Drosophila, we present the first high-resolution genetic study of variation within species for female mating discrimination that is enhanced by natural selection. We show that reinforced mating discrimination is inherited as a dominant trait, exhibits variability within species, and may be influenced by a known set of candidate genes involved in olfaction. Our results show that the genetics of reinforced mating discrimination is different from the genetics of mating discrimination between species, suggesting that overall mating discrimination might be a composite phenomenon, which in Drosophila could involve both auditory and olfactory cues. Examining the genetics of reinforcement provides a unique opportunity for both understanding the origin of new species in the face of gene flow and identifying the genetic basis of adaptive female species preferences, two major gaps in our understanding of speciation.Reinforcement occurs when natural selection strengthens behavioral discrimination to prevent costly interspecies matings, such as when matings produce sterile hybrids. This evolutionary process can complete speciation, thereby providing a direct link between Darwin's theory of natural selection and the origin of new species. Here, by examining a case of speciation by reinforcement in  Mating discrimination between two species of Drosophila is more pronounced where they hybridize and genes related to odor appear responsible for this "reinforcement," thus providing insights into the genetics of speciation During reinforcement, mating discrimination is strengthened by natural selection in response to maladaptive hybridization between closely related taxa . Althoug-allele models, alleles conferring mating discrimination spread if they become genetically correlated with alleles reducing hybrid fitness. However, the evolution of such a correlation is opposed by recombination because alleles conferring discrimination in a given species do not confer discrimination in the other species. Consequently, two-allele models require either very strong selection, or tight linkage  between alleles conferring mating discrimination and alleles reducing hybrid fitness  or different (two) alleles conferring mating discrimination spread in the emerging species (Felsenstein 1981). In two fitness . In cont fitness  and so m fitness .Drosophila and present for the first time a high-resolution genetic study of variation within species in female mating discrimination, including a set of candidate reinforcement genes and a discussion of the evolutionary implications of our findings.In addition to providing fundamental information for theoretical models, discerning the genetics of reinforcement will also develop our understanding of both the physiological basis of and forces governing changes in female preference. Furthermore, because the strengthening of female preference is driven by natural selection, the genetics of reinforcement will provide unique insights into the genetics of adaptation, another unsettled issue in evolutionary biology. Finally, high-resolution genetic studies of reinforcement can identify candidate speciation genes with effects on mating discrimination, information almost nonexistent in speciation studies but see, . Here, wDrosophila pseudoobscura and D. persimilis hybridize in nature and produce sterile male hybrids. While D. pseudoobscura occurs alone in non-coastal western United States and Central America, the two species co-occur in California and the Pacific Northwest. Males court females from both species indiscriminately  while females derived from populations where D. persimilis is present exhibited strong mating discrimination (hereafter “reinforced mating discrimination”). This difference in mating discrimination is likely the evolutionary consequence of maladaptive hybridization where the two species coexist: reinforcement has strengthened mating discrimination in the D. pseudoobscura populations co-occurring with D. persimilis. These observations and the recent completion of the genome sequence of D. pseudoobscura (BCM-HGSC 2004) make these species an ideal system to genetically dissect the enhancement of mating discrimination in sympatry.The North American fruitflies minately , but femD. pseudoobscura, or in any system, the genetic basis of other traits contributing to the species' reproductive isolation  is known in detail. All traits contributing to reproductive isolation between D. pseudoobscura and D. persimilis, including traits for basal discrimination, map primarily or exclusively to regions bearing fixed chromosomal inversion differences between the species  and mating discrimination creates the necessary genetic correlations to advance divergence in the presence of gene flow. However, we do not know whether the genetic basis of reinforced mating discrimination corresponds to this picture, and specifically, whether chromosomal inversions are fundamental to this process. Comparing these genetic architectures will provide the most comprehensive view yet on the genetics of mating discrimination contributing to the formation of new species in the face of interspecies gene flow.Although the genetics of reinforcement has not been studied in  species , 2001b. D. pseudoobscura females derived from sympatry (with D. persimilis) exhibited stronger mating discrimination against D. persimilis males than did D. pseudoobscura females derived from allopatry. This pattern holds for both inbred and outbred lines. Also, our data show that both sympatric-derived lines and allopatric-derived lines vary considerably in their degree of discrimination , suggesting some within-population variation in female mating discrimination, both basal and reinforced.D. persimilis males when exposed to sympatric or allopatric D. pseudoobscura females. To exclude this possibility, we measured the copulation latency and number of attempted copulations by D. persimilis males towards D. pseudoobscura females derived from sympatry or allopatry, and found no significant differences between groups . Finally, we investigated the mode of inheritance of the phenotype and observed that F1 females from crosses between sympatric and allopatric flies discriminated as strongly as their sympatric parent, suggesting that reinforced female mating discrimination is inherited as a dominant trait in both inbred and outbred lines . Taken together, these results suggest that reinforced mating discrimination in D. pseudoobscura is exclusive to females derived from areas of sympatry with D. persimilis, is inherited as a dominant trait, and is not markedly affected by inbreeding.Apparent reinforced mating discrimination could result from behavioral differences in ines see . This F1D. pseudoobscura populations. In each case, we performed a male-parent backcross in which a mixture of whole chromosomes from sympatry and allopatry (F1 genome) was substituted into an allopatric background (F2 backcross genome) . For example, sympatric X and fourth chromosomes derived from Mather, California , contribute significantly to reinforced mating discrimination , while the same chromosomes show no detectable effect on reinforced mating discrimination when derived from Mt. St. Helena, California   see A and 2B.D. pseudoobscura despite extensive gene flow within and between populations , and a line derived from Flagstaff, Arizona , and genotyped 275 to 1,500 individuals for 70 markers dispersed along the four major chromosomes in D. pseudoobscura. Our initial single-marker analyses revealed significant associations between reinforced mating discrimination and three regions defined by markers located on the right and left arms of the X chromosome (XR and XL, respectively)  and the fourth chromosome (4034 marker, p < 0.0001, n = 1,434). We were not able to detect an effect of any single region on the third chromosome even though nine markers were surveyed. Effects identified on XR and Chromosome 4 reinforced mating discrimination when the sympatric allele was present (positive), while the effect from XL was negative. After our initial scan, we used composite interval mapping (CIM) to account for any inflated estimates in the absence of background correction. In addition, several markers were genotyped around the X021 and 4034 regions with the goal of refining the segments containing the quantitative trait loci (QTLs). We measured female mating discrimination against Coy-1, is estimated to lie between two additional markers, X021-A1 and X021-A4 . According to the recently obtained genome sequence of D. pseudoobscura, there are seven genes between these two markers, one of which, bru-3, accounts for one-third of the sequence length of this region. In addition, CIM also identified another QTL, hereafter referred to as Coy-3, located between markers X021 and X021-B2, although with a weaker effect (CIM LOD score = 2. 84). There are approximately 200 kb and 30 genes between these markers and a recombination fraction of 3.5 cM. Finally, a third QTL was found near the XL telomere and, in contrast to the X021 region, showed a negative and weak additive effect (CIM LOD score = 2. 45). We tested this model for the X chromosome using multiple interval mapping and found that the strongest support is for Coy-1, followed by Coy-3. We were unable to recover any support for the QTL on XL. No epistatic interactions were detected among any QTLs.A more careful examination of the X021 region showed that the QTL location (CIM LOD score = 5. 16), hereafter referred to as Coy-2 and Coy-4, respectively. These QTLs show the strongest effects (CIM LOD scores of 7.7 and 7, respectively). As with Coy-1 and Coy-3, these QTL are additive and contribute positively to the degree of mating discrimination of F2 females. Coy-2 is located next to marker 4034-A8. This marker is within a 300-kb region homologous to a D. melanogaster region containing a p-element insertion disrupting normal olfactory behavior (see Discussion for details) . However, a more careful examination of the genes shows that their sequence overlap results from the inability of BLAST homology searches to distinguish the members of this gene family, suggesting that there may be only one or few UDP-glycosyltransferase genes here. Consequently, the number of candidate genes in the region may be reduced from nine to five genes, at least one of which is involved in olfaction. As before, we tested this model using multiple interval mapping and recovered significant support for Coy-2 under stringent conditions and no evidence of significant epistasis among previously identified QTLs.Dissection of the 4034 region using CIM split the effect into two QTLs for reinforced mating discrimination; we refer to them as details) , 2003. TCoy-1 and Coy-2, and that Coy-3 and Coy-4 are suggestive QTLs.Based on these results and those for the X chromosome, we suggest that the strongest evidence for QTLs contributing to reinforced discrimination in sympatry lies with Drosophila pseudoobscura. The resolution of our approach is novel to genetic studies of behavioral discrimination in that we have surveyed the genome with 70 microsatellite markers for chromosomal regions contributing to increased mating discrimination and have narrowed some of these regions to intervals containing as few as five genes. The role of these genes in reinforcing mating discrimination is supported by indirect evidence from D. melanogaster mutants: two of the major QTLs identified in our mapping experiments bear genes identified in smell impairment screenings of p-element mutants (CG13982 (D. melanogaster Chromosome 2L), appears to up-regulate a second gene located in the other interval, bru-3 (D. melanogaster X chromosome). Furthermore, we have shown that the chromosomal contributions to reinforced mating discrimination vary among strains of D. pseudoobscura. Finally, the chromosomal effects on mating discrimination are inherited in a dominant fashion, consistent with general theories on the evolution of adaptive characters  show that all QTLs for this trait map unequivocally to two inverted chromosomal regions separating it from D. persimilis ((DPS4003) just 400 kb away from the QTL identified on the fourth chromosome seems to have introgressed recently between D. pseudoobscura and D. persimilis  was striking in this regard, supporting a potential role of olfactory response in reinforcement in these species.Our results suggest, albeit not conclusively, that reinforced mating discrimination is related to differences in response to olfactory cues. We have shown here that candidate regions on the fourth chromosome bear an unusual excess of olfactory genes, and some of these have been associated with specific olfactory responses in other D. melanogaster variation.We also observed differences among strains in the genetic architecture of reinforced mating discrimination. Such variation in genetic control may be common when populations exchanging genes differ in phenotype because of selection. Multiple alleles from different loci may have increased in frequency because of selection for discrimination, and these alleles sometimes spread into allopatry or are replaced by the allopatric alleles in sympatry. When sampling from single lines, we capture only a fraction of the genetic variation in mating discrimination, and sometimes a high-discrimination allele is even sampled from allopatry (as we observed in the QTL on XL). This observation should be typical in many QTL mapping studies that utilize strains within species with extensive gene flow among populations, as in the many studies of D. pseudoobscura is a composite trait, and there is genetic variation within species in reinforced mating discrimination.In brief, these results show that basal and reinforced discrimination are different, species discrimination in 1 female offspring of crosses between allopatric and sympatric populations of D. pseudoobscura are as reluctant to mate with D. persimilis males as are D. pseudoobscura females from sympatric populations. This result implies that high discrimination can be expressed in heterozygous individuals, suggesting a dominant basis for the phenotype. In contrast, “basal” female mating discrimination seems to be a recessive trait . Taken D. pseudoobscura, (1) high discrimination in sympatry is inherited in a dominant fashion, (2) there is within-species variability for high female mating discrimination as evidenced by the different genetic architectures recovered in the male-parent backcross experiments, (3) there are multiple genes, possibly involved in olfaction, contributing to enhanced female mating discrimination, (4) some candidate genes for reinforcement identified here have been previously identified in p-element mutant screenings for smell impairment in D. melanogaster, (5) the genetic architecture of basal female mating discrimination is different from that of reinforced mating discrimination, and (6) inversions seem to play no direct role in creating or maintaining the genetic differences directly responsible for increased female mating discrimination in sympatry. However, these inversions seem to play a crucial role, as evidenced by previous studies , (2) identify chromosomal regions contributing to reinforced mating discrimination  and compare them to regions conferring basal mating discrimination, and (3) provide a set of candidate genes for increased mating discrimination.Our approach is based on measuring in one species the effects of substituting chromosomal segments from a highly discriminant genome into a less discriminant genome. In particular, we (1) test for within-species variation in the genetic architecture of female mating discrimination , Mt. St. Helena, California (1997), Flagstaff, Arizona (1993 and 1997), and Mesa Verde National Park, Colorado (2001). Isofemale lines were established by rearing the offspring of individual females previously mated in the wild. All lines were maintained under a constant regime of temperature (20 °C) and humidity (85%) in diurnal/nocturnal cycles of 12 h and reared on a mixture of agar, dextrose, and yeast.D. pseudoobscura isofemale lines from each of two populations were crossed: Mather (1997) 52 × 10  and Flagstaff (1997) 16 × 17 , respectively. Virgin F1 females from these crosses as well as D. persimilis males were routinely collected during afternoons and confined for 8 d. On the morning of the eighth day, individual females were confined with individual D. persimilis males. The rationale of this no-choice design is based on behavioral observations suggesting that females tend to copulate more often in the presence of single males than when multiple males approach them (Noor, unpublished data). Therefore, no-choice experiments should provide a more conducive setting for mating. The flies were observed for 10 min. If the male attempted fewer than three copulations, the pair was not scored, and the data were discarded. Otherwise, the pair was scored for successful copulation versus not . These protocols are the same used in D. pseudoobscura lines sympatric versus allopatric to D. persimilis. Comparisons between allopatric and sympatric populations were performed both for outbred lines and inbred lines and only between lines that were tested for the phenotype at the same time, thus controlling for environmental error. Our comparisons between the two allopatric lines and between the two sympatric lines were not temporally controlled, and therefore may have been subject to some environmental heterogeneity. We used pairs of D. pseudoobscura inbred lines that significantly differed in their degree of female mating discrimination against D. persimilis in our mapping experiments (see below).Pairs of D. persimilis males of F1 females resulting from crosses between sympatric and allopatric D. pseudoobscura lines. If F1 females discriminated as strongly as the parent derived from sympatry, then we concluded that higher (reinforced) mating discrimination was inherited as a dominant trait. Fisher exact tests where performed to evaluate this hypothesis  and the multipoint-linkage approach implemented in MapMaker version 3.0 (Microsatellite markers include those reported previously  and 100  project . Microsasion 3.0 .n1 = 900 and n2 = 600 flies) were used to determine the chromosomal basis of reinforced mating discrimination and its natural within-species variation. Crossing over does not occur in male Drosophila, and they thus transfer whole chromosomes to their offspring , and its DNA was subsequently extracted. Lines used in each backcross were: for backcross 1, Mather  17 and Flagstaff (Arizona) 1993, and for backcross 2, Mt. St. Helena  7 and Mesa Verde (Colorado) 17. We consider strains derived from California as sympatric and strains derived from Arizona or Colorado as allopatric to D. persimilis.Two male-parent backcrosses  accession numbers for the genes discussed in this paper are bru-3 (FBgn0036379) and CG13982 (FBgn0031811).The Flybase (


The researchers isolated lung lymphocytes from the samples and used two-color flow cytometry to phenotypically characterize the cells. They found that lymphocytes taken from patients with emphysema expressed more CCR5 and CXCR3 receptors, which are associated with a particular type of T cell called T helper 1 (Th1), than did those from control individuals. By contrast, expression of CCR4 receptors, which are found on T helper 2 (Th2) cells, was very low in both control and emphysema groups.In a separate experiment, Kheradmand's team showed that lung lymphocytes taken from patients with emphysema secreted more of three other proteins—interferon gamma, monokine induced by interferon (MIG), and interferon-inducible protein 10 (IP-10)—than control patients. MIG and IP-10 are known to be produced by injured epithelial cells and are ligands for CXCR3 receptors, which are expressed by Th1 cells. Importantly, the researchers were also able to show that isolated peripheral lung macrophages secreted matrix metalloproteinase-12 (MMP12), an enzyme that degrades elastin—a protein important for lung elasticity—in the lungs, in response to IP-10 and MIG. Together these findings, say the authors, indicate that Th1 cells, but not Th2 cells, are required for producing the elastin-destroying lung environment of emphysema.The researchers now intend to investigate the antigens that drive the Th1-based inflammation that underlies emphysema. “Ultimately, we seek to understand the biochemistry of tobacco smoke that triggers inflammation in the first place, and whether such insight might explain other environmentally triggered lung diseases,” explains Kheradmand. “To understand such detailed immune mechanisms, we really need an improved experimental model of disease, and this we are currently working on.”


The neuroprotective effect of xenon has been demonstrated for glutamatergic neurons. In the present study it is investigated if dopaminergic neurons, i.e. nerve-growth-factor differentiated PC-12 cells, are protected as well against hypoxia-induced cell damage in the presence of xenon.2-saturated atmosphere, a treatment that induced release of dopamine, reaching a maximum after 30 min. By determining extracellular lactate dehydrogenase concentration as marker for concomitant cellular damage, a substantial increase of enzymatic activity was found for N2-treated cells. Replacement of N2 by xenon in such a hypoxic atmosphere resulted in complete protection against cellular damage and prevention of hypoxia-induced dopamine release. Intracellular buffering of Ca2+ using the Ca-chelator 1, 2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA) reduced the neuroprotective effect of xenon indicating the essential participation of intracellular Ca2+-ions in the process of xenon-induced neuroprotection.Pheochromocytoma cells differentiated by addition of nerve growth factor were placed in a NThe results presented demonstrate the outstanding property of xenon to protect neuron-like cells in a hypoxic situation. Many studies have been performed on the mechanisms of glutamate-induced neuronal damage ,2 but real cells  possessial cells . In thesal cells ,11.Numerous approaches have been undertaken to reduce hypoxia-induced neurotoxicity ,12. The Cells kept under normoxic conditions did not release dopamine during the time period studied. If, however, they were kept in an atmosphere consisting of 100% nitrogene, considerable amounts of dopamine were found in the extracellular space reaching a maximum at 30 min of incubation, followed by a subsequent decrease. If under the same conditions nitrogen was replaced by xenon, no such increase in dopamine concentration was found Fig. . The levIn order to test if such hypoxia damaged the cells, extracellular LDH was determined after a two-hour period of treatment. A low level of LDH was found in cells kept under normoxic conditions whereas cells kept under nitrogen showed a significant release of LDH indicating severe cellular damage Fig. . If instHypoxia-induced extracellular increase of dopamine could be caused either by elevated release of dopamine or by a reduced, or even inhibited, dopamine uptake. If hypoxia caused faster release but did not interfere with uptake, uptake-inhibitors would cause a higher concentration of dopamine in the extracellular space. On the other hand, if the release was constant but the re-uptake inhibited by hypoxia, additional inhibition of uptake by inhibitors would have no or little effect. In the presence of 5 nM of the dopamine reuptake inhibitor GBR 1209 the extracellular dopamine concentration did not change in a normoxic or xenon environment. However, in nitrogen the extracellular dopamine concentration did not reach exactly the same value as in pure nitrogen, the dopamine level was slightly but significantly reduced, thus supporting the view that hypoxia-induced extracellular dopamine increase was caused by an enhanced release of dopamine and – to a lesser extent – an interference with the uptake mechanism . Surprisingly, such chelation reduces significantly the neuroprotective effect of xenon, indicating an essential role for intracellular Ca2+ for this effect to occur. A slight but significant reduction in cellular damage is observed when BAPTA-treated cells are incubated in nitrogen-saturated buffer.In order to test if changes in intracellular CaTo exclude that the results obtained were limited to the PC-12-system itself, hypoxia-induced dopamine release and cell damage was investigated in rat embryonic primary mesencephalic cell cultures that are known to contain 0.5 – 1.5% of dopaminergic cells . As show2+-regulated events was obtained when it was shown that xenon blocked cells in metaphase and that the block could be lifted by artificial small intracellular Ca2+- increases [2+-dependent regulatory events [2+-dependent systems. One of those may be the Ca2+/calmodulin-activated calcineurin system that has been implicated in the regulation of monoamine release [2+-dependent events required to occur in hypoxia-induced cell damage. Such a scenario is suggested by our demonstration that the neuroprotective effect of xenon is strongly reduced if PC-12 cells are loaded with BAPTA. Thus, at present all evidence obtained by us [2+-regulating and -regulated systems up to the activaton of transcription factors whereby such targets are probably not essentially and sequentially linked to each other.In hypoxia/ischemia a key feature of secondary damage after the primary neuron-damaging event is the over-release of neurotransmitters . Consequncreases . Since tncreases . It is wncreases -22 as wey events . Thus, i release . Alternaed by us ,14,18,19ed by us ,16 estabTo summarize briefly our main findings: (1) The presence of xenon blocks hypoxia-induced dopamine release in dopaminergic cells. (2) Hypoxia-induced dopamine increase is caused by an enhanced release of dopamine rather than a reduced uptake of dopamine. (3) When measuring LDH release as a marker of cellular damage, xenon was found to block such release, which suggests that xenon reduces hypoxia-induced cellular damage. (4) Increased extracellular dopamine can damage dopaminergic cells directly. This is mainly mediated by D1 receptor agonism rather than D2. (5) Such direct extracellular dopamine-induced damage can be reduced by the presence of xenon, even when the increase in extracellular dopamine has not been caused by an episode of cellular hypoxia. (6) The above described protective effects of xenon depend on the presence of calcium ions.Further studies will show if indeed in the hypoxic cell multiple intracellular targets exist for xenon and how they are orchestrated together to result in cellular protection.Based on the present results obtained with NGF-differentiated PC-12 cells and on the literature cited in this paper, xenon appears to be a neuroprotectant for a broad spectrum of neuronal cells; given its proven non-toxicity based on its long clinical use, it may come close to fulfilling the requirements for an ideal or "gold standard" neuroprotectant.2. For experiments, cells were seeded in 24-well plates at a density of 1 × 105 cells/well and nerve-growth-factor (Promega, Heidelberg, Germany) was added (0.4 μg/ml) whereupon cells entered differentiation. They were used on day five after the addition of growth factor [Rat pheochromocytoma cells (PC-12) were maintained in RPMI 1640 medium containing 5% fetal calf serum, 10% horse serum, at 37°C, 5% COh factor . PrimaryHypoxia-treatment was performed as described . Samplesbis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) was purchased from Molecular Probes, (Leiden, The Netherlands), and all standard chemical products were obtained from Merck (Berlin, Germany).Gases were supplied by AGA-Linde (Berlin, Germany). 1,2-p values less than 0.05 were considered statistically significant.All experiments were repeated at least five times, i.e. in five different plates on five different days. The data were presented as means ± SEM. The results of multiple groups were analyzed using one-way ANOVA with Dunnett's multiple comparison post test or two-way ANOVA with Bonferroni posttests using GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego California USA. Differences with CP conceived the study, participated in its design and coordination, carried out the cellular studies involving the various gas treatments, participated in the preparation of the cells, and drafted the manuscript. PB and WS participated in the design of the experiments and the gas applications, JM performed neurotransmitter analysis and LDH determinations, and WK participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.


Microarrays have emerged as the preferred platform for high throughput gene expression analysis. Cross-hybridization among genes with high sequence similarities can be a source of error reducing the reliability of DNA microarray results..We have developed a tool called XHM (cross hybridization on microarrays) for assessment of the reliability of hybridization signals by detecting potential cross-hybridizations on DNA microarrays. This is done by comparing the sequences of the probes against an extensive database representing the transcriptome of the organism in question. XHM is available online at Using XHM with its user-adjustable parameters will enable scientists to check their lists of differentially expressed genes from microarray experiments for potential cross-hybridizations. This provides information that may be useful in the validation of the microarray results. The two main platforms are: cDNA microarrays, where PCR products of individual cDNA fragments are immobilized on glass slides , and oliare used ,3. In a One of the major concerns of cDNA microarrays is cross-hybridization of the labeled RNA (or cDNA) to non-target homologous probe sequences on the array ,5. CrossSome studies have suggested that cross-hybridization can be a source of errors in cDNA microarray experiments ,4,7,9,10Cross-hybridization may occur for both oligonucleotide and cDNA microarrays. An advantage with oligonucleotide arrays is that the complete sequences of the probes are known. Because cDNA arrays are constructed from PCR products of cDNA clones sequenced from 3'- or 5'-ends, the complete sequences of the spotted probes may not be available.We have not been able to identify programs or web servers capable of doing flexible cross-hybridization analysis. Programs with related functionality have been described, including ProbeWiz , OligoWiIn the present study we limit ourselves to the following problem: Given a probe (or a set of probes), identify the targets to which it can hybridize given user-defined criteria.The exact criteria to be used to decide whether two genes with high sequence similarity can cross-hybridize will depend on a number of factors, including the set of probes used and the experimental protocols – in particular hybridization and washing stringencies. We propose to use generic forms of criteria allowing the user to choose parameters in order to define the criteria for cross-hybridization.We investigate whether BLAST can be used to detect targets satisfying a set of criteria for cross-hybridization, based on previous experimental findings ,4, and aBelow we describe the developed XHM (cross hybridization on microarrays) system. We also include analysis performed in order to identify appropriate analysis methods and databases. Furthermore we describe the web server for XHM. In the final section we report some example results using our system on three cDNA probe sets for human, mouse and rat, and one oligonucleotide probe set for mouse.The XHM system queries the given nucleotide sequences  against a database representing all possible targets (transcripts) in the organism being studied, and produces a list of all targets that can hybridize to each input sequence under the defined criteria.In this section we evaluate databases for use in the XHM system and we evaluate whether BLAST can be used to identify potential cross-hybridizing genes.For the calculation of melting temperature, the following formula is used :Tm = 81.5 + 16.6 * log [Na+] + 41 * (numG + numC)/n - 500/nNa+] is the Na+concentration, numG and numC is the number of Gs and Cs in the given alignment, and n is the total number of nucleotides aligned.where [We differentiate between two kinds of sequence similarity cutoffs for cross-hybridization, based on the situations described for oligonucleotide microarrays :Type A similarities for sequence segments of a certain minimum length with a defined minimum percentage identity .• Type B similarities for identical segments of some minimum length (short perfect match).• blastn are used unless specified otherwise. This includes using the DUST filter for masking lowcomplexity regions.To perform the searches in the different databases, NCBI BLAST version 2.2.1 (Mon Jul 9 14:02:00 EDT 2001)  was usedHomo sapiens (Hs) and the 14 k Rattus norvegicus (Rn) clone sets are I.M.A.G.E. Consortium [LLNL] cDNA clones [Mus musculus (Mm) clone set is from NIA [In this study we used three cDNA clone sets (corresponding to three cDNA sets spotted on microarrays). The 40 k A clones ,18 obtaifrom NIA . In addifrom NIA  have bee• RefSeq Version 2 – RefSeq mRNA collection . Number • UniGene – UniGene Unique, build 129/123/164 (mouse/rat/human) . Number • TIGR gene index  – Only t• BeGIn – Bergen Gene Index version 1.0. Number of sequences: 100654/49285 (mouse/rat).BeGIn is an alternative database we have developed (using tools described in ), by proIn order to achieve high specificity and sensitivity, we favored the databases where the highest number of probes were represented (completeness) and multiple matches per probe were minimized (minimum redundancy). Our criteria for choosing an appropriate database for a given organism was based on the results obtained for our probe sets. The presence of alternative splice forms in either the probe or the target set may complicate analysis. We do not consider this explicitly in the current study.type A or type B matches. The XHM system presents to the user a list of matches satisfying the criteria, and in addition information on GC-content in similar regions, estimated melting temperature (Tm) and position on the sequence (proximity to 3' or 5' end). Although the estimated Tm may not be completely accurate under microarray hybridization conditions, particularly on solid surfaces, it can give a relative measure of the impact of the potential cross-hybridization detected. An expert user may use the information to assess the likelihood and importance of the cross-hybridization effect.The basis of the XHM system is a BLAST search  for a number of probes (P1...nP). The simulated targets were ensured to satisfy the criteria described in [i Twas contained in iP's hitlist.Experiments were performed to assess whether BLAST is able to identify potentially cross-hybridizing targets. The analysis was done by generating simulated targets  cutoffs on E-value in order to cover the intended targets. Part of the reason for this is that BLAST is designed to identify homologous sequences and the sequences satisfying the cross-hybridization criteria need not necessarily receive significant scores or E-values. Our conclusion was that BLAST can be used, but to achieve optimal results, non-default parameters for initial word size and mismatch penalty should be applied. This was taken into consideration in the web-server.We found that  with two entry points for accessing cross-hybridization information  and minimum length for type B hits (default is 15 bp), based on the findings described in [+ concentration (default is 0.1 M), and size of GC-clamp (default is 10). The Na+ concentration is used to calculate melting temperature, and the size of the GC-clamp is used to plot the GC-content throughout the query sequence.Some of the parameters are shared among the two main versions of the XHM system. These include minimum length and minimum percentage similarity for ribed in , Na+ conIn addition, the real-time BLAST version also contains BLAST specific user-adjustable parameters. Main parameters include threshold on E-value (default is 10), number of alignments in the output from BLAST and whether DUST (low complexity) filter should be used. The user can adjust other BLAST specific parameters including gap opening and extension penalties, initial word size, mismatch penalty and whether or not to allow gapped alignments.The output from the XHM system consists of different parts , XHM presents the name and identifier of the hit, identity tuples , start and end position on the probe, calculated Tm, percentage GC in alignment and type of hit (A or B).type A similarities, in the rat clone set the Rat Gene Index from TIGR appears to be the best choice. For the mouse clone set it appears that the BeGIn database is the best choice, and for the human clone set it seems that RefSeq or UniGene Unique may be two good choices. These evaluations were based on completeness (high number of probes represented) and minimum redundancy of the database. We used only the tentative consensuses (TCs) of the Gene Indices databases from TIGR. Using the "full" versions decreased the number of probes having zero hits, but substantially increased the number of probes having two or more hits (results not shown).Experiments using the three cDNA sets and the oligonucleotide set as input to the XHM system yielded the results shown in Table type A similarities, and 25 nucleotides for the type B similarities. For the oligonucleotides we used 70% identity over the whole oligonucleotide sequence (type A) and 20 nucleotides perfect identity (type B). Choosing the parameters is not trivial, and these thresholds, which are in the lower range of % similarities leading to cross-hybridizations, are meant as examples of a possible configuration. The XHM tool allows for full flexibility to experiment with the settings. The reason why we chose to consider a 200 nucleotides stretch for the cDNA probes is that sometimes these sequences contain errors, especially toward the ends.As a practical experiment, we tested the XHM system on the cDNA probe sets for human, mouse and rat, and the mouse oligonucleotide set from QIAGEN. For the cDNA sets we used the following thresholds: 70% over 200 nucleotides for type A hit, 809  had two or more hits, indicating potential cross-hybridization. In most cases, a single hit represents the probe sequence itself, and does not represent a potential cross-hybridization. Looking at a more complete database, the TIGR Rat Gene Index, only 700 (5.2%) probes had no hits, and 2712 (21.3% of the probes represented in the database) had two or more hits. A conclusion from this was that the number of rat probes having potential cross-hybridizing partners was between 16% and 21%.Results are shown in Table type A hits was 3101 or 34.2% of the probes represented in the database. RefSeq is relatively incomplete, so this number may be a conservative estimate on the cross-hybridization occurrence. For the BeGIn database only 725 probes had no hits (5.3%) and 5211 probes (about 40% of the probes in the database) had two or more hits.For the mouse cDNA probes the cross-hybridization numbers appeared to be higher. Using RefSeq, the number of probes having two or more Even though as many as 47.3% of the mouse probes were not found in the TIGR Gene Index, the proportion of probes found in the database that had two or more hits was 59%. This number is most likely an artifact caused by redundancy in the database.The difference in number of possible hybridizations found in mouse versus rat is most likely due to the fact that more mouse sequences are available.The human cDNA probes seems to have a lower cross-hybridization potential than the mouse probes. In UniGene Unique and RefSeq, looking only at the human probes that were found in the databases, just above 30% of them had two or more hits. The results using TIGR Human Gene Index suggested that almost 70% of the probes found in the database could cross-hybridize, but this is most likely an artifact caused by redundancy.The mouse oligonucleotide set is clearly less prone to cross-hybridization. Using the UniGene Unique database, we observed that 14% of the probes found in the database had two or more hits, whereas the corresponding number for the 15 k mouse cDNA probe set using UniGene Unique was 45%.A flexible tool for assessing the cross-hybridization potential of microarray probes has been developed and made available. Several transcriptome databases can be used for searching, and more may be added upon request. Using the XHM tool, analysis of three cDNA microarray probe sets and one oligonucleotide probe set revealed that a high proportion of the cDNA probes can potentially cross-hybridize with one or more other transcripts in the organism. As expected, compared to the cDNA probes, a smaller percentage of the mouse oligonucleotide probes showed a potential for cross-hybridization. This is because the oligonucleotide probe sequences are much shorter (69-mers), designed from specific regions of cDNAs to minimize cross-hybridizations. Despite increasing use of the more specific oligonucleotide arrays, cDNA sequences including full-length clone sets ,26 are wtype A similarities (at least 70% identity), 15–45% of the cDNA probes showed hybridization with two or more apparently different transcripts (disregarding the results using the TIGR Gene Indices). This high percentage of potentially cross-hybridizing genes suggests that it is essential to carefully validate results from microarray experiments, particularly where cDNA clones are used to prepare arrays.Depending on the completeness and redundancy levels of the transcriptome databases used, with the chosen cutoff for Although cross-hybridization is known as one of the main sources of errors of cDNA microarrays, the high proportions of cross-hybridizing genes detected in our test are likely to be overstated by the possible redundancies in the databases . Also one may argue that the 70% identity threshold is somewhat low.Whether two different genes that are candidates for cross-hybridization actually lead to erroneous results in hybridization experiments will depend on factors such as the level of sequence identity, the stringency of the hybridization and the relative abundances of the transcripts. For example, two potentially cross-hybridizing genes do not necessarily pose a problem unless both are expressed in the tissue or cell-line analyzed. Quick inspection of the hit-list produced by the XHM tool for a typical input of differentially regulated genes will help in identifying significant noise from cross-hybridizations. A main advantage of the XHM tool is that it allows the user to perform searches at various stringencies to detect potential cross-hybridizations. Candidate genes from microarray analysis that show potential cross-hybridizations using the database search may then be further checked using other methods such as RT-PCR and Northern blot.We have shown that a significant proportion of probes used in cDNA microarray analysis may show cross-hybridization with non-target sequences. We have developed a flexible tool, XHM, suitable for detecting potential cross-hybridization artifacts during microarray data analysis. The tool may also be used to select specific probes for preparation of microarrays.. Program code for academic use can be supplied upon request to the author.The XHM system is freely available at KF implemented the system and made a draft of the manuscript. FY and AL contributed with ideas and proofread the manuscript. IJ has formulated and supervised the work, and edited the manuscript. All authors have read and approved the final manuscript.


The wealth of information on protein structure has led to a variety of statistical analyses of the role played by individual amino acid types in the protein fold. In particular, the contact propensities between the various amino acids can be converted into folding energies that have proved useful in structure prediction. The present study addresses the relationship of protein folding propensities to the evolutionary relationship between residues.The contact preferences of residue types observed in a representative sample of protein structures are converted into a residue similarity matrix or inter-residue distance matrix. Remarkably, these distances correlate excellently with evolutionary substitution costs. Residue vectors are derived from the distance matrix. The residue vectors give a concrete picture of the grouping of residues into families sharing properties crucial for protein folding.Inter-residue distances have proved useful in showing the explicit relationship between contact preferences and evolutionary substitution rates. It is proposed that the distance matrix derived from structural analysis may be useful in aligning proteins where remote homologs share structural features. Residue vectors derived from the distance matrix illustrate the spatial arrangement of residues and point to ways in which they can be grouped. The first such analysis was performed by Miyazawa and Jernigan -3, wherean et al , and shoan et al . An analan et al . A more an et al . It is r, where ij Nis the number of possible pairings between residue type i and residue type j and ij Cis the number of these parings corresponding to residues in contact. Only non-neighbouring residues on the protein chain are considered and a pair of residues is defined to be in contact if any of their side chain atoms are within a given distance of each other. The difference in contact propensities for two amino acid types can be measured by their rms difference and we define  as our amino acid difference measure or distance matrix.The observation that evolutionarily close residues appear to have similar contact propensities leads us to postulate that the extent of similarity between the contact propensities corresponding to two particular amino acids is related to the ease with which these amino acids can be mutated into each other. We define the contact propensity as D(P) will be difficult to mutate into one another and vice versa. This is because the factors involved in determining mutation rates are dominated by those affecting the structural integrity of the protein. Such factors are residue hydropathy, size, charge and etc. Substitution matrices such as PAM and Blosum are determined from mutation rates in aligned protein sequences [ij Dabove.If we have really got a measure of the distances between residue types then it should follow that residues sharing physical properties are close together. More crucially, we expect that residues that are distant according to equences ,9. We ca, where S is the substitution rate matrix. We show below that D(P) is indeed strongly correlated with D(S). It must be stressed that D(P) and D(S) are independently derived, with one based on structure and the other on sequence alignment. Their strong correlation is indicative of the validity of our definition of inter-residue distance.That is, Relating amino acids through a structurally defined distance measure should provide a useful tool for aligning remotely homologous protein sequences. Also, a distance measure naturally leads us to look for a vector representation of the amino acids. In much the same way as average hydropathy plots are useful in structural analysis we expect that average vector profiles will also pick out various structural features. Given a vector for each residue type we can visualise the residues in some abstract space and look for natural groupings of residues and thereby find ways of reducing the effective number of residues.A representative set of crystal structures was compiled from the PDBselect25 database , which cD(P). If this matrix is really a measure of residue similarity then we should be able to correlate it with an equivalent matrix constructed from an evolutionary substitution rate matrix. In what follows we will take PAM250 as the substitution matrix. In Figure D(S) in the top triangle and D(P) in the bottom triangle for a contact cut-off of 4.5Å, where the pearson correlation (r) is maximal, with r = 0.82. See P and D(S). The correlation can be seen explicitly in Figure D(P) is consistently less well correlated with D(S), peaking at r = 0.64 for a cutoff of 8Å, see Figure As explained above the contact propensities can be converted to a distance matrix , where  are the residue vectors. Explicitly, the vectors are defined such that  is minimal. When these vectors are derived it becomes clear that Cysteine is quite separate from the other residues in this property space and this maybe due to the unique role played by Cysteine in stabilising folds. Though it must be made clear that the distance matrix is independent of the frequency of an amino acid contacting its own kind and therefore does not count Cysteine bridges in the structures. Without Cysteine the distance matrix can be projected onto a plane i.e. the vectors can be taken to be two-dimensional and this vector space is illustrated in Figure D(P) residues with opposite hydropathies tend to be further apart. This is consistent with hydropathy playing a pivotal role in protein folding. In contrast, the MJ energy matrix vector space is shown in Figure We have defined inter-residue distances and this implies that there must be a vector representation for the residues. In this case the distance matrix will be di are defined such that  is minimal. We find that these distance matrix derived scalars have a correlation of 0.65 with the Kyte-Doolittle hydrophobicity scale [The dominant driving force of folding, at least in defining the crude fold, is hydrophobicity and it is apparent that residues with similar hydrophobicities are grouped together. It also seems that residues of similar size tend to be close in this space. To make a direct comparison between existing residue scales and our vectors we can project the residue vectors onto a line. Here the amino acid scalars, one-dimensional vectors, ty scale  and a coty scale . It is cPij = λeiej, where λ is the principal eigenvalue and i eis the principal eigenvector, and consequently our distance matrix has a scalar representation, . It is not surprising that the eigenvector is closely related to our scalar, in fact r(e,d) = 0.98. There are many hydrophobicity scales in the literature [r = 0.95 for Wertz & Scheraga scale [It is worth noting that a scalar reduction of the distance matrix can be got by a principal eigenvector analysis. In a principal eigenvector reduction of the contact propensity matrix we have terature  and somega scale . HoweverWe have generated full atom residue:residue contact propensity profiles for intra-molecular interactions from a non-redundant crystal structure database. Recasting the contact propensity matrix as a distance matrix we see that close residues are those with a low evolutionary substitution cost. The structure derived distance measures can serve as additional scores when aligning proteins where remote homologs share structural features. The distance matrix led us naturally to derive effective residue vectors. We found that residues sharing similar physical characteristics, such as hydrophobicity and volume, are grouped together. In contrast to the MJ matrix analysis, we find that a scalar representation for the residues is inadequate to capture the complexity of the propensity distance matrix. The most successful scalar representation for the amino acid residues has been the hydropathy scale. Representing a sequence as a smoothed hydropathy profile through wavelet analysis or simple averaging has resulted in many effective analytical tools, such as periodic structure prediction , remote In this study we have shown that inter-residue distance matrices and residue vectors allow us to make an explicit connection between amino acid interaction preferences observed in protein structures and amino acid evolutionary substitution costs. When problems are encountered with aligning structurally related proteins that are remote homologs then the structurally defined distance matrix may prove to be an effective supplement to existing substitution rate derived matrices. The distance matrix leads naturally to an amino acid vector representation. Projecting the vectors onto a two-dimensional plane illustrates ways in which the amino acids can be grouped and their effective number thereby reduced.i with amino acid type j, ij Nwere counted together with the number of these pairings corresponding to a pair with side chain atoms within a given distance of each other, ijC. The contact propensity matrix is given by . The residue vectors were defined such that  is minimal. The minimisation was carried out by a standard Newton-Raphson steepest descent iteration.The database used in the present study was compiled from the PDBselect25  list of Contact propensities and distance matrix derived from the structural database with contact cut-off set at 4.5Å.Click here for fileTwo dimensional residue vector components derived from the contact propensity distance matrix, the PAM250 distance matrix and the MJ distance matrix.Click here for file


Pneumocystis jiroveci pneumonia (PCP) is an important opportunistic infection among immunosuppressed patients, especially in those infected with human immunodeficiency virus (HIV). The clinical presentation of PCP in immunosuppressed patients have been well-reported in the literature. However, the clinical importance of PCP manifesting in the setting of an immunorestitution disease (IRD), defined as an acute symptomatic or paradoxical deterioration of a (presumably) preexisting infection, which is temporally related to the recovery of the immune system and is due to immunopathological damage associated with the reversal of immunosuppressive processes, has received relatively little attention until recently.10 copies/ml to 3.1 log10 copies/ml) was observed in HIV-positive patients who developed PCP. A similar upsurge in peripheral lymphocyte count was observed in our patients preceding the development of PCP, as well as in other non-HIV immunosuppressed patients reported in the literature.We aim to better define this unique clinical syndrome by reporting two cases of PCP manifesting acutely with respiratory failure during reversal of immunosuppression in non-HIV infected patients, and reviewed the relevant literature. We searched our databases for PCP cases manifesting in the context of IRD according to our predefined case definition, and reviewed the case notes retrospectively. A comprehensive search was performed using the Medline database of the National Library of Medicine for similar cases reported previously in the English literature in October 2003. A total of 28 non-HIV (excluding our present case) and 13 HIV-positive patients with PCP manifesting as immunorestitution disease (IRD) have been reported previously in the literature. During immunorestitution, a consistent rise in the median CD4 lymphocyte count (28/μL to 125/μL), with a concomitant fall in the median HIV viral load (5.5 logPCP manifesting as IRD may be more common than is generally appreciated. Serial monitoring of total lymphocyte or CD4 count could serve as a useful adjunct to facilitate the early diagnosis and pre-emptive treatment of this condition in a wide range of immunosuppressed hosts, especially in the presence of new pulmonary symptoms and/or radiographic abnormalities compatible with the diagnosis. Pneumocystis jiroveci (Pj) (previously known as Pneumocystis carinii f. sp. hominis) was first identified as a pathogen in premature infants suffering from interstitial plasma cell pneumonia in European countries during and after World War II, occasionally occurring in epidemics [pidemics -3. Sincepidemics . The incpidemics , widesprpidemics , and thepidemics ,8.Nevertheless, with the rising number of patients receiving immunosuppressive therapies for malignancies, solid organ transplantations and autoimmune diseases, PCP has been increasingly recognized in non-HIV immunosuppressed hosts -15. For Paradoxically, the clinical symptoms of PCP were often unmasked in HIV-negative immunosuppressed patients during the reversal of immunosuppression, often at the time when the dose of steroids was tapered ,17,21-24th August 2002 for investigation of jaundice. Investigations revealed deranged liver function tests with cholestatic pattern. A diagnosis of drug-induced hepatotoxicity was entertained, and azathioprine was stopped after admission. As her autoimmune disease was under control, her steroid dosage was reduced from 25 mg to 15 mg daily within the next 14 days. Her CXR taken on admission was normal.This is a fifty-one year old female patient with history of diabetes mellitus and systemic lupus erythematoses (SLE) complicated by lupus nephritis. Although we have included her case in our previous publication , we haveth Sept revealed new infiltrate over the left mid-zone, suggestive of pneumonia. She was started on intravenous ceftazidime 1 gram eight hourly and oral clarithromycin 500 mg twice daily. Serial CXR performed three days later showed increasing bilateral pulmonary infiltrates and worsening hypoxemia. There was an upsurge of total lymphocyte count from 0.7 × 109/L  at the time of admission to 5.6 × 109/L  at the time of clinical deterioration. Bronchoscopy with transbronchial biopsy performed on the same day revealed Pneumocystitis jiroveci by methenamine sliver stain. Workup for other opportunistic pathogens including cytomegalovirus and aspergillus was negative. She was commenced on intravenous pentamidine (4 mg/kg/day) and corticosteroids for severe PCP infection. Despite active treatment she developed progressive respiratory failure and required admission to intensive care unit. She subsequently recovered after a stormy hospital course, and upon discharge from hospital, her total lymphocyte count had returned to her baseline of 0.86 × 109/L.Soon after her immunosuppressive therapy was tapered, she developed fever and non-productive cough. A repeat CXR performed on 9A thirty-three year old gentleman initially presented to Prince of Wales Hospital with a diagnosis of SLE/dermatomyositis overlap syndrome. He was treated with steroid and hydroxychloroquine 200 mg twice daily since 1997. He had a flare up of disease in May 1998 with active vasculitis and myositis, for which he was put on prednisolone and azthioprine 50 mg and 100 mg daily respectively. Upon reassessment one month later, disease activity was under control, and the dosage of prednisolone was reduced to 45 mg daily.staphylococcus aureus; he was treated with cloxacillin 1 g intravenously every 6 hourly, together with incision and drainage of the buttock abscess. In view of the underlying active pyogenic infection, the steroid dosage was rapidly tapered from 45 mg to 15 mg daily within the next four days. However, he was noted to have persistent fever associated with mild unproductive cough. A repeat chest radiograph showed new infiltrates over the right upper and left lower zones, and he was empirically treated with intravenous ceftazidime 1 gram every 8 hours, cloxacillin 1 gram every 6 hours and netimicin 100 mg every 8 hours. As there was no clinical response after 5 days of treatment, bronchoscopy and bronchoalveolar lavage (BAL) was performed, which was positive for Pneumocystis jiroveci. Investigation for the presence of co-existing opportunistic pathogens such as cytomegalovirus and aspergillus was negative. On the day after bronchoscopy, he was commenced on intravenous cotrimoxazole 1.3 grams every 6 hours. He remained stable initially with fever on downward trend. However, on the 3rd day of treatment, he developed sudden desaturation with resurgence of high fever, and required supplemental oxygen therapy. Repeat chest radiograph showed increased perihilar hazziness in both lung fields. There was also an upsurge of total lymphocyte count from 0.6 × 109/L  on admission, to 1.3 × 109/L  at the time of clinical deterioration. He was treated with high dose prednisolone (80 mg daily), and his condition improved promptly afterwards. He was subsequently discharged, and on follow up at the clinic one month later, his total lymphocyte count had returned to his baseline level of 0.6 × 109/L.Twelve day after reducing the immunosuppressive regimen, he was admitted to hospital for treatment of left buttock abscess. The CXR taken on admission was unremarkable. An aspirate of the pus from the lesion grew methicillin-sensitive Pneumocystis carinii", "Pneumocystis jiroveci", "HIV", immunosuppression", "immunosuppressive", "steroid", and "corticosteroid" were used to select cases. All the case reports and case series with clinical details were included in this study if they fulfilled the above definition of IRD. When appropriate, the cited bibliographies were also retrieved for further analysis. As for statistical analysis, we used the Wilcoxon Signed Rank test, a non-parametric test for comparing paired samples, to analyze the serial changes in lymphocyte counts and HIV viral loads before and during the development of IRD. A two-tailed p-value of less than 0.05 was considered significant. All statistical analyses were performed using SPSS version 11.5 for Windows.Immunorestitution disease (IRD) has been described in both HIV and non-HIV immunosuppressed hosts previously -31. In tIncluding our present case, a total of 29 cases of PCP in non-HIV immunosuppressed hosts fulfilling our definition of IRD have been reported in the literature table  22-27,3,327,32. 10 copies/ml (range 5.0 to 5.9 log10 copies/ml) to 3.1 log10 copies/ml (range 2.9 to 4.5 log10 copies/ml) before and during IRD respectively, and the differences observed in both the CD4 counts and viral loads before and during IRD reached statistical significance . Antimicrobials, steroids, or both for PCP were reintroduced for IRD in 4, 1, and 6 cases respectively. Only 2 cases were treated conservatively. One case required mechanical ventilation for severe respiratory distress. None of the patients died.Among HIV-positive patients, 13 cases with newly diagnosed PCP were reported in the literature, in which IRD occurred shortly after the introduction of HAART table  28-31].-31.28-31PCP manifesting as a form of IRD is not a rare phenomenon. As shown in our previous study, it happens in 7 out of 10 (70%) of HIV-negative immunosuppressed hosts infected with Pj . HoweverRapid reduction of immunosuppressive therapy such as steroids has been implicated as a predisposing factor for the development of PCP in HIV-negative patients ,17,23,24Among HIV positive patients, PCP manifesting acutely during the initiation anti-retroviral therapy is a well-recognized phenomenon. The underlying immunopathological nature of this condition, which is reminiscent to IRD occurring in non-HIV infected patients, has been confirmed by histological examination of the lungs and transbronchial biopsy specimens, which demonstrated mixed inflammatory infiltrates including macrophages, neutrophils, lymphocytes, and plasma cells. Almost all infiltrating lymphocytes found in the tissues were of the T cell lineage, shown by immunophenotyping to be predominantly CD4 and CD8 cells . In anotIn our own experience, as well as from the review of published literature, it appears that a surge of absolute lymphocyte count, especially the CD4 lymphocyte count in HIV-positive patients, could potentially act as a surrogate marker for immunopathological damage during IRD in both HIV-negative and HIV-positive patients. In our recent publication , 7 out oFrom the result of this review, it appears that HIV-positive patients with PCP are at risk of clinical deterioration due to IRD if HAART therapy is started within 1 to 2 weeks after the initiation of treatment for PCP  declare that they have no competing interests.RAL and DSH were involved in the clinical evaluation and treatment of patients. BST and IFH helped with literature searching and review. AKW and VCC drafted and refined the manuscript. KYY conceived the study, participated in its design and coordination, and supervised the preparation of the manuscript. All authors have read and approved the final draft of the manuscript before submission.The pre-publication history for this paper can be accessed here:


PHACCS uses the contig spectrum from shotgun DNA sequence assemblies to mathematically model the structure of viral communities and make predictions about diversity.Phages, viruses that infect prokaryotes, are the most abundant microbes in the world. A major limitation to studying these viruses is the difficulty of cultivating the appropriate prokaryotic hosts. One way around this limitation is to directly clone and sequence shotgun libraries of uncultured viral communities . PHACCS PHACCS builds models of possible community structure using a modified Lander-Waterman algorithm to predict the underlying contig spectrum. PHACCS finds the most appropriate structure model by optimizing the model parameters until the predicted contig spectrum is as close as possible to the experimental one. This model is the basis for making estimates of uncultured viral community richness, evenness, diversity index and abundance of the most abundant genotype.PHACCS analysis of four different environmental phage communities suggests that the power law is an important rank-abundance form to describe uncultured viral community structure. The estimates support the fact that the four phage communities were extremely diverse and that phage community biodiversity and structure may be correlated with that of their hosts. By killing prokaryotes, phages can strongly impact microbial community biomass [Most environmental viruses are phages (a.k.a., bacteriophages) that infect prokaryotic cells, both Bacteria and Archaea. On average there are about ten phage particles per host cell . Extrapo biomass . DespiteTraditionally, the study of environmental phage diversity, dynamics, and ecology requires growing prokaryotes on microbiology plates and infecting them with phages. However this standard technique is limited by the fact that only a small fraction of environmental microbes are readily cultured  and thatBiodiversity is composed of richness, or total number of different species , and eveq (number of sequences in the group) [A new approach to accessing natural microbial diversity is through the creation of shotgun sequence libraries from environmental metagenomes  -14, so te group) . The strIn this work, we present PHACCS (PHAge Communities from Contig Spectrum), an online computational tool to assess the diversity and structure of environmental viral communities from the contig spectrum of shotgun sequence data. The PHACCS program and its predictions are first described and then used to analyze four environmental viral communities.The standalone core mathematics for PHACCS consists of Matlab (MathWorks Inc., Natick, MA.) scripts that are partly based on the previous works -12. A CGq-contigs (groups of q overlapping sequences) from the in silico assembly of random shotgun DNA fragments. Detailed information about the way to get viral metagenomes and their contig spectrum can be found in [The input for PHACCS is the contig spectrum, a vector containing the number of found in -12. Briefound in  and clonfound in -12). Thefound in . The strfound in . The numL of the genome, ii) the number N of DNA fragments studied, iii) the average size s of these fragments, and iv) the minimum overlap length o for the sequence assembly [PHACCS uses a modified version of the Lander-Waterman algorithm  to prediassembly . Given tp of an overlap: p = 1 - eNx/L -with x = s - o• Probability wq for a fragment to be part of a q-contig (overlap of q fragments):• Probability wq = qpq - 1 (1 - p)2cq that are part of a q-contig: cq = Nwq• Expected number of fragments • Contig spectrum: M different genotypes  [i.The modified Lander-Waterman algorithm is a generalization of the original algorithm to a group of mmunity) . The precq part of a q-contig:• Expected number of fragments wqi is the probability for a fragment to be part of a q-contig for the genotype i and ni is the expected number of fragments for the genotype i.In this modified algorithm, since there are several genotypes, an assumption about their underlying distribution within the community in terms of abundance has to be made.PHACCS offers six basic functional forms of relative rank-abundance for biological populations: the power law, logarithmic, exponential, broken stick, niche preemption, and lognormal distributions.The first three functional forms are empirical models that were designed to describe an asymptotic drop-off in the abundance :ni = aib -for 1 ≤ i ≤ M• Power: ni = a(log(i + 1))b -for 1 ≤ i ≤ M• Logarithmic: ni = aeib -for 1 ≤ i ≤ M• Exponential: a represents the abundance of the most abundant genotype, b is a parameter related to the evenness, and M is the number of different genotypes in the community.The parameter Two ecological models are based on a partitioning of resources between species ,19: for 1 ≤ i ≤ M• Broken stick: ni = Nk(1 - k)i - 1 and nM = N(1 - k)M - 1 for 1 ≤ i ≤ M - 1• Niche preemption: M, and assumes a random distribution of resources, whereas in the niche preemption function, each species takes only a fraction k of the remaining resources in the environment.The broken stick function has only one parameter, σ into M equal area slices and associating an abundance ni with the i-th slice by calculating an average value for the abundance within the slice. The result is:The sixth functional form is the lognormal distribution. It is the most commonly used species distribution, with numerous theoretical justifications in the literature ,21. The erf is the error function and erf1 its inverse.where b for the power law, logarithmic, exponential and lognormal distributions and k for niche preemption) are used to predict a contig spectrum using the modified Lander-Waterman algorithm. To determine the model fitness, the error between the actual and the predicted contig spectrum is calculated as the variance-weighted sum of squared deviations, L being the contig spectrum vector length and cq' the experimental number of fragments that belong to a q-contig:The PHACCS algorithm is represented in Figure The best descriptive model for a community structure is defined as the one with the smallest error. For each rank-abundance function tested, the global minimum for the error is found by optimizing the value of the function parameters.0.1 which is about 11:10 between the two models. This means that the model with the smallest error is about 10% more likely to give rise to the observed data.The values of the error can be roughly interpreted as logarithms of odds ratios of the observed contigs being seen from community distributions of the specified forms. Thus a value of 0.1 for the difference in errors between two models corresponds to an odds ratio of eS is estimated as equal to the number of different genotypes M found in the community structure model. The abundance of the most abundant genotype is also directly determined from the model as the highest rank-abundance value. The Shannon-Wiener index, which is a measure for diversity, is calculated using the relative rank-abundance values ri = ni/N of all individual genotypes i [For each rank-abundance form, the best model is used by PHACCS to assess diversity. The richness otypes i :H' (in nats): • Shannon-Wiener index H' [The evenness is derived from H' :E: E = H'/Hmax = H'/ln S• Evenness As a case study, four viral metagenomes obtained from previous studies and belonging to different ecosystems were tested. Two of these were phage community samples of near-shore surface seawater from Scripps Pier (SP) and Mission Bay (MB), San Diego, California, USA . The twoThe errors obtained from the contig spectrum analysis of the different samples are presented in Table The different diversity indicators and the rank-abundance curves obtained by using the best descriptive model for each sample are summarized in Figure  and the source code is freely available [see PHACCS is publicly accessible at able see . The bioPHACCS analyses are computer intensive. On a dual-Opteron™ server, the computation for the SP sample takes ~5 minutes. The broken stick and lognormal rank-abundance forms account for most of the computation time (data not shown). Increasing the range of genotypes to search dramatically increases the time needed to complete the analysis (data not shown).PHACCS estimations about the virus community are: i) structure – best descriptive rank-abundance form, model equation and error, and ii) diversity – richness, evenness, abundance of the most abundant genotype, and Shannon-Wiener index. Graphic representations of the community structure and of the error minimization can also be displayed. The error provides information about which model has the best fit relative to the others for a given contig spectrum. For each type of distribution, the user is informed if the best model  has not been found using the given computation parameters.Predictions by PHACCS are dependent on the quality of the contig spectrum input. The difference in error between two models can be small Table  and usinμm is collected. Larger viruses and RNA viruses are not represented in the shotgun library and in the resulting contig spectrum. The contig spectrum assembly parameters (98% identity on at least 20 bp for phages) are stringent enough to limit the number of false-contigs (contigs between DNA fragments from different genotypes), but may on the other side omit some true-contigs . Additionally, the present implementation of the Lander-Waterman algorithm assumes that all DNA fragments and all the genotypes have the same size. For these reasons, PHACCS estimates should be considered approximations.The way the contig spectrum is obtained leads to approximations of the viral diversity. In the samples analyzed here, only the DNA from viruses smaller than 0.22 The comparative analysis of the four phage communities showed that the power law seems overall to be a powerful rank-abundance distribution to model phage community structure Figure . A recenFirst, the extreme diversity of the sediment viral community may reflect the higher diversity of the microbial communities found in sediments using automated rRNA intergenic spacer analysis (ARISA)  in compaPHACCS is a web-based service that predicts community structure and diversity using the contig spectrum from metagenomic random shotgun sequence data. This methodology allows PHACCS to determine the mathematical model that most accurately reflects the underlying genotype abundance distribution  and use it to makes estimates about the diversity of the communities, (i.e., richness, evenness, Shannon-Wiener index and abundance of most abundant genotype). Using uncultured environmental viral samples, PHACCS has been used to confirm that phage biodiversity is higher than in any previously observed community and that the structure of viral communities may closely follow that of their hosts. PHACCS is designed for biologists to mathematically analyze their viral shotgun libraries and gain insights about viral ecology and population dynamics.Project name: PHACCS – PHAge Communities from Contig Spectrum• Project home page: • Operating system(s): Unix based system for PHACCS and its web interface. Platform independent for PHACCS core.• Programming language: Matlab (for the core scripts) and Perl• Other requirements: For the interface: CGI.pm Perl module, ppmtogif, webserver program (to use PHACCS as a web service)• License: GNU GPL• FA developed the PHACCS main program and its interface. BRB helped with the programming. BRB, DB, PMN, PS, BF, JN and JM developed the modified Lander-Waterman algorithm and implemented it with Matlab. FR and MB helped write the manuscript and provided the test datasets. All authors read and approved the final manuscript.This file contains the script files part of PHACCS. These files are either standard text or picture files.Click here for file