 μl serum protein at a dilution of 1:500 (PBS), were fixed to wells of 96 well polystyrene microculture plate (Corning) by incubation overnight at 4 degrees C. Excess AAT/ serum was dumped from wells, and all wells were blocked by washing 3X with 300 μl blocking solution . One hundred microliters of primary antibody (mouse Mab to AAT (Biomedia), diluted 1:500 with PBS, was added to all wells and plate was incubated for 2 hours at 37 degrees C. All wells were washed 3 times with PBS/tween. One hundred microliters of affinity purified alkaline phosphotase conjugated goat anti mouse IgG (Biorad), diluted 1:5000 with PBS, were added to all wells and incubated for 45 minutes at 37 degrees C. All wells were washed 5 times with PBS/tween. One hundred microliters of AP substrate (Biorad) were added to all wells and the plate was incubated at room temperature until significant color change in positive control (10–15 minutes). Optical density was measured using ELISA Reader (Biorad).Purified AAT (Sigma), and controls were incubated 1:1 with loading dye (with beta mercaptoethanol) at 95 C for 5 minutes. Protein was run on SDS-Page (18%) (BioRad), at 100 volts, 1 μg/lane.Proteins were transferred to nitrocellulose at 30 volts, overnight at 4 C. Nitrocellulose was flooded with blocking solution (1% casein/PBS) and rocked gently overnight.Ten ml of primary antibody  was flooded over appropriate nitrocellulose sheets and incubated for 2 hours at room temperature with gentle rocking. Sheets were washed 3 times using PBS/tween. Five minutes for each wash, rocking gently at room temperature. Sheets were flooded with 10 ml of affinity purified goat anti-mouse IgG conjugated with Horse Radish Peroxidase (HrP) (Biorad), diluted 1:5000 with PBS. Sheets were washed 5 times with PBS/tween, five minutes each wash, rocking gently at room temperature. Sheets were flooded with 10 ml of HrP substrate at room temperature until significant color change.Polymerase chain reactions was performed with standard buffer . in a total volume of 100 μl containing: 250 ng of genomic DNA, 0.25 pM each of the oligonucleotide primers, 0.2 mμ of dNTPs and 2.5 U of Taq polymerase. Primers used 11detectingtheSmutationare:5′-TGAGGGGAAAC-TACAGCACCTCG-3′ and 5′AGGTGT-GGGCAGCTTCTTGGTCA-3′; primers detecting the Z mutation are: 5′-ATAAGGCTGTGCTGAC-CATCGTC-3′ and 5′-TTGGGTGGGATTCAC-CACTTTTC-3′. Temperature cycling conditions were: initial 10 min denaturation at 94 °C, 30 cycles of 2 min at 94 °C, annealing for 2 min at 55 °C, extension for 3 min at 72 °C, and final extension for 10 min at 72 °C. Twenty microlitres of amplification product were digested for 3 h at 65 °C in a 50 μl volume containing 20 U of TaqI restriction enzyme, in the appropriate buffer. The digested DNA was electrophoresed in a 3% agarose minigel for 3 h at 90 volts, stained with ethidium bromide and visualized under 254 nμ uv trans-illumination.Seventy-one family members from 16 families of individuals with autism, obtained from the Autism Genetic Resource Exchange , and 18 controls (parents with no family history of autism), obtained from National Disease Research Interchange  were tested for their serum concentration of alpha-1 antitrypsin and AAT genotype.Using an indirect ELISA, serum from individuals was tested using anti-AAT monoclonal IgG and compared to varying concentrations (1 μg–0.001 μg/) of purified AAT and negative control (PBS). Specificity of monoclonal anti-AAT IgG was established using western blotting . ResultsForty-five of the 71 individuals from the 16 families had AAT concentrations less than 85 mg/dL serum, making them low producing individuals . WhereasComparing AAT levels in parents and autistic children with a variety of demographics , a signiUsing medical history reports (gathered by AGRE), we analyzed the phenotypic characterization of the probands of family members tested in this study . OverallThree of the 23 affected individuals who answered the related questions had respiratory distress syndrome (RDS) (p < 0.05). Two of these three were born after short gestation (29 weeks). All three required oxygen supplimentation. None of these individuals had hyperbilirubinemia. Two of these three had deficient levels of AAT.Six other affected siblings (not including the three with RDS) reported respiratory problems later in life. Five of these 6 had deficient levels of AAT. Although none of the individuals had emphysema, two reported multiple respiratory problems—tracheomalacia and sleep apnea, stridor and laryngomalacia. One reported reactive airway disease. One reported multiple incidences of pneumonia and the other two did not report a specific disorder.Seven of 22 affected siblings reported gastrointestinal problems. One of these did not answer the question. Five of the remaining six had deficient levels of AAT (p < 0.05). Three of the six reported chronic diarrhea, one reported constipation, one colic and the other, projectile vomiting.If we predicted the genotype of all 71 members of the 16 autistic families based on their AAT serum concentrations , most (4Using a PCR/Restriction Digest we genotyped 52 members from 12 of the 15 families who had been measured for serum AAT concentration (above) (DNA was not available for three of the families). Expected product demonstrating specific MM, MS, SS, MZ and ZZ genotypes for AAT expression is shown in A significantly high number of family members (37) had the MZ genotype. A significant number of these heterozygotes (35 of 37) had correspondingly low levels of serum alpha-1 antitrypsin (less than 100 mg/dL) .Approximately 75 allelic variants have been described in the AAT gene locus resulting in a very complex genetic classification based upon the phenotypic features of the circulating AAT protein. The most common variant, PI M, is present in approximately 95% of the Caucasian U.S. population and is regarded as the normal variant associated with normal serum levels of functional AAT. The predominant deficiency alleles, such as PI Z and PI S, may result in decreased levels of circulating AAT but with completely normal functioning proteins. The MM phenotype is therefore designated as manifesting 100% concentration of circulating AAT (85–250 mg/dL). The heterozygous combinations MZ and MS yield 35% and the ZZ allele 10%–25% of this normal MM value. Approximately 95% of all AAT deficiency states leading to clinical manifestations are made up of PI ZZ homozygotes. Certain alleles, such as the S allele, either in the homozygous state or associated with the M allele, do not appear to be associated with the abnormally polymerized molecules within the endoplasmic reticulum and have not been incriminated in the development of liver disease unless combined with the Z allele.30The association of AAT deficiency and liver disease in children was first described in 1969.31Cederlund and Gillberg (2004),Asperger first recorded the link between celiac disease and behavioral psychosesIn inflammatory conditions, AAT levels are normally elevated. In this study, however, the results demonstrate low alpha-1-antitrypsin serum levels, which are probably due to genetic factors and not excess protein loss in stool. This might contradict the theory that autistic patients have inflammatory bowel disease because if there was chronic inflammation due to enterocolitis, the alpha-1-antitrypsin level should be elevated. However, there is evidence that increased inflammation is associated with AAT deficiency, such as GI inflammation in patients with ulcerative colitis, associated with the PiZ carriers,43In this preliminary study, a significant number of autistic family members have low serum levels of AAT, which is, at least in part, caused by the PiMZ genotype. Low serum AAT in autistic children supports the findings of Walker-Smith and colleagues, and strongly suggests an association between autism and AAT deficiency.A high number of autistic children with low levels of AAT in our study, also had neonatal hyperbilirubinemia (8/22), respiratory problems (9/23) and digestive disorders (7/22). Since AAT deficiency may be associated with these problems, this also supports our hypothesis of an association between AAT deficiency and autism.Our study also suggests an association between autistic children with regressive disease and AAT deficiency. This suggests that, besides genetic predisposition, environmental factors may be influencing levels of serum AAT in these individuals.Our observation, however, that some non-autistic siblings inherit AAT deficiency, and parents of autistic children who also have low levels of serum AAT, suggests that AAT deficiency alone is not a causative agent for ASD, but may make a subset of autistics susceptible to inflammatory disease.Knowing that low levels of alpha-1 antitrypsin is inherited, and that low levels of AAT may be associated with GI disease, genotyping autistic children may help identify those autistics susceptible to developing digestive problems. And a better understanding of the incidence of AAT deficiency in autistic families and its potential relationship to liver, lung and bowel diseases, may lead to better therapeutic strategies.


There is increasing evidence that septic complications, occurring after major hepatectomies, may be caused by gram negative bacteria, translocating from the gut. We investigated in rats, the effect of extended hepatectomy on the structure and morphology of the intestinal mucosa as well as on the translocation of intestinal bacteria and endotoxins. We also examined the effect of nonabsorbable antibiotics on reducing the intestinal flora and consequently the phenomenon of translocation by administering neomycin sulphate and cefazoline. Hepatectomy was found to increase translocation, while administration of nonabsorbable antibiotics decreased it significantly. In addition, hepatectomy increased the aerobic cecal bacterial population, which normalised in the group receiving antibiotics. Among the histological parameters evaluated, villus height demonstrated a significant reduction after hepatectomy, while the number of villi per cm and the number of mitoses per crypt, remained unchanged. Our results indicate that administration of nonabsorbable antibiotics presents a positive effect on bacterial and endotoxin translocation after extended hepatectomy, and this may be related to reduction of colonic bacterial load as an intraluminal effect of antibiotics.