The recently created professional science master's degree may be the answer to the increasing need for science-savvy employees in the business world Sloan Foundation to cultivate science managers. Sloan's ultimate goal is to make science careers more attractive to talented young people like Herzer, a 2003 PSM graduate in Applied Biosciences from the University of Arizona, who believes his PSM makes him more marketable to science-based businesses. “I am at the true junction of science and business,” he says.Fortunately for Herzer, the business of science is booming. Jobs for scientists and engineers grew four times faster than the United States national average since 1980, and should outpace the market until at least 2010. Surprisingly to many academics, most of these jobs are in industry. In 1999, the last year with complete data, two out of three employed science and engineering (S&E) graduates worked in industry, including the great majority of bachelor's and master's degree holders, and 40% of doctorates. In other words, industry, not academe, now drives American S&E employment, and will for the near future.Like academia, industry needs scientifically literate personnel; unlike academia, industry wants employees with business savvy as well. However, in the past, graduate students received either science or business instruction, not both. “Industry simply hired regular master's-degreed people, or MBAs, or more likely PhDs, and just expected them to learn their weaknesses on the job,” says Eleanor L. Babco, Executive Director of the Commission on Professionals in Science and Technology, a nonprofit corporation with funding from the Sloan Foundation to assess PSM graduates.For science-based businesses, then, the American S&E doctorate—viewed by many as the worldwide gold standard for science education—is too specialized for their needs see . But a mThe length of time to obtain a biological science doctorate has increased……The number of postdocs and part-time faculty in the biological sciences is increasing……And the proportion of doctorates in academia is decreasing…http://www.nsf.gov/sbe/srs/seind04/] and the National Science Foundation Science Resources Statistics Division's “1995 and 2001 Survey of Doctorate Recipients” [http://www.nsf.gov/sbe/srs/infbrief/nsf04328/table1.xls].) program.Like all graduate programs, PSMs offer advanced coursework in a (science or math) specialization, usually in an emerging or hybrid field such as bioinformatics. Most PSMs also provide business courses—including finance, project management, regulatory affairs, and intellectual property law—and information technology classes as well. PSMs are “industry relevant” by design, with external advisory committees populated by local business leaders, weekly colloquia led by corporate representatives, special arrangements for employed students, and industry internships or final projects exploring realistic business scenarios see .Only programs meeting most of the following requirements may earn the official moniker “professional science master's.”Two years of science or math graduate-level coursework, taught by regular faculty, characterized by interdisciplinary studies and a focus on informaticsTraining in business fundamentals—such as finance, marketing, project management, communication, and team building—and exposure to industry professionalsFinal project reflecting a realistic workplace issue and/or industry internshipAdvisory board of industry professionalsTargeted recruitment and admissions separate from other degree programsCommitment to tracking graduates through first five yearsLong-term sustainability http://www.sciencemasters.com/affiliation.html.)Wall Street Journal, “Harvard tries to create leadership in industry, academics and government, and our philosophy is we don't think that with a master's degree people can fill that role very easily.”Although most scientific careers demand a graduate degree, a professional master's in many hard sciences still encounters entrenched academic opposition. According to Lee-Jen Wei, then acting chair of the Department of Biostatistics in the Harvard School of Public Health, quoted in the The government appears to agree with this view. While most doctoral candidates receive federal funds for tuition and other expenses, there is little money for master's students, who disproportionately end up in industry regardless of specialization. PSM students are especially affected by this problem because “interdisciplinary” equals “expensive.” Similarly, “interdisciplinary” can also mean “hard to find”—companies with targeted recruitment often miss PSM students, who are not “in” any particular department—and “confusing”—differences in these new, still somewhat vaguely defined programs can make hiring comparisons difficult. But perhaps the most conspicuous drawback to PSMs is their newness, and resulting obscurity: almost half of graduates say they are “not sure” employers will value their PSM, or the unique skill set it affords.Professionalizing Graduate Education: The Master's Degree in the Marketplace, argues that converging market forces will lead to the success of the professional master's degree in science. These forces include: rapid technological change; the rise of alternative learning channels such as online and distance education, corporate universities, and hi-tech certification programs; the proliferation of degrees in general, and in multidisciplinary fields specifically; and a fundamental societal shift away from public service and toward entrepreneurship, profitability, and competition.Still, many observers of higher education support the PSM concept. Judith Glazer-Raymo, author of the forthcoming book Kenneth R. Smith, former dean of the Eller College of Business and Public Administration at the University of Arizona, and others make the case that PSMs may protect students' careers from outsourcing to foreign countries. The American S&E labor pool is shrinking, and industry has already responded by transferring much of its research and development overseas; however, companies are mostly moving lab scientists, not strategic analysts. Cross-training in both science and business could thus provide an edge for domestic workers in the near-term employment environment; in fact, PSM programs have a higher proportion of US citizens and residents than S&E doctoral programs.Further, in its 2003 report, the National Science Board urged the government to better align S&E graduate education with “expected national skill needs,” including “interdisciplinary skills.” The report also recommended federal funding for a wider range of educational options and more attention on the real economic concerns of students—code words for support of professional master's degree initiatives. In the same vein, top universities now advocate “interconnections” between their professional schools and traditional departments, as a way of strengthening the overall academic mission, and many countries are sponsoring initiatives to stimulate university–industry links, to maximize marketing of technological innovations.For advocates, then, the PSM both advances the cause of science education reform and addresses changing employment conditions with one big idea: reinvention of the two-year graduate credential for an entrepreneurial age. Herzer, for one, now a technology development representative at the Scripps Research Institute, has staked his future on the potential of professional master's degrees. “Scientists rarely understand business dealings, and business personnel rarely comprehend scientific discoveries,” he says. “The overlay of the two is crucial for any successful business transaction of scientific origins.”


EIF2B2, 4 and 5.Premature Ovarian Failure (POF), defined as the development of hypergonadotropic amenorrhea before the age of 40 years, occurs in about 1% of all women. Other than karyotype abnormalities, very few genes are known to be associated with this ovarian dysfunction. Recently, in seven patients who presented with POF and white matter abnormalities on MRI (ovarioleukodystrophy) eight mutationswere found in EIF2B genes in POF, we screened 93 patients with POF who did not have identified leukodystrophy or neurological symptoms. We evaluated these eight mutations and two additional mutations that had been found in patients with milder forms of eIF2B-related disorders. We used restriction enzymes and direct sequencing.To further test the involvement of known mutations of EIF2B genes, either homozygous or heterozygous, were identified in our 93 patients with pure 46,XX POF. The upper 95 % confidence limit of the proportion 0/93 is 3.2%.None of the known mutations in We conclude that eIF2B mutations, already described in cases of POF associated with white matter abnormalities, are an uncommon cause of pure spontaneous premature ovarian failure. Premature Ovarian Failure (POF) can present as a primary or secondary amenorrhea and is associated with elevated gonadotropins before 40 years of age. POF affects 1% of all women and occurs in 0.1% before the age of 30 years . While cEIF2B genes (EIF2B2, 4 and 5) were reportedly involved in seven patients who presented with POF and white matter abnormalities on MRI (ovarioleukodystrophy) [spectrum can range from a rapid course leading to death in severe congenital forms to asymptomatic MRI findings in adult patients [EIF2B genes.Recently three of the five strophy) . These gpatients ,11. Ovarpatients . TherefoEIF2B mutations in 93 unrelated and well-characterized women with POF. An institutional review board approved the study and all participants gave a written informed consent. Referring physicians made the diagnosis of premature ovarian failure based on the following criteria: development of at least 4 months of amenorrhea before age 40 associated with two serum FSH levels in the menopausal range. Women with premature ovarian failure as a result of surgery, radiation, chemotherapy, or known karyotype abnormalities were not included in the study. There were 6 Asians, 12 Blacks, 4 Hispanics and 71 Caucasians. The median age at the onset of menstrual irregularity was 24.5 years (range 13 to 39). Eighteen women had a family history of POF. All women underwent a history and physical examination and laboratory screening to confirm the diagnosis of POF and all had a normal karyotype. None of the women had evidence of a neurological disorder.In the current study, we evaluated the presence of Genomic DNA was extracted from peripheral blood using standard procedures.EIF2B2, 4 and 5 which contain mutations found in POF patients or in milder forms of eIF2B-related disorders were amplified by the polymerase chain reaction (PCR) as previously described  mutation in the escribed .EIF2B4) described in milder forms of eIF2B-related disorders were not found in this series of 93 patients with POF. The upper 95 % confidence limit of the proportion 0/93 is 3.2%.None of the eight mutations already described in ovarioleukodystrophy were detected in our 93 patients with pure 46,XX POF, neither in a homozygous nor in a heterozygous state. In addition, the mutations C728T and C1120T , 2 to 5 years (group 2) and > 5 years (group 3) . Group 3In these six eIF2B-mutated families, neurological symptoms with abnormalities of the cerebral white matter on MRI were associated with primary or secondary amenorrhea due to POF . A correEIF2B mutations. In the present study, we tested for EIF2B mutations a series of 93 patients with pure, karyotypically normal POF without identified signs of cerebral dysfunction.The mutated eIF2B may be responsible for both increased apoptosis of ovarian follicles leading to POF, and a defect in glial cell development causing abnormal formation of white matter structures. In ovarioleukodystrophy, a phase of amenorrhea without neurological symptoms can be observed, suggesting that an apparently isolated case of POF might be due to EIF2B5 gene) and A638G (EIF2B2 gene) are found in 71% of families with late onset forms of eIF2B-related disorders (group 3) [EIF2B mutations in women with POF who have no apparent neurological signs.In eIF2B-related disorders, a correlation exists between genotype and disease onset . The mutgroup 3) . In ovarEIF2B mutations is not clinically indicated.For patients presenting with POF without neurological signs or MRI abnormalities, the routine screening of the The author(s) declare that they have no competing interests.AF and FGB carried out the molecular genetics studies, including enzyme restrictions (AF) and sequencing (AF and FGB). AF drafted and conceived of the study. RS participated in the coordination of the study. VHV, VKB, LMN recruited and evaluated the patients, collected DNA samples, participated in the design and coordination of the study, and helped in drafting the manuscript. OBT conceived of the study, and participated in its design and coordination. All authors participated in the writing of the manuscript and have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Drosophila melanogaster genes involved in hybrid incompatibility have been identified but none has yet been shown to have functionally diverged in accordance with the Dobzhansky-Muller model. By introducing transgenic copies of the X-linked Hybrid male rescue (Hmr) gene into D. melanogaster from its sibling species D. simulans and D. mauritiana, we demonstrate that Hmr has functionally diverged to cause F1 hybrid incompatibility between these species. Consistent with the Dobzhansky-Muller model, we find that Hmr has diverged extensively in the D. melanogaster lineage, but we also find extensive divergence in the sibling-species lineage. Together, these findings implicate over 13% of the amino acids encoded by Hmr as candidates for causing hybrid incompatibility. The exceptional level of divergence at Hmr cannot be explained by neutral processes because we use phylogenetic methods and population genetic analyses to show that the elevated amino-acid divergence in both lineages is due to positive selection in the distant past—at least one million generations ago. Our findings suggest that multiple substitutions driven by natural selection may be a general phenomenon required to generate hybrid incompatibility alleles.Interspecific hybrid lethality and sterility are a consequence of divergent evolution between species and serve to maintain the discrete identities of species. The evolution of hybrid incompatibilities has been described in widely accepted models by Dobzhansky and Muller where lineage-specific functional divergence is the essential characteristic of hybrid incompatibility genes. Experimentally tractable models are required to identify and test candidate hybrid incompatibility genes. Several  Transgenic experiments show that the HMR gene has functionally diverged in Drosophila melanogaster and its sibling species and causes the death of hybrid offspring in interspecific crosses A from one species causes the incompatibility while the ancestral allele a from the other species does not.Reproductive isolation is the most commonly used criterion to define species. Hybrid incompatibilities (HIs) such as hybrid sterility and lethality are widely observed examples of reproductive isolation. The Dobzhansky-Muller (D-M) model explains how the genes causing deleterious phenotypes in hybrids can evolve  Figure A. The moA (or X) but not allele a (or x) will affect the HI phenotype. For example, increasing the dosage or activity of allele A should decrease hybrid fitness, while identical manipulations of allele a should not. In contrast, downstream effector genes such as d1 in Drosophila melanogaster.This model makes clear and testable genetic predictions, namely, that experimental manipulation of allele If the genetic changes that cause HI are rare, then genes that have undergone extensive divergence may be more likely to cause HI simply because there is more chance that they have experienced rare HI-causing mutations. But the D-M model itself offers no suggestions about the mode of evolution that leads to this divergence. One possibility is that HI genes accumulate genetic changes over time as they evolve neutrally. Alternatively, many models have suggested that speciation may be driven by molecules that are undergoing natural selection, for example, under the pressure of ecological divergence  or sexuaD. melanogaster can hybridize with three closely related species that we refer to collectively as its sibling species: D. simulans, D. mauritiana, and D. sechellia. Crosses between D. melanogaster females and sibling-species males result in invariantly lethal hybrid sons and temperature-dependent lethal hybrid daughters  gene has a major effect on the fitness of hybrids from this cross. This point is most strikingly demonstrated by the fact that D. melanogaster Hmr loss-of-function mutations such as 1Hmr suppress the lethality of both hybrid males and females (In(1)AB rescue mutation  from the sibling species. Hmr gene, which encodes a predicted DNA-binding protein similar to the ADF and MYB family of transcriptional regulators, and proposed that D. melanogaster HI may be caused by transcriptional misregulation.aughters . The Hyb females , as doesmutation . Increasiability . These sHmr is that all genetic manipulations were done only with the D. melanogaster allele. These studies therefore cannot determine whether Hmr is an HI gene as modeled by Dobzhansky and Muller  sites, a remarkable finding considering that Hmr encodes a predicted protein over 1,400 amino acids in length. Two other Drosophila genes that are involved in HI show elevated divergence that appears to have evolved under positive selection, but the divergence is confined to a small region of each gene AB mutations (+D. melanogaster Hmr kills hybrids, with the 1Hmr and In(1)AB mutations rescuing hybrids by reducing or eliminating +Hmr activity. The transgenic copies of +D. melanogaster Hmr thus suppress rescue by increasing +Hmr activity in hybrids. We therefore asked whether or not sibling-species +Hmr has the same activity. We reasoned that if Hmr is functionally diverged between the species, as in +Hmr would not have the property of suppressing hybrid rescue. On the other hand, if Hmr is not functionally diverged, as in +D. melanogaster Hmr transgenes and suppress hybrid rescue.In the absence of available nogaster . It has utations . Those e+D. melanogaster Hmr transgenic construct and that exceed the minimal +Hmr region previously defined AB in D. melanogaster/D. simulans hybrids AB-dependent rescue of hybrid female sterility (+D. melanogaster Hmr transgenes (+D. simulans Hmr transgenes had little or no effect on egg counts in D. melanogaster/D. simulans hybrids. sim,w mIn(1)AB,w/X females heterozygous for the insertion +t8.6}2-4P{Dsim\Hmr averaged 10.0 ± 8.9 eggs (n = 41) while their non-transgene-carrying sisters averaged 8.0 ± 12.4 eggs (n = 28). Using a second, independent transgenic line, we found that sim,wIn(1)AB,w/X females heterozygous for the insertion +t8.6}4-1P{Dsim\Hmr had 12.2 ± 13.7 eggs (n = 24) while their non-transgene-carrying sisters had 17.7 ± 17.3 eggs (n = 31).To maximize the chances that the sibling-species transgenic lines would function normally, we made constructs that are similar to the largest ined see A. We ass hybrids . In all terility . In contansgenes , we foun+Hmr transgenes are expressed  site N)(D between D. melanogaster and its three sibling species (Hmr between D. melanogaster and D. simulans and compared it to compiled datasets containing over 250 genes from these two species (Hmr has one of the highest levels of nonsynonymous divergence (0.089); only four other non–Accessory gland protein (Acp) genes have a higher nonsynonymous divergence, and two of these include expressed sequence tag comparisons less than 350 bases in length. The remaining two loci, both X-linked, are mei-218 and Odysseus. In contrast, the average divergence per synonymous site (Ds) of 0.110 for Hmr is lower than the mean value of 0.125 for this dataset (again excluding Accessory gland protein genes).It has been shown previously that  species . To unde species . PairwisHmr raises a number of important questions: (1) Is divergence high on the lineage of either D. melanogaster or its sibling species, or both? (2) Was the divergence on either or both lineages caused by positive selection consistent with the time scale of speciation? (3) Can we identify specific regions and codons subject to positive selection on these lineages, and how does this compare to the few other known candidate speciation genes? (4) Is divergence on one or both lineages potentially responsible for causing the HI phenotype of Hmr?The exceptional divergence of Hmr began to diverge rapidly after the D. melanogaster–sibling-species divergence by isolating and assembling an outgroup Hmr orthologous sequence from the D. melanogaster subgroup species D. erecta, which is estimated to have diverged from D. melanogaster between 6 and 15 million years ago , then one would expect that the ratio of nonsynonymous to synonymous divergence between species would be similar to the ratio of nonsynonymous to synonymous polymorphisms found within these species (π and synonymous S) and D. simulans  relative to other loci . This significant variation across the locus can be further refined by examining the ratio of nonsynonymous to synonymous variation across the locus and is visually displayed for D. melanogaster and D. simulans using sliding window analyses in ND/SD ratios and low Nπ/Sπ ratios, in other words, much more amino-acid divergence than polymorphism. These contrasting ratios do not suggest that regional variation in substitution rates among the two classes of sites is due solely to mutation (and drift) but rather suggest that selection is contributing to the divergence pattern across the gene. Although this sliding window plot is highly suggestive of selection, we wished to obtain independent and statistically supported evidence for regional selection. We therefore tested rates of divergence relative to polymorphism for each exon in a manner similar to the McDonald-Kreitman test (MK test) described in the previous section. The data were partitioned a priori using each exon as a unit (rather than picking an arbitrary window size). Given the size of the sampled region, there is considerable power to address regional variation. These MK tests for each exon revealed that the fourth and fifth exons of Hmr appear to contribute the most to the overall MK test . It is interesting to note that these regions are not homologous to any other proteins known in D. melanogaster (or in any other species), including other MADF domain–containing proteins. We conclude that multiple regions of Hmr show strong evidence for positive selection.Polymorphism and divergence vary substantially for the separate exons and the DNA binding domains. In contrast to the neutral expectation, we found that there is highly significant heterogeneity in the ratio of polymorphism to divergence across the five exons of Hmr locus appear to have evolved rapidly since D. melanogaster and the sibling species diverged, relative to both synonymous coding sites and introns. The ratios of divergence to polymorphism between D. melanogaster and D. simulans for the 5′ and 3′ flanking regions combined (including the UTRs) are significantly different from those of the coding and intron regions  . These observations suggest that adaptive fixations have occurred at a number of noncoding sites.We also observed that the flanking regions of the  0.0001) , but thiHmr might offer a unique opportunity to apply these approaches to identify codons that exhibit positive selection and that could be contributing to HI. We identified 25 amino-acid positions that may have diverged due to selection, 20 of which fit possible models for the evolution of the D. melanogaster–sibling-species incompatibility . Based on these values we expected to find approximately three to four shared polymorphic sites for Hmr among the 54 segregating sites in D. melanogaster and 94 segregating sites in D. simulans, but instead found zero. Although the expectation of three to four shared polymorphisms is not a strong test, these findings are consistent with Hmr alleles not having been exchanged between these taxa since at or near the time of species divergence and/or with selection events having occurred throughout the Hmr gene and having removed recurrent mutations.Loci directly involved in reproductive isolation may reflect the true species history of divergence more accurately than a “random” locus sampled from the genome, because these loci cease exchanging alleles among species earlier than other loci . Both reears ago , and manears ago . Howeverears ago , we founHmr is due to positive selection. These findings raise the critical question of whether selection events occurred recently or at the time of speciation. The number of years separating D. melanogaster and the sibling-species lineage makes this a challenging task. We therefore concentrated on the more tractable problem of looking for evidence to exclude the possibility that Hmr divergence reflects the action of contemporary or recent selection. In other words, while it may not be possible to prove that selection occurred at Hmr at the time of speciation, it is nevertheless important to demonstrate that available data are, at the least, not inconsistent with more ancient selective events. Examination of the frequency spectrum of polymorphism within both D. melanogaster and D. simulans showed no evidence of recent sweeps, as the distribution of per-site heterozygosities did not differ from expectations of neutrality (Tajima's D is −0.61 and 0.75, respectively, which is not significant). However, the power for detecting a selective sweep with this method is not high (75%) even if the event occurred in the very recent past, and diminishes to 25% as early as 0.8N generations ago (where N= population size) (Hmr, we reasoned that we could apply more powerful Bayesian approaches that use not only frequency spectrum information but other summary statistics of polymorphism and recombination to estimate the time at which these events happened (X-linked locus is on average 3N generations ago. We determined that in D. melanogaster, a recent selective sweep (within the last 0.25N generations) is clearly not consistent with the polymorphism data, whereas adaptive mutations occurring more than N generations ago are consistent with the polymorphism data, and selective sweeps more than 1.5N generations ago are possible  . Becausehappened . To estipossible . Similarvolution  but haveD. simulans or +D. mauritiana Hmr have no effect on hybrid fitness, in contrast to the strong deleterious effects previously observed with +.D. melanogaster Hmr These transgenic experiments demonstrate that Hmr has functionally diverged between D. melanogaster and its sibling species and thus meets the experimental criteria defined by the D-M model. To our knowledge this is the first demonstration of functional divergence for a D. melanogaster HI gene. Although transgenic technologies have limitations—for example, genes with large and complex regulatory regions may not function correctly when transformed—they have the clear advantage of providing unambiguous analyses of single genes. Transgenics was previously used  is a gene duplication that is present in X. maculatus but appears to be absent from X. helleri). Functional divergence can also be inferred in other HI systems where multigene regions are transferred between species by repeated backcrossing. This approach has been used to show that Odysseus (Ods) has functionally diverged: X-chromosome introgressions from D. mauritiana into D. simulans that contain the D. mauritiana Ods region are sterile, while related introgressions that lack D. mauritiana Ods remain fertile (We have shown here that transgenes carrying sly used  to show Weis and ). Functi fertile .Hmr and Ods, as well as Nup96, which has recently been implicated in causing lethality in D. melanogaster/D. simulans male hybrids ((Ods and Nup96) or most (Hmr) of their coding regions. If generally true, this finding may reflect the fact that a high rate of substitution is required in order to generate a rare HI-causing mutation. Alternatively, it may be an indication that the combined effects of multiple substitutions are required to generate an HI allele. Our analysis of Hmr suggests two additional characteristics of HI genes. One is that while the D-M model requires divergence along only one lineage in order to generate HI, we have strong statistical support for accelerated divergence of Hmr along both lineages since D. melanogaster and the sibling species split.Both  hybrids , show hiHmr that are candidates for causing the incompatibility. The simple model shown in melHmr and sib,A implies that any codons in melHmr that have diverged from the ancestral allele could potentially be causing the incompatibility; these codons could also diverge in the sibling-species alleles as long as they remain different from the D. melanogaster allele. There are 137 amino acids in Hmr (plus two sites of D. melanogaster–specific insertions) that fit these criteria. However, considering the extensive divergence we have detected at Hmr along both lineages, a more realistic model is shown in Hmr and gene A go through multiple changes in both D. melanogaster and the sibling species. This model raises a possibility described by sibA may become incompatible with anc,Hmr which means that melHmr will also be incompatible if it retains the interacting residues present in anc.Hmr Since sibHmr must be compatible with sib,A the candidate codons are those where sibHmr differs from ancHmr and mel,Hmr and melHmr remains identical to anc.Hmr There are 49 amino acids that fit this model, all of which are different from those identified in the previous model. Distinguishing between these models may be possible by using our transgene assays on +Hmr constructs containing site-directed changes at candidate amino acids.One consequence of this extensive divergence is that it becomes unexpectedly complicated to identify the divergent codons in Hmr as well as Ods and Nup96. Adaptive fixations and reproductive isolation at Hmr have clearly shaped the pattern of polymorphism relative to divergence and swept away any shared polymorphisms that may have been present. This characteristic raises the general question of what forms of selection are responsible and whether genes involved in certain traits or phenotypes that are under strong directional selection may preferentially contribute to HI. In allopatric models, where speciation occurs between two populations in geographical isolation, HI between species is strictly a secondary consequence of divergence of HI genes that has occurred within each species. The target of selection must therefore be sought by looking at gene function within species. We do not yet know the function of 1Hmr; Hmr mutants are viable and fertile, but this allele is clearly hypomorphic  ancestor, respectively.The subscripts RT-PCR was performed from total RNA as described in Hmr constructs were derived from D. simulans and D. mauritiana lambda genomic libraries  to make the p94 plasmid. An approximately 9.4-kb NotI/XbaI fragment of the p94 insert was cloned into the corresponding sites of pCasper4. End sequencing of the insert demonstrated that this construct extends from approximately 2.8 kb 5′ of Hmr to approximately 1.8 kb 3′ of Hmr. This construct also contains the complete predicted coding regions of Rab9D and CG2124. The formal designation of this construct is +mc Dmau\Rab9D+t9.4 Dmau\Hmr+t9.4 Dmau\CG2124+t9.4=Dmau\Hmr+t9.4}.P{wA D. erecta Hmr was isolated by screening a gridded fosmid library  with D. melanogaster Hmr cDNA clone RE54143 as a probe. An approximately 8-kb XbaI fragment was subcloned from fosmid 11D-19 into pBSII KS(+) and sequenced using a GPS-1 Genome Priming System (New England Biolabs, Beverly, Massachusetts, United States) and custom sequencing primers.Hmr ortholog from the more distant D. pseudoobscura, but an unalignable repetitive region in the second exon made unambiguous calculations of amino-acid and silent divergence impossible.We also identified an D. melanogaster, the ortholog of CG2124 is located 5′ of Hmr in opposite orientation in both D. erecta and D. pseudoobscura, demonstrating that we have correctly identified the ortholog of Hmr in both of these species.As is found in D. melanogaster Hmr alleles were sequenced from a collection of iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe . We also included in our analyses four D. melanogaster alleles and one each from the three sibling species reported previously , Hmr coding region from D. mauritiana contained in plasmid p94 (AY573924), Hmr coding region from D. simulans contained in plasmid p92 (AY568391), D. melanogaster Hmr alleles sequenced from iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe (AY568380–AY568389), and D. simulans Hmr alleles sequenced from highly inbred isofemale lines collected in Wolfskill, California, United States (AY568392–AY568396).The GenBank accession numbers of the genes discussed in this paper are


Peach is being developed as a model organism for Rosaceae, an economically important family that includes fruits and ornamental plants such as apple, pear, strawberry, cherry, almond and rose. The genomics and genetics data of peach can play a significant role in the gene discovery and the genetic understanding of related species. The effective utilization of these peach resources, however, requires the development of an integrated and centralized database with associated analysis tools.The Genome Database for Rosaceae (GDR) is a curated and integrated web-based relational database. GDR contains comprehensive data of the genetically anchored peach physical map, an annotated peach EST database, Rosaceae maps and markers and all publicly available Rosaceae sequences. Annotations of ESTs include contig assembly, putative function, simple sequence repeats, and anchored position to the peach physical map where applicable. Our integrated map viewer provides graphical interface to the genetic, transcriptome and physical mapping information. ESTs, BACs and markers can be queried by various categories and the search result sites are linked to the integrated map viewer or to the WebFPC physical map sites. In addition to browsing and querying the database, users can compare their sequences with the annotated GDR sequences via a dedicated sequence similarity server running either the BLAST or FASTA algorithm. To demonstrate the utility of the integrated and fully annotated database and analysis tools, we describe a case study where we anchored Rosaceae sequences to the peach physical and genetic map by sequence similarity..The GDR has been initiated to meet the major deficiency in Rosaceae genomics and genetics research, namely a centralized web database and bioinformatics tools for data storage, analysis and exchange. GDR can be accessed at  Malus), pear (Pyrus), raspberries/blackberries (Rubus), strawberries (Fragaria), and stone fruits (Prunus) such as peach/nectarine, apricot, plum, cherry and almond [Arabidopsis and other characteristics: a relatively short juvenile period (2–3 yrs) and extensive genetics and genomics resources such as molecular marker maps, interesting mutants and clone library resources [In the United States and temperate regions throughout the world, the family Rosaceae ranks third in economic importance. The most important fruit producing crops include apple  has been initiated. The goals of the GDR are (1) to develop an organized and integrated web resource for peach genomics data to facilitate the gene discovery in other member species by a comparative mapping approach, (2) to collect and integrate all Rosaceae genomics data, (3) to develop online tools and resources for the Rosaceae community. In this paper, we describe the structure and content of the database, we review the database access utility and tools and we report a case study where we mapped publicly available Rosaceae sequences to the peach physical and genetic maps by sequence similarity.The volume and the complexity of the data being produced by these peach genomics projects, in addition to the rapidly accumulating genomics and genetics data for other important rosaceous species, necessitate the development of a properly curated and integrated scientific database. Such a database will help scientists to efficiently access, analyze, integrate and apply the data to their own research in a timely manner. RoseDB, which focused on apple genetics and cherry genomics, has been decommissioned and now only exists as a mirror site at INRA. To meet the major need for a centralized and integrated web-database for genomics and genetics research in Rosaceae, the Genome Database for Rosaceae  and the singletons that have either unique protein matches or no known significant matches. Peach ESTs were further annotated by Gene Ontology (GO) assignment based on the single "best hit" match against the SWISS-PROT database. Of the 1552 sequences from the putative peach unigene set that had matches with the SWISS-PROT database, 1439 could tentatively be assigned GO classifications. Additional sequence annotation includes computational analysis for simple sequence repeats (SSR) and open reading frame (ORF) on both the filtered clone library and the contig library. SSR analysis was preformed using a modified version (CUGISSR) of a Perl script SSRIT [. FLIP is a UNIX C program that finds/translates ORFs (open reading frames) in sequences. Using the FLIP output, CUGISSR selects the longest ORF as the putative coding region and reports the location of SSRs in relation to the putative coding region.The peach and almond ESTs are being processed at the Clemson University Genomics Institute (CUGI) utilizing publicly available software, integrated in a fully automated in-house developed script (CUGIEST). The processing occurs in three stages: trace file processing to identify a filtered, high quality clone library, assembly of high quality sequences to produce longer transcripts and reduce redundancy, and sequence annotation. Annotation consists of pairwise comparison of both the filtered clone library and the EST contig consensus sequences against the GenBank nr protein database using the fastx3.4 algorithm . The tenpt SSRIT  with parpt SSRIT  program -9) following a monthly homology search of GenBank nr protein database using the fastx34 algorithm.In addition to the peach and almond ESTs processed by CUGI, all the publicly available Rosaceae EST data are daily downloaded from GenBank dbEST and annotated with the top ten most significant matches . The SVG viewer plug-in can be freely downloaded from the Adobe Website  and the system requirements can be found at their website . Our map viewer program accesses our underlying relational database to dynamically generate an integrated genetic and transcript map with a direct link to the WebFPC physical map from each marker.Web interfaces for database query and the query result pages are mostly developed using Java Server Page (JSP). JSPs are more efficient, easier to use, more powerful and more portable than traditional CGI and many alternative CGI . BAC conThe GDR website is composed of general information pages, database query/browse interfaces and other tools such as map viewer and sequence similarity server. The GDR web pages are extensively linked such that users can easily access the data of interest regardless of the navigation starting point. For example, the EST detail pages have links to the BAC detail pages, marker detail pages or map viewer for the ESTs that are anchored to BACs, markers or maps. Similarly, the BAC detail pages have links to EST detail page, marker detail page, WebFPC and map viewer. Users can also access the data detail pages from the map viewer or WebFPC/WebChrom. Instead of displaying the entire EST or BAC data in one page, we used the right hand side navigation bars to help users find specific information easily and quickly. A general GDR navigation tool bar is also included in each page to help provide a more user-friendly interface.The generic search site allows users to select data types such as EST, BAC and Marker, and search by name. Users can also follow the link to perform more detailed search for each data type. In the EST search site, users can search either the CUGI peach EST database or Rosaceae EST database downloaded from GenBank. ESTs can be searched by their name(s) and annotation features such as whether the EST belongs to a contig, is a unigene, is used as a probe and has SSRs, or any combination thereof. The EST details page, instead of displaying all the details in one page, initially displays the clone information and the sequence with a side bar containing links to library detail, assembly/unigene information, sequence homology, SSR information, Map position and anchored BACs. Each page linked from this side bar has the same side bar for easy navigation between the features. The Sequence homology page shows the most significant matches (EXP < 1e-9) in the Genbank nr protein database from the fastx sequence similarity search. The SSR information page shows the sequence along with the computationally derived SSRs to help users in the primer design for SSR marker development. The longest putative open reading frame (ORF) is also marked in color in the sequence along with the SSRs. SSRs in the non-coding region tend to be more polymorphic and those in the coding region tend to be more transferable among species, so the information of SSR position in a gene structure will be useful for marker development. The Map position page allows users to view the ESTs' map position using our Map Viewer. Users can retrieve the anchored BAC clones for the EST of interest and all the other ESTs and markers that hybridized to the same BAC through the anchored BACs page. The assembly/unigene information page displays the assembly results which include the contig name and the unigene clone that best represents the contig. The contig name is linked to a contig page that displays the contig sequence with a side bar containing links to the comprising ESTs, sequence homology and SSR information. As ESTs with no match to the GenBank proteins or with no SSRs can still assemble into contigs with a match or SSRs, users may get further annotation results of their ESTs of interest by visiting the contig detail site. In addition, contigs may have longer sequences surrounding the SSRs, allowing more flexibility in the primer design for marker development.In the BAC search site, users can search BACs by name or by probe specifications used for BAC hybridization. The search results site and the linked sites provide users with all the data about the BAC, such as the BAC contigs that the BAC belongs to, other BACs in the contig, probes that hybridized to the BACs, the detailed data about the probes, and link to the WebFPC physical map. Markers can be searched by name or features such as map name, type, and source organism. Similarly with the BAC search result sites, the maker search results sites leads users to pages with the marker information, anchored BACs, other markers and ESTs hybridized to the same BACs and link to the Map Viewer and the WebFPC physical map.Prunus map and each linkage group has a link to a page where the developing contigs are located by the linkage group. Each contig has a direct link to WebFPC in which the individual BAC clones are displayed  and various search parameters. Users can upload batch sequence files and the parsed results from the search are formatted in a spread sheet and sent to users by email. When the query sequence has a match to the annotated GDR sequences, users can retrieve all the information such as putative function, SSRs, and the anchored map positions via a hyperlink in the excel spreadsheet. Our sequence similarity server, specifically designed for Rosaceae researchers, will help users utilize the developing peach resources in the studies of other Rosaceae species. For example, as described in the case study below, sequences derived from other Rosaceae species could be immediately anchored to the various Rosaceae maps and the physical map when the query sequences show significant similarity to the mapped peach ESTs.Prunus sequences such as apricot (Prunus armeniaca), almond (Prunus dulcis) and sour cherry (Prunus cerasus). This was expected as peach also belongs to the genera Prunus. The 259 query/match pairs consisted of 209 Rosaceae sequences and 61 mapped peach ESTs. The matching of multiple Rosaceae sequences to single peach sequences was expected since the Rosaceae sequences were not assembled and therefore potentially contains multiple sequences representing the same gene. The 209 Rosaceae sequences were anchored to 38 different loci in four different Rosaceae maps. The number of sequences from each Rosaceae species that anchored to each map is shown in Figure .We report here a case study illustrating the utility of our sequence similarity server and other integrated GDR web resources. In this study, we performed a sequence similarity search using the FASTA algorithm with the non-peach Rosaceae sequences against the mapped peach ESTs to annotate Rosaceae sequences with map positions. A fasta formatted file with a total of 16258 publicly available non peach Rosaceae sequences was uploaded to the GDR FASTA server. We selected the mapped peach database and used the default parameters. The search results returned from the server are formatted in a spread sheet for easy browsing and the match names are hyperlinked to both GenBank and the GDR web site (Figure Prunus genomics projects. Data to be added in the near future include apple ESTs, strawberry ESTs, rose ESTs and apricot map/marker data from collaborators. When the genomics projects from Prunus are finished, we will host 10–15,000 unique ESTs from a variety of vegetative and reproductive tissues of peach and almond, the complete peach physical map with anchored genetic markers and unique ESTs. In addition to adding new data, future development efforts will focus on improving the tools and functionality of the web interface such as an advanced search site with options for search/display categories, full sequence processing facilities for Rosaceae researchers, a newsgroup for the Rosaceae community, a site for Rosaceae literature, and more analysis tools such as an interactive contig viewer and a comparative map viewer.We plan to incorporate more Rosaceae genomics and genetics data from researchers worldwide as well as data from the ongoing The GDR is initiated to support the genomics and genetics research in Rosaceae, which contains numerous economically important fruit trees and horticultural plants. Currently GDR contains all the genomics data for the Rosaceae model peach, maps and markers of Rosaceae species and all the publicly available Rosaceae ESTs. Our integrated database provides users with easy access and retrieval of the annotated data, and the web tools enable them to further analyze their data. With future plans, including more data acquisition and tool developments, GDR will play an important role in the timely and efficient analysis of the data, the exchange of results and ideas among researchers worldwide, the support of Rosaceae labs worldwide with Bioinformatics tools and the utilization of the data from the model species in the study of other Rosaceae species. The methodology and tools applied to develop GDR should be easily applied to develop other comparative genomic databases for different families..The GDR is publicly available and can be accessed at AFLP: Amplified Fragment Length PolymorphismBAC: Bacterial Artificial ChromosomeCGI: Common Gateway InterfaceEST: Expressed Sequence TagEXP: EXpectation ValueHTML: HyperText Markup LanguageHTTP: HyperText Transfer ProtocolINRA: Institut National de la Recherche AgronomiqueRFLP: Restriction Fragment Length PolymorphismSSR: Simple Sequence RepeatXML: eXtensible Markup LanguageSJ performed web interface design and programming for html pages and JSP search pages, participated in the database construction, carried out database curation, designed the Map Viewer and performed the case study. CJ wrote scripts for database upload and sequence processing, programmed Map Viewer and implemented WebFPC and WebChrom. MS participated in the database construction and wrote scripts for data upload and search pages for Genbank Rosaceae sequences. SF performs database administration. IC developed the application prototype for servlet-database connection and participated in the database construction. AA conceived of the project and participated in its design and coordination. ZD developed the sequence similarity server application. JT participated in the coordination of the project. DM conceived and supervised the project, participated in the database construction, carried out EST analysis, and provided input in the web interface design. All authors read and approved the final manuscript.


PLoS Biology, volume 2, issue 12.In 10.1371/journal.pbio.00203809 beads per g of cells” contains an error. The correct amount of beads is 8 × 108, i.e., ten times less.In Materials and Methods, the sentence “The magnetic beads were added to the extract to a ratio of about 8 ×10Published February 15, 2005


The ongoing battle between hosts and pathogens has long been of interest to evolutionary biologists. N) often greatly exceed those of synonymous substitution per site , as expected if most mutations are fixed because they increase fitness  reported to date . In part fitness .At the host level, most studies of the selection pressures acting on immune system genes have concentrated on genes implicated in the adaptive immune response against microbial pathogens, particularly those producing antibodies , or geneN greater than dS in many comparisons , so that this nucleotide mutates to uracil (U). This is essential for various aspects of cellular function. APOBEC1, for example, is involved in the C→U editing of apolipoprotein B mRNA (therein christening the family), while another family member, the activation-induced deaminase, has a vital role in adaptive immunity in that it assists in the diversification of antibodies. Two more enzymes, APOBEC3G and APOBEC3F, form part of the innate immune system; they function as antiviral agents and are being intensively studied in the context of infection with the human immunodeficiency virus (HIV), the cause of AIDS. In particular, APOBEC3G targets the reverse transcription step of the HIV life cycle, in which the viral genomic RNA is converted into proviral DNA, which is then integrated into the host genome . APOBEC310 virions are produced in a single infected individual  that counters APOBEC3G . Hence, The intense selective pressure on the defensins and APOBEC3G illustrates that although the innate immune response is generalist in its action, it is as highly and intricately evolved as its better-studied ally, the adaptive immune system. Rather than being an evolutionary remnant, it is a dynamic and continually adapting system. Less clear is whether other host proteins act in the same manner as APOBEC3G. In particular, the most common and destructive pathogens faced by humans and other mammals are RNA viruses, such as influenza A, yellow fever, and hepatitis C. In most cases, our ability to survive these viral infections is simply a combination of good luck and good breeding; with the right combination of MHC alleles, itself a function of population history and what we by chance inherit from our parents, some individuals may be more able to fight off viral infections than others. The ubiquity of RNA viruses hints that our genomes might also contain an innate, yet highly adapted, defence system that targets this abundant class of pathogens by manipulating their mutation rate. Although utilizing lethal mutagenesis might one day be an important way to design new drugs against a variety of viral pathogens, it would come as no surprise if nature got there first.Adaptive immune system. The pathogen-specific part of the vertebrate immune system. It is comprised of two major arms, antibodies  and T-cells (the cellular response), both of which lay down an immunological memory for future defence.Innate immune system. The nonspecific part of the vertebrate immune system. It has a wide variety of components, ranging from lysozymes in saliva to cytokines, defensins, interferons, and natural killer cells in a variety of tissues.Endogenous retroviruses. The  dead remnants of functional retroviruses that are now passed on through the germ line like normal genes. It has been estimated that approximately 5% of the human genome is composed of endogenous retroviruses.


In February 2003, the first (and so far only) epidemic of severe acute respiratory syndrome (SARS) started in Guangdong Province, China. A respiratory illness that begins with a high temperature and can develop into life-threatening pneumonia, SARS is spread by close person-to-person contact. Before the end of the month, a Guangdong doctor had inadvertently taken the infection to Hong Kong. A woman staying in the same Hong Kong hotel as the doctor then carried the disease to Toronto. In March, the World Health Organization issued a global alert and warned against unnecessary travel to affected areas. Because of these and other containment efforts, 8,098 people became ill with SARS, rather than the predicted millions; 774 people died. The last case of the epidemic was reported in Taiwan in June 2003, and since then there have been only two cases in Singapore and nine in China.By May 2003, a coronavirus had been identified as the cause of SARS, and the full genome sequence of this new human pathogen, which may have jumped from civet cats to people, had been published. From the viral genome, researchers have deduced the sequences and structures of the viral proteins, hoping to use this information to develop treatments and vaccines for SARS. But could the structure of the RNA genome itself also be a target for antiviral drugs?The genome of the SARS virus is a single strand of RNA that folds into regular repeating patterns to form secondary structures such as helices. These then fold and bend in three dimensions to form complex tertiary structures. William Scott and colleagues have used X-ray crystallography to measure the exact positions of individual ribonucleotides and the interactions between them in a small segment of the SARS virus genome called the s2m element. This element sits at one end of the viral genome, and, as the researchers show, its sequence is highly conserved in related coronaviruses. Furthermore, unlike the rest of the SARS genome, which changes rapidly, the s2m element is absolutely conserved in SARS variants obtained from patients during the SARS epidemic. This strong sequence conservation indicates that the tertiary structure of s2m could be important for viral function, and when the researchers solved the three-dimensional crystal structure of the element, they found that it had a unique tertiary structure. In particular, there was a right-angle kink in its helical axis and a tunnel with a net negative charge.The biological role of a new protein can often be deduced by comparing its shape with that of proteins with known functions. Scott and colleagues used this approach to hypothesize that the function of the s2m element involves interaction with a conserved host factor during the SARS life cycle. Finding a similar 90° kink in a region of ribosomal RNA that binds factors necessary for the initiation of protein synthesis, the researchers speculate that the SARS virus may use the s2m element to hijack its host cell's protein synthesis machinery. This and other putative roles need to be tested experimentally, but given that the s2m element is absent in the human genome, its unusual structural features could be an attractive target for the design of antiviral therapeutic agents.


Neisseria meningitidis, as a consequence of the insertion of a restriction endonuclease-encoding gene, the 'dam replacing gene' (drg) at the dam locus. Here, we report the results of a survey to assess the prevalence of drg in a globally representative panel of disease-associated meningococci.DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of drg was absent in all representatives of the ST-8 and ST-11 clonal complexes tested, but uniformly present in the representatives of the other hyper-invasive lineages present in the isolate collection . The patterns of sequence diversity observed in drg were consistent with acquisition of this gene from a source organism with a different G+C content, at some time prior to the emergence of present-day meningococcal clonal complexes, followed by spread through the meningococcal population by horizontal genetic exchange. During this spread a number of alleles have arisen by mutation and intragenic recombination.Of the known meningococcal hyper-invasive lineages investigated, drg gene may contribute to the divergence observed among meningococcal clonal complexes, but does not have a direct mechanistic involvement in virulence.These findings are consistent with the idea that possession of the  Neisseria meningitidis, the causative agent of meningococcal meningitis and septicaemia, is a common inhabitant of the human nasopharynx, being asymptomatically carried by approximately 10% of the population [pulation . There ipulation -7 but, dpulation . These lpulation  and multpulation .DNA adenine methyltransferase (Dam) is an enzyme involved in the mismatch repair system of bacteria. During DNA replication, only the parental strand is fully methylated by the enzyme, since methylation is not immediate, allowing a mismatch on the newly synthesised strand to be excised and replaced . DisruptN. meningitidis has been contentious. Bucci et al., 1999 [siaD capsule gene, resulting in an increased virulence of some strains. In addition, the latter work suggested that all pathogenic isolates lacked Dam activity, the dam gene inactivated by the insertion of a putative restriction endonuclease, named 'dam replacing gene' (drg), with the genotypes dam+/drg- and dam-/drg+ being mutually exclusive. However, two subsequent studies [drg gene encodes a restriction enzyme, NmeBII that is similar to the Streptococcus pneumoniae DpnI restriction endonuclease which cleaves at GmeATC, but not at GATC, sequences [Dam methylation has been implicated in modifying the virulence of a number of bacterial pathogens -26, but l., 1999  suggeste studies ,29 have equences ,31.drg and its association with disease-associated isolates of N. meningitidis was examined by a survey of its distribution in a collection of isolates chosen to represent the known diversity of pathogenic meningococci [drg is present in some lineages but not others, this gene has spread in the meningococcal population by horizontal genetic exchange, possibly after the introduction of the drg gene from an exogenous source.In the present study the prevalence of ngococci . The resdam and the absence of drg, and vice-versa. Of the 84 isolates tested, 23 were indicated to be dam+/drg- and 61 to be dam-/drg+ by restriction analysis and PCR amplification  present in this collection. The ratio of non-synonymous to synonymous substitutions (dN/dS, 0.11) was also similar to that seen in the MLST gene fragments. Three drg gene fragments (drg-13, drg-14 and drg-15) exhibited polymorphisms resulting in single base frameshifts, two resulting in stop codons  had an insertion corresponding to a 24-base direct repeat. The G+C content of drg at 42.4% was lower than that of the housekeeping genes, which were in the range 51.1–57.4%.The level of nucleotide diversity, as represented by number of alleles and polymorphic sites, along with the p-distances seen within ST Table , with a drg fragment sequences generated a star phylogeny with some net-like phylogenetic structure involving the drg-2 allele  of disease-associated isolates and 75% (11/15) of carrier isolates possessing the drg insertion. The isolate collection employed by the present investigation was chosen to be globally representative of meningococcal disease in the latter part of the 20th century, with multiple examples of each of the major hyper-invasive meningococci identified over this period. When analysed by clonal complex, which identifies hyper-invasive lineages, it was apparent that the drg insertion was absent from all of the isolates representing the ST-8 and ST-11 clonal complexes, both major hyper-invasive lineages but, conversely, drg was present in all representatives of the hyper invasive lineages represented by the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes. This association was independent of the serogroup expressed by the isolates and provided a likely explanation of the earlier observations [dam locus. At a given point in time they may be dominated by drg containing meningococci, while at other times isolates without this insertion might dominate. For example, as both the ST-1 and ST 32 complexes contain the drg insertion, this insertion would be prevalent among the disease isolates recovered by the pandemic outbreaks caused by these clonal complexes [drg insertion. This observation highlights the importance of assembling genetically defined isolate collections with a prescribed sampling frame if comparisons of meningococci with potential for virulence are to be undertaken.The data presented here, however, do not support the contention that the rvations . As meniomplexes ,38; convomplexes ,40 woulddrg gene has been introduced into Neisseria from an exogenous source [drg gene compared to the meningococcal housekeeping genes was consistent with this idea; however, the levels of diversity among the drg alleles, which was similar to that observed in the meningococcal housekeeping genes, along with the reported occurrence of drg in other Neisseria species [dN/dS), which was also similar to that observed in meningococcal housekeeping genes, suggested that the drg gene had been subject to stabilising selection for conservation of function since its putative introduction into the meningococcal population, although the possession of frame shift mutations in some of the drg alleles demonstrated that, as with N. lactamica, not all meningococci possessing the drg insertion expressed the endonuclease [It has been suggested that the s source . The dif species , suggestnuclease . In othenuclease .Neisseria and may be related to the transformable nature of these organisms [drg in the dam locus, replacing a methylase with an endonuclease, may provide a potent barrier to genetic exchange from dam+ to drg+ meningococci; indeed, while there was evidence for the occasional horizontal genetic exchange of drg alleles among the genetically diverse meningococci that possess the insertion, there was no evidence for the transformational loss of drg from a given clonal complex among the isolates examined here. This concept of genetic isolation among lineages receives some support from the possession of porB2 alleles by members of the dam+ ST-8 and ST-11 complexes and porB3 alleles by the clonal complexes with the drg insertion [The presence of multiple methylation systems is a characteristic of the rganisms . Whole grganisms ,44. It irganisms  and in tnsertion ,39, but drg insertion may influence meningococcal population structure, the data presented here do not support a direct association of this genotype with meningococcal virulence.While it is possible that the possession of the Neisseria meningitidis [).The 84 isolates used in this study see  were a sngitidis . The colDpnI, DpnII, and Sau3AI, followed by separation of the digestion products by agarose gel electrophoresis. Each enzyme specifically recognises a cognate target site of GATC and cleaves this site depending on whether it is methylated or not: DpnI cleaves this target sequence only when N6-methyladenine is present within the recognition sequence; DpnII cleaves only unmethylated sites, and Sau3AI cleaves both methylated and unmethylated target sequences.The methylation status of the chromosomal DNA of each isolate was determined by restriction digestion with the restriction endonucleases dam gene was determined by polymerase chain reaction (PCR) performed using dam specific primers, DamF1 (5' – TAAAATGGGCAGGCGGCA – 3') and DamB2 (5' – CGTAAGGGGGATCGCAAT – 3'). These amplify a 534 bp fragment from the 5' end of the dam gene in dam+ strains but not from dam-strains. The presence of drg was determined by PCR using primers DrgF1 (5' – CATGAATTTATTTTTCGATA – 3') and DrgB2 (5' – AATTTGCAACTGTTGGCG – 3') that bind to drg internal sites and produce a 705 bp fragment in isolates containing the drg gene. PCR amplification was also performed using primer pairs Drg5F (5' – TGTCTAAAGAACTCAAAG – 3') / DrgB3 (5' – CGGTATCGAAAAATAAAT – 3') and Drg3F (5' – ATCCATCCAATTTCCCCA – 3') / DamB5 (5' – AAATGCCGTCTGAA – 3') based on dam and drg coding regions in order to confirm that dam inactivation was due to drg insertion.The presence of the drg gene were purified by precipitation with poylethylene glycol and sodium chloride as described previously [drg sequence was assigned an arbitrary allele number in order of discovery.Amplicons corresponding to the eviously  and theidrg gene fragments was performed using SPLITSTREE, version 3.1 [Percentage G+C was calculated using the program START . Split dsion 3.1 . The prosion 3.1  using thsion 3.1 .KAJ carried out the nucleotide sequencing and analysis of the data. LS performed the PCR screening of the isolates. ERM and MCJM conceived of the study and participated in its design and coordination.Bacterial isolates used in the studyClick here for file


The book "Understanding the Human Machine, A Primer for Bioengineering" embraces various aspects of biomedical engineering as an essential resource book for physical scientists, engineers and biomedical students. The book interfaces physiologic systems with engineering principles to capture the important concepts from a plethora of facts in field of biomedical sciences. Humors, exercise, history of biomedical discovery and elements of humility continuously emerge throughout the book; thereby, enhancing the self-taught approach for the new comers.The Introduction reflects the author's philosophy and advice for the readers from his accumulated wisdom and experience in teaching bioengineering. In Chapter 2, the author used the system approach to intertwine cardiovascular, renal, respiratory, gastrointestinal, endocrine, nervous and muscular systems. The organization is timely and effective in the era of systems biology and the emerging research towards predictive and preventive medicine.The system approach in Chapter 2 paves the way for detecting signal output from the physiologic systems. Biosensors are illustrated to measure electric signals from the visual, auditory and olfactory systems as well as the heart, brain, and muscle. The integration of electric circuits using the classic examples such as the Wheatstone bridge and operational amplifiers provides the frame work for signal acquisition and processing. Finally, the author completed the book with a touch of bioinformatics in the post genomic era.In general, the book is comprehensive and succinct, readable and interesting. The presented material spans from the fundamental principles to the applications of bioengineering.Certain areas will improve the quality of this book for the readership of undergraduates and new comers to bioengineering. Some figures are difficult to understand as a result of small captions (eg. figs 2.11 and 3.19). Others are old and lack clear labeling . Tissue engineering deserves a paragraph. Analogous to nanotechnology, a link to a tissue engineering website would be helpful for the readers. A paragraph on molecular engineering involving proteins, DNA and RNA molecules would enhance the balance between classic bioengineering and the emerging fields. The readers would be intrigued if the book ends with a paragraph introducing Bio-MEMS , bionanotechnology, molecular imaging, and surface chemistry.Overall, "Understanding the Human Machine, A Primer for Bioengineering" is a very useful book that highlights the fusion between organ systems and engineering principles. I would recommend this book as an introduction to bioengineering and a reference for students from physical science, engineering and life science.


Little is known about the prevalence of khat-induced psychotic disorders in East African countries, where the chewing of khat leaves is common. Its main psycho-active component cathinone produces effects similar to those of amphetamine. We aimed to explore the prevalence of psychotic disorders among the general population and the association between khat use and psychotic symptoms.In an epidemiological household assessment in the city of Hargeisa, North-West Somalia, trained local interviewers screened 4,854 randomly selected persons from among the general population for disability due to severe mental problems. The identified cases were interviewed based on a structured interview and compared to healthy matched controls. Psychotic symptoms were assessed using the items of the WHO Composite International Diagnostic Interview and quantified with the Positive and Negative Symptoms Scale. Statistical testing included Student's t-test and ANOVA.Local interviewers found that rates of severe disability due to mental disorders were 8.4% among males (above the age of 12) and differed according to war experiences . The clinical interview verified that in 83% of positive screening cases psychotic symptoms were the most prominent manifestations of psychiatric illness. On average, cases with psychotic symptoms had started to use khat earlier in life than matched controls and had been using khat 8.6 years before positive symptoms emerged. In most cases with psychotic symptoms, a pattern of binge use (> two 'bundles' per day) preceded the onset of psychotic symptoms, in contrast to controls of the same age. We found significant correlations between variables of khat consumption and clinical scales , and between the age of onset of khat chewing and symptom onset .per se but rather early onset and excessive khat chewing seemed to be related to psychotic symptoms. The khat problem must be addressed by means other than prohibition, given the widespread use and its role in Somali culture.Evidence indicates a relationship between the consumption of khat and the onset of psychotic symptoms among the male population, whereby not the khat intake  The leaves of the khat shrub alpha-aminopropiophenone) . Cathino disease ,25. The  disease  may also disease .The first goal of this study was to verify the impression of an unusually high prevalence of psychotic disorders in the city of Hargeisa via an epidemiological survey carried out in a representative sample of households. In addition, we wished to study the association between khat abuse and psychotic disorders. If khat abuse does induce psychotic disorders, a higher prevalence of psychotic diagnoses, mainly in men (as women rarely consume khat), was to be expected in Hargeisa compared to localities with less khat use. In addition, a case-control study served to examine whether individuals identified in the survey suffering from psychotic symptoms presented a pattern of khat consumption different from matched controls.In order to screen for households with mentally ill members, 612 households with 4,854 members were randomly selected as representative of the city of Hargeisa (approximately 400,000 inhabitants). For household selection the town was first subdivided into 30 sections of approximately equal populations. For this purpose we used the same sections as UNICEF in their assessment of vaccination coverage for children in October 2002, which were defined in a joint approach by UNICEF and the municipality of Hargeisa. A section had to be subdivided into square-shaped clusters if it was L-shaped, had a natural boundary within its limits (e.g. a steep hill) or had a much greater length than depth. A map for each section or cluster was produced using an aerial photograph. For random selection the geographical center of each cluster was determined and a random direction was chosen using a compass, a watch and a list of random numbers between 1 and 12 . Following the random direction until the border of the cluster was reached, all houses on the right side within a range of 15 meters were numbered. The first household to be approached was selected by a second set of random numbers. The next houses were selected by door-to-door distance until the predefined number was reached. Trained local staff interviewed the heads of the 612 households. If the head of household was not available, another adult member of the household was asked to answer to the questions. If no household member could be interviewed, the next house was selected according to the selection procedure.The following question was used to determine whether a person severely disabled due to mental health problems resided in the household: "Are there any members of your household who currently have mental problems that are so severe that the person has been unable to provide income or has been unable to help in the household for at least four weeks?" This criterion was fulfilled for 169  cases. These individuals will be referred to as 'positive screening cases'. A subsample of 52 'positive screening cases' was then randomly selected and examined in a clinical interview. In this group there were 44 males and 8 females. These individuals will be referred to as 'examined cases'.For each 'examined case' a matched control, who did not meet the criteria for disability due to mental health problems, was identified. Controls were matched by gender, age, and educational level. These 'case controls' were subjected to the same clinical interview as the examined cases. Forty-nine of the 52 examined cases and one control were diagnosed with psychiatric or neurological disorders (on this basis we estimated a sensitivity of 0.98 and a specificity of 0.94 for the screening procedure). We included only those forty-three examined cases (82.7%) in the further analysis who – in addition to the impairment of function – showed as main manifestations of mental problems at least one very severe or two moderate positive psychotic symptoms in the absence of organic somatic damages that might explain them; from hereon these are termed 'cases with psychotic symptoms'. The disorders of other examined cases were stroke (2), traumatic brain injury 1), mental retardation (2), and dementia (1). The case control with a positive diagnosis reported hallucinations with intact reality testing during khat intoxication and was replaced by a healthy individual. Socio-demographic characteristics for the two studied groups are summarized in Tables , mental All interviews were carried out in October and November 2002. Prior to the interview, participants were informed about the background of the study and the survey procedure. All participants signed a written informed consent, which was first read and explained to them (as most of the participants were illiterate). In the case of mentally challenged participants, background and procedure were also explained to the responsible caretakers whose written informed consent was a prerequisite for their participation. Interviews were approved by the local authorities and the National Demobilization Committee of Somaliland. Representatives of the different communities were informed and invited to observe the assessment in the field. Newspaper advertisements, daily radio programs and flyers informed the population about and helped them to understand the real purpose of the assessment . Consequently, the level of cooperation was extremely high. Out of 73 households approached under the supervision of the first author only three refused their participation. Therefore, we estimate that in total less than 5% of all households refused participation.Local interviewers were recruited among NGO and hospital staff who already had experience in working with mentally disabled persons. They participated in a two-week training course, which involved teaching the basic psychiatric concepts (e.g. psychotic symptoms), interviewing skills, training on the screening-instrument and field work under close supervision of experts. After the end of the course, fourteen of the seventeen trainees were employed for the duration of the study. Five interviewer teams, each comprising one male and one female interviewer, and four local supervisors did the field work. The local supervisors received additional training on the random sampling method. During the first weeks of interviewing the first author closely supervised the teams in the field.The screening interview assessed individual socio-demographic information, khat consumption and experiences in the civil war (subjects were grouped as either active war participants, civilian war survivors, or without any war experiences). Khat consumption (number of bundles/week) was assessed for the week prior to the interview. The household economic situation was approximated as the sum of four significant assets  and type of housing Table .Clinical interviews were carried out by some of the authors (MO, MS, CC, BL), all trained in the assessment of psychotic disorders and PANSS rating. Socio-demographic information and war-history were detailed. A standard event list was used to quantify the number of traumatic event types a person had experienced; the list included 11 types of events: active combat experience, accident, natural disaster, witness murder or killing, rape, sexual molestation, physical assault, being kidnapped or captured, torture, other, or suffered shock because a close person had experienced a traumatic event. In a short semi-structured qualitative interview the main areas of psychological condition and functioning were assessed. Additionally, psychotic symptoms were assessed using the items of the Composite International Diagnostic Interview (CIDI), Section G . The indClinical interviews were administered in the English language with the help of trained local interpreters, who had participated in the same training course as the interviewers. After the interview, the interviewing psychologist rated the current psychopathology using the Positive and Negative Syndrome Scale, PANSS .All items of the standardized interviews were translated from English into Af Somali by mixed teams of clinical experts and bilingual staff. Several steps of independent back-translations and corrections were necessary until a satisfactory translation was achieved. Content validity was assured by the involvement of clinical experts at all levels of the translation process. Also, interpreters were introduced to the concepts targeted by the respective questions.2-tests (or Fisher's exact tests), binomial testing, Student's t-test or Wilcoxon, and ANOVA or Krsucal-Wallis test . Univariate Analysis of Variance (ANOVA) was used to explore the effects of war-trauma on the khat intake. Means and standard deviations are reported.Differences between cases and controls were confirmed using chiOf the sample of 4,584 inhabitants of Hargeisa the screening interview disclosed 169 positive screening cases (i.e. 3.5%). Positive screening cases were more often male than female  than in male civilian war survivors of the same age (p < 0.001, Table Psychotic symptoms meeting our criteria were determined for 83% (43) of the examined cases. Retrospective investigation suggested that the onset of psychotic signs occurred at the age of 23.4 ± 9.9 years .PANSS ratings of these 43 cases showed a substantially higher magnitude of current psychotic symptoms compared to a sample of 240 medicated schizophrenic patients  in the fFifteen of the 43 cases with psychotic symptoms (35%) were under current medication at the time of assessment; another nine (21%) had received medication in the past. A variety of drugs had been given, ranging from neuroleptics (12 patients) to prometazine (6), benzodiazepines (3), tricyclic antidepressants (3), carbamazepine (1) and other unknown drugs (10).Uncontrollable (disruptive, violent) behavior had led to restraint of cases with psychotic symptoms by chaining them to an object in 28 of the 38 men (i.e. 74%) and 3 of the 5 women (i.e. 60%) (p = 0.608). Additionally, 9 men (24%) and 3 women (60%) had been locked up to control their behavior (p = 0.589). On average, the 31 cases with psychotic symptoms who had ever been chained had spent 4.2 ± 5.2 years in chains  and the 10 cases who had ever been locked up spent on average additional 5.2 ± 6.5 years restrained .The proportion of khat user was higher among cases with psychotic symptoms than among matched controls: all except one of the 38 male psychotic cases, in contrast to 25 of the 38 male controls, had used khat (p < 0.001). In the cases with psychotic symptoms, regular khat consumption had started at an earlier age (16.6 ± 4.8 years) than among their matched controls , General Psychopathology , Anergia (r = - 0.39, p = 0.022, N = 34) and Thought Disorder (r = - 0.42, p = 0.014, N = 34), while the amount of khat consumed prior to onset of psychotic symptoms correlated significantly with the subscale Paranoia/Belligerence (r = 0.49, p = 0.006, N = 30) and the Supplemental scale (r = 0.50, p = 0.005, N = 30). The amount of khat consumed in the week before the interview correlated with the PANSS subscale Anergia (r = - 0.35, p= 0.029, N = 38).2 = 0.620). In Figure The number of traumatic event types experienced did not differ between cases with psychotic symptoms and matched controls . Also, the age when the first traumatic event was experienced did not differ between them . In order to estimate the relationship between number of experienced traumatic events and khat intake we used univariate ANOVA with khat intake before onset of psychotic symptoms (for controls at the age of symptom onset of matched pair) as dependent variable, and number of traumatic events in three categories (no or one event, two or three, four or more) and being a case or a control as independent variable. We found significant effects for the number of traumatic events  and for the variable 'case or control'  and a significant effect of their interaction   In a representative subsample of male inhabitants of Hargeisa (older than 12 years) we found 8.4% severely disabled due to mental problems; of these, 83% had severe psychotic symptoms. Compared to the prevalence rates of psychotic disorders reported for male samples elsewhere ,32 this (2) Khat abuse showed a significantly earlier onset and was more frequently excessive in male cases displaying psychotic symptoms than in matched controls.(3) There is a higher vulnerability to disability due to mental disorders in those groups of society who were directly involved in combat in the past.(4) Among the positive screening cases examined and their matched controls we found that khat intake before the onset of psychotic symptoms (respectively at the same age for matched controls) was higher when more traumatic events had previously been experienced.These results are intriguing, as we may have detected only the very tip of the iceberg with our sub-optimal screening procedure, leaving less severe cases of mental and neurological disorders undiscovered.per se but rather specific patterns of it that are related to psychosis, especially early onset in life and the excessive intake (> two bundles per day). Both seem to be related to active participation in war, e.g. child ex-combatants used khat while fighting in order to counteract fear and enhance performance [The findings of the present study suggest that there is an association between khat consumption and psychosis; however the existence of a causative relationship and its direction between the two remains unclear. Furthermore, it seems that it is not the consumption of khat formance . Excessiformance . Our datKhat consumption might be related to the outbreak of psychosis in various ways. Considering psychoses as the result of genetic and acquired vulnerability and additional stress factors, the early onset of khat abuse may have substantially increased the risk in already vulnerable individuals. In animal studies, damage imposed on the developing brain, e.g. by drugs, increases the potential of amphetamine-like agents to change neuro-chemical systems and to induce psychotic-like behavior . In humaMoreover, khat consumption may affect the course of a psychotic disorder and the maintenance of symptoms. In contrast to the high khat consumption prior to the onset of psychotic symptoms, the amount of current khat consumption did not differ between cases with psychotic symptoms and controls and a significant number of patients did not have current access to the drug. Drug-effects on the course of illness may be attributed to sensitization  or to laSome methodological shortcomings of the study are related to the nature of field work in this specific post-conflict setting, which involved restricted freedom of movement due to the security situation and limited resources. However, although a perfect design is not possible in a rather complex field situation, the importance of such studies is evident from the fact that little or no information from post-war Somalia is available. First, we could not control whether our sample was comparable to the population from which it was drawn due to the lack of recent statistics in Somalia.Second, the validity of our screening and clinical interview can be questioned because of various points, e.g. whether the descriptions of symptoms and disability might be adequate for the Somali culture, or whether factors such as interpretation or the interviewing of a whole family rather than a single patient might distort the information. In our approach we decided to use disability as a selection criterion and a descriptive rather than a normative approach to identify the reasons for it. Thus, we decided to use the more unspecific terms 'cases with psychotic symptoms' or 'psychotic symptoms' rather than 'schizophrenia' or 'drug-induced psychosis'. However, the fact that our local interviewers found 123 of the 169 positive screening cases (72.8%) restrained, i.e. chained or locked up, and the overall high average PANSS scores of the examined cases, show that there are 'hard' criteria, which justify the use of the term 'psychosis' and validate our screening. Nevertheless, we recognize that with our method we must have failed to discover persons suffering from psychotic and other mental disorders . But assuming that this error is somehow the same for the whole sample, our finding that the group of society that engages most in khat chewing  most often suffers severe impairment due to mental disorders shows evidence that khat is related to mental health problems.Thirdly, the fact that we could not choose our matched controls from the pool of negative screening cases – the resources that would have been necessary to (repeatedly) contact and arrange appointments with them exceeded our capacities – leads without any doubt to an overestimation of the specifity of our detection procedure.Furthermore, the retrospective third-person assessment of the onset of psychotic signs, as well as the retrospective and subjective assessment of khat use, will have introduced some error variance in the data. However, we believe that especially in the Somali culture khat intake cannot be compared to any food intake, of which the retrospective assessment is problematic. In the Somali culture khat has a special significance, which also comes from existing traditional knowledge about its dangers. Therefore we suppose that retrospective assessment of khat intake is more valid than that of food intake. Also, the effects of social desirability might have affected both the detection of positive screening cases and the assessment of khat use. The fact that dozens of families approached the compound of our collaborating partner agencies in order to find help for their mentally ill members (often they brought them in chains to the compound) shows the great need for mental health services. Thus, the tendency to hide family members from our interviewer teams can be estimated as marginal. On the contrary, during the interviews our greater fear was that households would over-report the presence of mentally challenged family members. For this reason, we counted only those persons as positive screening cases when their existence in the household could be validated . The assessment of khat consumption is another point where effects of social desirability can be assumed. Whereas the information given by examined cases could be validated by the information of family members, we were well aware of the danger that the matched controls might have under-reported their khat intake. In order to allow for this in the interview we spent much time and effort to identify the real khat intake . Nevertheless, the high correlations validate the assessment procedures.Also, the sample size was too small to answer some of the questions in detail, especially the small number of female examined cases. However, the results were nonetheless statistically very robust.In sum, our design could not determine the existence of a causal relationship between khat and psychosis – this would have been too ambitious a goal. However, our main findings do not contradict the hypothesis that khat might cause psychosis.Future research should focus on the question of causality. Unfortunately, the Horn of Africa is probably the region where stimulant abuse currently reaches highest levels on a world-wide scale. This circumstance offers a unique research opportunity for cross-sectional and repeated measurement studies, which would enable us better to understand the relationship between schizophrenia and drug effects and to fill the gap in knowledge related to khat use. At the same time, the alarmingly high prevalence of khat chewing among persons who suffer from psychotic symptoms in one of the countries of its highest use, and the magnitude of human suffering associated with it, demands the development of adequate community-based prevention and treatment interventions. The usefulness of standard procedures derived from developed countries and brought to the Horn of Africa must be viewed in a "service-research attitude". We beliNot khat intake per se but rather specific patterns of it are linked to the development of psychotic symptoms, like early onset in life, excessive chewing (> 2 bundles/day) and use as self-medication for war trauma related symptoms. Although we cannot establish a causal relationship between khat and psychosis, we find some evidence that the prevalence of psychotic disorders is increased among the male adult population of Hargeisa. Ex-combatants are the group in society who are most affected by severe mental disorders, among them we find the highest abuse of khat. The way of khat use in Hargeisa is changing, indicated by e.g. the development of a group of heavy users who show consumption patterns similar to amphetamine addicts in Western countries. Measures of prevention and treatment of and further research on khat-related severe mental disorders have to be undertaken, however, taking into consideration that khat is an integral part of the Somali culture, which cannot simply be prohibited.The author(s) declare that they have no competing interests.Development of the study design and selection of instruments were accomplished by MO, FN, TE, MS, BR and HeH. MS, TE and MO performed pilot studies. Training and supervision of local interviewers was provided by MO, HaH, FN and BL. Clinical interviews were conducted by MO, MS, CC and BL. Statistical analysis was performed by MO and TE. The article was composed and revised by MO, TE, BR, FN and HeH.The pre-publication history for this paper can be accessed here: