We often think of ourselves as either a day person or a night person—one who rises with the sun, raring to go, or one who prefers to stay up through the night to get things done. Regardless, we each have our regular waking and sleeping cycles. It's been known for some time that variations in sleep and wakefulness are part of our circadian rhythm, or molecular clock. A portion of the brain called the hypothalamic suprachiasmatic nucleus (SCN) regulates this biorhythm. When this area of the hypothalamus is destroyed in animal models, the circadian rhythm is disrupted. Two transcription factors (proteins that regulate gene expression) called Bmal1 and Clock regulate aspects of circadian rhythm, possibly by regulating neurons in the SCN.Other aspects of human physiology are also regulated in a circadian manner. Besides altering sleep and wakefulness patterns, ablation of the SCN alters the ability to regulate sugar levels. Sugar (glucose) levels must be maintained within fairly narrow limits for survival. This regulation is controlled in part by a balance between blood sugar level and insulin production (insulin lowers the blood sugar level). In people and in mouse models, both glucose level and insulin level are subject to circadian rhythms. It isn't clear, however, if this is a behavioral effect, whereby the disruption of the SCN might alter our feeling of being well fed—that is, being sated—as eating has a profound effect on blood sugar levels.Garret FitzGerald and colleagues tested the effect of the molecular clock genes in glucose regulation (homeostasis) by examining mice in which Clock and Bmal1 were impaired. In normal mice they observed a peak in glucose levels early in the day. This diurnal regulation was lost in the mutant mice. Furthermore, whereas the normal mice could fairly easily return their glucose levels to normal when they were artificially treated with insulin, this ability was severely impaired in the mutant mice. What's more, a high-fat diet amplified this circadian variation in the normal animals, but the rhythm was abolished in the mutants on a high-fat diet. Thus, the authors demonstrated that circadian control of blood glucose levels is due directly to the presence of these transcriptional factors rather than due to some other behavioral effect that ablation of the hypothalamus might have caused. It's possible, therefore, that besides what we eat, our internal circadian clock could also be an important regulator of blood sugar levels.What is still left to be explored is whether the change in glucose that results from disruption of the Clock and Bmal1 genes is due to the transcription factors' effect as circadian regulators or to an activity of these transcription factors that is unrelated to circadian rhythm generation. But the study does raise the possibility that when you eat may be as important to your health as what you are eating.


Anopheles gambiae populations is important for the current tactics in mosquito control and for the proposed strategy using genetically-modified mosquitoes (GMM). Genetically-isolated mosquito populations on islands are a potential site for testing GMM. The objective of this study was to determine the genetic structure of A. gambiae populations on the islands in Lake Victoria, western Kenya.Understanding the genetic structure of island A. gambiae populations from five islands on Lake Victoria and six villages from the surrounding mainland area in the Suba District were examined using six microsatellite markers. The distance range of sampling sites varied between 2.5 and 35.1 km.The genetic diversity and the population genetic structures of 13 ST = 0.019) and between the island and mainland populations (FST = 0.003). A total of 12 private alleles were found, and nine of them were from the island populations.A similar level of genetic diversity between island mosquito populations and adjacent mainland populations was found. The average number of alleles per locus was 7.3 for the island populations and 6.8 for the mainland populations. The average observed heterozygosity was 0.32 and 0.28 for the island and mainland populations, respectively. A low but statistically significant genetic structure was detected among the island populations (FA level of genetic differentiation between the island and mainland populations was found. Large extent of gene flow between the island and mainland mosquito populations may result from wind- or human-assisted dispersal. Should the islands on Lake Victoria be used as a trial site for the release program of GMM, mosquito dispersal between the islands and between the island and the mainland should be vigorously monitored. Plasmodium [Despite 50 years of malaria vector control efforts, malaria remains a major public health threat in tropical and subtropical countries -3. In reasmodium  has signasmodium .A. gambiae cell lines were successfully transformed with the Hermes element [Minos transposable element bearing an exogenous gene was efficiently integrated into the genome of Anopheles stephensi [A. gambiae [A. gambiae genome sequences will greatly facilitate identification and cloning of parasite-inhibiting genes [One potential alternative malaria control strategy is based on the genetic disruption of mosquito vector competence -13. This element ,15, and tephensi ,17. Gene gambiae , and gen gambiae . Availabng genes .A. gambiae population genetic structure in the African continent has been examined [A. gambiae; however, no significant genetic structure was detected for mosquito populations between western Kenya and West Africa. The minimum area associated for a deme of A. gambiae in western or coastal Kenya is larger than 50 km [et al. [Anopheles arabiensis populations from the high plateau of Madagascar and those from Réunion and Mauritius islands (FST ranges from 0.080 to 0.215). Population substructure was also detected on the island of São Tomé, West Africa [The success of the transgenic mosquito approach depends on the spread and even fixation of parasite-inhibiting genes into natural populations. Presently, releasing transgenic mosquitoes to the field is premature. Isolated islands have been suggested as an ideal natural site for testing transgenic mosquito release strategies and spatial spreading of transgenes ,21,22. Iexamined -28. Thesan 50 km . Simard  [et al.  found a A. gambiae mosquitoes from five islands on Lake Victoria and the surrounding mainland in western Kenya. This information is valuable for selecting field sites to test transgene release strategies and evaluating the spread of transgenes in nature.The present study examined the genetic diversity and the population genetic structures of A. gambiae mosquitoes are the major malaria vectors in this region [Anopheline female mosquitoes were collected from seven villages on five islands in Lake Victoria and from six villages in the mainland Suba District, western Kenya Fig. . The sams region .A. gambiae sensu lato (s.l.) specimens were separated from other anophelines according to the identification key provided by Gillies and Coetzee [At least 170 anopheline mosquitoes were collected from four to 28 houses within each village using the pyrethrum spray collection method . Mosquit Coetzee  and thenA. gambiae species complex cannot be identified by morphology alone [A. gambiae s.l. females per village were tested. If the initial PCR testing failed to amplify for a sample, then the PCR analysis was repeated once or twice until successful amplification was achieved. If a sample could not be identified after three PCR amplifications, it was scored as unknown.PCR analysis was conducted for species identification using the rDNA-PCR method because individual species within the gy alone . About 1A. gambiae populations from the Lake Victoria islands and the surrounding mainland in western Kenya).Six microsatellite markers were used for specimen genotyping, including AGXH1D1 and AGXH131 of Chromosome X, AG2H46 and AG2H79 of Chromosome 2, and AG3H29C and AG3H33C of Chromosome 3 ,18,22,232, 1.0 μg BSA, 1.0 unit Taq polymerase and about 20 ng genomic DNA. Cycling conditions in a MJ Research PTC-220 thermocycler were 35–40 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 45 seconds. Allele sizes were determined using Gene ImagIR computer software [A Li-Cor Model 4200 Automated DNA Analyzer (Li-Cor Inc., Lincoln, NE) was used for gel electrophoresis. For the apparatus to detect PCR products, one primer in every pair of microsatellite primers must be fluorescently labelled. To reduce the cost associated with synthesis of fluorescently labelled primers, we used the "tailed primer" method ,35; thatsoftware . The allIS statistics and probability test were used to determine whether distortion from HWE resulted from heterozygosity deficiency or excess using. Because the probability test is robust to low allele frequencies, rare alleles were not pooled. Variations in heterozygosity among the populations were analyzed following Weir's method [ST) using FSTAT 2.8 [ST statistic appears to be more sensitive to detect intraspecific differentiation than RST [Microsatellite polymorphism was measured by the number of alleles and heterozygosity at each locus. Using the probability test available in the GENEPOP computer program , conforms method , using tSTAT 2.8 . FST stathan RST ,41. The than RST  were calthan RST . A dendrST/(1 - FST) against the natural logarithm of straight-line geographical distance between population pairs [The isolation-by-distance model of population genetic structure was tested by linear regression of pair-wise Fon pairs . Statiston pairs .A moderate to high level of polymorphism was found in six loci across the 13 populations See the . The thrA. gambiae populations [A total of 14.1% loci (11 out of 78 tests) showed significant departure from Hardy-Weinberg equilibrium, all due to heterozygote deficiency. This was caused entirely by heterozygote deficiency in the locus AG2H46, a locus known for the presence of null alleles in western Kenyan ST = 0.019, P < 0.001) was almost twice as high as the six mainland populations (FST = 0.010, P < 0.001). Genetic differentiation between island and mainland populations was also small (FST = 0.010, P < 0.001). Pair-wise comparisons between all populations revealed that only seven pairs (Kibougo/Kamsengere, Kasgunga/Kamsengere, Takawiri/Ruri, Sena/Ruri, Utajo/Ruri, Ngodhe/Ruri and Ngodhe/Gingo) exhibited significant FST values, and six of them were between an island and a mainland population.A low, but significant, genetic structure was detected among the seven island and the six mainland populations Table . The genST/(1 - FST) (P < 0.001), suggesting that the population genetic structure of A. gambiae populations from the island and mainland is consistent with the isolation-by-distance model. When the Ruri population is removed from the analysis, the correlation was still statistically significant (P = 0.015). Therefore, the population genetic structure of our study populations is consistent with the isolation-by-distance model. The cluster analysis revealed that the Ruri population, located farther inland from the other populations, was out-grouped from other populations with a significant bootstrap value, while other mainland and island populations were intermixed with non-significant branch bootstrap values  and among the six villages in the mainland in an area of approximately 40 × 20 km2 (FST = 0.010). The