If you're an evolutionary biologist, the tired old saw, “You can tell a lot about a person by the company they keep,” represents a fresh new approach to a longstanding problem. Especially if the company in question is a parasite—say, for example, lice—and the problem is tracing the path of human evolution.Homo sapiens interbred with archaic humans. While genetic data have provided insight into recent human evolution, fossils remain the only available evidence for many archaic human species—and the fossil record is notoriously spotty, leaving the data open to multiple interpretations. Two prominent models and a subset of variants have emerged, differing mainly on the question of gene flow: one asserts that modern humans emerged from an archaic ancestor in Africa about 130,000 years ago and then replaced archaic forms in Africa, Asia, and Europe with no gene flow between them; the other proposes gene flow between modern human populations as well as interbreeding between modern and archaic forms in different parts of the world. Both models find support in the available data, but neither can claim a perfect fit with all the data, leaving the possibility of interbreeding an open question.One of the hottest debates in the study of human origins centers around whether modern Faced with a relative paucity of human fossil and genetic data, scientists have been forced to rely on other data sources. Mounting evidence suggests that parasites with an established coevolutionary history with their hosts can serve as a proxy for host evolutionary history, an especially handy tool in the event of insufficient host data. Following this approach in a new study, David Reed and colleagues circumvent the lack of human data by analyzing the next best thing: head lice.As host-specific, obligate parasites—that is, occurring on a single species and not able to survive off that host—lice require direct physical contact between hosts for transmission. As human parasites, lice harbor in their genetic sequence hints of the slings and arrows of evolutionary fortune (and touches of grace, for that matter) that strike their host. Recent studies of the evolutionary history of other human parasites , for example, fall in line with fossil and genetic data that place our origins in Africa.Pediculus humanus) and pubic (Pthirus pubis). Head and body lice obviously occupy different habitats, but are not genetically distinct. Interestingly, P. humanus contains two ancient lineages, offering the opportunity to shed light on this murky period in human evolution. To do this, Reed and colleagues had to reconstruct the evolutionary history of P. humanus, which they did using morphological and genetic analyses of this and other species of lice. They confirmed that P. humanus comprises two lineages—one contains both head and body forms and has worldwide distribution; the other contains only the head louse and is restricted to the New World—but discovered that P. humanus originated long before its H. sapiens host.Two louse species parasitize humans, head/body  of a single host. More likely, the New World louse evolved on an archaic form of humans and then cast its lot with a modern version.H. sapiens and H. neanderthalensis was too recent (about 700,000 years ago) to support a concurrent split between the worldwide and New World lice lineages, the split between H. sapiens and H. erectus (about 1.8 million years ago) could. Reed and colleagues propose a scenario in which H. sapiens and H. erectus carried distinct types of lice owing to a million years or so of isolation. As the first waves of modern humans left Africa about 100,000 years ago and modern humans replaced archaic forms, the two forms engaged in enough contact—whether in the form of fighting, swapping clothes, or interbreeding—for archaic lice to make the switch to modern human hosts. Tackling the question of interbreeding, the authors suggest, might best be pursued by studying P. pubis, which requires sexual contact for transmission.While the split between


Caenorhabditis elegans.An mRNA-tagging method was used to selectively isolate mRNA from a small number of cells for subsequent cDNA microarray analysis. The approach was used to identify genes specifically expressed in ciliated sensory neurons of  Caenorhabditis elegans using the mRNA-tagging method, in which poly(A) RNA was co-immunoprecipitated with an epitope-tagged poly(A)-binding protein specifically expressed in sensory neurons. Subsequent cDNA microarray analyses led to the identification of a panel of sensory neuron-expressed genes.It is not always easy to apply microarray technology to small numbers of cells because of the difficulty in selectively isolating mRNA from such cells. We report here the preparation of mRNA from ciliated sensory neurons of  Recent advances in technologies for analyzing whole-genome gene-expression patterns have provided a wealth of information on the complex transcriptional regulatory networks and changes in gene-expression patterns that are related to phenotypic changes caused by environmental stimuli or genetic alterations. Changes in gene expression are also fundamental during development and cellular differentiation, and differences in gene expression lead to different cell fates and eventually determine the structural and functional characteristics of each cell type. Comparative analyses of gene-expression patterns in various cell types will therefore provide a framework for understanding the molecular architecture of these cells as cellular systems.Caenorhabditis elegans is an ideal model organism for investigating development and differentiation at high resolution, because adult hermaphrodites only have 959 somatic nuclei, whose cell lineages are all known. About 19,000 genes were identified by determination of the C. elegans genome sequence [sequence -5, largesequence , systemasequence -9, and dsequence -12, are C. elegans genes have been constructed and used in global gene-expression analyses under a wide variety of developmental, environmental and genetic conditions [Genome-wide gene-expression profiling using DNA or oligonucleotide microarray technology has also been applied to this organism. Microarrays containing more than 90% of nditions -15. Genonditions ,17. MutaTo analyze gene-expression patterns in various cells, particularly those forming small tissues, selective isolation of mRNA from these cells is necessary. As an example of this approach, mRNA was prepared from mechanosensory neurons after cell culture of their embryonic precursors followed by selection of the cells by flow cytometry . AlthougC. elegans modifies its behavior by sensing environmental cues such as food, chemicals, temperature or pheromones. These cues are recognized by approximately 50 sensory neurons positioned in the head and tail. Although the overall functions of the chemosensory or thermosensory neurons have been examined by laser-killing experiments, the molecular mechanisms that underlie the functions of each sensory neuron have not yet been fully explored. Profiling of genes that are expressed in sensory neurons might therefore provide insights into the genes required for the specific functions of neurons.C. elegans.To identify sensory neuron-expressed genes, we adopted the mRNA-tagging method . In thisin situ crosslinking of RNA and proteins, followed by affinity purification of the tagged PABP from lysates of these animals, is expected to co-precipitate all the poly(A)+ RNA from cells expressing the tagged PABP  tails of mRNA , in situP Figure . This meed genes , but wheed genes , to the pab-1 gene in C. elegans. Nematode strains expressing FLAG-tagged PAB-1 from transgenes were generated using tissue-specific promoters. To prepare mRNA from sensory neurons, we generated the JN501 strain  in which the transgene was expressed in most of the ciliated sensory neurons using a che-2 gene promoter [acr-5::PABP), was generated to prepare mRNA from another subset of neurons using an acr-5 promoter, which is active in B-type motor neurons, as well as unidentified head and tail neurons [myo-3::PABP), which expressed the transgene in non-pharyngeal muscles using the myo-3 promoter [acr-5::PABP strain compared to that in sensory neurons in the che-2::PABP strain. As a measure of the functional integrity of FLAG-PAB-1-expressing cells, responses of the che-2::PABP strain to the volatile repellent 1-octanol, which is sensed by ASH amphid sensory neurons was tested. The sensitivity of the che-2::PABP animals was indistinguishable from the wild type (data not shown). The ability of the exposed sensory neurons to absorb the lipophilic dye diQ was also tested. Amphid sensory neurons in the head stained normally, whereas phasmid neurons, PHA and PHB, in the tail showed weak defects in dye-filling (90% staining of PHA and 91% staining of PHB, compared to 100% in wild type for both neurons). The acr-5::PABP and myo-3::PABP strains appeared to move normally, suggesting overall functional integrity of motor neurons and body-wall muscles, respectively.PABP is encoded by the promoter . A secon neurons . A thirdpromoter , was gentax-2, which is expressed in a subset of sensory neurons [che-2::PABP. The mRNA for odr-10, which is expressed in only one pair of sensory neurons [che-2::PABP. On the other hand, mRNA for acr-5 and del-1, both of which are expressed in B-type motor neurons [acr-5::PABP. The mRNA for unc-8, which is expressed in motor neurons and ASH and FLP sensory neurons in the head [che-2::PABP and acr-5::PABP. The mRNA for unc-54, which is expressed in muscles [myo-3::PABP. Representatives of housekeeping genes, eft-3 [lmn-1 [odr-10 transcript in RNA from che-2::PABP was 39-fold higher than that from acr-5::PABP, and mRNA for gcy-6, which is expressed in only a single sensory neuron [acr-5 was enriched eightfold in RNA from acr-5::PABP compared with that from che-2::PABP. mRNA for the pan-neuronally expressed gene snt-1 [acr-5::PABP and che-2::PABP. Therefore, selective enrichment of sensory neuron-, motor neuron- and muscle-expressed genes in RNA from che-2::PABP, acr-5::PABP and myo-3::PABP strains, respectively, have been achieved as intended. Of these, the enrichment of motor neuron-expressed genes appeared less efficient, because weak bands were sometimes seen for these genes in RT-PCR from che-2::PABP or myo-3::PABP RNA.Poly(A) RNA/FLAG-PAB-1 complexes were pulled-down from whole lysates of these transgenic worms using anti-FLAG monoclonal antibodies. Poly(A) RNA was then extracted and concentrated. The amounts of known tissue-specific transcripts were examined by reverse transcription PCR (RT-PCR) Figure . The mRN neurons , was enr neurons , was als neurons , was enrthe head , was con muscles , was enrs, eft-3  and lmn-3 [lmn-1 , were dey neuron , was enrne snt-1  was equache-2-expressing ciliated sensory neurons with that from acr-5-expressing cells. RNA purified from che-2::PABP was labeled with Cy5 and that from acr-5::PABP was labeled with Cy3. The two types of labeled RNA were mixed and hybridized to the cDNA microarray and the che-2::PABP/acr-5::PABP (Cy5/Cy3) ratio was calculated for each cDNA spot. The cDNA microarray contained 8,348 cDNA spots corresponding to 7,088 C. elegans genes. Two sets of independently prepared RNA samples were hybridized to two separate arrays. The logarithm of the hybridization intensity ratio for each spot, log2(che-2::PABP/acr-5::PABP), was calculated and values from the two experiments were averaged. This calculation allowed us to order the genes represented on the microarrays according to the log2(che-2::PABP/acr-5::PABP) value . Genes specifically expressed in che-2-expressing cells should have higher rank orders in this list, whereas those expressed in acr-5-expressing cells should have lower rank orders.We used a cDNA microarray to compare the properties of mRNA prepared from To evaluate the results of the microarray experiments, we searched for genes that are known to be expressed in amphid sensory neurons, but not in ventral cord motor neurons, or vice versa, using the WormBase database (WS94). Of these, 20 sensory neuron-specific genes and five motor neuron-specific genes were present on the arrays . These genes showed a highly uneven distribution, with sensory neuron-specific genes concentrated in the highest rank orders and motor neuron-specific genes distributed in lower rank orders Figure . Muscle-daf-19 encodes a transcription factor similar to mammalian RFX2. Several genes expressed in ciliated sensory neurons and essential for ciliary morphogenesis, such as che-2 and osm-6, are under the control of daf-19 and have one or more copies of the cis-regulatory element X-box in their promoter regions [ regions . We therche-2::PABP. However, only a few genes were previously known to be expressed in these tissues. In fact, the expression patterns for most top-ranked genes in our list were not known. To determine which of these genes were actually expressed in sensory neurons, we examined the expression patterns of 17 genes with the highest rank orders using translational green fluorescent protein (GFP) fusions. The expression patterns for these genes had not been reported previously.The above analyses showed that sensory neuron-expressed genes were enriched in the mRNA population purified from We did not observe any GFP fluorescence for two clones, K07B1.8 and C13B9.1, probably because the promoter region we selected did not contain all the functional units or expression was below the level of detection. GFP-expressing cells were identified for all the remaining 15 genes Figure , Table 1spp-5). Expression in the ventral nerve cord was observed for Y44A6D.2, while only weak expression in the intestine was observed for T08A9.9 (data not shown). These results also suggested that our procedure was somewhat less effective in enriching motor neuron-expressed genes than sensory neuron-expressed genes  annotations, top-ranked genes showed a significantly larger fraction with a 'nucleic acid binding' functional capacity (che-2::PABP-derived mRNA fraction was enrichment of genes homologous to nephrocystins. Nephrocystins are responsible for a hereditary cystic kidney disease, nephronophthisis, and to date, nephrocystin 1 (NPHP1) through nephrocystin 4 (NPHP4) have been identified [C. elegans homologs of NPHP1 and NPHP4 were ranked at positions 15 and 25 in our list, suggesting a link between these disease genes and the functions of worm sensory neurons.Another prominent characteristic of the entified -35. C. eC. elegans [We prepared poly(A) RNA from a subset of neurons using the mRNA-tagging technique. The genome-wide identification of muscle-expressed genes demonstrated that mRNA tagging is a powerful technique for collecting tissue-specific transcripts in  elegans . The metin situ formaldehyde crosslinking. Although this procedure reduced the recovery of immunocomplexes, it ensured minimal contamination by mRNA from non-target cells. As there are many characterized promoters that can deliver FLAG-PAB-1 to small numbers of neurons in C. elegans, profiling of the gene-expression pattern of each type of neuron should be possible with this technique.The procedure of mRNA tagging is based on immunoprecipitation of poly(A)-RNA/FLAG-PAB-1 complexes. A potential problem with this technique is that once the cells are broken, poly(A) RNA released from non-target cells might bind unoccupied FLAG-PAB-1. To reduce this possibility, we adopted stringent washing conditions in addition to Another potential problem with this method is that PABP might have different binding affinities for different transcript species, rendering some tissue-specific transcripts difficult to recover. Although PABP binds tightly to the poly(A) tails of most mRNA , RNA speC. elegans . Genes expressed in the intestine were enriched in the acr-5::PABP-derived mRNA preparations. FLAG-PAB-1 was weakly expressed in both the che-2::PABP and acr-5::PABP strains in intestine, with the latter showing higher level of expression (data not shown). Low-level expression of artificially manufactured genes in the intestine seems to be quite common, either due to readthrough transcription from the vector or the 3' regulatory sequences. Our results may suggest that in future applications one must be very careful about this type of low-level expression of FLAG-PAB-1.Previously known sensory neuron- and motor neuron-expressed genes were used to evaluate the results of our microarray analyses. Most genes were enriched in our cterized , and botche-2::PABP-derived mRNA preparations. Thirteen of 15 genes that showed clear expression patterns of GFP reporters were expressed in multiple sensory neurons. None of these genes has previously been characterized. In addition, quantitative PCR analysis shows that genes expressed in only one or two neurons, gcy-6 and odr-10, respectively, can be enriched. Therefore, our procedure is effective for identifying genes that are preferentially expressed in a particular subset of cells. On the other hand, the presence of small fractions of genes that are predominantly expressed in tissues other than sensory neurons was also evident. Therefore, mRNA-tagging technology should be regarded as enrichment of candidate cell-specific genes and the real expression pattern of each gene should be verified independently.We determined the expression patterns of genes highly enriched in the che-2::PABP-derived mRNA preparations proved to be sensory neuron-expressed, characterization of the enriched genes as a set should lead to molecular characterization of the sensory neurons of C. elegans. A prominent feature of the genes enriched in the che-2::PABP-derived mRNA preparations is that they include nematode-specific genes more often than the rest of the genes, as judged from inter-species BLASTP comparisons, suggesting that many of the genes identified have functions unique to nematode sensory neurons. The existence of many nematode-specific gene families has previously been noted, and was proposed to be related to the nematode-specific body plan [Since most of the genes enriched in the ody plan . Since wcis-acting elements commonly found in the promoter regions of the sensory neuron-expressed genes. The enrichment of X-boxes in the che-2::PABP fraction suggests that this might be plausible. In addition, other reports have identified cis-acting elements in genes expressed during particular developmental stages or in particular neurons . Howche-2::PABP gene set. This might indicate that C. elegans sensory neurons have specialized regulatory mechanisms for gene expression, although it remains to be seen which of these 'chromatin' genes are actually expressed in a sensory neuron-specific manner. It was also apparent from visual inspection or computer searches that two homologs of nephrocystins are included in the highest rank orders. It has recently been shown that nephrocystin 1 and nephrocystin 4 interact with each other and are both components of cilia. These studies have led to the hypothesis that the kidney disease nephronophthisis is caused by malfunctions of cilia on the tubular epithelium [C. elegans ciliated sensory neurons also have prominent ciliary structures [C. elegans homologs (bbs-1, 2, 7 and 8) of the human genes responsible for Bardet-Biedl syndrome, which is also thought to be a ciliary disease, are specifically expressed in ciliated sensory neurons [C. elegans homologs of as yet unidentified ciliary disease genes.By surveying GO annotations and protein motifs, genes whose predicted functions are related to nucleic acids and/or chromatin were found to be enriched in the ithelium -35,43. Cructures , but non neurons . It is tThe present study demonstrates that a combination of mRNA tagging and microarray analysis is an effective strategy for identifying genes expressed in subsets of neurons. Systematic reporter expression analyses following this approach will facilitate the accumulation of information regarding gene expression patterns. In particular, profiling of the gene expression patterns of subsets of neurons, in combination with analyses of neural functions, might provide insights into understanding the distinct roles of cells within the neural network.pab-1, yk28d10, was replaced with a linker composed of two complementary oligonucleotides, 5'-AATTGCTAGCATGGATTACAAGGATGATGACGATAAGT-3' and 5'-CTAGACTTATCGTCATCATCCTTGTAATCCATGCTAGC-3', in which the underlined sequence encodes an initiation codon followed by a FLAG peptide. The resulting epitope-tagged gene was cloned into the pPD49.26 vector (donated by Andy Fire, Stanford University). The promoter of che-2 [acr-5 [myo-3 [rol-6 allele, as a marker [Is [che-2p::flag-pab-1 pRF4]; JN502: Is [acr-5p::flag-pab-1 pRF4]; and JN503: Is [myo-3p::flag-pab-1 pRF4].The initiation codon of a cDNA for of che-2 , acr-5 [2 [acr-5  or myo-35 [myo-3  was insea marker . Stable a marker . Each inin vivo, worms were treated with 1% formaldehyde in M9 for 15 min at 20°C with gentle agitation. The formaldehyde was then inactivated by 125 mM glycine for 5 min at 20°C and washed out by replacing the buffer with four changes of TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl). At this point, worms were dispensed into 0.4 g aliquots, placed in 2-ml microtubes and stored frozen until lysate preparation.To purify poly(A)-RNA/FLAG-PAB-1 complexes from subsets of neurons, we modified a protocol for chromosome immunoprecipitation . Transgeg for 20 min. Five hundred microliters of supernatant, with the protein concentration roughly adjusted to 20 mg/ml, was incubated with 50 μl of anti-FLAG M2 affinity gel beads (Sigma) for 2 h. The affinity beads were sequentially washed three times with lysis buffer supplemented with PMSF, twice with wash buffer  and once with TE (10 mM Tris-HCl pH 7.5, 0.5 mM EDTA). Lysate preparation and purification of RNA-protein complexes were performed at 4°C. Precipitated materials were eluted with 100 μl elution buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS, 20 mM RVC) by incubation for 5 min at 65°C. Elution was repeated and the two supernatant fractions were combined. The eluted RNA/FLAG-PAB-1 complexes were incubated for 6 h at 65°C to reverse the formaldehyde crosslinks. Proteins were digested with proteinase K and removed by phenol-chloroform extraction. Nucleic acid was recovered by ethanol precipitation. Typically, 100 ng nucleic acid was obtained from 0.5 ml cleared lysate of myo-3::PABP. Under the above washing conditions, binding of free poly(A) RNA to PABP was severely impaired (data not shown).Worms were resuspended in 0.45 ml lysis buffer , 1 mM DTT, 0.2 mM PMSF, protease inhibitor cocktail (Complete-EDTA, Roche) at the recommended concentration) supplemented with 20 mM ribonucleoside vanadyl complexes (RVC, Sigma) and 1000 U/ml of human placental ribonuclease inhibitor (Takara). Animals were disrupted by vigorous shaking with 2 g acid-washed glass beads (Sigma), and worm debris was removed by centrifugation at 18,000 che-2::PABP worms at L4 to young adult were observed. Cells were identified by their positions and the percentage of stained cells was scored. Responses of the che-2::PABP strain to 1-octanol was assessed as described [For staining of living animals with lipophilic dye, we followed the procedure described before  except tescribed  except teft-3 as a reference, and the results of three independent experiments were averaged. The primers used for the amplification of each gene were: lmn1-52: 5'-CGTTCACCACCCACCAGAA-3' and lmn1-32: 5'-CAAGACGAGCTGATGGGTTATCT-3' for lmn-1; eft3-52: 5'-ATTGCCACACCGCTCACA-3' and eft3-32: 5'-CCGGTACGACGGTCAACCT-3' for eft-3; tax2-54: 5'-GATTAATCCAAGACAAGTTCCTAAATTGAT-3' and tax2-34: 5'-TTCAATTCTTGAACTCCTTTGTTTTC-3' for tax-2; unc8-52: 5'-TCTCAGATTTTGGAGGTAATATTGGA-3', and unc8-32: 5'-GATCTCGCAGAAAAGTTCTGCAA-3' for unc-8; unc54-52: 5'-AACAGAAGTTGAAGACCCAGAAGAA-3', and unc54-32: 5'-TGGTGGGTGAGTTGCTTGTACT-3' for unc-54; snt1-51: 5'-GAGCTGAGGCATTGGATGGA-3' and snt1-31: 5'-CCAAGTGTATGCCATTGAGCAA-3' for snt-1; acr5-52: 5'-AATCGATTTATGGACAGAATTTGGA-3' and acr5-32: 5'-ATGTTGCAAAAGAAGTGGGTCTAGA-3' for acr-5; odr10-51: 5'-TCATTGTGTTTTGCTCATTTCTGTAC-3' and odr10-31: 5'-ATATTGTTCTTCGGAAATCACGAAT-3' for odr-10; del1-51: 5'-TAAACTGCCTCACGACAGAAG-3' and del1-31: 5'-GCCATCAAGTTGAACCAAGAAT-3' for del-1. All primers were designed to include one intron in the PCR product amplified from the genomic DNA for each gene, such that the length and melting point were different from the product amplified from the cDNA. In Figure eft-3 was amplified for 25 cycles, lmn-1, snt-1 and unc-54 for 30 cycles and tax-2, unc-8, odr-10, del-1 and acr-5 for 35 cycles. Amplified DNA was visualized by electrophoresis followed by staining with ethidium bromide.Fifty nanograms of RNA was converted to cDNA using an RNA PCR Kit (AMV) Ver. 2.1 (Takara) according to the manufacturer's protocol. One-tenth of the cDNA from each sample was subjected to a gene-specific PCR reaction in a total volume of 20 μl. Quantification of the PCR products was performed using a FastStart DNA Master SYBR Green I Kit (Roche) with the Light Cycler system (Roche). Serial dilutions of cDNA prepared from poly(A) RNA of wild-type worms were used to generate a standard curve. The ratio of expression levels for each gene was calculated using the amount of Microarrays were prepared using a 16-pin arrayer constructed according to the format of Patrick Brown (Stanford University ) on CMT-A genomic DNA fragment for each gene was amplified by PCR such that it contained an upstream promoter region followed by a partial or full-length predicted coding region. The 3' PCR primers were designed to introduce a restriction site in-frame with GFP in the vectors. The 5' PCR primers were designed to anneal to a sequence 0.6-5.0 kilobases (kb) upstream of the predicted coding region of each gene. The upstream-predicted gene was essentially not included in the promoter fragment. The amplified PCR fragments were cloned into pPD95.70, pPD95.75 (donated by A. Fire) or a Gateway vector (Invitrogen) to create GFP fusions . The resulting reporter plasmid for each gene was introduced into wild-type animals. Transgenic worms at all developmental stages were observed under a differential interference contrast (DIC)-fluorescence microscope. Cells were identified according to their positions  by compaC. elegans genome using BLAT [C. elegans was obtained from the Gene Ontology Consortium [cDNA clones were mapped to the ing BLAT  and BLASing BLAT  and protnsortium .et al. [et al. observed that X -boxes were present about 100 bp upstream of the initiation codons [For Figure et al. . Only ben codons .Caenorhabditis briggsae, whose draft genome sequence has recently been released, was downloaded from WormBase [Drosophila melanogaster, Schizosaccharomyces pombe and Saccharomyces cerevisiae were obtained from NCBI [C. elegans gene.The proteome data set for WormBase . Proteomrom NCBI , and BLA2(che-2::PABP/acr-5::PABP) value for all genes that carried the motif was calculated. To avoid artifactual results due to gene families with close sequence similarities (such as major sperm proteins), groups of genes whose mRNA are expected to cross-hybridize were treated as a single imaginary gene with the average log2(che-2::PABP/acr-5::PABP) value.Protein motifs that preferentially appeared in genes at the higher rank orders were searched for as follows. For all genes represented in our microarrays, the protein motifs contained in each gene product were obtained from the AcePerl server. For each motif, deviation of the average logThe following additional data are available with the online version of this article. Additional data file a table listing the results of the microarray experimentsClick here for additional data fileGenes expressed in sensory neurons, motor neurons, muscles and the intestine, and those with X-boxes shown in Figure 3Click here for additional data fileThe primers and vectors used for reporter constructionsClick here for additional data fileThe legends to the above three tablesClick here for additional data file


Austin Bradford Hill's landmark 1965 paper contains several important lessons for the current conduct of epidemiology. Unfortunately, it is almost exclusively cited as the source of the "Bradford-Hill criteria" for inferring causation when association is observed, despite Hill's explicit statement that cause-effect decisions cannot be based on a set of rules. Overlooked are Hill's important lessons about how to make decisions based on epidemiologic evidence. He advised epidemiologists to avoid over-emphasizing statistical significance testing, given the observation that systematic error is often greater than random error. His compelling and intuitive examples point out the need to consider costs and benefits when making decisions about health-promoting interventions. These lessons, which offer ways to dramatically increase the contribution of health science to decision making, are as needed today as they were when Hill presented them. One of the most cited papers in health research is Austin Bradford Hill's "The Environment and Disease: Association or Causation?" , Hill's Hill outlined a systematic approach for using scientific judgment to infer causation from statistical associations observed in epidemiologic data, listing nine issues to be considered when judging whether an observed association is a causal relationship. Despite widely distributed and clearly elaborated advice to the contrary , Hill's Hill never used the term "criteria" and he explicitly stated that he did not believe any hard-and-fast rules of evidence could be laid down, emphasizing that his nine "viewpoints" (p. 299) Causal criteria are an intriguing subject for the history of science, including the question of why Hill's list seems more popular than others -10 and wThis brief analysis of Hill's "criteria" and what has been made of them can add little new to that topic . Our purpose is to call attention to the seldom-cited last page and a half of the article, which presents lessons that remain overlooked today.t table diverts attention from the inadequacies of the fare" [Hill eloquently warned about overemphasis on statistical significance testing, writing "the glitter of the he fare" (p. 299).he fare" , and litOne implication of Hill's advice is well understood. Emphasis on the p-value  has been soundly denounced for decades ,17. EstiA further inadequacy of the fare is less well appreciated, stemming not from the question of p-values versus confidence intervals, but from systematic errors. No statistical test of random sampling error informs us about the possible impacts of measurement error, confounding, and selection bias. Methods for quantifying such errors (and perhaps more importantly, arguments for why we need to do so) have been developed in epidemiology, particularly over the last five years -25. HillEven as modern epidemiologic analysts become less dazzled by the t-table, replacing significance testing with confidence intervals and introducing quantification of systematic errors, there is still a tendency to completely overlook Hill's other important insight. Hill sought to address the question how to decide whether to take action once causal inferences are made. In his last few paragraphs, he offers an important commentary on the policy recommendations that flow from decisions regarding cause and effect in epidemiology. Since "our object is usually to take action" (p. 300),Even when policy implications are presented explicitly, they are seldom carefully analyzed. Analyzing the implications of a health research finding for decision making is often not terribly difficult, but making recommendations without such analysis can lead to absurd suggestions . One episome health hazard, and act based on the expected gains and losses, with or without statistical certainty. Hill, who was educated in economics, argued that in order to take policy action, we ought to pay attention to the absolute costs and benefits of potential actions. It would clearly be reading too much into the text to suggest that he had a prescient vision of modern probability-weighted cost and benefit based policy analyses and decision theory (those fields were in their early stages at the time of his writing and he never used any of those terms). But, in another memorable phrase, he did make the case for having "differential standards before we convict" (p. 300),Hill points this out (in an example sufficiently ill-chosen that it may have contributed to his important message being ignored): "On relatively slight evidence we might decide to restrict the use of a drug for early-morning sickness in pregnant women. If we are wrong in deducing causation from association no great harm will be done. The good lady and the pharmaceutical industry will doubtless survive (p. 300).Setting aside the impolitic dismissal of women's preferences and the unsupported assertion that there is no great harm at stake (as well as the irony of the popularity, withdrawl, and rehabilitation of the morning sickness drug, Bendectin) the underlying point might be his most important lesson: Policy actions that appear to create a net benefit  should be taken, even without statistical "proof" of an association, while actions that entail great costs should only be taken with sufficient certainty of substantial benefit.Hill goes on to strengthen his argument: "On fair evidence we might take action on what appears to be an occupational hazard, e.g. we might change from a probably carcinogenic oil to a noncarcinogenic oil in a limited environment and without too much injustice if we are wrong. But we should need very strong evidence before we made people burn a fuel in their homes that they do not like or stop smoking the cigarettes and eating the fats and sugar that they do like (p.300)."Hill clearly stated that the science and data analysis should not be influenced by what is at stake. But health researchers should recognize that the stakes matter, and incorporate a consideration of them into their work. The alternative to carrying out the policy analysis is to leave the weighing of costs and benefits to an unreliable post-science political process.The observation that the costs and benefits matter, despite being rather obvious, is frequently – indeed, typically – overlooked in public health discussions. The popular decongestant phenylpropanolamine was banned on weak evidence without regard to the high cost to consumers ; dietaryMaking a good decision does not depend on having studies with confidence intervals that exclude the null. A best decision can be based on whatever information we have now, and indeed a decision will be made – after all, the decision to maintain the status quo is still a decision ,38. HillThe pursuit of the low p-value  leaves our society postponing apparently useful policy choices while we do more research to try to show what we already believe to be true. It also creates the incentive to use dubious methods (e.g., unstated multiple hypothesis testing, choosing models or transforming data to maximize the effect size ) in ordeThis would release us from the trap of letting ignorance trump knowledge. Regulators often fail to act because we have not yet statistically "proven" an association between an exposure and a disease, even when there is enough evidence to strongly suspect a causal relationship. There is a growing movement to escape this mistake by making a similar mistake in the other direction: adopting precautionary principles, which typically call for restrictions until we have "proven" lack of causal association – a decision based on ignorance that merely reverses the default. If we can escape from the false dichotomy of "proven vs. not proven," facilitated by the nonexistant bright line implied by statistical hypothesis testing and by the notion that causality can be definitively inferred from a list of criteria, then we can make decisions based on what we do know rather than what we don't.The uncritical repetition of Hill's "causal criteria" is probably counterproductive in promoting sophisticated understanding of causal inference. But a different list of considerations that can be found in his address is worthy of repeating:• Statistical significance should not be mistaken for evidence of a substantial association.• Association does not prove causation (other evidence must be considered).• Precision should not be mistaken for validity (non-random errors exist).• Evidence (or belief) that there is a causal relationship is not sufficient to suggest action should be taken.• Uncertainty about whether there is a causal relationship (or even an association) is not sufficient to suggest action should not be taken.These points may seem obvious when stated so bluntly, but causal inference and health policy decision making would benefit tremendously if they were considered more carefully and more often. The last point may be the most important unlearned lesson in health decision making.In fairness to those who do not appreciate these points even today, it overinterprets Hill's short paper to claim that he clearly laid out these considerations, or that he was calling for modern decision analysis and uncertainty quantification. But the fundamental concepts were clearly there (and the overinterpretation is not as great as that required to derive a checklist of criteria for determining causation). Several generations of advancement in epidemiology and policy analysis provide much deeper exposition of his points. But Hill still offers timeless insightful analysis about how to interpret our observations. Strangely, these forgotten lessons, which are only slowly and grudgingly being appreciated in modern epidemiology, are hidden in plain sight, in what is possibly the best known paper in the field.1. Interestingly, there are more extreme cases of a scholar's name being immortalized for something contrary to his beliefs. The "Coase Theorem" in economics, from one of the most cited article in the economics and legal literatures  . Cur


Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.The complete sequence of the 1,267,782 bp genome of  Wolbachia provides insights into the origins of mitochondria, as well as the ecology and evolution of endosymbiosisThe genome sequence of  Wolbachia are intracellular gram-negative bacteria that are found in association with a variety of invertebrate species, including insects, mites, spiders, terrestrial crustaceans, and nematodes. Wolbachia are transovarialy transmitted from females to their offspring and are extremely widespread, having been found to infect 20%–75% of invertebrate species sampled , feminization , male-killing , and cytoplasmic incompatibility   .Wolbachia have been hypothesized to play a role in host speciation through the reproductive isolation they generate in infected hosts (Wolbachia could be utilized to either suppress pest insect populations or sweep desirable traits into pest populations (e.g., the inability to transmit disease-causing pathogens)  . Moreoveult worm .Wolbachia and their invertebrate hosts. This is partly due to the difficulty of working with an obligate intracellular organism that is difficult to culture and hard to obtain in quantity. Here we report the completion and analysis of the genome sequence of Wolbachia pipientiswMel, a strain from the A supergroup that naturally infects Drosophila melanogaster  elements, four likely retrotransposons, and four families without detectible similarity to known elements but with many hallmarks of mobile elements (flanked by inverted repeats, present in multiple copies) . Thus, many of these repetitive elements may be useful markers for strain discrimination. In addition, the mobile elements likely contribute to generating the diversity of phenotypically distinct Wolbachia strains (e.g., mod− strains [The most striking feature of the families , were idfamilies . These w copies) . One of  copies) . However copies) , and the strains ) by alteWolbachia sp. wKue . The other two are closely related to and exhibit extensive gene order conservation with the WO phage described from sp. wKue  .The irregular pattern of GC skew in ith this   and that of many hypothetical and conserved hypothetical proteins.Despite evident genome reduction in  species . Many ofwsp, which is a standard gene for strain identification and phylogenetic reconstruction in Wolbachia (wsp (WD0159), wMel encodes two wsp paralogs (WD0009 and WD0489), which we designate as wspB and wspC, respectively. While these paralogs are highly divergent from wsp (protein identities of 19.7% and 23.5%, respectively) and do not amplify using the standard wsp PCR primers (Wolbachia strains. This has apparently occurred in one study of Wolbachia strain wKueYO, for which the reported wsp gene (gbAB045235) is actually an ortholog of wspB  and not strains .Buchnera, Rickettsia, Chlamydia, and Wigglesworthia, that all have very low levels of repetitive DNA and mobile elements. The recently sequenced genomes of the intracellular pathogen Coxiella burnetti , we propose that the acquisition and maintenance of these repetitive and mobile elements by wMel have played a key role in shaping the evolution of Wolbachia.We propose that the mobile DNA in layi see . The lacchia DNA , 1998 orWolbachia  has been suggested previously for intracellular bacteria, based in part on observations that these bacteria have higher evolutionary rates than free-living bacteria  in wMel is particularly relevant since this pathway is one of the key steps in regulating mutation rates in other species. In fact, wMel is the first bacterial species to be found with two mutL homologs. Overall, examination of the predicted DNA repair capabilities of bacteria (wMel and Buchnera spp., have kept the genes that likely regulate mutation rates. In addition, some free-living species without high evolutionary rates have lost some of the same pathways lost in intracellular species, while many free-living species have lost key pathways resulting in high mutation rates (e.g., Helicobacter pylori has apparently lost mismatch repair [Another possible explanation for inefficient selection is high mutation rates. It has been suggested that the higher evolutionary rates in intracellular bacteria are the result of high mutation rates that are in turn due to the loss of genes for DNA repair processes e.g., . This isbination . The onl species . However species . The prebacteria  suggestsh repair ). Given wMel involve population-size related factors, such as genetic drift and the occurrence of population bottlenecks. Such factors have also been shown to likely explain the high evolutionary rates in other intracellular species  the free-living α-Proteobacteria, and (iv) mitochondria within α-Proteobacteria. Since amino acid content bias was very severe in these datasets, protein LogDet analyses, which can correct for the bias, were also performed. In LogDet analyses of the concatenated protein alignment, both including and excluding highly biased positions, mitochondria usually branched basal to the Wolbachia–Rickettsia clade, but never specifically with Rickettsia  do not even branch within the α-Proteobacteria, although these genes almost certainly were encoded in the ancestral mitochondrial genome  and two conserved hypothetical proteins (WD1005, WD0724). The α-Proteobacterial origin of these genes suggests that at least some of the genes of the de novo nucleotide synthesis pathway in eukaryotes might have been laterally acquired from bacteria via the mitochondria. The presence of such genes in other Proteobacteria suggests that their absence from Rickettsia is due to gene loss . We used the ene loss . This fiWolbachia are excellent candidates for such transfer events since they live inside the germ cells, which would allow lateral transfers to the host to be transmitted to subsequent host generations. Consistent with this, a recent study has shown some evidence for the presence of Wolbachia-like genes in a beetle genome , riboflavin biosynthesis, pyrimidine metabolism (i.e., from PRPP to UMP), and chelated iron uptake (using a single ABC transporter). The two Rickettsia species have a relatively large complement of predicted transporters for osmoprotectants, such as proline and glycine betaine, whereas wMel possesses only two of these systems.Many of the predicted metabolic properties of avengers . This liwMel genome is predicted to encode few proteins for regulatory responses. Three genes encoding two-component system subunits are present: two sensor histidine kinases (WD1216 and WD1284) and one response regulator (WD0221). Only six strong candidates for transcription regulators were identified: a homolog of arginine repressors (WD0453), two members of the TenA family of transcription activator proteins (WD0139 and WD0140), a homolog of ctrA, a transcription regulator for two component systems in other α-Proteobacteria (WD0732), and two σ factors (RpoH/WD1064 and RpoD/WD1298). There are also seven members of one paralogous family of proteins that are distantly related to phage repressors (see above), although if they have any role in transcription, it is likely only for phage genes. Such a limited repertoire of regulatory systems has also been reported in other endosymbionts and has been explained by the apparent highly predictable and stable environment in which these species live . The second operon contains virB3, virB4, and virB6 as well as four additional non-vir CDSs, including three putative membrane-spanning proteins, that form part of a 15.7 kb operon (WD0859–WD0853). Examination of the Rickettsia conorii genome shows a similar orga-nization  is also present in the wMel genome. WD0818 is quite divergent from virB8 and, as such, does not appear to have resulted from a recent gene duplication event. RT-PCR experiments have failed to show expression of this CDS in wMel-infected Drosophila (data not shown). PCR primers were designed to all CDSs of the wMel Type IV secretion system and used to successfully amplify orthologs from the divergent Wolbachia strains wRi and wAlbB (data not shown). We were able to detect orthologs to all of the wMel Type IV secretion system components as well as most of the adjacent non-vir CDSs, suggesting that this system is conserved across a range of A- and B-group Wolbachia. An increasing body of evidence has highlighted the importance of Type IV secretion systems for the successful infection, invasion, and persistence of intracellular bacteria within their hosts , it is not likely to have been acquired from other species. However, it is possible that these genes can be transferred among Wolbachia strains via the phage, which in turn could lead to some variation in host–symbiont interactions between Wolbachia strains.Genes involved in pathogenicity in bacteria have been found to be frequently associated with regions of anomalous nucleotide composition, possibly owing to transfer from other species or insertion into the genome from plasmids or phage. In the four such regions in wMel has 23 ankyrin repeat-containing genes, the most currently described for a prokaryote, with C. burnetti being next with 13. This is particularly striking given wMel's relatively small genome size. The functions of the ankyrin repeat-containing proteins in wMel are difficult to predict since most have no sequence similarity outside the ankyrin domains to any proteins of known function. Many lines of evidence suggest that the wMel ankyrin domain proteins are involved in regulating host cell-cycle or cell division or interacting with the host cytoskeleton: (i) many ankyrin-containing proteins in eukaryotes are thought to be involved in linking membrane proteins to the cytoskeleton  angulation ; (iii) s repeats ; and  has provided insights into the biology of Wolbachia, helped identify genes that likely play roles in the unusual effects Wolbachia have on their host, and revealed many new details about the evolution of Wolbachia and mitochondria. Perhaps most importantly, future studies of Wolbachia will benefit both from this genome sequence and from the ability to study host–symbiont interactions in a host (D. melanogaster) well-suited for experimental studies.Analysis of the wMel DNA was obtained from D. melanogaster yw67c23 flies that naturally carry the wMel infection. wMel was purified from young adult flies on pulsed-field gels as described previously (gmax for 30 min at 4°C to remove undigested agarose. The supernatant was concentrated on a Centricon YM-100 microconcentrator (Millipore, Bedford, Massachusetts, United States) after prerinsing with 70% ethanol followed by TE buffer and, after concentration, rinsed with TE. The retentate was incubated with proteinase K at 56°C for 2 h and then stored at 4°C. wMel DNA for gap closure was prepared from approximately 1,000 Drosophila adults using the Holmes–Bonner urea/phenol:chloroform protocol (eviously . Plugs weviously , aiding protocol  to prepaDrosophila Genome Project site (http://www.fruitfly.org/blast/) showed that 3,912 of the 10,642 contigs were likely contaminants from the Drosophila genome. To aid in closure, the assemblies were rerun with all sequences of likely host origin excluded. Closure, which was made very difficult by the presence of a large amount of repetitive DNA (see below), was done using a mix of primer walking, generation, and sequencing of transposon-tagged libraries of large insert clones and multiplex PCR  and wMel (1.2 Mb).The complete genome sequence was determined using the whole-genome shotgun method . For theplex PCR . The finWolbachia and their hosts may undergo lateral gene transfer events  to analyze the phylogenetic profile , and (iii) to determine the presence of homologs in different species. Orthologs between the wMel genome and that of the two Rickettsia species were identified by requiring mutual best-hit relationships among all possible pairwise BLASTP comparisons, with some manual correction. Those genes present in both Rickettsia genomes as well as other bacterial species, but not wMel, were considered to have been lost in the wMel branch  toanch see . Genes panch see . For theMarchantia polymorpha and Reclinomonas americana , leaving 32 genes for investigation: atp6, atp9, atpA, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad9, rpl16, rpl2, rpl5, rpl6, rps1, rps11, rps12, rps13, rps14, rps19, rps2, rps3, rps4, rps7, rps8, yejR, and yejU. Using FASTA with the mitochondrial proteins as a query, homologs were identified from the genomes of seven α-Proteobacteria: two intracellular symbionts (W. pipientis wMel and Rickettsia prowazekii) and five free-living forms (Sinorhozobium loti, Agrobacterium tumefaciens, Brucella melitensis, Mesorhizobium loti, and Rhodopseudomonas sp.). Escherichia coli and Neisseria meningitidis were used as outgroups. Caulobacter crescentus was excluded from analysis because homologs of some of the 32 genes were not found in the current annotation. In the event that more than one homolog was identified per genome, the one with the greatest sequence identity to the mitochondrial query was retrieved. Proteins were aligned using CLUSTALW . PCR products were cloned into pGem-T vector (Promega, Madison, Wisconsin, United States) as previously described (wsp genes for the same Wolbachia strains (Genbank accession numbers AF020070, AF020059, and AJ252062). The four partial wsp sequences were aligned using CLUSTALV  and cDNA synthesized using SuperScript III RT (Invitrogen) using primers wspBR, WD0817R, WD0853R, and WD0852R. RNA isolation and RT were done according to manufacturer's protocols, with the exception that suggested initial incubation of RNA template and primers at 65°C for 5 min and final heat denaturation of RT-enzyme at 70°C for 15 min were not done. PCR was done using rTaq (Takara, Kyoto, Japan), and several primer sets were used to amplify regions spanning adjacent CDSs for most of the two operons. For operon virB3-WD0853, the following primers were used: (virB3-virB4)F, (virB3-virB4)R, (virB6-WD0856)F, (virB6-WD0856)R, (WD0856-WD0855)F, (WD0856-WD0855)R, (WD0854-WD0853)F, (WD0854-WD0853)R. For operon virB8-wspB, the following primers were used: (virB8-virB9)F, (virB8-virB9)R, (virB9-virB11)F, (virB9-virB11)R, (virB11-virD4)F, (virB11-virD4)R, (virD4-wspB)F, and (virD4-wspB)R. The coexpression of virB4 and virB6, as well as WD0855 and WD0854, was confirmed within the putative virB3-WD0853 operon using nested PCR with the following primers: (virB4-virB6)F1, (virB4-virB6)R1, (virB4-virB6)F2, (virB4-virB6)R2, (WD0855-WD0854)F1, (WD0855-WD0854)R1, (WD0855-WD0854)F2, and (WD0855-WD0854)R2. All ORFs within the putative virB8-wspB operon were shown to be coexpressed and are thus considered to be a genuine operon. All products were amplified only from RT-positive reactions .Click here for additional data file.Figure S2wMel codon usage. Multivariate analysis of codon usage was performed using the CODONW package (available from http://www.molbiol.ox.ac.uk/cu/codonW.html).Proteins with fewer than 100 residues are excluded from this analysis because their effective number of codon (ENc) values are unreliable. The curve shows the expected ENc values if codon usage bias is caused by GC variation alone. Colors: yellow, hypothetical; purple, mobile element; blue, others. Most of the variation in codon bias can be traced to variation in GC, indicating that the mutation forces dominate the (289 KB PDF).Click here for additional data file.Figure S3wMel. Transporters are grouped by predicted substrate specificity: inorganic cations (green), inorganic anions (pink), carbohydrates (yellow), and amino acids/peptides/amines/purines and pyrimidines (red). Transporters in the drug-efflux family (labeled as “drugs”) and those of unknown specificity are colored black. Arrows indicate the direction of transport. Energy-coupling mechanisms are also shown: solutes transported by channel proteins (double-headed arrow); secondary transporters (two-arrowed lines, indicating both the solute and the coupling ion); ATP-driven transporters (ATP hydrolysis reaction); unknown energy-coupling mechanism (single arrow). Transporter predictions are based upon a phylogenetic classification of transporter proteins (Overview of the predicted metabolism (energy production and organic compounds) and transport in proteins .(167 KB PDF).Click here for additional data file.Table S1(649 KB DOC).Click here for additional data file.Table S2(147 KB DOC).Click here for additional data file.Table S3(718 KB XLS).Click here for additional data file.Table S4(116 KB DOC).Click here for additional data file.Table S5(24 KB DOC).Click here for additional data file.Table S6(26 KB DOC).Click here for additional data file.Table S7(34 KB DOC).Click here for additional data file.Table S8(117 KB DOC).Click here for additional data file.Table S9(47 KB DOC).Click here for additional data file.wMel has been given GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession ID number AE017196 and is available through the TIGR Comprehensive Microbial Resourceat http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=dmgThe complete sequence for Wolbachia sp. wAlbB outer surface protein precursor wsp gene), AF020070 (Wolbachia sp. wRi outer surface protein precursor wsp gene), AJ252062 (Wolbachia endosymbiont of D. immitis sp. gene for surface protein), AJ580921 , AJ580922 , and AJ580923 .The GenBank accession numbers for other sequences discussed in this paper are AF020059 (


Dysphagia and feeding intolerance are common in neurologically handicapped children. The aim is to determine the etiologies of feeding intolerance in neurologically handicapped children who are intolerant of tube feedings.Eighteen neurologically handicapped children, followed in the Tube Feeding Clinic at the Children's Hospital of Wisconsin who were intolerant of gastrostomy feedings. The charts of these 18 patients were reviewed. Past medical history, diagnoses, history of fundoplication and results of various tests of gastrointestinal function including barium contrast radiography, endoscopy and antroduodenal manometry were documented.Five of 11 children had abnormal barium upper gastrointestinal series. Seven of 14 had abnormal liquid phase gastric emptying tests. Two of 16 had esophagitis on endoscopy. All 18 children had abnormal antroduodenal motility.In neurologically handicapped children foregut dysmotility may be more common than is generally recognized and can explain many of the upper gastrointestinal symptoms in neurologically handicapped children. Oral pharyngeal dysphagia due to disordered swallowing has become increasingly recognized in children with cerebral palsy and other neurodevelopmental disorders. This has led to the increasing use of enteral tube feedings either for full or supplemental nutritional support. Symptoms of foregut dysmotility, such as vomiting, retching gagging and bloating, are often associated with tube feeding in neurologically handicapped children -4. PreviIn order to elucidate the mechanisms behind these symptoms we reviewed the charts of a group of children followed in the Tube Feeding Clinic at the Children's Hospital of Wisconsin with neurological dysfunction, who were intolerant of tube feedings and who had undergone antroduodenal motility studies as part of their evaluations.2 b1receptor antagonists are summarized in Table The charts of 18 neurologically handicapped children  with dysphagia and symptoms of foregut motility were reviewed. The symptoms, underlying disorders, feeding route, the presence of fundoplication, and the use of prokinetic agents and HFollowing an overnight fast, antroduodenal motility studies were performed using a multilumen catheter with 8 recording ports spaced 2.5–5 cm apart, passed through the gastrostomy either under fluoroscopic guidance or endoscopically, connected to a low compliance, pneumohydraulic capillary infusion system  and a computerized motility system . Fasting activity was recorded for 3–4 hours. Erythromycin (1 mg/kg) was given intravenously over 10 minutes and the recording continued for another hour. Octreotide (0.5 mcg/kg) was then given intravenously over 5 minutes. 45 minutes later a liquid meal was given followed by an additional 2–3 hour recording period. The meal varied and consisted of the usual formula and volume given at home. Patients receiving jejunal feedings or TPN were given a bolus gastrostomy feeding. In the one patient receiving TPN, the TPN was discontinued during the study. Prokinetics were stopped at least 48 hours prior to study.Phase 1 of the MMC was defined as motor quiescence. Phase 2 was defined as the time between Phases 1 and 3 and is characterized by random contractions of varied amplitude and frequency. Phase 3 of the MMC is characterized by an aborally propagating cluster of repetitive contractions with a frequency of 11–13/minute in the duodenum and 3/minute in the antrum with a duration of 3–10 minutes. The tracings were analyzed by visual inspection.This study was approved by the Research and Publications Committee/Human Rights Review Board of the Children's Hospital of Wisconsin and the Institutional Review Board of the Medical College of Wisconsin.Eleven children had had recent upper GI series. Of these 5 were normal, 3 had gastroesophageal reflux and 3 had paraesophageal hernias , 1 had a bezoar and 1 had esophageal dysmotility. Fourteen patients had liquid phase gastric emptying studies. Of these 7 were normal, 6 had delayed emptying and 1 had rapid emptying. Two of 16 patients who had had recent endoscopies had esophagitis, 14 were normal. The diagnoses and clinical histories of the patients are summarized in Table No patients had a normal antroduodenal motility study Figures ,2,3. In Following erythromycin eight had a normal response consisting of antral contractions with a frequency of 3/min followed by phase 3 like activity in the duodenum. Three patients had no response and seen had abnormal responses consisting of abnormal clusters in three, no antral response in three and no duodenal response in three.Fourteen patients had a normal response to octreotide consisting of cessation of antral activity and the development of phase 3 activity in the duodenum. Two patients had continued antral contractions and two did not develop phase 3 duodenal activity. These patients had non-propagating duodenal clusters.Fourteen patients had normal postprandial phase 2-like activity. Eight developed premature phase 3 activity within 30 minutes following the meal. Seven patients failed to develop phase 3 activity during the 2–3 hour postprandial monitoring period and seven had no antral contractions in the postprandial monitoring period. One patient had a retrograde MMC.Three patients developed severe pain or irritability associated with antral or duodenal contractions following erythromycin  or octreForegut dysmotility is common in children with neurodevelopmental disorders such as cerebral palsy -4. Up toRavelli and Milla have shown that gastric electrical activity as measured by the electrogastrogram (EGG) was abnormal in 31/50 neurologically handicapped children . Eleven In a previous study we compared gastric electrical activity as measured by EGG in a group of neurologically handicapped children who were tolerant of their tube feedings to a group that were intolerant or symptomatic during tube feedings . The perThere have been few reports of antroduodenal motility, which have focused on neurologically handicapped children with feeding intolerance. DiLorenzo and colleagues reported that 25/28 children who remained symptomatic following fundoplication had abnormal antroduodenal motility .Due to the nature of our patients there are some limitations in the design and interpretation of our study. Because many of our patients had had multiple formula changes, we decided to use the formula, which the child was receiving at the time of the study, so that any symptoms occurring during the studies could not be attributed to a formula change. Bolus feeds were given to all patients during the study even those who had been receiving drip feeds. The fasting state could not be recorded for 4 hours in all patients, thus it is possible that absence of phase 3 of the MMC in these patients might not be abnormal. Since normal manometry data have not been published for children, non propagating clusters may or may not be normal in our patients.Since only 12 of our 18 patients had undergone fundoplication, we agree with DiLorenzo et al  that theIn this study we have confirmed that the incidence of foregut dysmotility is very high in neurologically handicapped children with feeding intolerance. Prokinetics and acid suppression did not resolve the symptoms in our patients. Twelve of our 18 patients had had fundoplications and two had undergone two fundoplications in unsuccessful attempts to control what had been thought to be reflux symptoms. While there is no way to know how much abnormal antroduodenal motility contributed to our patient's feeding disorders, following antroduodenal manometry a number of our patients were treated successfully with jejunal feeding, suggesting that while they had foregut dysmotility, midgut motility is normal.In neurologically handicapped children foregut dysmotility may be more common than is generally recognized and can explain many of the upper gastrointestinal symptoms in neurologically handicapped children. Thus in this patient population generalized foregut dysmotility may mimic reflux and the decision to perform a fundoplication should be made very cautiously and only after a complete evaluation of foregut motility particularly in children with gagging retching and forceful vomiting.None declared.The pre-publication history for this paper can be accessed here:


High-throughput genomic research tools are becoming standard in the biologist's toolbox. After processing the genomic data with one of the many available statistical algorithms to identify statistically significant genes, these genes need to be further analyzed for biological significance in light of all the existing knowledge. Literature mining – the process of representing literature data in a fashion that is easy to relate to genomic data – is one solution to this problem.We present a web-based tool, MILANO (Microarray Literature-based Annotation), that allows annotation of lists of genes derived from microarray results by user defined terms. Our annotation strategy is based on counting the number of literature co-occurrences of each gene on the list with a user defined term. This strategy allows the customization of the annotation procedure and thus overcomes one of the major limitations of the functional annotations usually provided with microarray results. MILANO expands the gene names to include all their informative synonyms while filtering out gene symbols that are likely to be less informative as literature searching terms. MILANO supports searching two literature databases: GeneRIF and Medline (through PubMed), allowing retrieval of both quick and comprehensive results. We demonstrate MILANO's ability to improve microarray analysis by analyzing a list of 150 genes that were affected by p53 overproduction. This analysis reveals that MILANO enables immediate identification of known p53 target genes on this list and assists in sorting the list into genes known to be involved in p53 related pathways, apoptosis and cell cycle arrest..MILANO provides a useful tool for the automatic custom annotation of microarray results which is based on all the available literature. MILANO has two major advances over similar tools: the ability to expand gene names to include all their informative synonyms while removing synonyms that are not informative and access to the GeneRIF database which provides short summaries of curated articles relevant to known genes. MILANO is available at  Saccharomyces cerevisiae, there are excellent curated databases, such as the Yeast Proteome Database [In the post-genomic era, biologists encounter a flood of information derived mainly from microarray experiments. The blessing of this wealth of information is accompanied by a great difficulty in identifying the biologically significant findings, which are often embedded in irrelevant information. Currently, there are several approaches to deal with this problem. One approach is to identify a category of genes which is overrepresented in the microarray output. This approach can be carried out using the Gene Ontology project (GO) which describes gene products in terms of their associated biological processes, cellular components and molecular functions . The advDatabase  and the Database , which cWe have developed an analysis tool that combines the advantages of all the mentioned approaches and overcomes some of the disadvantages. Our tool (MILANO – Microarray Literature-based Annotation) uses an automatic search of literature databases for performing custom annotation of the list of genes obtained from a microarray output. This is done by generating dynamic annotations for genes, built according to terms provided by the researcher. The program receives as input a list of gene identifiers obtained from any microarray experiment and a set of custom search terms. The program expands each gene identifier to its informative synonyms and searches literature databases for co- occurrences of every gene on the list with each of the custom terms. The program's output is an annotation table with the numbers of publications for each gene-term combination (hit-counts). This novel annotation format can be easily used within a web browser or a spreadsheet program to quickly identify genes within the list that are related to the terms provided by the researcher, and may be easily extended, as every hit-count in the annotation is a hyperlink to the query's results. The great advantage achieved by this method over standard static annotations, such as Gene Ontology (GO) annotations, is that the annotations are generated based on terms provided by the researcher, and therefore help in addressing the specific scientific question the researcher is pursuing.The program is able to search two literature databases, GeneRIF  and MedlTo demonstrate the power of this strategy, we have analyzed a list of 148 genes affected by over-expression of p53 . Our anaMILANO is accessed through a familiar web form Fig . A CGI . Although expansion of the search terms is a useful tool to increase the number of articles retrieved for each gene it also adds many irrelevant articles due to the fact that some of the gene symbols are not informative as Medline query terms. For example one of the aliases of the gene aquaporin_1 is CO, a term that is mostly mentioned as an abbreviation for Carbon mono-oxide, and one of the aliases of the gene CD36_antigen is FAT, which is found in over 100,000 articles, unrelated to CD36. In order to diminish this problem we filtered out from the list of gene symbols any term that was shorter than three characters and any term that is an English word. In order to check our name expansion strategy we conducted a Medline search for 16862 well-known human genes , using three search strategies: using only the official symbol for each gene (Symbol), using the official symbol together with all its aliases and the gene product (Expanded) and using only the informative terms (Filtered). Using the Expanded search allowed the identification of literature information on about ~1900 additional genes over a query using the official symbol only (Table One of the major problems in the automation of literature searches is the ambiguity in gene names . MultiplAfter expanding the search terms, MILANO performs an automatic search of literature databases, and retrieves the number of hits each query returned. MILANO performs Boolean searches in which one can search for co-occurrence of each of the primary terms (the expanded gene name) with user defined secondary terms Figure . The proIn order to assist in further evaluation of the results, we have built the annotation table such that each number in the table is a hyper-link to the literature database and thus clicking on it will perform this specific search again and will open a window containing the actual abstracts found by this combination of search terms.The MILANO program can search two databases Figure  – the fuTo demonstrate the power of our literature-based annotation strategy, we analyzed a list of 148 genes affected by over-expression of p53 . This liOur first aim was to identify the known p53 target genes that were affected by p53 overproduction in this experiment. By using specific secondary terms, we were able to trim down the list of 148 genes to a much shorter list of genes highly enriched for known p53 target genes Figure . In ordeP53 is involved in apoptosis, cell cycle arrest and cancer. It is interesting to find out which of the genes affected by p53 is involved in these processes. Using MILANO we easily identified genes known to be involved in these processes Figure , which hRecently, few literature mining tools has been developed, using a similar approach to the one presented here -17, howest and the 100th query in an unbiased manner. The MILANO program uses all the published information for the annotation of each gene according to its co-occurrence in the literature with a user defined secondary search term. These features of MILANO makes it especially suitable for analyzing microarray results, since it can be used to annotate the results with terms defined by the user and not limited by preset terms such as the GO terms based annotation.MILANO is a simple and intuitive literature search tool. It allows automatic Medline and GeneRIF searches followed by a quick survey of the results. Using this tool dramatically reduces the time needed to query literature databases. Moreover, due to its systematic nature, it assists in treating the 1We have demonstrated the power of our program by the analysis of a list of 148 genes that were deregulated in cells that overproduced the p53 tumor suppressor gene . FrequenSeveral literature mining approaches have been developed to integrate multiplex biological datasets into the context of published medical literature. A good example of such an approach is the PubGene program , which sMILANO's aim is to further improve the literature based automatic annotation approach by adding two essential features that address these limitations:Each gene symbol is expanded to all its aliases, while removing non-informative terms, and the gene product name is added to the query. This addresses the synonym problem, while omitting many of the irrelevant results, thus reducing the polysemy problem (words with multiple meanings). The advantage of our synonym expansion scheme over the existing tools is demonstrated by the comparison presented in table In contrast to the existing tools, MILANO is able to search not only the Medline database, but also the GeneRIF database, which contains short summaries of articles relevant to known genes. The curation of GeneRIF is done by the National Library of Medicine's MeSH indexing staff, who have advanced degrees in the life sciences and use the full text of articles for the indexing process . Using t, a literature mining tool that can help in annotating microarray results in light of all available literature using experiment-specific terms. In designing MILANO we focused on the accuracy of the search results by providing two novel features: i) Expansion of gene names to include in the literature searches all their informative synonyms, while removing non-informative synonyms; ii) Searching two literature databases – Medline and GeneRIF. While Medline encompasses all the literature and provides the most comprehensive results, it also contains many irrelevant articles. GeneRIF provides a subset of Medline articles that are relevant to known genes and thus avoids most of the irrelevant results often found in Medline searches.We present MILANO The usefulness of MILANO is demonstrated by the automatic analysis of a list of 148 p53 target genes. The use of literature mining dramatically reduced the time and effort required for a task such as identifying the known p53 target genes within this list. A search in GeneRIF immediately discovered the full list of target genes, with no false hits.. The author will provide data, scripts and programs used on demand. We encourage users to install the software on their own servers, as we provide no assurance to the privacy or accuracy of the results.All software and databases are freely available and may be executed online at our web site: RR designed and programmed the software. IS managed the project and drafted the manuscript.


If there is life on distant worlds, how would we go about finding it? Star Trek that can detect life forms even from orbit. Unfortunately, we don't have a clue how a tricorder might work, since life forms don't seem to have any observable property that distinguishes them from inanimate matter. Furthermore, we lack a definition of life that can guide a search for life outside Earth. How can we find what we can't define? An answer may lie in the observation that life uses a small, discrete set of organic molecules as basic building blocks. On the surface of Europa and in the subsurface of Mars, we can search for alien but analogous patterns in the organics.I need a “tricorder”—the convenient, hand-held device featured on The obvious diversity of life on Earth overlies a fundamental biochemical and genetic similarity. The three main polymers of biology—the nucleic acids, the proteins, and the polysaccarides—are built from 20 amino acids, five nucleotide bases, and a few sugars, respectively. Together with lipids and fatty acids, these are the main constituents of biomass: the hardware of life , p 21. TOur lack of data is reflected in our attempts to define life. The simplest, but not the only, proof of life is to find something that is alive. There are only two properties that can determine if an object is alive: metabolism and motion. (Metabolism is used here to include an organism's life functions, biomass increase, and reproduction.) All living things require some level of metabolism to remain viable against entropy. Movement (either microscopic or macroscopic) in response to stimuli or in the presence of food can be a convincing indicator of a living thing. But both metabolism (fire) and motion (wind) occur in nature in the absence of biology.The practical approach to the search for life is to determine what life needs. The simplest list is probably: energy, carbon, liquid water, and a few other elements such as nitrogen, sulfur, and phosphorus . Life reOrbital images, such as the canyon in On Jupiter's moon Europa, the cracks and icebergs on the surface of the ice indicate water beneath the ice, but not necessarily at the present time. Present water on Europa is indicated by the magnetic disturbance Europa makes as it moves through Jupiter's magnetic field, not unlike the way coins in the pocket of a passenger will set off an airport metal detector. Europa has a large conductor, and this is most likely a global, salty layer of water.The Viking missions to Mars in the late 1970s were the first (and as yet, the only) search for life outside Earth. Each Viking conducted three incubation experiments to detect the presence of metabolism in the Martian soil. Each lander also carried a sophisticated Gas Chromatograph Mass Spectrometer for characterizing organic molecules. The results were unexpected , 1999. TIf considered alone, the Labeled Release results would be a plausible indication for life on Mars. However, the Gas Chromatograph Mass Spectrometer did not detect the presence of any organic molecules in the soil at level of one part per billion . It is dLife on Mars is also likely to be the same at the top of the table: at the ecological level. Primary production in a Martian ecosystem is likely to be phototrophic, using carbon dioxide and water. Heterotrophs are likely to be present to consume the phototrophs and in turn to be consumed by predators. Darwinian evolution would result in many of the same patterns we see in ecosystems on Earth. While it may be similar at the top  and bottom  levels, life on Mars could be quite alien in the middle, in the realm of biochemistry. Future missions to Mars might find microfossils in sedimentary rocks such as those at Meridiani Planum. Microbes don't readily form convincing fossils; the one exception may be the strings of magnetite formed by magnetotactic bacteria . As inteIf we were to find organic material in the subsurface of Mars or on the ice of Europa, how could we determine whether it was the product of a system of biology or merely abiotic, organic material from meteorites or photochemistry? If this life were in fact related to Earth life, this should be easy to determine. We now have very sensitive methods, such as PCR and fluorescent antibody markers, for detecting life like us. This case would be the simplest to determine, but it would also be the least interesting. If the life turned out to be truly alien, then the probes specific to our biology would be unlikely to work. What, then, could we do to determine a biological origin?The question is open and possibly urgent. On space missions already being planned, we may have the opportunity to analyze the remains of alien organics on the surface of Europa or frozen below ground on Mars. The instruments that will make this analysis must be designed in the next couple of years.d- instead of l-amino acids.One approach appears promising. I call it the “Lego Principle.” It is based on the patterns of the molecules of life. Biological processes, in contrast to abiotic mechanisms, do not make use of the range of possible organic molecules. Instead, biology is built from a selected set. Thus, organic molecules that are chemically very similar to each other may have widely different concentrations in a sample of biological organics. An example of this on Earth is the 20 amino acids used in proteins and the use of the left-handed version of these amino acids. The selectivity of biological processes is shown schematically in This approach has immediate practical benefit in the search for biochemistry in the Solar System. Samples of organic material collected from Mars and Europa can be easily tested for the prevalence of one chirality of amino acid over the other. More generally, a complete analysis of the relative concentration of different types of organic molecules might reveal a pattern that is biological even if that pattern does not involve any of the familiar biomolecules. Interestingly, if a sample of organics from Mars or Europa shows a preponderance of D-amino acids, this would be evidence of life, and at the same time would show that this life was distinct from Earth life. This same conclusion would apply to any clearly biological pattern that was distinct from that of Earth life.Organic material of biological origin will eventually lose its distinctive pattern when exposed to heat and other types of radiation, , but at the low temperatures in the Martian permafrost, calculations suggest that there has been no thermal alteration . An inteOn Europa, the organic material for our tests might be collected right from the dark regions on the surface. On Mars, there is ice-rich ground in the cratered southern polar regions , which p


Cercopithecus mitis) make up an abundant, widespread and morphologically diverse species complex in eastern Africa that naturally harbors a distinct simian immunodeficiency virus (SIVsyk). We carried out a retrospective serological survey of SIV infection from both wild and captive Sykes' monkeys from Kenya. We compared two commercially available, cross-reactive ELISA tests using HIV antigens with a novel SIVsyk antigen-specific Western blot assay and analyzed the data by origin, subspecies, age and sex.The Sykes' monkey and related forms . Females were infected more often than males in both wild and captive populations. Seropositive infants were common. However, no seropositive juveniles were identified.We have developed a specific and sensitive Western blot assay for anti-SIVsyk antibody detection. Sykes' monkeys are commonly infected with SIVsyk, but with extremely variable prevalence in the wild. Higher infection prevalence in females suggests predominantly sexual transmission. High infection prevalence in infants, but none in juveniles, suggests maternal antibodies, but little or no vertical transmission. Pan troglodytes troglodytes) and West African sooty mangabey monkeys (Cercocebus atys), respectively [Macaca mulatta) and is pathogenic in that species, producing an acquired immunodeficiency syndrome-like condition [Both human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) have been evolutionarily linked to zoonotic transmissions of naturally occurring simian immunodeficiency viruses (SIVs) from Central African chimpanzees  belongs to an abundant, widespread and morphologically diverse species complex found throughout East Africa [C. mitis have previously been shown to have antibodies that cross-reacted with SIVsmm and SIVagm [The Sykes' monkey  serum samples from the same animals taken at different times. From these sera, the Innotest HIV-1/HIV-2 antibodies test (Innogenetics, Belgium) detected a total of 132 positive samples and 144 negative samples. The Genescreen HIV-1/2 version 2 test (Biorad, Japan). detected 129 positive samples and 147 negative samples. Five samples gave disparate results using these two tests, four of these samples were positive using the Innotest kit, but negative using the Genscreen test and one sample was positive using the Genscreen test, but negative using the Innotest kit.Two hundred and seventy six serum samples from both wild and captive To confirm these results and compare the sensitivity and specificity of these assays, all 276 samples were screened by an SIVsyk viral antigen-specific Western blot assay. Serum samples were considered positive by SIVsyk Western blot assay if they showed reactivity to ENV, GAG or CA proteins. The SIVsyk Western assay identified a total of 138 samples as positive and 138 as negative. Ten samples that were negative by the Genscreen assay and seven samples that were negative by the Innotest assay were identified as positive by the SIVsyk Western assay . This includes both sexes and all age classes. Ten of 17 captive adult highland Sykes' and 3 of 4 captive adult lowland Sykes' were positive, yielding prevalences of 59.0% and 75.0%, respectively for these two subspecies. No known captive Blue Sykes' were available for testing. Among captive adult animals, 4 of 8 (50.0%) males and 9 of 13 (69.2%) females were positive. Four of 16 (25.0%) captive infant males and 7 of 19 (36.8%) captive infant females were positive. However, no captive juveniles (0 of 6 (0.0%) males and 0 of 5 (0.0%) females) were positive. Tables 4/67. ThiThe overall captive seroprevalence was 35.8%, which is slightly lower than the overall wild seroprevalence of 46.8%. Infection prevalence was highest in adults at 69.8% for wild populations and 61.9% in captive populations. Adult females were infected at a higher prevalence than adult males in both the wild (74.1% (20/27) for wild adult females and 65.4% (17/26) for wild adult males) and captive populations (69.2% (9/13) for captive adult females and 50.0% (4/8) for captive adult males). Infants were also found to have a high seroprevalence at 66.7% (2/3) from wild populations and 31.4% (11/35) from captivity, although few wild infants were surveyed. No seropositive juveniles were identified from either wild (0/3) or captive (0/11) populations. A Fisher's exact (2 × 2) contingency test for sex-related differences in the combined wild and captive populations yielded a p value of 0.455. This indicates that while the there is a clear trend in each data set showing higher prevalence among females, the small numbers of samples do not show a statistically significant difference between infection in males and females. An exact 3 × 2 contingency test for age-related differences in the combined wild and captive populations yielded a p value of 0.00001, indicating that age-related differences in the combined populations are highly statistically significant.Cercopithecus mitis have used assays relying on cross-reactivity between antibodies against SIVsyk and other HIVs or SIVs. To assess the relative effectiveness of the use of cross-reactive tests, we compared serological results using two commercially available HIV-1/HIV-2 ELISA kits with an authentic SIVsyk antigen Western blot assay. The cross-reactive ELISA assays were very specific and in only one out of 276 samples gave a false positive result compared to the SIVsyk antigen Western assay. Curiously, this one particular captive juvenile female was repeatedly positive by both ELISAs but repeatedly negative by Western. This animal may possibly have been infected in captivity with a different SIV that produces cross-reactive antibodies to HIV-1 and HIV-2, but not to SIVsyk. On the other hand, the cross-reactive ELISAs failed to detect between 4.2 to 6.1% of the animals that were positive by the SIVsyk antigen Western assay. This result may be due to increased sensitivity using a Western blot format, but is also possibly due to increased sensitivity to the authentic SIVsyk viral antigens.Previous SIV serosurveys of Variable SIV infection prevalences have previously been reported in this species. However, information on age, sex and captive or wild status was not provided in any previous survey. Lowenstine et al, 1986  found noTo avoid age-related bias, a comparison between captive and wild populations should be made using only adult animals because the captive population contained many more samples from infants and juveniles than did the wild population  includes coastal and eastern regions of Kenya [C. mitis kolbi Neumann) includes the upland central portion of the country [C. mitis stuhlmannii Matschie) includes western Kenya and the border with Uganda [The sera used in this study were obtained between 1981 and 1996 from animals that had been previously trapped in the wild or from previously sampled captive animals housed at the Institute of Primate Research in Nairobi, Kenya. Serum samples were stored continuously at -20°C. All animals were captured, housed and sampled in accordance with humane animal use guidelines established by the Kenya Institute of Primate Research. Wild born monkey samples used in this study were derived from eight locations. In general, these sites were rural or suburban areas that bordered forested areas with natural flowing water. These sites are typical of areas in which humans have encroached on natural habitats and now may come in contact with these monkeys as they forage for food. The geography of the sites varied considerably from heavily forested areas in the highlands of Kenya to drier forests along the lowland coast of Kenya. The range of the lowland Sykes' (of Kenya ,13. The  country ,13. The h Uganda ,13. SomeSerum samples were tested using the Innotest HIV-1/HIV-2 Antibodies test (Innogenetics, Ghent, Belgium) and Genscreen HIV1/2 version 2 (Biorad, Tokyo, Japan). The Innotest ELISA kit uses synthetic peptides representing HIV-1 subtype M envelope, HIV-2 envelope, and HIV-1 group O envelope antigens. The Genscreen uses purified gp160 envelope and p25 nucleocapsid recombinant protein of HIV-1 and a peptide that mimics the immunodominant epitope of HIV-2 envelope. Each kit was used according to the manufacturers' recommendations. Photometric measurements were performed with a Dynatech MRX plate reader at 450 and 650 nm wavelengths. Positive and negative determinations were based upon cutoff values as determined by the manufacturers' protocols.CD4+ CEMx174 cells were transduced with a CCR5-encoding vector (pBABE-puro-CCR5, a gift of David Dorsky, University of Connecticut) as previously described . The resSerum samples were screened by Western blot using SIVsyk-specific viral antigens. SIVsyk-1.2 infected cells were centrifuged for 5 min at 1,000 × g and resuspended in lysis buffer containing 1% triton, 50 mM tris pH 7.5, 2 mM EDTA. Cell debris was pelleted for 2 min. at 12,000 × g and the supernatant was added to loading buffer containing 50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromophenol blue. Viral antigens were separated on a 12% polyacrylamide gel and subsequently transferred to a PVDF membrane. Membrane strips were blocked with a solution of PBS-T containing 5% dry milk. Serum samples were diluted 1:500 and incubated for one hour at room temperature with individual membrane strips. Primary serum antibodies were detected with a 1:4,000 dilution of goat-anti-rhesus HRP conjugated secondary antibody (Nordic Immunology, Tilburg, Netherlands). Colorimetric detection was performed using the Immunopure Metal Enhanced DAB Substrate Kit (Pierce Endogen, USA).The author(s) declare that the have no competing interests.BRE and EM conducted the ELISA and Western blot experiments and BRE wrote the draft manuscript. DE and JR cultured the SIVsyk virus. MGO provided serum samples and oversaw the ELISA experiments. SFM assisted with and oversaw the Western blot experiments and prepared the final manuscript. All authors read and approved the final manuscript.