degree of genetic differentiation between the island populations in this study was less than for the island A. gambiae populations of São Tomé, western Africa (FST = 0.032) [ST estimates in the populations in this study were probably caused by shorter distance between islands (3–15 vs. 23–38 km) [ST estimates for the mainland populations in this study were comparable to other studies on the western Kenya populations (FST = 0.0033) [ST = 0.003). Thus, there is a very small degree of genetic isolation between island and mainland populations. This estimation is consistent with the private allele distribution in the studied populations, in which nine of the 12 private alleles were from the island populations. Further evidence for a small degree of genetic differentiation between island and mainland populations is from pair-wise population comparisons in which six out of the seven pairs that exhibited significant genetic differentiation were between an island population and a mainland population.A small but statistically significant genetic structure was detected for = 0.032) . The low3–38 km)  and a la 0.0033) ,27. The A. gambiae is usually less than 1 km [et al. [A. gambiae mosquitoes was related to the predominant wind direction at night, suggesting that wind assisted the dispersal of mosquitoes from their breeding site. A. gambiae have been shown to fly up to 7 km with the assistance of wind [The low level of genetic differentiation between island and mainland mosquito populations implies large gene flow between the two areas (83.1 migrants per generation). The normal flight range of han 1 km . The dis [et al.  found th of wind ,53. ThisAedes polynesiensis populations from islands found no significant effect of geographic distance on the population genetic structure, but detected a significant correlation between gene flow and commercial traffic by planes and/or boats between islands [A. arabiensis to the Mascarene islands and Madagascar was thought to be caused by human transportation by steamship lines [A. gambiae larvae were collected at the bottom of a wooden fishing boat [Mosquito migration may also be assisted by human activities. A study on  islands . The intip lines ,56. In Ling boat . RushingA. gambiae populations have implications for the ecological safety evaluation of the transgenic mosquito release program. During the initial field test of environmental safety and public health consequences by transgenic mosquito release, ideal sites would be islands that are totally genetically isolated from other islands and the mainland, with a sufficient number of human inhabitants and active malaria transmission on the island. Such an island may be extremely difficult to find, so islands with some genetic isolation from the mainland may have to be chosen. If so, the Lake Victoria islands could be used as field test sites; however, due to potential gene flow between the islands and between the islands and the mainland, mosquito dispersal between the islands and between the islands and the mainland should be vigorously monitored. After the release of the genetically modified mosquitoes, long-term monitoring programs should be launched to evaluate the spread of the transgenes to any unintended areas. In addition, methods to minimize the negative effects of transgene leak need to be developed prior to the field trial of transgene release [The results of this study of the population genetic stricture of island and mainland  release .This study showed that a low level of genetic differentiation existed between the island and mainland populations and no any genetically-isolated population was found among the 13 mosquito populations. If the islands on Lake Victoria were used as a trial site for the program to release genetically-modified mosquitoes, short-term and long-term mosquito dispersal between the islands and between the island and the mainland should be vigorously monitored.HC conducted species identification using PCR, microsatellite analyses and drafting the manuscript. NM was responsible for sample collection, and participated in species identification and drafting the manuscript. JB and GY supervised the study, and assisted data analysis and manuscript preparations.A. gambiae populations from the Lake Victoria islands and the surrounding mainland in western Kenya.A table of sample size, allelic number, heterozygosities and breeding coefficient of 13 Click here for file


Fetal growth restriction is associated with significantly increased risks of neonatal death and morbidity and with susceptibility to hypertension, cardiovascular disease and NIDDM later in life. Human birth weight has a substantial genetic component, with at least a quarter of the variation attributable to additive genetic effects.One hundred twenty-five subjects (83 control and 42 case) were selected using stringent inclusion/exclusion criteria. DNA sequencing was used to identify 26 single nucleotide polymorphisms in the pituitary growth hormone gene (GH1) at which all subjects were genotyped. Association with fetal growth restriction was tested by logistic regression for all sites with minor allele frequencies greater than 5%.Logistic regression identified significant association with fetal growth restriction of C alleles at sites -1 and +3 (relative to the start of transcription) that are in complete linkage disequilibrium. These alleles are present at higher frequency (6% vs. 0.4%) in fetal growth restricted subjects and are associated with an average reduction in birth weight of 152 g in normal birth weight and 97 g in low birth weight subjects.There is suggestive association between fetal growth restriction and the presence of C alleles at sites -1 and +3 of the pituitary growth hormone gene. Fetal growth restriction (FGR) is a major risk factor for illness in the perinatal period and throughout life, with the smallest 7.5 percent of infants accounting for two-thirds of infant deaths . Term, lIt is difficult to distinguish the genetic and environmental components of human birth weight variation, but recent studies support a major genetic component to birth weight variation. Clausson et al.'s  study ofThe five genes of the human growth hormone locus reside within about 45 kb on chromosome 17 . PituitaHere we use DNA sequencing to identify and to determine the frequencies of both 12 newly-identified single nucleotide polymorphisms (SNPs) in the promoter and coding region of the GH1 gene and 15 previously reported SNPs ,18. Usinth percentile of size for gestational age [th percentile of female and lowest 6.5 percentile of male deliveries [DNA was extracted from placental tissue from 125 live births  at Baystate Medical Center (Springfield, MA) in a case-control study of the genetic predisposition to fetal growth restriction. All subjects were Caucasian, both Hispanic and non-Hispanic. Classification of newborns as fetal growth restricted (or IUGR, intrauterine growth retarded) followed the definition of the American College of Obstetricians and Gynecologists as those newborns below the 10onal age . Our cutliveries . StringeThe region from -624  to +1,726 (197 nucleotides after the termination codon) of GH1 was amplified in two overlapping fragments: -624 to +541 (GHN-1F 5' AGGGACCTGGGGGAGCCCCAGCAA 3', GHN-1R 5' TCACCCCTTCCTGCCACCCCTGAT 3') and +450 to +1,726 . Approximately 50 ng of DNA was used as a template in a polymerase chain reaction with 30 cycles of 95°C (1 minute), 62°C (1 minute), and 72°C (2 minutes 30 seconds). PCR products were purified from agarose using a QIAquick PCR Purification kit (Qiagen) and sequenced directly with BigDye v2.0 chemistry (Applied Biosystems) and run on either an ABI Prism 377 or 3100 automated DNA sequencer. PCR products were sequenced with the PCR primers and additional internal primers: 5' AAGCACAGCCAATAGATTG 3', -459 to -441; 5' GCACAAGCCCGTCAGTGGCC 3', -108 to -89; 5' GGATTTTAGGGGCGCTTACC 3', +71 to +90; 5' CATCTCCCTGCTGCTCATC 3', +931 to+949; 5' GCGCTTGGGYACTGTTCCCT 3', +1280 to +1299.Sequence traces were aligned and assembled into contigs by the program Polyphred . ContigsThe five genes of the human growth hormone locus exhibit high sequence similarity, and the paralogous regions corresponding to the portion of GH1 sequenced in this study (-624 to +1,726) were multiply aligned. Nucleotide positions , as defined in the methods. Of the invariant sites, 5 are polymorphic in GH1, while 21 of the variant sites are polymorphic. A comparison of the proportion of polymorphic sites at invariant and variant sites by Fisher's Exact Test is highly significant (P <<0.001). This result indicates that there is a strong bias for polymorphisms in GH1 to occur at the minority of sites that exhibit sequence divergence among the paralogous genes. The high correspondence between the sequence of minor alleles in GH1 and nucleotides present in paralogs of GH1 supports previous assertions that the unusually high polymorphism of the GH1 gene is driven by gene conversion ,18.A proportion of this bias may be explained by selective constraint, in that sites that are polymorphic within GH1 may be under less selective constraint and thus more free to exhibit sequence divergence among paralogs. However, to entirely explain the bias towards polymorphism at sites of divergence, one must assume that about 1,804 (91%) of the invariant sites are selectively constrained and not free to vary. Given that a substantial proportion (814 bp, ~36%) of the sequence surveyed in this study is composed of introns, this assumption seems unreasonable.Logistic regression Table  was perfAlthough the A/C polymorphism at site -1 was not significant in a reduced model, we decided to investigate this site for three reasons. First, the C allele at site -1 is in complete linkage disequilibrium with C at site +3. Second, both of these sites are located at the start of transcription, making them good candidates for affecting the level of transcription of GH1. Third, the only paralog that shares C at these sites is CS-5, a pseudogene, consistent with the possibility that C at sites -1 and +3 is disadvantageous. The C allele at both sites exhibits a much higher frequency in low birth weight (6%) versus normal birth weight 0.4%) subjects. Restricting examination only within the normal or low birth weight subjects, the AC genotype is associated with an average reduction of birth weight of 152 g and 97 g in normal and low birth weight subjects, respectively. However, this difference does not achieve significant in an ANOVA on the A/C polymorphism and gestational age  did among British army recruits (0.4% vs. 0.3%) but that among the low birth weight subjects the frequency of the C allele is substantially higher (6%). Comparing all the other allele frequencies . There are two possible explanations for this observation. First, site -1 could be a hotspot for nucleotide mutation, with transversions from A to C/T occurring often. Second, GH1 may be the recipient of gene conversion events from more than one paralog within the GH locus. Among the four other genes of the GH locus, all three alleles observed at site -1 occur Table . Given tIt must be pointed out that the common wisdom is that GH1 plays no role in regulating fetal growth because the GH receptor is expressed fairly late in gestation and because anencephalic infants or those born without a pituitary achieve nearly normal length . ImportaIn a stringently selected set of subjects, C alleles at sites -1 and +3 relative to the start of transcription of GH1 have a higher frequency (0.4% vs. 6%) in fetal growth restricted newborns. These two alleles are in complete linkage disequilibrium and their presence is associated with a reduction in birth weight of 152 g in term, normal birth weight subjects and 97 g in term, low birth weight (<2,500 g) subjects. In combination with environmental, behavioural and other genetic factors, these alleles may contribute to fetal growth restriction.The author(s) declare that they have no competing interests.RMA conceived the project and directed its design and execution. CC and RV performed the molecular genetic work and participated in preliminary analyses. TKB performed the subject selection.The pre-publication history for this paper can be accessed here:


Over 250 million years ago (mya), all the continents of Earth formed a single land mass called Pangaea. Some 50 million years later, this supercontinent began to split in two, forming Laurasia—now North America, Asia, and Europe—and Gondwana—present-day Antarctica, Australia, South America, Africa, and India. After another 50 million years, Gondwana, too, broke up. At the end of the Cretaceous period, New Zealand split off (about 80 mya), then South America and Australia separated from Antarctica (about 35 mya). Fairy-tale quality aside, the story of continental drift fits comfortably with the geological and fossil record and feeds our understanding of current distributions of plant biodiversity.Nothofagus—the southern beech tree, a plant whose 80-million-year-old fossil history goes back to the days of Gondwana—has polarized views on the nature of Southern Hemisphere biogeography.Although we know how and when Pangaea broke apart, the distribution of fossils of the same species on many different continents, separated by vast ocean waters, challenges us to explain how they got there. Plant life on New Zealand, for example, shares striking similarities to that on other Southern Hemisphere land masses, but scientists have yet to agree on how this came to pass. In particular, one genus, Nothofagus seeds are not considered ocean-worthy vessels, many believe vicariance is the only possible explanation.One theory suggests that geographic barriers  would have prevented species expansion after the break-up of the continents, so similar contemporary species must have already existed in both places before New Zealand broke away from Gondwana. In this scenario, called vicariance, ancestors of existing lineages drifted with the repositioned land masses. Another hypothesis, born of existing distributions and fossil data, suggests that long-range oceanic dispersal is more likely. But since Peter Lockhart and colleagues argue that a clear picture of the divergence dates of various southern beech species could help clarify the relative contributions of vicariance versus dispersal. But they would need significant lengths of DNA sequences to reliably characterize the evolutionary history of each species.Nothofagus subgenera—Lophozonia, Fuscospora, and Nothofagus—from South America, Australia, and New Zealand. Reconstructing the trees' evolutionary relationships (phylogeny) based on analyses of their chloroplast sequences, the authors discovered a nuanced evolutionary history that supports vicariance for some species and dispersal for others.Consequently, Lockhart and colleagues analyzed a 7.2-kilobase fragment of the chloroplast genome  for 11 species of three Assuming that beech was present throughout Gondwana (which fossil data support), the sequence of the Gondwana breakup should be reflected in the beech's phylogeny. New Zealand beeches should be more distantly related to both Australian and South American species, because of the greater period of separation—65 million years compared to 30 million years. Yet Australian and New Zealand beeches are more closely related to each other than to South American species, which reflects more recent relationships. Given that fossils of all beech subgenera extend back to the New Zealand Cretaceous period, the dating of splits and the nature of the relationships indicate extinction of beech lineages within current subgenera in New Zealand, and possibly in Australia and South America.Lophozonia and Fuscospora species are too recent to have roots in Gondwana, indicating a role for transoceanic dispersal. The evolutionary relationship between the Australasian and South American Fuscospora lineages, however, is consistent with vicariance. These divergence results, the authors conclude, indicate that current distributions of Nothofagus cannot be explained solely by continental drift (followed by extinction of some species) and that contemporary New Zealand Nothofagus species are not direct descendants of the beeches thought to have reached the island after the split from Antarctica.Lockhart and colleagues' analyses suggest that the relationships of the Australian and New Zealand Nothofagus seeds—and stresses the need to consider more complex hypotheses to explain something as dynamic and complex as the evolutionary history of biodiversity.Taken together, the results highlight the need for caution in evaluating fossil evidence. The fossil record doesn't necessarily capture when a species first appeared, and a continuous fossil presence can mask extinctions and reinvasions. The authors conclude that their molecular data make the case for investigating possible mechanisms of long-range dispersal—especially the dispersal properties of


Fusobacterium nucleatum genome sequence we have examined the evolutionary relationships of Fusobacterium genes by phylogenetic analysis and comparative genomics tools.The phylogenetic position and evolutionary relationships of Fusobacteria remain uncertain. Especially intriguing is their relatedness to low G+C Gram positive bacteria (Firmicutes) by ribosomal molecular phylogenies, but their possession of a typical gram negative outer membrane. Taking advantage of the recent completion of the Fusobacterium has a core genome of a very different nature to other bacterial lineages, and branches out at the base of Firmicutes. However, depending on the method used, 35–56% of Fusobacterium genes appear to have a xenologous origin from bacteroidetes, proteobacteria, spirochaetes and the Firmicutes themselves. A high number of hypothetical ORFs with unusual codon usage and short lengths were found and hypothesized to be remnants of transferred genes that were discarded. Some proteins and operons are also hypothesized to be of mixed ancestry. A large portion of the Gram-negative cell wall-related genes seems to have been transferred from proteobacteria.The data indicate that Many instances of similarity to other inhabitants of the dental plaque that have been sequenced were found. This suggests that the close physical contact found in this environment might facilitate horizontal gene transfer, supporting the idea of niche-specific gene pools. We hypothesize that at a point in time, probably associated to the rise of mammals, a strong selective pressure might have existed for a cell with a Clostridia-like metabolic apparatus but with the adhesive and immune camouflage features of Proteobacteria. Fusobacterium, together with some close relatives such as Leptotrichia, forms an ecologically and physiologically coherent group [Fusobacterium nucleatum has received particular attention being a key component of the human dental plaque that also has considerable pathogenic potential. In fact after Bacteroides, Fusobacterium is responsible for most human anaerobic infections, producing abscesses at different locations and aspiration pneumonia among other serious conditions [The genus nt group . They sent group . They arnt group ). The spnditions ,5.Bacteroides and other Gram negative anaerobes, their association became conflicting when, after the extensive gene sequencing carried out by the mid 80's, it became clear that Bacteroides showed a clear relationship to other aerobic Gram negatives such as Flavobacterium or Cytophaga [Fusobacterium appeared as a separate cluster only distantly associated to the low G+C Gram positives [Phylogenetically speaking the fusobacteria have become somewhat of a puzzle . Originaytophaga -9 while ositives ,11. Howeositives -14).Fusobacterium nucleatum genome [Clostridium species (low G+C Gram positives) genomic analysis showed also a strong proximity to Proteobacteria. Based on the ERGO chromosomal clustering tool, F. nucleatum had more "clusters" of genes with the same gene order in common with Escherichia coli than with Enterococcus or Staphylococcus, although less than with Clostridium or Bacillus [F. nucleatum thrives is an ecosystem with a large bacterial biodiversity. In a recent survey using 16S rDNA sequences from sub gingival plaque samples, 347 species or phylogroups were identified, and the best estimate of the total species diversity in the oral cavity is approximately 500 species [F. nucleatum interacts with a great deal of them, because it plays a crucial "bridge" role between early and late colonizers of the tooth surface [F. nucleatum interacts and aggregates (including spirochaetes, proteobacteria, bacteroidetes, firmicutes, and even fungi) there is a great potential for HGT.The publication of the m genome  did not Bacillus . As expeBacillus . On the  surface  and form surface -21. BecaF. nucleatum, using a variety of bioinformatics tools, in an attempt to clarify the phylogenetic position of the Fusobacteria and the relative contributions of vertical descent and horizontal transfer in shaping the genome of this highly specialized organism. In addition, our study aims at providing material for further discussions on evolution of the gram-negative cell wall, and on the evolution of bacterial communities in micro-environments.We have reanalysed the fully sequenced genome of Fusobacterium with the cyanobacteria and the elongation factor tufA with the proteobacteria. Other cases are also unclear. DNA pol III is a complex holoenzyme formed by 10 subunits in E. coli [Thermotoga and Aquifex. The RNA-directed DNA polymerase and a RNA helicase seem related to archaeal counterparts. In Clostridium, for example, to which most informational genes of Fusobacterium have a best match, all subunits of both polymerases cluster clearly into the Firmicutes (data not shown). Summarizing, the fusobacteria appear as an independent taxon with remote relatedness to the Firmicutes. Although the phylogenetic signal for many genes was considerably weak, the rRNA genes and the ribosomal proteins were very congruent and therefore their trees  and Bacteroides thetaiotaomicron (the Gram-negative species to which it shows the highest number of homologs). Due to differences in mutational biases between the leading and lagging strands, it is common to find the GC skew value (G-C/G+C) with opposite signs on each replichore, the change in sign indicating the origin or terminus of DNA replication [F. nucleatum display a standard plot, with mainly positive values over the right replichore and negative values on the left replichore. Fusobacterium, however, does not show a clear pattern, with constant shifts in GC-skew values across the genome. This situation could be caused by horizontal gene transfer (HGT) incorporating xenologous sequences with a different GC-skew across the recipient chromosome, distorting a clear-cut plot. In favour of this, many GC skew oscillations coincide with clusters of putative xenologous origin . Although other related genomes are not available for comparison and the potential inversions could have happened prior to the split of both subspecies, the suggestion is that the oscillating GC-skew plot is not due to multiple inversions. Finally, the GC-skew plot of F. nucleatum could be partly due to multiple replication origins constantly shifting the values, but this situation has not been observed in any bacterial species.The observed GC-skew could also arise from chromosomal inversions (see, for example, the genome of  pestis -). Howeveobserved ,31 and aobserved . Moreoveincentii  show an A sequence similarity search performed by BLASTP  against Fusobacterium chromosome and, in many cases, they are clustered  also hints at a secondary acquisition by HGT. Therefore, the interpretation of F. nucleatum as a gram-positive bacterium with gram-negative clothing [Looking into more detail at the gene clusters with a best hit outside Firmicutes it was found that many genes were involved in typical gram-negative features, mainly membrane-associated functions Table . For exaclostridiales and streptococci), which share the mouth and dental plaque ecosystem [F. nucleatum with most sequenced genomes, a weak phylogenetic signal may remain for many trees. The branching pattern in trees is also influenced by other variables like different rates of evolution for different genes, method of alignment or number of species included. Like in the sequence similarity method, the data presented are inferences based upon the data, and the limitations of each method should be kept in mind.BLAST analysis has the advantage of giving a closest similarity match for almost every gene. It is however a crude method that can give as the top sequence similarity hit a species that is not the closest from a phylogenetic point of view  and it ccosystem . The phyF. nucleatum protein-coding genes belonged to clusters of 2 or more genes that had some order conservation in other bacteria. From these, 35% had the same order as most Firmicutes  best sequence similarity match, respectively. The tree analysis confirms that the β chain is likely to have a proteobacterial origin. Interestingly, although the β chain is located in a proteobacterial cluster (at the edge of cluster 12), it is contiguous to the Firmicutes related α chain gene, separated by a very short spacer without a promoter. This exemplifies how selection may have put together two functionally related genes, presumably to ease cotranscription, even though their phylogenetic origin appears to be different.To explore the possibility that the chimeric nature of bacteria . An inteMethanosarcina acetivorans, to which it presents the highest sequence similarity, whereas the second and third operon appear to be a blend of genes with relatedness to firmicutes, proteobacteria, spirochaetes and Thermotoga. These genes are present in many Gram negatives including Helicobacter and other Proteobacteria [F. nucleatum, this time dispersed along the genome. Although one of the sets has best matches to Firmicutes species, another one appears to be of spirochaete source , whereas the other three are, according to sequence similarity, gene order and tree reconstruction a mixture of genes with archaeal, Firmicutes and proteobacterial origin. A third case can be seen in the three copies of a hemin transport system located away from each other and formed by a hemin receptor and the genes hmuT, hmuU and hmuV. Although the different taxonomical origin analysis methods are not always consistent, two of the hemin operons are probably of proteobacterial origin  and a potential transferred gene, whereas only 8 appeared between core genes. Since intergenic spacer regions are known to increase in length as a result of genomic rearrangements and pseudogene formation [-5) to one another and to other five long spacers scattered throughout the genome. In all cases except one, these long spacers are flanked by outer membrane proteins of Gram-negative origin, suggesting that they may represent remnants of old membrane-associated genes. A similar case is that of the long spacer located after the hemolysin activator protein precursor (FN1818), which shows high sequence similarity to a hemolysin activator located someplace else in the genome (and to another spacer and a short ORF with unknown function).An indication of massive gene transfer events comes from looking at intergenic spacer regions of ormation , many ofvincentii), spanning 450 sequences that represent 20% of the genome. On average, these orphan genes are extremely short , glycine permease, periplasmic-like proteins, etc.Another potential signature of ancient transfers subsequently erased is the high number of ORFs without a match in the complete, non-redundant NCBI database . As it is unlikely that all these short ORFs are highly expressed, we believe that this biased codon utilization is reflecting very divergent pseudogene fragments. Thus, the picture that emerges is that of massive gene transfer leaving many non-coding segments that are remnants of unnecessary genes and genomic rearrangements.In addition, some short ORFs appear to be degraded fragments of bigger genes. For example, there are 3 sequences with similarity to HIPA proteins, one of which is less than half the length of the other two. As it also has a very biased codon usage, it is likely that it represents a degraded remnant of this protein. The 3 copies of integrases scattered across the genome show another case. Two of them are around 900 bp long and have a normal codon usage. The third copy (FN0402) is only 177 bp long, is flanked by a long spacer and has a very skewed codon usage. In general, the codon usage of these orphans is very biased  points to HGT as the most likely origin of these genes. Even less apparent, our work suggests multiple episodes of gene transfer to or from phylogenetically-related bacteria, like certain Firmicutes species (such as the cariogenic bacterium S. mutans or some Clostridia), that might be confounded with vertically inherited traits.The genome of  example ). One poFusobacterium requires special consideration. Some type of Gram-negative cell wall seems to be the default phenotype in Bacteria (see, for example [Sporomusa and Desulfotomaculum) with Gram-negative cell wall structures [Fusobacterium does not show any primitive trait and its outer membrane and transport mechanisms show all the characteristics of any sophisticated Gram-negative cell wall. In addition, many of the outer membrane proteins are closest to specific taxa  and not equally dispersed among species with a Gram-negative cell wall. Thus, many of the genes involved in the construction of the Gram-negative outer membrane have probably been horizontally transferred. The extent of this transfer deserves further examination. If we assume that Fusobacterium evolved after the Gram-positive/negative divergence on the low-GC Gram-positive lineage, massive HGT is the most likely explanation for the formation of the outer membrane. On the other end of possible explanations, most genes of the outer membrane would already be present in the common ancestor of fusobacteria and Firmicutes, where a massive loss would be responsible for the differences observed today.The origin of the Gram-negative cell wall found in  example ), being ructures ,59. On tructures . It mighructures . HoweverThermotoga is paradigmatic, a hyperthermophilic bacteria that is assumed to have recruited genes from the archaeal hyperthermophiles to reach its unusual (for bacteria) thermotolerance. Here (as in the case of Methanosarcina, a mesophilic anaerobe) there is not such an obvious explanation. F. nucleatum natural habitat seems to be the dental plaque of mammals, a rather unique and special environment that probably requires very special features to survive. Strong adhesion mechanisms, such as those found often in the Proteobacteria, probably represent an essential ability for survival in the early stages of plaque formation, particularly for non-motile cells. Also the mucose-associated immune system that prevails in the mouth of mammals could have acted as a strong selective pressure favoring the Gram-negative envelopes that are often less immunogenic and easier to disguise thanks to the LPS polysaccharide O chain [Fusobacterium and its interaction with the environment, such as peptide transport systems, cell adhesins and outer membrane components have probably been acquired by gene transfer. It is therefore not only the number of horizontal transfers but also their contribution to niche adaptation that makes the HGT mechanism of dramatic impact on genomes. It is interesting that some of these genes are shared by different organisms inhabiting the dental plaque. From an applied point of view, some of these highly transferred genes are likely to provide a critical advantage in the establishment and adaptation of the bacteria to their niche, and could be used as potential targets for antimicrobial agents.Recently, the idea of gene pools that are characteristic of certain environments has been advanced to explain the large number of common genes among groups of thermoacidophiles distantly related by ribosomal phylogeny . The pre O chain . Thus, iThe different rRNA and conserved protein data sets were analyzed with Bayesian methods using the program MrBAYES 3 . For theRickettsia prowazekii, Rickettsia conorii, Wigglesworthia brevipalpis, and Buchnera aphidicola, and with the addition of Bacteroides thetaiotaomicron and and Desulfovibrio vulgaris. An alignment was generated for each ribosomal gene, using the Clustalw software with default parameters [Fusobacterium nucleatum, S1 is absent/truncated in the Mollicutes subgroup of the low-GC gram-positives, L25, L30, L31, L32, L33 contained a high number of paralogs and/or were absent in several key species.The amino-acid sequences of ribosomal genes S1–S20 and L1–L35, excluding S1, S14, L24, L25, L30, L31, L32 and L33, were retrieved from the KEGG website from a total of 60 different bacteria. The bacteria chosen were all those represented in the KEGG ribosomal genes ortholog table , except rameters . When twrameters , excludirameters . Exclusirameters , L24 is Fusobacterium nucleatum subsp. nucleatum ATCC 25586 were retrieved from . Peptide sequence database of all non-redundant GenBank CDS translations + PDB + SwissProt + PIR was retrieved from . We performed an all against all BLASTP [Fusobacterium nucleatum subsp. nucleatum ATCC 255586 against peptide sequence database. We then recorded the top hit for each protein sequence with an E-value of 10-5, filtering the hits whose sequence identity and length was lower than 30 and 50%, respectively. We categorized all the hits into 8 categories as belonging to the CFB group, Firmicutes bacteria, α,β,γ-Proteobacteria, δ,ε-Proteobacteria, Spirochaetes, other Bacteria, Archaea and Eukaryotes/no hit. Hits to Firmicutes (the group to which Fusobacterium appear to be more closely-related) were refined by further BlastP analysis between F. nucleatum and the 31 sequenced bacteria available from this group. If the gene had a homolog in only one genus from all the available low-GC gram-positive species, it was considered a HGT event from/to this group. If it was present in more than one genus it was considered vertically inherited and consistent with the ribosomal phylogeny. There were 61 cases of genes found in more than one genera from a single subgroup of this taxon . These can be equally explained by HGT or by common descent and were conservatively assigned to the vertical inherited category.The protein sequences of l BLASTP  search oF. nucleatum gene, the protein sequences of up to 50 best blast hits with e-value lower than e-5 were retrieved (the hits were identified by the "Blast method" described above). All sequences were then automatically clustered with the Clustalw alignment tool with default parameters. A neighbor-joining tree with 1000 bootstrap replicates was generated from the resulting alignment, using Clustalw with default parameters. The trees were visualized with NJPLOT [F. nucleatum genes to a taxonomic group was done using the following criteria:For each h NJPLOT . In all F. nucleatum gene was determined to originate from the firmicutes if it was found in the tree most closely associated with at least 5 different species from that group, or with at least 3 species from 2 different subgroups . If the F. nucleatum gene branched at the base of the firmicutes, the gene was assigned as being consistent with phylogeny; otherwise, it was assigned as a potential horizontal gene transfer (HGT) from the firmicutes.A F. nucleatum genes with trees fulfilling this criteria were assigned as HGTs from proteobacteria. Note that a distinction was made between the grouping of the alpha-, beta-, and gamma- proteobacteria, and the grouping of the epsilon- and delta- proteobacteria whenever possible.Same as described above (low-GC gram-positives), the subgroups in this case were:alpha-, beta-, gamma-, gamma-entero-, delta-, and epsilon-proteobacteria. In the case of the proteobacteria, all F. nucleatum had to be closest to at least 3 species, and there had to be a clear association between the two groups, i.e. the branches were relatively short, and the tree topology did not resemble a "star phylogeny".To be assigned as originating from the archaeales, the F. nucleatum gene had to be found closest in the tree to at least 3 different species, there had to be a clear association  and there should have been no obvious evidence of gene transfer from the Fusobacteria. Evidence of transfer from the Fusobacteria would be when, apart from the association to some species of the high-GC gram positives , the tree placed the F. nucleatum gene in agreement with the accepted species phylogeny (just outside of the firmicutes).To be assigned to these groups, the F. nucleatum gene had to be found closest to at least 1 species from that group . A clear association was necessary, as well as no evidence of transfer from Fusobacteria (see above).The F. nucleatum gene had to be closest to at least 2 species from that group, or 1 species with a clear association, and no evidence of transfer from Fusobacteria.The F. nucleatum gene, it was then assigned as "unknown/not resolved".If the tree contained less than four hits other than eukaryotes and other Fusobacteria, the gene was not considered for further analysis. In cases where it was not possible to clearly associate a taxonomic group to the F. nucleatum and other genomes, all available amino-acid protein sequences sets for all replicons of all published bacterial and archaeal genomes at the time (may 1st 2004) were downloaded from the NCBI ftp website . All Orthologs of F. nucleatum genes  were detected between the two replicons (F. nucleatum and replicon X). Clusters of consecutive orthologs were found (consecutive orthologs are determined in terms of the numbered position in both F. nucleatum (exactly consecutive) and replicon X (possible gap of 2 genes in between the orthologs), and for each cluster, a score was assigned as follows:In order to identify clusters of at least two genes with conserved order between )*(1-(deletions/numberOfGenesInCluster)) *Mean(Identity%)*Mean(Length%)*numberOfGenesInCluster/10000F. nucleatum gene the orthologs that were part of clusters were ordered by their score, and an excel table was generated for manual investigation. If the gene order of a given gene cluster was not preserved in Firmicutes species but maintained in another procaryotic group, the genes were assigned as HGT from/to the group with the highest score (highest gene-order conservation). If the order was preserved in at least one species from two or more groups of low-GC gram-positives  the cluster was assumed to be ancestral to the divergence of fusobacteria and Firmicutes, and consistent with the ribosomal phylogeny. If gene order was preserved in one or more species from only one of the low-GC gram-positive groups the cluster was classified as HGT from low-GC gram-positive bacteria.Where "totalCost" was determined by the program derange2 ,72 with F. nucleatum genes by function was based on the TIGR Gene Attribute Annotation [Classical GC-skew plots were done using the formula (G-C)/(G+C) in 5000 bp windows, following Lobry's methods ,26. The notation .AM carried out the gene-order analysis of gene transfer, and the study of chimeric operons and short ORFs. FRV conceived the study, and FRV and AM drafted the manuscript. RP and BAL did the bioinformatics work. RP and AM carried out the BLAST analysis of gene transfer and made the figures, except the phylogenetic trees. BAL developed the phylogenetic and gene-order method and did the ribosomal proteins tree. DM did all Bayesian trees. All authors read and approved the final manuscript.Fusobacterium nucleatum based on phylogenetic analysis and comparative genomics. Supplementary material published as additional information in the manuscript Mira et al. 2004. Evolutionary relationships of The file contains three additional figures. It is available in pdf format and includes figure legends.Click here for file


Keratocyte loss by apoptosis following epithelial debridement is a well-recognized entity. In a study of corneal buttons obtained from patients of corneal ulcer undergoing therapeutic keratoplasty, we observed loss of keratocytes in the normal appearing corneal stroma, surrounding the zone of inflammation. Based on these observations, we hypothesized that the cell loss in the inflammatory free zone of corneal stroma is by apoptosis that could possibly be a non-specific host response, independent of the nature of infectious agent.Acanthamoeba keratitis. The corneal sections were reviewed for morphologic changes in the epithelium, stroma, type, degree and depth of inflammation, loss of keratocytes in the surrounding stroma . TUNEL positivity was evaluated in the corneal sections, both in the zone of inflammation as well as the surrounding stroma. A correlation was attempted between the keratocyte loss, histologic, microbiologic and clinical features.To test our hypothesis, in this study, we performed Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labelling (TUNEL) staining on 59 corneal buttons from patients diagnosed as bacterial, fungal, viral and Acanthamoeba (8) keratitis. The morphological changes in corneal tissues noted were: epithelial ulceration (52, 88.1%), destruction of Bowman's layer (58, 99%), mild to moderate  to severe inflammation . Morphologic evidence of disappearance or reduced number of keratocytic nuclei in the corneal stroma was noted in 49 (83%) cases; while the TUNEL positive brown cells were identified in all cases 53/54 (98%), including cases of fungal (19), bacterial (14), viral (13), and Acanthamoeba keratitis. TUNEL staining was located mostly in the deeper stroma and in few cases the peripheral stroma. TUNEL positivity was also noted with the polymorphonuclear infiltrates and in few epithelial cells (10 of 59, 17%) cases, more with viral infections .The corneal tissues were from 59 patients aged between 16 years and 85 years (mean 46 years) and included fungal (22), viral (15), bacterial (14) and We report apoptotic cell death of keratocytes in the corneal stroma in infectious keratitis, a phenomenon independent of type of infectious agent. The inflammatory cells in the zone of inflammation also show evidence of apoptotic cell death. It could be speculated that the infective process possibly triggers keratocyte loss of the surrounding stroma by apoptosis, which could possibly be a protective phenomenon. It also suggests that necrotic cell death and apoptotic cell deaths could occur simultaneously in infective conditions of the cornea. Acanthamoeba keratitis and had proposed that one of the mechanisms for this loss was apoptosis of keratocytes [Infections of the cornea are potentially blinding diseases and may be caused by fungi, bacteria, viruses and a few protozoans. Despite best efforts with early diagnosis and specific antimicrobial treatment about one-third of cases require surgical intervention . Those watocytes . We haveApoptosis is an active process orchestrated by a series of events starting from activation of genes, release of enzymes, specific morphologic changes and ultimately leading to "an environmental-friendly cell death" whereby the dead cells are removed without affecting the bystander cells -15. ThisThe corneal buttons removed during therapeutic keratoplasty for uncontrolled infections could possibly throw some light on these events. This study was designed to look for the evidence of apoptotic cell death of keratocytes, along with the morphological changes in corneal tissues in infective conditions.Acanthamoeba (8) keratitis in the study. The diagnosis of fungal, bacterial and Acanthamoeba keratitis was based on the microbiological investigations as described below. The etiologic diagnosis was consistent with histopathology of the corneal buttons excised during therapeutic penetrating keratoplasty. Clinical information was available in 43 cases from the ulcer database of our institute. The cases with missing clinical data on a specific parameter were excluded while calculating the statistical significance of that parameter. The clinical data was analyzed for duration of symptoms, size of ulcer, location, depth of infiltrates, presence of perforation, thinning, anterior chamber infiltration and duration of treatment.Corneal tissues from patients diagnosed as infectious keratitis undergoing therapeutic keratoplasty between 1998 and 2002 were reviewed. The corneal sections of those cases, in which the normal stroma surrounding the zone of inflammation and necrosis was visible, were included in the study. We included 59 patients diagnosed for fungal (22), viral (15), bacterial (14) and Acanthamoeba. Corneal scrapings were subjected to Grams, Giemsa and Calcofluor white stain for direct smear examination and culture on blood agar, chocolate agar, brain heart infusion broth, thioglycollate broth, Sabouraud's dextrose agar and non-nutrient agar with Escherichia coli overlay. Immunofluorescent assay for HSV-1 was done on cold acetone fixed corneal scraping smears using anti-rabbit HSV-1 polyclonal antibody and FITC conjugated swine antirabbit immunoglobulin (DAKO, Denmark). In addition, polymerase chain reaction using primers specific for HSV-1 glycoprotein D gene was done on DNA extracted from corneal scraping with commercially available DNA zol solution (Helena Bio Sciences, UK)All patients had undergone microbiological investigations by processing of their corneal scrapings for bacteria, fungus, The excised corneal buttons of the 59 patients were subjected to routine histopathology processing. The paraffin embedded sections were stained with hematoxylin eosin, periodic acid Schiff's and special stains like Gomori's methenamine silver stain and Grams stains. The sections were studied for histologic changes viz. ulceration, inflammation, vascular channels, necrosis, keratocyte loss and other details. Inflammation was graded in a semi-quantitative way as mild, moderate or severe based on the density of the inflammatory cells and the visibility of the stromal collagen in the background. We looked for the absence or the reduced number of the keratocytic nuclei in the corneal stroma surrounding the zone of inflammation.Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labeling (TUNEL) staining was performed on all the 59 sections, as per the manufacture's instructions using In-Situ Cell Death Detection Kit, POD (Roche Diagnostics, Manheim, Germany). In brief, the sections were de-paraffinized, re-hydrated and incubated with proteinase K (20 ug/ml) for 10 minutes at room temperature. After rinsing with phosphate buffered saline (PBS) the sections were blocked with 3% hydrogen peroxide in methanol for 10 minutes. The sections were then incubated in the permeabilization solution consisting of 0.1% triton × 100 in 0.1% sodium citrate for 20 minutes. After washing the sections were incubated with TUNEL reaction mix at 37°C in a humidified chamber for 2 hours followed by washing and incubation with POD converter for 2 hours, the sections were stained with diaminobenzidine and counter stained with hematoxylin and observed under the microscope. Sections from the normal corneal tissues obtained from the eyebank corneas were used as control slides. Statistical analysis was done using Chi-Square test and Fisher Exact test.Acanthamoeba keratitis. The mean age of the patients was 46 years (range 15–85 years) with male to female ratio of 1 (22): 1.7 (37). Based on microbiologic and histologic findings, the buttons were diagnosed as fungal keratitis in 22; bacterial in 14, viral in 15 and Acanthamoeba keratitis in 8 cases.We studied the excised corneal buttons of 59 patients who underwent therapeutic penetrating keratoplasty for fungal, bacterial, viral and The clinical features of 43 patients at the time of presentation are given in Table The histologic changes were observed in the sections of the excised corneal tissues are described in Table Acanthamoeba etiology. In addition, TUNEL positive cells were seen in the epithelium in 10 cases,  declare that they have no competing interests.This work was conceived and planned by Geeta K Vemuganti. Clinical and microbiological support was provided by Prashant Garg and Savitri Sharma respectively. The bench work and analysis was done by Kishore Reddy and Ghazala Iftekhar.The pre-publication history for this paper can be accessed here:


Some currently available birth weight for gestational age standards are customized but others are not. We carried out a study to provide empirical justification for customizing such standards by sex and for whites and blacks in the United States.We studied all male and female singleton live births and stillbirths  in the United States in 1997 and 1998. White and black singleton live births and stillbirths were also examined. Qualitative congruence between gestational age-specific growth restriction and perinatal mortality rates was used as the criterion for identifying the preferred standard.The fetuses at risk approach showed that males had higher perinatal mortality rates at all gestational ages compared with females. Gestational age-specific growth restriction rates based on a sex-specific standard were qualitatively consistent with gestational age-specific perinatal mortality rates among males and females. However, growth restriction patterns among males and females based on a unisex standard could not be reconciled with perinatal mortality patterns. Use of a single standard for whites and blacks resulted in gestational age-specific growth restriction rates that were qualitatively congruent with patterns of perinatal mortality, while use of separate race-specific standards led to growth restriction patterns that were incompatible with patterns of perinatal mortality.Qualitative congruence between growth restriction and perinatal mortality patterns provides an outcome-based justification for sex-specific birth weight for gestational age standards but not for the available race-specific standards for blacks and whites in the United States. Birth weight-specific perinatal mortality curves among male and female births intersect to produce a paradox: overall perinatal mortality rates and perinatal mortality rates at lower birth weights are relatively higher among male births, while at higher birth weights perinatal mortality rates are relatively higher among female births . This puIn this paper, we demonstrate the paradoxical crossover of birth weight-specific perinatal mortality curves among male and female births and show how this phenomenon is resolved using the fetuses at risk approach. We also explore issues related to fetal growth restriction among males and females using the same approach. This latter issue is particularly important from a conceptual and clinical standpoint because the current literature on birth weight for gestational age standards  is confusing. Some standards provide unisex reference values -24, seveWe used the fetuses at risk approach to contrast growth restriction and perinatal mortality rates among males and females in order to provide empirical justification for sex-specific (vs unisex) birth weight for gestational age standards. We also constructed and compared gestational age-specific growth restriction and perinatal mortality curves among whites vs blacks in order to evaluate currently available birth weight for gestational age standards (single standard vs separate standards for whites and blacks in the United States).We used data on all reported live births and stillbirths in the United States in 1997 and 1998 . Live births and infant death records for these years have been previously linked and gestational duration has been calculated based on the last menstrual period (LMP). Missing or inconsistent information on gestational age has been imputed or replaced in a small fraction (approximately 7 percent) of records by the National Center for Health Statistics (Hyattsville, Maryland). Gestational age was imputed from the month and year of the LMP when the exact LMP day was missing . LMP-basAnalyses were restricted to singleton live births and stillbirths ≥22 weeks gestational age and ≥500 g birth weight in order to eliminate potential problems arising from regional differences in birth registration. Male and females births were first contrasted in terms of their gestational age and birth weight distributions. Birth weights were categorized into 500 g intervals for this purpose (500–999 g, 1,000–1,499 g, 1,500–1,999 g and so on). Birth weight-specific perinatal mortality rates, calculated within these birth weight categories, were computed as per convention by dividing the number of stillbirths and early neonatal (0 to 6 days) deaths in any birth weight category by the number of total births (stillbirths and live births) in that birth weight category. Similarly, gestational age-specific perinatal mortality rates among male and female births were contrasted, with rates computed by dividing perinatal deaths at any given gestation by the number of total births at that gestation.The numbers of fetuses at risk for stillbirth and early neonatal death at each gestation were then used to calculate a second set of perinatal mortality rates. Under this fetuses at risk formulation, the stillbirth rate at 28 weeks gestation was computed by dividing the number of stillbirths at 28 weeks by the number of live births and stillbirths at 28 or more completed weeks of gestation. This implies that fetuses who delivered at 29, 30, 31 and 32 or more weeks gestation were also at risk of stillbirth at 28 weeks -19,41-44th percentile cut-off from a birth weight for gestational age standard based on live births in the United States [th percentile values provided by this standard [th percentile of birth weight for gestational age  compared with appropriate-for-gestational age births at the 50th percentile of birth weight for gestational age . We also examined gestational age-specific growth restriction differences among males and females using rate ratios  and contrasted these with gestational age-specific differences in stillbirth and neonatal mortality rates . This was done to ascertain the relationship between patterns of growth restriction and patterns in the two components of perinatal mortality .We also examined gestational age-specific patterns of fetal growth restriction using the fetuses at risk approach ,17-19. Td States . Gestatistandard  to evaluComparisons of male and female gestational age-specific growth restriction and gestational age-specific perinatal mortality patterns were contrasted with similar comparisons according to maternal race. Specifically, live births and stillbirths ≥22 weeks of gestational age and ≥500 g birth weight in the United States in 1997 and 1998 were used to compare gestational age-specific growth restriction and perinatal mortality rates among whites vs blacks.Identification of SGA live births among blacks and whites was carried out using a single standard for both races  and alsoDifferences in rates were assessed using rate ratios and excess risks. Taylor series 95% confidence intervals were calculated on all rate ratios. All p values presented are two-sided. Sensitivity analyses were carried out to assess the potential effect of gestational age errors on patterns of growth restriction and perinatal mortality among males and females. Specifically, we reassessed growth restriction and mortality patterns among males and females after excluding all births for whom menstrual-based gestational age was either imputed or replaced by the clinical estimate of gestation.There were 3,905,694 singleton male births in the United States in 1997 and 1998 . The low birth weight (<2,500 g) rate among male live births was 5.5%, and 10.5% of male live births were born preterm (<37 weeks). There were 3,723,153 female births in the United States during the same period and relative to males, female live births had a higher rate of low birth weight (6.4%, p < 0.0001) but a lower rate of preterm birth (9.4%, p < 0.0001). Males had a 14%  higher perinatal mortality than females; perinatal mortality rates among males and females were 6.78 and 5.95 per 1,000 total births, respectively.The gestational age distribution of male live births Figure  was 'shiGestational age-specific perinatal mortality rates calculated using the fetuses at risk approach showed that perinatal mortality rates increased with increasing gestational age Figure . Males hhigher growth restriction rate than females at the same gestational week (sex-specific standard) and this was qualitatively congruent with a 17 percent  higher perinatal death rate among males compared with females at 35 weeks gestation. On the other hand, when a unisex standard was used to identify growth restricted live births, males had a lower rate of growth restriction at all gestational ages and this was not qualitatively congruent with the higher gestational age-specific pattern of perinatal mortality among males .Figure s Figure . For insOverall growth restriction rates based on a sex-specific standard showed that rates were 3% (95% CI 2 to 3) higher among males. Stillbirth and early neonatal mortality differences (rate ratios) among male vs female births both favored females , despite a significantly higher perinatal mortality rate among blacks at 40 weeks gestation . On the other hand, rates of gestational age-specific growth restriction were qualitatively congruent with patterns of gestational age-specific perinatal mortality when growth restriction among blacks and whites was defined using a single birth weight for gestational age standard . Growth restriction (based on a single standard for both races) and perinatal mortality rates were substantially higher among births to black mothers as compared with births to white mothers at all gestational ages  for identifying fetal growth restriction at all gestational ages. Our findings challenge the former assumption and show that in fact fetal growth restriction rates are better viewed as increasing with advancing gestational age . This may be a consequence of the use of sex-specific birth weight for gestational age standards or sex-specific ultrasound-based fetal growth standards and, as mentioned, probably also reflects higher rates of suspected fetal compromise among pregnancies with male fetuses. Despite the marginally higher rates of labor induction among pregnancies with male fetuses, however, mortality differences persist. Research should be directed at ascertaining whether excess neonatal mortality among males can be successfully reduced through explicit recognition of male sex as a factor for altering the threshold for obstetric intervention.Although contemporary birth weight for gestational age standards have substantial face validity ,47,48, tOur study has limitations that are typical of studies that use large data bases. Errors in gestational age information are inevitable, although the magnitude of these errors is likely to be similar among male and female births. The overall rate of missing gestational age was low, however (0.9 percent among white live births and 0.8 percent among black live births). Our estimates of gestational age-specific fetal growth restriction rates are approximate. Ideally, estimation of the incidence of fetal growth restriction requires identification of fetal growth restriction on a longitudinal basis among continuing pregnancies . The altThe fetuses at risk approach resolves the paradox of intersecting perinatal mortality curves. Male births have higher rates of gestational age-specific perinatal mortality than female births. There is empirical justification for using sex-specific standards of birth weight for gestational age since gestational age-specific growth restriction patterns based on such standards correspond qualitatively with gestational age-specific perinatal mortality patterns. On the other hand, a single birth weight for gestational age standard for whites and blacks in the United States appears more appropriate than currently available race-specific standards since gestational age-specific growth restriction patterns among blacks and whites (based on a single standard) are qualitatively congruent with gestational age-specific patterns of perinatal mortality.The author(s) declare that they have no competing interests.KSJ proposed the study, carried out the analyses and drafted the manuscript. The results of the analyses were presented and discussed at a meeting of the Fetal and Infant Health Study Group of the Canadian Perinatal Surveillance System. All authors contributed to revising the manuscript for intellectual content. All authors read and approved the final version.The pre-publication history for this paper can be accessed here:


Biliary atresia (BA) is a neonatal cholestatic disease of unknown etiology. It is the leading cause of liver transplantation in children. Many similarities exist between BA and graft versus host disease suggesting engraftment of maternal cells during gestation could result in immune responses that lead to BA. The aim of this study was to determine the presence and extent of maternal microchimerism (MM) in the livers of infants with BA.Using fluorescent in situ hybridization (FISH), 11 male BA & 4 male neonatal hepatitis (NH) livers, which served as controls, were analyzed for X and Y-chromosomes. To further investigate MM in BA, 3 patients with BA, and their mothers, were HLA typed. Using immunohistochemical stains, the BA livers were examined for MM. Four additional BA livers underwent analysis by polymerase chain reaction (PCR) for evidence of MM.By FISH, 8 BA and 2 NH livers were interpretable. Seven of eight BA specimens showed evidence of MM. The number of maternal cells ranged from 2–4 maternal cells per biopsy slide. Neither NH specimen showed evidence of MM. In addition, immunohistochemical stains confirmed evidence of MM. Using PCR, a range of 1–142 copies of maternal DNA per 25,000 copies of patients DNA was found.Maternal microchimerism is present in the livers of patients with BA and may contribute to the pathogenesis of BA. Biliary atresia (BA) is a cholestatic disease of infancy characterized by the destruction of the biliary tree. -3 Both tMany hypotheses on the etiology of BA exist. Two leading hypothesis are that BA occurs as a result of ductal plate malformations occurring during development of the liver, or as a result of an immune mediated process triggered by yet to be determined stimulus.  As more + T – helper (Th) 1 cells. [Graft versus host disease (GVHD), seen after allogeneic hematopoietic stem cell transplantation, is an immune mediated process triggered by the transfer of alloimmune cells from a donor to a host. GVHD shares many similarities with BA. Both BA and GVHD are characterized by a lymphocytic infiltration around the portal triad and damage to the biliary ducts.  The pred1 cells. -14 As wi1 cells. Given the similarities between BA and GVHD, we hypothesized that a contributing etiology of BA could be an alloimmune reaction such as in GVHD triggered by maternal microchimerism. Maternal microchimerism occurs when a small number of maternal cells are transferred to the offspring during pregnancy. This is known to occur in up to 40% of normal pregnancies. ,16 In adThis study was performed with the approval of the Committee of Human Research at USCF (H9048-20247-01). Cases of BA were identified through a data search in the Anatomic Pathology CoPath system by diagnosis or during patient medical visits to the UCSF Division of Pediatric Gastroenterology, Hepatology, and Nutrition. An explanation of the study was given to each study candidate. Written consent was obtained from each study participant.We modified a previously reported method of FISH,  using X ®, Brown Deer, WI, USA).Patients with BA and their mothers were HLA typed. The HLA typing HLA-A, -B, and -DRB1 alleles of the child and mother were determined by sequence specific PCR (SSP)  were fixed in acetone for 10 minutes at 4°C then washed in PBS (5 minutes centrifugation × 3). Sections were blocked with Protein Block (Dako, Carpinteria, CA, USA) for thirty minutes at room temperature. Sections were then incubated with anti-HLA-B14 mouse monoclonal antibody  then washed in PBS. Sections were incubated in goat anti-mouse conjugated with FITC for 30 minutes at room temperature. Sections were rewashed and then counterstained with DAPI. Slides were then examined using a fluorescent microscope.Using the HLA types of the patients with BA and their mothers, and a previously reported method of kPCR, evidence of maternal DNA was explored within the liver biopsy specimens of explanted BA livers.  The minoUsing FISH, we examined 11 male BA liver biopsies and 4 male NH liver biopsies. After initial digestion and staining of the liver specimens, we were able to interpret 8 of the BA and 2 NH liver biopsy specimens (Table -, with an HLA-B14+ mother. Specimen A (positive control) has a bright signal for HLA-B14 while there is only background staining for HLA-B14 in specimen B (negative control). Specimen C, the test specimen, has isolated scattered bright signals suggesting maternal microchimerism.To further investigate our findings, three patients with BA and their mothers were HLA typed. Using the HLA marker B14, we examined the BA livers for evidence of maternal microchimerism using immunohistochemistry techniques. An HLA-B14 positive patient served as our positive control Figure . SpecimeFinally, using polymerase chain reaction, in conjunction with the HLA typing of patients and mothers, we were able to affirm that maternal microchimerism occurs in the BA livers. An additional four fresh frozen liver specimens of BA infants, obtained at the time of transplantation, were examined for maternal DNA. All the livers had evidence of maternal microchimerism, with the range of 1 to 142 copies of maternal DNA detected per 25000 copies of patients' DNA. Figure In all eleven of twelve patients with BA had evidence of maternal microchimerism by FISH or kinetic PCR, while neither of the two neonatal hepatitis patients had evidence of maternal microchimerism (p = 0.03).BA is a progressive cholestatic disease of infancy, which is characterized by a mild to moderate lymphocytic infiltration within the extra and intrahepatic bile ducts. It is plausible, that maternal microchimerism may play a role in the etio-pathogenesis of BA by causing an alloimmune reaction given the similarities between BA and GVHD. We have shown that maternal microchimerism is present within the livers of patients with BA.Maternal microchimerism occurs when maternal cells reside in the body of offspring. Maternal-fetal lymphocytic transfer is known to occur during pregnancy starting as early as the tenth week of gestation and continuing up to delivery.  The numbDisease has been shown to occur in mothers who have had fetal microchimerism, and children/adults who have had maternal microchimerism. Children who have immunodeficiencies are at greater risk for graft verses host disease owing to engraftment of maternal lymphocytes. In both SCID and DiGeorge syndrome, maternal microchimerism and GVHD have been well described. In a study by Susanne Müller et al., 121 patients with SCID were evaluated for maternal microchimerism using HLA typing. Maternal cells were found in 48 patients, with 19 patients showing signs of GVHD. GVHD manifested itself in the skin and in the liver.  There haThe histologic and immunologic similarities of BA and GVHD are striking. The site of damage in BA and GVHD after bone marrow transplantation is the same. In each, lymphocytes congregate around the bile ducts. The damage occurs to both the intra-and extrahepatic biliary tracts. In a mouse model of acute GVHD, Nonomura et al. showed that transfer of allogeneic cells set along minor HLA mismatch can cause damage to both the intra and extrahepatic biliary ducts. Interestingly, the timeframe in this mouse model of acute GVHD, in terms of lymphocytic infiltration and fibrosis, appear similar to that of BA.  Initiall+ T lymphocytes. The genes in BA livers showed differential lymphocytic function, with activation of osteopontin, a regulator of cell-mediated immunity in Th 1 cells, and interferon gamma. [+ T lymphocyte. Although a variety of complex mechanisms are involved in causing the end organ damage in GVHD, evidence exists showing similarities in effector mechanisms. In a SCID mouse model of acute GVHD, an increase in interferon-gamma secretion with a synchronized increase in activated Th cells was seen during acute GVHD. [The lymphocytic infiltrates in both BA and GVHD are predominantly CD4n gamma.  Similarlte GVHD.  The inflte GVHD. ,25,26Maternal microchimerism is not a rare occurrence in healthy individuals, but is usually not associated with disease. The high occurrence of maternal microchimerism in healthy individuals has been suggested to have a tolergenic effect that may contribute to long-term microchimerism. AdditionaMaternal microchimerism occurs in the livers of patients with BA. Given the previously well-described relationship between microchimerism and GVHD, and the similarities between BA and GVHD, these findings indicate a potential etio-pathogenisis for BA. Further investigations into the role of maternal microchimerism in BA are warranted.None declared.DLS conceived of the hypothesis and contributed to the design and coordination of the study; he also drafted the manuscript. PR and MBH participated in the design of the study and manuscript preparation. DK carried out the kPCR and HLA typing analyses. GM performed the FISH analyses. LBL carried out the kPCR and HLA typing analyses and participated in the study design. MOM participated on the study design and coordination, immunohistochemistry stains and the manuscript preparation.The pre-publication history for this paper can be accessed here:


The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune and immunecomplex-mediated skin diseases. For preservation of tissue-bound immunoreactants, biopsies are usually fresh-frozen in liquid nitrogen or transported in Michel's fixative. But even optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate IgG fluorescence of the epidermal basement membrane zone is involved. In such cases false negative results are easily obtained due to the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates.In the present study we have compared the use of normal saline (0.9% NaCl) with liquid nitrogen and Michel's fixative as transport medium for skin biopsies. From 25 patients with an autoimmune skin disease (pemphigus, pemphigoid, lupus erythematosus and vasculitis) four matched skin biopsies were obtained and transported in either saline for 24 and 48 hours, liquid nitrogen, or Michel's fixative for 48 hours.Direct IF microscopy showed significant reduction of background fluorescence (p < 0.01) and relatively enhanced desired specific (IgG, IgA) staining in biopsies transported in saline. A conclusive or tentative IF diagnosis was reached in 92% after 24 h saline, 83% after 48 h saline, 68% after freezing in liquid nitrogen, and 62% after 48 h Michel's medium (n = 25).We conclude that transporting biopsies without freezing in normal saline for 24 hours is an adequate and attractive method for routine IF diagnosis in autoimmune and immune complex-mediated dermatoses. The superior results with saline incubation are explained by washing away of IgG background in dermis and epidermis. The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune skin diseases. ReliablThe present study was initiated by the unexpected finding of significant increase of the signal-to noise ratio in a skin biopsy submitted for DIF and accidentally kept overnight in normal saline. The biopsy, obtained from a patient suspect of pemphigoid, showed substantial reduction of IgG background fluorescence and relatively bright specific IgG fluorescence along the BMZ. This finding encouraged us to compare diagnostic results of DIF in matched skin biopsies using standard snap-freezing, Michel's fixative and normal saline.The 25 patients included in this study were selected on the basis of previously confirmed positive direct immunofluorescence (IF) in skin biopsies transported in liquid nitrogen. The final diagnosis was reached by clinical, routine laboratory, histological and direct IF findings. In case of bullous autoimmune diseases, serum samples were characterized by indirect IF on 1.0 M NaCl-split skin ,9, immun2 to minimize the risk of local variation of immunoreactants. The matched skin specimens from each patient were immediately placed in one of the following transport media: (a) liquid nitrogen, (b) Michel's fixative 48 hours with appropiate pH, (c) saline 24 hours and (d) saline 48 hours. We used 5 ml screw-capped polypropylene vials for transporting biopsies in Michel's fixative and saline.From each patient four skin specimens were obtained by punch biopsy (4 mm) using lidocaine as the local anaesthetic. The biopsies were taken from perilesional skin within an area of 2 cmBiopsies were placed in aluminum vials, snap-frozen in liquid nitrogen and stored at -80°C until further processing within two weeks for DIF (see below).1 Biopsy specimens were kept in 5 ml Michel's fixative for 48 hours (Mi48) at room temperature, followed by washing for 30 minutes in Michel's buffer solution. The specimens were then blotted on filter paper to remove excess moisture, and stored at -80°C until further processing.Michel's fixative and buffer solution were prepared monthly according to the original description.We used normal saline solution (0.9% NaCl in aqua dest.) without addition of calcium or magnesium. Skin specimens were kept in 5 ml saline solution for 24 hours (S24) and 48 hours (S48) at room temperature. Preliminary experiments with saline time of 6 hours did not result in improvement of the signal-to noise ratio. The specimens were then blotted on filter paper and stored at -80°C until further processing for DIF.2 antibodies against human IgG, IgA and IgM (Protos Immunoresearch, Burlingame CA), and rabbit antibodies against human C3c and fibrinogen/fibrin . Proper conjugate dilutions were made in phosphate-buffered saline (0.01 M PBS, pH 7.3) supplemented with bovine serum albumin 1%. After washing in 1000 ml PBS for 30 min, the sections were coverslipped under fresh PBS/glycerol (50% v/v). The slide preparations were kept at 4°C until microscopic examination within two days.For comparative purposes, matched skin specimens of each patient were processed for direct IF microscopy at the same occasion. Cryosections of 4 μm thickness were mounted on polysine™ glass slides, air-dried for 30 min before a fan, and encircled with a hydrophobic emulsion . The sections were then stained for 30 min in a moist chamber at room temperature, using fluorescein (FITC)-labeled, Fc-specific goat F(ab')12;13.: (a) epidermal in vivo ANA, (b) epidermal intercellular spaces, (c) basement membrane zone , and (d) blood vessel walls. In addition, the background staining (USS plus NSS) of the upper dermis was scored. All sections were read blindly by the same experienced observer (MCJMdJ). The diagnosis made by direct IF was regarded as conclusive if the DSS score of the relevant target structure was at least weak but definite (Table). The diagnosis was regarded as tentative in case of weak staining that was not consistently distributed at the relevant target structure. A case was regarded as non-diagnostic if target structures showed negative to doubtful DSS scores.The sections were examined with a Leica DMRA microscope (Leica, Wetzlar, Germany) for selective incident light fluorescence using a xenon arc (XBO 75W) as light source and PL Apo ×40/0.80 dry objective. The fluorescent staining was graded as follows: - (negative), ± (doubtful), + (weak), ++ (moderate), +++ (bright). The desired specific staining (DSS) of the following target structures was scoredStatistical analysis of IgG and IgA background fluorescence in matched biopsies was done by the McNemar test.A total of 95 biopsies from 25 patients were examined; three biopsies were missing, and two biopsies proved unsuitable at cryosectioning. Cutting of 4 μm cryosections was easiest with biopsies transported in saline and hardest with biopsies transported in Michel's fixative.Biopsies kept in saline for 24 and 48 hours showed statistically significant reduction of background fluorescence in the dermis, especially with IgG, as compared with fresh-frozen and fixed biopsies p < 0.01). After 24 hours, this resulted in enhanced signal-to noise ratios and accordingly more easy detection of immunoreactants at target structures, in particular the epidermal basement membrane zone (BMZ) and subepidermal blood vessel walls . We know that our visual perception is sensitive for light contrast and not for intensity . The staA disadvantage of saline is the limited time of transport 24 h) for consistently reliable results. If biopsies are kept longer than 24 hours in saline, decreased fluorescence of tissue-bound immunoreactants may be encountered, although we have observed bright specific fluorescence of the BMZ in biopsies kept in saline for at least 5 days. Michel's fixative seems to have a similar limitation: Skeete and Black found that biopsies stored in this fixative should be received within 1 day of biopsy for consistently reliable results [ h for coin vivo ANAs, possibly due to extraction or degradation of nuclear antigens. Another disadvantage of saline might be, from a histopathological point of view, morphological disturbances such as hydropic degeneration [The second disadvantage of saline is the loss of at least some epidermal neration , and splneration . That isneration . Neitherneration ,6,20. Honeration .Besides the diagnostic benefit, the preference would also go to the use of saline because it is a ready available, inexpensive, and convenient transport medium, and it certainly improves cutting properties of skin biopsies compared to biopsies fixed in Michels' medium. Saline can be used as standard medium at room temperature for express postal delivery of IF biopsies to the laboratory, if delivery is guaranteed within 24 hours. Transport of biopsies by express postal delivery or in-house airtube post is much more cost-effective than courier delivery necessary for biopsies transported in liquid nitrogen. If it is expected to tide over a longer period we advise to store the biopsy in saline for 24 hours and then to place it in Michel's fixative for further transport.Practically, place the biopsy in a screw-capped 5 ml polypropylene tube filled to the top with saline. The saline does not have to be sterile. If the specimen arrives in the laboratory the same day after biopsy, we advise to keep it overnight in saline at room temperature, followed by snap-freezing and  storage at -80°C the next day.The use of normal saline offers an attractive alternative to liquid nitrogen and Michel's fixative for diagnostic IF in autoimmune and immune complex-mediated dermatoses, but is only consistently reliable if the specimens are received within 24 hours after biopsy.None declared.BMZ: basement membrane zoneBP : bullous pemphigoidDH: dermatitis herpetiformisDIF: direct immunofluorescence microscopyDSS: desired specific stainingEBA: epidermolysis bullosa acquisitaIF: immunofluorescence microscopyLAD: linear IgA dermatosisLE : lupus erythematosusMMP: mucous membrane pemphigoidNSS: non-specific stainingPF : pemphigus foliaceusPV : pemphigus vulgarisUSS: undesired specific stainingRV carried out the studies, participated in the design and data analysis and drafted the manuscript. MdJ participated in patient selection and carried out the immunofluorescence microscopy and imaging. HM performed the immunofluorescence technical work up. MW participated in material harvesting. HP participated in patient diagnosis. MJ conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: