Despite abundant bereavement care options, consensus is lacking regarding optimal care for bereaved persons.We conducted a systematic review, searching MEDLINE, PsychINFO, CINAHL, EBMR, and other databases using the terms (bereaved or bereavement) and (grief) combined with (intervention or support or counselling or therapy) and . We also searched citations in published reports for additional pertinent studies. Eligible studies had to evaluate whether the treatment of bereaved individuals reduced bereavement-related symptoms. Data from the studies was abstracted independently by two reviewers.74 eligible studies evaluated diverse treatments designed to ameliorate a variety of outcomes associated with bereavement. Among studies utilizing a structured therapeutic relationship, eight featured pharmacotherapy (4 included an untreated control group), 39 featured support groups or counselling (23 included a control group), and 25 studies featured cognitive-behavioural, psychodynamic, psychoanalytical, or interpersonal therapies (17 included a control group). Seven studies employed systems-oriented interventions . Other than efficacy for pharmacological treatment of bereavement-related depression, we could identify no consistent pattern of treatment benefit among the other forms of interventions.Due to a paucity of reports on controlled clinical trails, no rigorous evidence-based recommendation regarding the treatment of bereaved persons is currently possible except for the pharmacologic treatment of depression. We postulate the following five factors as impeding scientific progress regarding bereavement care interventions: 1) excessive theoretical heterogeneity, 2) stultifying between-study variation, 3) inadequate reporting of intervention procedures, 4) few published replication studies, and 5) methodological flaws of study design. Give sorrow words; the grief that does not speakWhispers the o'er fraught heart and bids it break.Macbeth IV, iii, 209Shakespeare, Grieving the death of a loved one has an ancient history: from time immemorial, cultures have provided the bereaved with advice and rituals to address – and express – the experience of grief . Over thGiven the abundance of care options, what is the best way to care for a bereaved person? Numerous studies measuring the impact of bereavement interventions have been published in diverse journals, yet no consensus has emerged in the medical, mental health, or social work communities regarding whether one form of treatment is preferable to another -5. We thbereaved or bereavement" and "grief", combined with secondary descriptors of "intervention or support or counselling or therapy" and "controlled or trial or design".To identify studies in the traditional medical literature as well as the complementary and alternative medicine literature, we searched the following databases: MEDLINE; PsychINFO; Cumulative Index to Nursing and Allied Health (CINAHL); BIOSIS Previews; ISI Science Citation Index Expanded and Social Sciences Index; Evidence Based Medicine Reviews (EBMR), including the Cochrane Database of Systematic Reviews (DSR), the Cochrane Controlled Trial Registry (CCTR), Database of Abstracts of Reviews of Effectiveness (DARE), and the American College of Physicians' (ACP) Journal Club Review; Sociological Abstracts; Alt HealthWatch; and Wilson Web from 1966 to 2003. We identified all relevant articles on bereavement care interventions by using the primary search terms of "Our inclusion criteria specified that each study: 1) addressed the treatment of bereaved individuals, and 2) included an evaluation of a selected method of therapy aimed at reducing the grief reaction due to bereavement. We considered only articles written in the English language. We then reviewed the titles and abstracts of all articles we retrieved through our initial database search, and obtained the full texts of all applicable studies. We also reviewed the references in all applicable studies for additional pertinent studies.The full articles of all studies that met inclusion criteria and passed subsequent title and abstract reviews were retrieved and examined independently by two of the authors. Each article was reviewed for measured outcomes, patient and decedent characteristics, and intervention characteristics. These measures included sample size, type of intervention, length of intervention, patient's relationship to the deceased, time since the bereaved death, and patient demographics. Data was extracted and any disagreements were resolved through discussion, clarification, and consensus within the research team.The initial literature search generated 737 citations. Elimination of duplicate citations yielded 340 references. 2 studies, written in Chinese and Spanish, were excluded. Reviewing the titles culled the sample to 243 citations, and a review of the abstracts found 87 of these to be potentially relevant. Of these, 9 were dissertations, 2 were irretrievable, 2 were duplicate publications of the same study, and 15 were ineligible because they did not meet our inclusion criteria. The resulting set of 74 articles was subject to review for data extraction. A list of all citations found, including those excluded from this analysis, is available see .Of the 74 studies that met inclusion criteria, almost 6,000 participants within these studies experienced a multitude of losses – of parents, spouses, children, and other loved ones who had died from a wide range of causes, both sudden and protracted. The therapies utilized and outcomes evaluated varied widely. Heterogeneity among both the outcomes and the measures used to assess similar outcomes precluded an effort to summarize data across studies, even in the form of generic effect-size measures. Furthermore, for a significant portion of the studies, concerns regarding the internal or external validity of the reported results cautioned against making quantitative summary statements regarding treatment effects.The 74 studies selected for detailed review evaluated diverse types of interventions designed to ameliorate the adverse physical and psychological outcomes associated with bereavement. These interventions can be classified according to various schemes, including their underlying theoretical framework , the format of the intervention , the timing of the intervention , the tasks assigned to the bereaved , or the population targeted for the intervention . We chose to organize this review on the basis of the social framework used to implement the intervention , as this attribute of the interventions emerged as the most verifiable and salient measure.pharmacotherapy, but only four compared active therapy to non-pharmacotherapy controls, and only one study clearly reported their random allocation method  report a bereavement intervention for Mexican-American widows . They foPsychotherapy-based treatments, another form of psychological interventions, can be done in different formats , and via different approaches. Of the 25 studies that use psychotherapy as an intervention, approaches included cognitive-behavioral, psychodynamic, psychoanalytical, and interpersonal approaches, as well as combinations of these and modality and social support  studied an intervention for parents bereaved by the violent death of their children .-79.73-79Ringdal and colleagues (2001) found no significant differences between those family members whose relative received palliative care and those who received traditional care . This inRobinson (1998) examined the psychological effect of witnessing resuscitation efforts of patients in the emergency room on bereaved relatives . They foper se, no consistent pattern of treatment benefit has been established across well-designed experimental studies.When reviewed systematically, the current bereavement intervention literature – notwithstanding the existence of many intriguing reports – yields few reliable conclusions to guide treatment. Good evidence supports the pharmacological treatment of depression occurring in the context of bereavement. For all other forms of intervention, however, and for all attempts to diminish grief Why – despite prevalence of bereavement, the intense dedication on the part of the bereavement research community, and the multitude of peer-reviewed published bereavement studies – does the field of bereavement care lack a formidable evidence base? In order to improve the effectiveness and quality of bereavement care, this question begs to be addressed. On the basis of our systematic review of the literature, we postulate the following five factors as hindering methodical scientific progress regarding bereavement care interventions.As the history of science and medicine suggests, successful scientific inquiry into a topic is typically a cumulative process undertaken by a community of investigators working within a shared scientific paradigm ,81. The Treatments featured in published studies vary almost as much as the authors who tested them. One can observe substantial variation across studies regarding the type of intervention generally or regarding the specific implementation of a specific type of intervention ; regarding characteristics of targeted patient populations; regarding outcome measurements and study design methodology. Scrutinizing the key outcome measures listed in the accompanying tables illustrates this remarkable heterogeneity. Although these differences have been due in part to diverse treatment-theory paradigms, even studies conducted within the same theoretical paradigm often differed markedly in terms of what potential benefit was being tested, and how it was being measured. Such substantial variation between studies stymies comparison or confirmation of treatment effects.Aside from the pharmacological studies, which reported the dosing of the intervention medication, very few reported studies describe the intervention procedures in sufficient detail for readers to envision clearly what tasks or activities intervention subjects were asked to perform. This under-specification prevents sensible analysis, within a class of treatments , of observed differences in treatment effects .Inadequate specification of intervention procedures, combined with other factors at work within the community of bereavement care investigators, may have resulted in the dearth of published replication studies. This lack of replication prevents the accumulation of a body of evidence that would confirm, refute, or refine prior estimates of treatment effects.ad hoc basis, and which may therefore have compromised measurement accuracy and inference validity. Lastly, studies that failed to demonstrate a statistically-significant difference for the main outcome measure often performed numerous post hoc subgroup analyses, a practice that negates the rigor of statistical hypothesis testing.A final factor inhibiting research progress in the realm of bereavement care interventions encompasses a number of recurring methodological flaws that greatly limit inferences regarding treatment effects. First and foremost is the omission of control groups. Control groups are essential for the valid evaluation of a bereavement intervention, particularly because of the typically self-limited course of grief: even absent any treatment, most bereaved people show "diminished pathological symptoms and fewer signs of disturbance within two years of the loss". PurportIf these five factors are indeed hampering progress towards improving bereavement care interventions and quality of care for bereaved individuals, then concrete actions could facilitate progress within the field of bereavement care, specifically: 1) Convening a consensus-building conference among key stakeholders and investigators to define a specific research agenda that would draw on a limited number of theoretical paradigms and delineate key elements of treatment theory ; 2) FocuNone declared.CG Control groupNT NortriptylineSSRI Selective serotonin reuptake inhibitorsTCA Tricyclic antidepressantsTRIG Texas Revised Inventory of GriefAF assisted in the design of the review, conducted data collection, data abstraction, and drafted the manuscript. MH assisted in the design of the review and critically reviewed several drafts of the manuscript. RP assisted in data collection and abstraction. CF conceived of and designed the review, assisted in drafting and revision of the manuscript, and provided support and mentorship through the process. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:All citations. This file contains citations to all of the studies identified by our literature search and screened for inclusion in this review, as well as other scholarly works consulted during the conduct of this review.Click here for file


Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium.M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain.To be able to study the role of the GPL in rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium.In this study, we affirm our results that M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug resistance can be studied in a consistent manner.We report the second allelic exchange system for  Mycobacterium avium is a frequent cause of disseminated infection among patients with end-stage AIDS [M. avium can also present with a similar spectrum of pulmonary and extra pulmonary syndromes as Mycobacterium tuberculosis [M. avium infection, there is only a rudimentary understanding of the bacterial factors involved in the pathogenesis of infection [age AIDS ,11,19. Mrculosis  includinrculosis . While snfection ,22.M. avium, there is evidence that the glycopeptidolipids (GPL), as the dominant lipid for this species, may negatively affect host immunity [M. avium pathogenesis has been limited by a lack of suitable genetic techniques to be able to create site directed knockouts. Further, as reviewed below, there is controversy as to which portion of GPL predominates in disease production.Numerous studies have implicated the cell wall lipids in mycobacterial pathogenesis. For immunity ,25. Studallo threonine-D-alanine-alaninol with a fatty acyl group N-linked to the phenylalanine residue and a methylated rhamnose modifying the terminal alaninol. The LP core is glycosylated at D-allo threonine with 6-deoxytalose  to form non-specific GPL (nsGPL) and is further glycosylated at 6dTal with a haptenic oligosaccharide to yield serovar-specific GPL (ssGPL). All serovars maintain a common α-L-rhamnopyranosyl-(1→2)-6dTal [The GPLs are comprised of a lipopeptide (LP) core of D-phenylalanine-D-Staphylococcus aureus coated with M. avium GPL promote phagocytosis and inhibit phagolysosomal fusion in relation to serovar [Historically, the predominance of serovars 1, 4, and 8, among patients with disseminated infection ,26 has b serovar . Other s serovar . SignifiM. avium, an allelic exchange mutant in rtfA was created for a pathogenic serovar 8 strain to yield a strain deficient in ssGPL. Homologous recombination was performed using a novel allelic exchange vector that incorporated a temperature-sensitive mycobacterial origin of replication (ts-oriM) and sacB as counter selective markers [xylE [rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wild type (wt) serovar 8 smooth opaque (SmO) parent strain. In addition to reaffirming our results for serovar 2 [rtfA encodes an enzyme responsible only for the transfer of Rha to 6d-Tal to form the serovar-8 specific oligosaccharide, this study delineates a second system of allelic exchange mutagenesis for M. avium.To study the role of the serovar-specific oligosaccharide moiety of GPL in the pathogenesis of  markers  and xylErs [xylE  as a poserovar 2  that rtfEscherichia coli strain DH5α was used as the host strain for plasmid construction and propagation. Wild type and recombinant M. avium and Mycobacterium smegmatis strains were grown in Middlebrook 7H9 broth or 7H11 agar supplemented with 10% OADC  at 37°C, except where indicated. M. smegmatis mc2155 [M. avium 920A6 is a serovar 8 bloodstream isolate cultured from a patient with AIDS [E. coli and M. smegmatis was performed as described [M. avium was performed according to the protocol of Lee et al. [E. coli, selection was carried out using ampicillin at 50 μg ml-1 and kanamycin at 25 μg ml-1. For M. smegmatis and M. avium, selection was accomplished using hygromycin at 100 μg ml-1, gentamicin at 100 μg ml-1, and kanamycin at 50 μg ml-1.s mc2155  was emplescribed ,24. Trane et al. . For E. ts-oriM, sacB, and the hygromycin resistance gene (hyg); and positive selection marker xylE. Construction of allelic exchange vector pVAP39 is shown in Fig. BamHI-XbaI fragment of pXYL4 containing the xylE gene [BamHI site of pPR27, containing a temperature-sensitive origin of replication of M. fortuitum plasmid pAL5000 and sacB [XbaI-XhoI fragment containing rtfA::hyg, isolated from pVAP37 [rtfA::hyg in pVAP38 was confirmed by PCR and Southern blot analysis as described [rtfA gene with HindIII overhangs into the HindIII site of plasmid pMVGFP . Plasmiositive, ).et al [Colonies of wt, mutant, and complemented strains were collected from 7H10 plates. Procedures for purification of alkaline stable GPLs, and alditol acetate analyses of sugar moieties by gas chromatography/mass spectrometry (GC/MS) were performed as described by Eckstein et al .xylE was first examined for M. avium since there is minimal published data on the use of this marker in mycobacteria. After construction, pVAP38 was first electroporated into M. smegmatis strain mc2155 to assess expression in a test system. All (100%) of gentamicin-resistant colonies expressed XylE as determined by a yellow color change after application of catechol. M. avium 920A6 SmO was then transformed with pVAP39 and selected at 32°C on 7H11 medium containing 100 μg ml-1 hygromycin. Yellow colonies were easily detected, indicating that xylE represents a suitable marker for M. avium. However, only 25–40% of hygromycin-resistant colonies yielded a yellow color, indicating a high rate of spontaneous hygromycin resistance when M. avium is transformed at 32°C, with a final efficiency of transformation of 1–8 × 102 transformants per μg of DNA. These results contrasted with our earlier observations that transformation of M. avium with non-temperature sensitive plasmids yielded less than 5% of spontaneously resistant colonies and an efficiency of transformation 1.6-log higher .M. avium 920A6 SmO/pVAP39 was inoculated into 7H9 medium for 3 weeks at 32°C to late log phase. Growth in hygromycin-free medium allowed for spontaneous loss of plasmid DNA. Moreover, expansion in hygromycin-free medium limited the appearance of spontaneous hygromycin resistance (unpublished data). Selection for allelic exchange mutants was performed at 39°C on 7H11 medium containing 100 μg ml-1 hygromycin and 2% (w/v) sucrose to isolate single colonies. Incubation at 39°C precludes the replication of the temperature-sensitive origin of replication of pVAP39. Hygromycin-resistant, XylE-positive colonies that arose at this non-permissive temperature represented single crossover mutants or illegitimate recombinants, whereas XylE-negative colonies represented either double crossover mutants or colonies that had lost plasmid DNA and had developed spontaneous hygromycin resistance. Growth on sucrose was used as a means to eliminate strains that retained plasmid DNA.To derive an allelic exchange mutant, a representative hygromycin-resistant, XylE-positive colony of 4 colonies screened, three XylE-negative colonies were identified of which only one (213R.4) colony with an SmO morphotype yielded a single 3.2 kb PCR product corresponding to the 1.9 kb rtfA gene interrupted with the 1.3 kb hyg cassette. Southern blot analysis confirmed that strain 213R.4 possessed only a chromosomal copy of rtfA::hyg  plasmid to complement rtfA in trans. Strain 233R.1 created by transformation of 213R.4 with pVAP52 and thus containing only a single copy of rtfA, demonstrated a pattern of ssGPL and nsGPL similar to wild-type, serovar 8 M. avium  expressed ssGPL but not nsGPL  has been used successfully to generate homologous recombinants of M. tuberculosis [xylE [Here we report on the generation of allelic exchange mutants using a double negative-selection system utilizing a temperature sensitive origin of replication of plasmid pAL5000 and the rculosis . The incctor pPR2 has beenis [xylE , providesacB or the inability of this gene to provide efficient counter selection as a single copy. The latter was consistent with our observation that katG expressed as a single copy-integrant did not confer INH-susceptibility to M. avium sufficient to serve as a counter selection marker [katG, as we have observed with the smooth transparent (SmT) morphotype of 920A6 (unpublished data). In this system, for INH-resistant strains, it may be prudent to perform selections as a two-step process, i.e., growth in broth at 39°C to eliminate plasmid replication followed by selection in solid medium containing hygromycin to increase the yield of double crossover mutants in relation to spontaneous hygromycin-resistant strains. Although sacB is a useful marker for M. tuberculosis, it appears to be minimally useful as a counter-selection marker for allelic exchange in M. avium. Also, this is the first reported instance of using xylE as a marker for allelic exchange in M. avium.We observed a high background of hygromycin-resistant, sucrose-resistant, XylE-positive colonies after selection at 39°C on sucrose-containing media suggesting a high frequency of a single crossover or illegitimate recombination. The high number of XylE-positive clones either represented a high degree of spontaneous mutation in n marker . AdditioM. avium. Studies have suggested a role for serovar-specific GPL in the pathogenesis of M. avium infection as highly antigenic molecules [rtfA gene via homologous recombination to block the addition of rhamnose as the proximal sugar common to ssGPLs resulting in construction of isogenic mutants expressing only non-specific GPL. Complementation of the rtfA gene as a single copy integrant in trans restored ssGPL synthesis and maintained nsGPL synthesis. Complementation of the ssGPL-null mutant with rtfA on an episomal plasmid, however yielded only serovar-8 specific GPL. In the latter case, all nsGPL components  were utilized as substrates for generation of ssGPL and thus were lost due to over-expression of rtfA. Also, since we do not observe any serovar-1 ssGPL  on TLC or GC analyses, this would suggest that the serovar-8 specific GPL disaccharide Rha-Gluc was generated prior to its addition to 6dTal for the generation of ssGPL.The glycopeptidolipids represent the most abundant cell wall component of olecules  affectinolecules  or have olecules ,3,5,25. ts-sacB double negative and xylE counter-selection system was reported for M. avium and we were able to construct isogenic mutants devoid of serovar-8 GPL. Due to limitations of various genetic manipulation techniques, this is the second only reported allelic exchange system for M. avium. With a few experimental modifications, this system of allelic exchange would be especially useful for M. avium strains that demonstrate high levels of INH drug resistance. Finally, through the construction of mutants in GPL (or any other cellular component) synthesis, the role of M. avium GPLs (and other components) in host-pathogen interaction, immunogenesis, and other qualities such as drug resistance can be determined.Insertion mutagenesis via the GPL: glycopeptidolipid6-d Tal: 6-deoxytalosensGPL: non-specific glyopeptidolipidssGPL: serovar-specific glycopeptidolipidrtfA: rhamnosyltransferasewt: wild typeLP: lipopeptideSmO: smooth opaqueRha: rhamnoseHyg: hygromycinGC/MS: gas chromatography/mass spectrometryPCR: polymerase chain reactionTLC: thin-layer chromatography3,4-O-diMe-Rha: 3,4-O-dimethyl-Rhamnose3-O-Me-6dTal: 3-O-Methyl-6-deoxytalose3-O-Me-Rha: 3-O-Methyl-RhamnoseSmT: smooth transparentINH: isoniazid hydrazide6dTal-Rha: 6-deoxytalose-rhamnoseRha-Gluc: rhamnose-glucose-1: microgram per milliliterμg mlrtfA mutant, and complemented strains, generation of pVAP52 and complemented rtfA mutant as well as selection techniques of the rtfA mutant.VRI: Writing and submission of this manuscript, molecular genetic analysis of the wt, rtfA mutant.SHL: Generation and initial characterization of plasmids and the serovar 8 TME, JMI, and JTB: Isolation and analysis of GPL, critical reading of the manuscript, and assistance in experimental techniques and study design.JNM: Principal Investigator in whose lab this research was conducted.


Escherichia coli FabA and subsequently in 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas sp. strain CBS. Since that time structural determinations have shown a number of other apparently unrelated proteins also share the Hotdog fold.The Hotdog fold was initially identified in the structure of Using sequence analysis we unify a large superfamily of HotDog domains. Membership includes numerous prokaryotic, archaeal and eukaryotic proteins involved in several related, but distinct, catalytic activities, from metabolic roles such as thioester hydrolysis in fatty acid metabolism, to degradation of phenylacetic acid and the environmental pollutant 4-chlorobenzoate. The superfamily also includes FapR, a non-catalytic bacterial homologue that is involved in transcriptional regulation of fatty acid biosynthesis.We have defined 17 subfamilies, with some characterisation. Operon analysis has revealed numerous HotDog domain-containing proteins to be fusion proteins, where two genes, once separate but adjacent open-reading frames, have been fused into one open-reading frame to give a protein with two functional domains. Finally we have generated a Hidden Markov Model library from our analysis, which can be used as a tool for predicting the occurrence of HotDog domains in any protein sequence.The HotDog domain is both an ancient and ubiquitous motif, with members found in the three branches of life. Escherichia coli β-hydroxydecanoyl thiol ester dehydratase (FabA), where Leesong et al. noticed that each subunit of this dimeric enzyme contained a mixed α + β 'hot dog' fold -V-x-[HF]-x(2)-[FY], where D is the active site residue, is found in the YbgC-like subfamily and in the 4HBT-I subfamily. 19 of the 29 members are found in the YbgC-like group and 3 in the smaller 4HBT-I group. The remaining 7 proteins are scattered in various clusters consisting of hypothetical or unknown proteins. We have found, using MASIA, that this motif is found in the entire YbgC and 4HBT-I subfamilies, extending the number of proteins containing this motif to 107. We have also identified a HGG motif in the 4HBT-II and PaaI subfamilies. This motif is HGGAS-x-ALAE in the 4HBT-II subfamily and HGG-x-IF-x-LAD in PaaI members. The active site residue, Glu73, is known for 4-hydroxybenzoyl-CoA thioesterase from Arthrobacter sp. Strain SU, however the active site for E. coli PaaI is not known and we suggest that it is Asp61 in the HGG motif above, which is 100 % conserved in all members of this subfamily .-15.To define subfamilies we clustered the results of an all-against-all search of the 1357 HotDog domain proteins using NCBI BLASTP and single linkage clustering at an E-value of 10Gene Context Tool) . A motif starts when at least 3 of 4 consecutive positions are more than 40% conserved and extend until at least 2 amino acids in a row are less than 40% conserved [onserved . Motifs Click here for fileSubfamily alignments. These alignments were constructed using the MAFFT alignment program [72] and rendered using the CHROMA software package [73]. Known active site residues are indicated below the subfamily alignments. The highly conserved Asp residue in the PaaI subfamily is proposed as an active site residue based on motif similarities between the 4HBT-II subfamily and the PaaI subfamily. Jpred predicted consensus secondary structures are indicated above the alignments [74].Click here for file


On the Origin of Species, biologists are still debating how new species emerge from old—and even the definition of species itself. Darwin demurred from offering a hard and fast definition, suggesting that such a thing was “undiscoverable.” One of the more enduring definitions characterizes organisms as distinct reproductive units and species as groups of individuals that can interbreed and produce viable, fertile offspring. The lack of genetic exchange between species, called reproductive isolation, lies at the heart of this definition. Environmental changes can create physical barriers between populations that preclude mating between the populations. Reproductive isolation can also involve changes at the genetic level, when molecular barriers prevent two recently diverged populations from producing viable or fertile offspring. Such factors limit gene flow between diverging species and allow the emergence of genetically novel yet sound populations—that is, new species.Nearly 150 years after Darwin published Drosophila melanogaster, none has been shown to display this functional divergence. Now, working with D. melanogaster and its sibling species D. simulans and D. mauritiana, Daniel Barbash, Philip Awadalla, and Aaron Tarone establish the functional divergence of a candidate hybrid compatibility gene and confirm its status as a true speciation gene.At the heart of reproductive isolation is a phenomenon called hybrid incompatibility, in which closely related species are capable of mating but produce inviable or sterile offspring. The classic example of hybrid incompatibility is the male donkey–female horse cross, which yields a sterile mule, but many other cases have been documented among mammals, and thousands of plant crosses produce infertile offspring. Much has been learned about the genetic architecture of hybrid incompatibility by studying the offspring of closely related, or “sibling,” fruitfly species in the lab. Sibling species are morphologically very similar, or even indistinguishable, but typically do not interbreed in nature. In the lab, their offspring are either sterile or inviable, a fate that varies depending on the gender of the offspring and species of the parents. To elucidate the molecular mechanisms of reproductive isolation, biologists must first identify candidate hybrid incompatibility genes. Species- or lineage-specific functional divergence is an essential trait of these genes.  While several such candidate genes have been identified in the fruitfly (A) and not the ancestral allele (a) causes the incompatibility phenotype, which means that experimental manipulations of A but not a should affect the hybrid incompatibility phenotype.Since the 1930s, investigations of reproductive isolation have been guided by the Dobzhansky-Muller model, which attributes hybrid incompatibility to the interactions between two or more genes that have evolved independently in two isolated populations. These independently evolving genes diverge functionally, and the interactions of these functionally divergent genes in a hybrid individual are responsible for the defective phenotypes observed (either inviability or sterility). If this is the case, the alleles, or versions, of the gene causing hybrid incompatibility should have distinct phenotypes in the two species. A corollary of the model says that the diverged allele Hybrid male rescue (Hmr) gene from each sibling species and observing the mutations' effects on the flies' hybrid offspring. In previous experiments the researchers had shown that loss-of-function mutations in the D. melanogasterHmr gene “rescue” hybrid individuals from the hybrid incompatibility phenotype  typically observed in the offspring of crosses between D. melanogaster and its sibling species, and that increased Hmr activity suppresses rescue and kills hybrids. If D. melanogasterHmr has functionally diverged between the species, then transgenes containing Hmr from sibling species should not cause the hybrid incompatibility phenotype caused by the D. melanogasterHmr. The researchers tested this hypothesis by introducing transgenic Hmr genes from sibling species into D. melanogaster. In all cases, the hybrid male offspring of D. melanogaster/D. mauritiana and D. melanogaster/D. simulans crosses “were at least as viable as their brothers without the transgene.”Barbash et al. tested the model's predictions by genetically manipulating the alleles of the D. melanogaster and D. simulans and found that the Hmr gene was among the most rapidly evolving genes. By examining the frequency of mutations that have accumulated between D. melanogaster and sibling species relative to the number of mutations accumulated within species, the authors show that the mutations between species were by and large not neutral and that they occurred after D. melanogaster diverged from its sibling species, indicating that the gene has been under positive natural selection. Barbash et al. have not only identified a bona fide speciation gene by demonstrating its functional divergence, they've also created a platform for investigating the mechanisms through which such genes cause hybrid incompatibility and lay the groundwork for speciation.To examine this divergence at the genomic level, Barbash et al. compared the divergence of 250 genes in


These phylogenetic studies did not, however, sample two additional groups traditionally classified within Rafflesiales (Apodantheaceae and Cytinaceae). Here we provide molecular phylogenetic evidence using DNA sequence data from mitochondrial and nuclear genes for representatives of all genera in Rafflesiales.The phylogenetic relationships among the holoparasites of Rafflesiales have remained enigmatic for over a century. Recent molecular phylogenetic studies using the mitochondrial Mitrastema, ascertained using mitochondrial matR, are congruent with results from nuclear SSU rDNA when these data are analyzed using maximum likelihood and Bayesian methods. The relationship of Cytinaceae to Malvales was recovered in all analyses. Relationships between Apodanthaceae and photosynthetic angiosperms varied depending upon the data partition: Malvales (3-gene), Cucurbitales (matR) or Fabales (atp1). The latter incongruencies suggest that horizontal gene transfer (HGT) may be affecting the mitochondrial gene topologies. The lack of association between Mitrastema and Ericales using atp1 is suggestive of HGT, but greater sampling within eudicots is needed to test this hypothesis further.Our analyses indicate that the phylogenetic affinities of the large-flowered clade and matR gene tree, thus providing compelling evidence for organismal relationships. Horizontal gene transfer appears to be influencing only some taxa and some mitochondrial genes, thus indicating that the process is acting at the single gene (not whole genome) level.Rafflesiales are not monophyletic but composed of three or four independent lineages (families): Rafflesiaceae, Mitrastemonaceae, Apodanthaceae and Cytinaceae. Long-branch attraction appears to be misleading parsimony analyses of nuclear small-subunit rDNA data, but model-based methods  recover a topology that is congruent with the mitochondrial  Combining gene sequences from multiple subcellular compartments continues to provide increasingly well-resolved flowering plant phylogenies  and thesRafflesia, whose meter-wide flowers are the largest among all angiosperms, and Pilostyles, whose flowers are less than a centimeter in diameter. Such wide morphological variation has resulted in classifications that comprise four families: 1) the "small-flowered clade" (Apodanthaceae) with Apodanthes, Berlinianche, and Pilostyles, 2) the "large-flowered clade" (Rafflesiaceae s. str.) with Rafflesia, Rhizanthes, and Sapria, 3) the "inflorescence clade" (Cytinaceae) with Bdallophyton and Cytinus, and 4) the "hypogynous clade" (Mitrastemonaceae) with Mitrastema [Rafflesiales are a fascinating and enigmatic group of holoparasitic plants that includes trastema ,8.matR to identify the closest photosynthetic relatives of two clades within Rafflesiales. Three genera, representing two of the four families in the order, were used in that study: Rafflesia and Rhizanthes (Rafflesiaceae s. str.) and Mitrastema (Mitrastemonaceae). Analyses of the matR data placed Rafflesiaceae s. str. within Malpighiales, an order that includes passionflowers (Passiflora), willow , and violet (Viola). Mitrastemonaceae was placed within Ericales, an order containing blueberries (Vaccinium), primroses (Primula), and tea (Camellia). The authors argued that these results were robust because they were congruent using different analytical methods  and were not affected by long-branch attraction artifacts [Recently, Barkman et al.  used DNArtifacts . MoreovematR, 2) mitochondrial atp1 and 3) a "3-gene" data set consisting of nuclear SSU rDNA plus two chloroplast genes: rbcL and atpB (the latter two only from nonparasites).In this study we expand upon the previous analysis  by inclumatR resulted in trees congruent with each other and with those previously generated [Rafflesia, Rhizanthes, and Sapria were placed with strong support in Malpighiales. Mitrastema was placed in Ericales sister to Vaccinium. The Cytinus and Bdallophyton clade (Cytinaceae) was strongly supported and this clade was sister to one composed of four genera of Malvales, an order that contains cotton (Gossypium), rockrose (Cistus) and chocolate (Theobroma). For Apodanthaceae, Apodanthes and Pilostyles were sister taxa and derived from within Cucurbitales, an order that contains squash/pumpkin (Cucurbita) and Begonia. For Berlinianche, sequences homologous to matR could not be obtained using several primer combinations.Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) analyses of mitochondrial enerated  , although this relationship does not receive strong support. This is remarkable given that 15 taxa from Ericales were included, yet a relationship with this order (as seen with matR) was not obtained with atp1. The large-flowered clade was strongly supported as monophyletic in all analyses, however, its position within the eudicots did not receive strong support. Parsimony analysis placed Pilostyles as sister to Pisum  and this clade was sister to Berlinianche, but both with low bootstrap support. Apodanthes was strongly suported (90% bootstrap) as sister to Polemonium  with MP but with ML this long-branch clade received lower support  to strong (BI) support.All three analytical methods of the e Figure . Clades t Figure . The twoPilostyles were sister to a clade composed of Pavonia and Gossypium, also with low bootstrap support. In constrast, BI analysis of the 3-gene matrix placed Mitrastema with Ericales and the large-flowered clade was a component of Malpighiales, the latter with strong support. The inflorescence clade  and the small-flowered clade (Pilostyles) were allied with Malvales  and reduced (77 taxon) 3-gene matrices were generally congruent and both resulted in all taxa of Rafflesiales being associated with Malvales  placed this clade in Malpighiales. Thus, the positions of the parasite clades inferred in four separate MP analyses matched the positions found for these clades in the single ML tree.MP analyses of SSU data sets from which all but one parasite group had been removed resulted in phylogenetic placements that matched those found in the ML tree. MP analysis of a data set from which all Rafflesiales except rbcL that are typically used to infer phylogenetic relationships among angiosperms but have not yet been amplified from any Rafflesiales and are presumed lost [Rafflesia and Cytinus [atp1 and matR were used, in combination with nuclear rDNA and chloroplast genes, to reliably place Hydnoraceae with Aristolochiaceae [Determining the photosynthetic relatives of Rafflesiales has long presented a challenge owing to the extreme reduction and/or modification of morphological structures that have accompanied the evolution of this lineage ,11. Molemed lost . Increasmed lost ,12. Simi Cytinus . DespiteRafflesia was several times longer than any other branch, and that this branch was attracted to the second-longest branch in the tree – the one between gymnosperms and angiosperms. For these reasons, they argued that nuclear SSU rDNA sequences are of limited utility for assessing the phylogenetic position of Rafflesia.Long-branch attraction, a bias in certain phylogenetic inference methods in which similarity due to convergent or parallel changes produces an erroneous phylogenetic grouping of taxa , is oftematR topology presented by Barkman et al. [Rafflesia is closely related to Malpighiales and Mitrastema is a member of Ericales , recovers a nearly monophyletic Rafflesiales containing all of the longest terminal branches in the tree  but he also considered other groups such as Aristolochiaceae , Sterculiaceae  and Cucurbitaceae . In general, different characters supported relationships with one or another group and therefore he left the subject as unresolved. Three proposed synapomorphies between Passifloraceae and Rafflesia were cited by Barkman et al. [Rafflesia), an androgynophore (gynostemium or column in Rafflesia), and an annular corona (diaphragm in Rafflesia). Whether these structures are homologous is not clear and will likely require further morphological studies, possibly examining the floral development genes themselves. These hypotheses require scrutiny because the apparent similarities in structure are not clear when examined in detail. For example, the androgynophore of Passiflora is composed of a stalk that bears the androecium and gynoecium. In Rafflesia, the ovary is inferior , hence the central column must involve other gynoecial parts. The corona of Passiflora is very different in structure and function from the diaphragm of Rafflesia [Passiflora caerulea and Euonymus [Rafflesia. Whether this association represents parasitism or not is a matter of semantics [Cissus and Opuntia growing on Yucca and Opuntia on Cercidium and Idria. In all of these cases, a true haustorium does not form and more likely these represent forms of grafting. It is difficult to state whether such rare occurrences have any bearing on the origin of parasitism in Rafflesiales or other parasitic flowering plants.Barkman et al.  suggestert Brown  represenn et al. : a hypanafflesia . The obsEuonymus  was discEuonymus  as a posemantics , for othMitrastema in Ericales, a result congruent with that obtained using mitochondrial matR. As noted by Barkman et al. [Mitrastema has bisexual, protandrous flowers with a collar-shaped, four-merous perianth tube. The stamens are connate into a tube (androphore) crowned by a fertile zone of pollen-bearing locules. The staminal tube, open at the top by a small hole, circumscissally separates from the flower as it is pushed up by the growing gynoecium. The apical portion of the staminal tube is sterile, but below this is a series of vertical rings of ca. ten minute, pollen sacs each. The gynoecium is hypogynous, one-locular, with a thick, conical stigma. Placentation is parietal with 8–15 (-20) unequal placental lobes filling the locule. The numerous ovules are small (190 by 120 μm), anatropous, unitegmic (but with two cell layers), and tenuinucellar. Although some floral morphological features of Mitrastema are not in conflict with those seen in Ericales, such as extrorse anthers and cellular endosperm, features such as decussate leaves, circumscissile fruit dehiscence, and parietal placentation are too general to draw specific associations. Given that Mitrastema is an achlorophyllous holoparasite and that one clade of Ericaceae (Monotropoideae) contains achlorophyllous mycotrophs, it is intriguing to ask whether these groups share a common ancestor or evolved independently. The most specialized morphological feature found in Mitrastemonaceae, the athecal androecium, is not found in Ericales but in Malvaceae, the only angiosperm family that shows the entire gamut from taxa with normal stamens, to taxa with stamens deviating only slightly from the common pattern [Maximum likelihood and Bayesian analyses of the 3-gene and nuclear SSU rDNA data partitions placed n et al. , this re pattern ,28, to a pattern .matR results. These results and those obtained from the simulation study indicate that the large-flowered clade and Mitrastema are artifactually attracted to Cytinaceae when parsimony is utilized.The most consistent phylogenetic signal that is seen across all data sets and types of analyses is a relationship between Cytinaceae and Malvales Table . BecauseRafflesia and Sapria (but not Rhizanthes), is lacking in Cytinaceae. Bdallophyton is dioecious and Cytinus is either dioecious  or monoecious (C. hypocistis). The perianth is tubular, composed of four to nine imbricate organs. The androecium is connate, forming a compact synandrium with extrorse anthers and the pollen is 2-, 3-, or 4-porate. The female flower is epigynous with a columnar style terminated by a globose or capitate, viscous stigma with commissural lobes [Unlike other Rafflesiales, members of Cytinaceae have multiple flowers arranged in an inflorescence. The floral structure called the diaphragm, seen in al lobes . The ovaPilostyles (the only Apodanthaceae for which SSU rDNA sequences are available) within Malvales, however, a sister relationship with Cytinaceae is not consistently obtained. A 3-gene alignment that included additional representatives of Malvales (16 taxa) gave similar results as shown in Figure Pilostyles on a clade separate from other Rafflesiales). These data, in conjunction with the results from the mitochondrial genes, support an evolution of Apodanthaceae independent from Rafflesiaceae s. str. The well-supported relationship between Pilostyles and Apodanthes using matR is expected given their very similar floral morphology [Begonia and Cucurbita). Contamination with host tissue is excluded because neither parasite is known to currently occur on a member of Cucurbitales. Apodanthaceae are grouped with Pisum  and Polemonium  on the atp1 tree, but no atp1 sequences from representatives of Cucurbitales were available from GenBank to test the matR result. The sister relationship between Apodanthes and Polemonium is strongly supported on the MP tree . Additional sampling within the eudicots will be required to better understand the atp1 gene tree topology.Maximum parsimony and likelihood analyses of the 3-gene data set and nuclear SSU rDNA sequences alone also place rphology , yet thiPilostyles and Apodanthes male flowers, a tubular synandrium surrounds and fuses with a central structure that could be interpreted as a sterile gynoecium. Support for the concept that such a central structure is a pistillode comes from Berlinianche where the upper portion of the synandrium is free from the central part. In female flowers of Apodanthaceae, there is no rudiment of an androecium, hence the central tissue is apparently entirely gynoecial.Morphological features shared between Apodanthaceae and Cytinaceae are: unisexual flowers, a connate androecium, an inferior ovary, and a unilocular ovary with four parietal placentae bearing numerous, anatropous, tenuinucellate ovules ,31. FlormatR data indicate Apodanthaceae are related to Cucurbitales, an order with seven families, 129 genera and 2300 species. Hosts for Apodanthaceae are generally legumes, although Apodanthes occurs most frequently on Casearia . Thus, neither recent HGT nor contamination explains this result. Apodanthaceae shares some morphological features with members of Cucurbitaceae, subfamily Cucurbitoideae: unisexual, five-merous flowers (Berlinianche); carpellate flower with a unilocular, inferior ovary with parietal placentation; anatropous, bitegmic ovules; staminate flower with connate filaments (monadelphous) and a rudimentary gynoecium (pistillode) [In contrast to the above discussion, the tillode) . ConflicA requirement of the molecular phylogenetic approach to inferring evolutionary histories of organisms is vertical transmission of genetic material from parent to offspring. In contrast, horizontal gene transfer (HGT) describes the movement of genetic material between organisms of no direct ancestor-descendant relationship. Although the frequency of HGT is currently not well understood among prokaryotic and eukaryotic organisms, it is clear that HGT can compromise accurate inference of genealogical history. In plants, lateral movement of genetic material has been documented for mobile genetic elements such as introns -37 but oIncongruence among gene trees derived from different data sets can derive from a number of factors such as technical causes , gene/genome-level processes, and organism-level processes  . HGT hasApodanthes and Pilostyles on the atp1 tree as sister to Pisum  represents a likely case of HGT. The atp1 data conflict with those from matR that associates Apodanthaceae with Cucurbitales. Moreover, we infer that the SSU rDNA tree better represents the organismal phylogeny because it seems less likely that nuclear genes would be influenced by HGT [We believe that incongruence between the the mitochondrial and the nuclear gene trees Table  stem notd by HGT ,45. The matR tree closely matched results from other molecular phylogenetic investigations of angiosperms. The present study confirms that HGT is not implicated for the two lineages studied by Barkman as well as Cytinaceae, but this process could be invoked for Apodanthaceae. More recent work by these authors [atp1. These included Dalea to Pilostyles, Tetrastigma to Rafflesia, and Lithocarpus to Mitrastema. In addition, HGT of another mitochondrial gene, nad1, has been reported for Rafflesia and Sapria, both of which occur on the same clade as their hosts (Tetrastigma) on a gene tree [matR gene in Pilostyles and Apodanthes. Moreover, present-day hosts of parasitic angiosperms do not represent the only conduit for HGT if host choice has shifted through time as the parasite lineage evolves. For example, Barkman et al. [Mitrastema only parasitizes Fagales  but this parasite has also been recorded from Aquifoliaceae, Asteraceae, Elaeocarpaceae, Juglandaceae, and Myrtaceae [In their study of Rafflesiaceae s. str. and Mitrastemonaceae, Barkman et al.  discount authors  identifiene tree . These en et al.  state thyrtaceae . Host lamatR tree  were found to be identical to these "parasite" sequences, strongly suggesting contamination. In the case of Rafflesia, the DNA was obtained from a bud still attached to the host vine, both of which had been sectioned longitudinally. Disruption of these tissues likely resulted in transfer of host sap to the bud region where the tissue was sampled. Other samples of R. tuan-mudae from the same population, obtained as floral bracts with no host tissue, resulted in matR sequences that were similar to the other two Rafflesia species.Contamination of parasite DNA with DNA from the host plant is an issue that must be given serious attention. Indeed, two sequences shown on the e Figure , TetrastBerlinianche, whose flowers are much smaller than those of Rafflesia (5 mm in diameter), extreme care (using a stereo microscope) was exercised to remove floral parts devoid of any host tissue. Despite this, the matR sequence obtained from the first sample was that of the host, Julbernardia. Later, silica gel dried samples of other populations of the parasite were extracted, again using extreme care in avoiding host contamination. PCR products were obtained using several mitochondrial matR primers, but none were found to be homologous to this gene following BLAST searches. This result shows that host DNA was not present in this sample in sufficient amounts to amplify and that the parasite matR gene, if present, is highly divergent at the priming sites used.For Pilostyles was extremely difficult. Despite PCR products of the correct sizes using a variety of primer combinations, the sequences obtained from genomic DNA derived from flowers were deemed contaminants following BLAST searches that showed them to be most similar to monocots, fungi, etc. Only when sequences from two accessions of Pilostyles  both were most similar to Malvales was this considered good evidence for their true phylogenetic affiliation. Retrospectively, it is likely that the sticky flowers had accumulated wind-dispersed pollen  and that this DNA, despite being in low concentration, had less divergent priming sites than the parasite target DNA, allowing PCR to preferentially amplify the contaminant DNA.For all three Apodanthaceae genera, the conical style in female flowers is papillate and heavily secretory . This stPilostyles: P. hamiltonii [P. thurberi [Cuscuta [Pilostyles suggest that intact, mature mitochondria are too large to pass through heteroplastic plasmodesmata, however, mitochondrial genomes or portions of the genome are certainly small enough for transmission. Once inside the parasite cell, there are various fates for the host gene. It could become incorporated into the parasite mitochondrial genome, and then either replace the parasite copy or exist as a duplicate, or the host gene could reside in the parasite nuclear genome. For the latter case, the gene would likely become a pseudogene given the requirement of mitochondrial-specific patterns of RNA editing. Two forms of atp1 are present in the primitive angiosperm Amborella trichopoda [matR mRNA would be useful to determine whether the gene is expressed and whether mitochondrial-specific RNA editing patterns are present.Given the accumulating molecular evidence for HGT from host to parasitic plant, it is worthwhile to consider potential mechanisms, along with their constraints, that may suggest further research. Relatively little information exists on the structure of the endophyte of Rafflesiales. Ultrastructural studies have been conducted on two species of miltonii  and P. tthurberi . These a[Cuscuta  and inde[Cuscuta . Given tichopoda , one of Rafflesia, Rhizanthes, and Sapria) representing the large-flowered clade are monophyletic and are related to Malpighiales. The monogeneric family Mitrastemonaceae, the only member of the order with a superior ovary, is related to Ericales. The first of the remaining two families that have previously not been sampled is Cytinaceae  which is strongly supported as a member of Malvales. The last remaining unsampled family, Apodanthaceae  is either related to Malvales or Cucurbitales. Our simulation studies indicate that Mitrastema, Bdallophyton/Cytinus, and Rafflesia/Rhizanthes/Sapria have branches that are long enough to mislead parsimony. All of these relatively long branches appear to be attracted toward the Cytinaceae clade within Malvales. When nuclear SSU rDNA sequences are analyzed with ML, results fully congruent with those previously reported for two Rafflesiales clades using mitochondrial matR are obtained. If the phylogenetic affinityof Apodanthaceae are with Malvales, the results from the mitochondrial matR gene must represent a case of horizontal gene transfer (HGT) from Cucurbitales. If this proves to be the case, this provides an example of HGT from a nonhost plant to a parasitic angiosperm.In this study we have used data derived from nuclear, mitochondrial and chloroplast DNA and a variety of analytical approaches to address long-standing questions about the holoparasitic flowering plant order Rafflesiales. We show that Rafflesiales are not monophyletic but composed of at least three and possibly four independent lineages. Rafflesiaceae . Comparisons among gene trees allows for the identification of HGT, a phenomenon that requires further investigation to determine its modes of action and frequency among taxa and through evolutionary time.® 377 automated DNA sequencer, Applied Biosystems) according to manufacturer's protocols.DNA was extracted, amplified, cloned, and sequenced by using methods formerly reported -54. The matR alignment incorporated all of the Rafflesiales parasites and the nonparasite sequences previously published [Ginkgo, Pinus, and Zamia), 28 monosulcates, 63 nonparasitic eudicots, and 15 Rafflesiales. For two taxa (Mitrastema and Rhizanthes), our sequences, as well as those previously published, were from the same species but different accessions to test for consistency. Taking into account codon information, an alignment of 2177 sites was constructed manually using SeAl version 2.0 [atp1 alignment was similarly constructed and included the same gymnosperm outgroup genera as above, 24 monosulcates, 32 nonparasitic eudicots and 12 Rafflesiales. All of the monosulcate genera in the atp1 alignment were also represented in the matR data set, whereas eudicot sampling for atp1 was constrained by sequences available on GenBank (12 of the same genera as with matR or placeholders from same family).The initial ublished  as well sion 2.0 . The fulrbcL and atpB was constructed that included 103 taxa . Sampling across angiosperm orders was very similar to the matR matrix, differing only by the presence of 11 placeholders and a second accession of Pilostyles. For the holoparasites, only nuclear SSU rDNA sequences were included; the chloroplast gene data for these taxa were coded as missing. The two chloroplast genes were included to add stability to the tree topology given that nuclear SSU has been shown to contain lower phylogenetic signal when used alone [matR, the 103-taxon matrix was truncated to 77 taxa by removing all but five monosulcate taxa to facilitate likelihood analyses. All alignments reported in this paper have been deposited with TreeBASE [study accession number S1177, matrix accession numbers = M2034–M2037.To test the position obtained for Rafflesiales taxa using mitochondrial genes with an independent data set derived from different compartments, a 4646-site "3-gene" matrix combining sequences from nuclear SSU rDNA and chloroplast ed alone . As withTreeBASE : study aAll three data sets were analyzed using maximum parsimony (MP) and maximum likelihood (ML) methods in PAUP* 4.0b10  and BayeBdallophyton + Cytinus, Mitrastema, Pilostyles or the large-flowered clade comprising Rafflesia, Rhizanthes and Sapria) was removed to determine the position of each parasite group in the absence of other long-branch parasite taxa in the analysis. This is a form of the test proposed by Siddall and Whiting [All MP searches were performed using 100 random addition sequence replicates with tree-bisection-reconnection (TBR) branch-swapping, holding ten trees at each addition step, with all sites equally weighted. For the 77-taxon SSU data set, a series of four MP analyses were performed in which all but one parasite group . This submodel employed four (rather than six) relative rate parameters: one for A-C transversions and A-G transitions, one for A-T and C-G transversions, one for C-T transitions, and one for G-T transversions; the PAUP* LSET option used for analysis was "RCLASS = (a a b b c d)". Likewise, the models chosen by ModelTest for the matR data set were TVM+Γ (hLRT) and TIM+Γ (AIC), but a simpler statistically equal model  was used for analysis. This model employed three relative rate parameters: one for A-C, A-G, and G-T substitutions; one for A-T and C-G substitutions; and one for C-T substitutions; "RCLASS = (a a b b c a)", with among-site rate heterogeneity modeled with a gamma distribution. These simplified models were chosen to reduce computational time and to avoid estimation of unnecessary parameters, which can lead to greater variance in parameter estimates and higher topological uncertainty.For ML analyses, a MP tree was used in PAUP* to evaluate 56 nucleotide substitution models. ModelTest 3.06  was usedA successive approximations approach was used for all ML analyses ,64. SubsmatR and atp1 data sets; 10 million generations, with the first 5 million discarded as burn-in and trees sampled every 500 generations for the 3-gene data set) [matR and atp1 data sets instead. A partitioned model was used for the 3-gene data set (see below). Two Bayesian runs were performed for all analyses in an attempt to determine if stationarity was reached, and plots of log likelihood and parameter convergence were also evaluated; log-likelihood plots alone are insufficient for monitoring chain mixing and convergence [Nodal support for all data sets was estimated using one or more of the following methods: equal-weights MP bootstrap analysis , ML bootstrap analysis  ,66, and ata set) . The GTRvergence ,68.atpB first, second and third codon positions; rbcL first, second and third codon positions. Appropriate substitution models for each data partition were chosen by computing likelihood scores for each partition on a MP tree for the 3-gene data set under 56 substitution models in PAUP* and comparing the scores in ModelTest. The GTR+Γ + I model was the best-fitting model for all partitions. The Bayesian analysis was performed with all model parameters (except branch lengths) unlinked across partitions.The 3-gene data set was also analyzed in MrBayes 3.0. A "fully partitioned" analysis was used in which the 3-gene data set was divided into seven partitions: nuclear SSU; For the nuclear SSU rDNA data, constrained analyses were also performed. A constraint tree for 63 nonparasitic taxa was constructed using the MP topology of the "B series" tree from Soltis et al.  with relTo investigate possible long-branch attraction in parsimony analyses of the SSU rDNA data set, two sets of simulations were performed. For the first set of simulations, a reduced data set of SSU rDNA sequences for 20 taxa  was constructed and analyzed under ML (GTR+Γ + I model) in PAUP*. The tree resulting from this analysis, with its associated ML branch lengths and model parameters, was used as the model tree on which 100 data sets of length 1766  were simulated in Seq-Gen 1.2.7 . For theΓ + I – gamma + invariant sites distributionatp1 – ATP synthase alpha subunitatpB– ATP synthase beta subunitBI – Bayesian inferenceGTR – general time reversible modelHGT – horizontal gene transfermatK – maturase KmatR – maturase RML – maximum likelihoodMP – maximum parsimonyrbcL – ribulose bisphosphate carboxylase/oxygenase, large subunitSSU – small subunitTBR – tree bisection-reconnection branch swappingatp1 and matR sequencing and revised the text regarding morphological character comparisons. YQ provided primers, introduced AB to the field of molecular systematics, and supervised his Ph.D thesis. RVR conducted the PCR experiments showing host contamination of Rafflesia DNA and generated the matR sequences for several taxa. FEA performed the phylogenetic analyses. All authors read and approved the final manuscript.DLN coordinated all aspects of the study, obtained many of the genomic DNAs, generated all the nuclear SSU rDNA, conducted the sequence alignments, and drafted the manuscript. AB conducted the majority of the mitochondrial matR MP strict consensus tree from mitochondrial Strict consensus of 200,000+ trees obtained from maximum parsimony (unconstrained MP) analysis of the 77-taxon mitochondrial matR matrix. Bootstrap percentages are shown above the lines. Rafflesiales taxa are shown in bold italics.Click here for fileatp1 Strict consensus MP tree from mitochondrial Strict consensus of 328 trees resulting from a MP analysis of the 71-taxon mitochondrial atp1 matrix. Rafflesiales taxa are shown in bold italics. Bootstrap percentages are given above the branches.Click here for fileMajority rule consensus BI tree from 3-gene data set Majority rule consensus of 20,000 trees  resulting from Bayesian analysis of the 77-taxon nuclear 3-gene matrix. Clades with Bayesian posterior probabilities are indicated above the clades. Rafflesiales taxa are shown in bold italics.Click here for fileStrict consensus constrained MP tree from nuclear SSU rDNA Strict consensus of 6 trees resulting from the constrained MP analysis of the 77-taxon nuclear SSU rDNA matrix. Rafflesiales taxa are shown in bold italics. Bootstrap percentages are given above selected nodes .Click here for fileTaxa used in this study MS Excel file giving taxon names and GenBank numbers for all genes used.Click here for file


Whether a person inherits a defective gene or acquires genetic damage by chance, two types of genes typically play a role in transforming a healthy cell into a cancer cell. Oncogenes and tumor suppressor genes are normally involved in cell growth, development, and cell differentiation. Both functions can be appropriated to ill effect by mutations. Single mutations in these genes rarely cause cancer on their own, but they predispose cells to additional insults that precipitate malignant transformation.Susceptibility to cancer depends, among other things, on age. Though cancer in children is rare, the most common childhood cancers strike the hematopoietic system (leukemia), nervous system, and skeletal muscle system, while solid tumors of the lung, breast, prostate, and colon are more common in adults. This age differential suggests that an oncogene's ability to cause cancer in a particular cell type might depend on that cell's developmental stage.  In a new study, Dean Felsher and colleagues show that age matters: activating oncogenes at different developmental time points in mouse liver cells produces different results.MYC oncogene to initiate liver cancer (hepatocellular carcinoma) in a transgenic mouse model varies with the age of the mouse.Typically, once a cell is transformed, it stays in its “differentiative” state, that is, it stays in whatever developmental stage it was in when it became a tumor cell. But in a previous study, Felsher and colleagues found that turning off oncogenes in tumor cells allowed them to differentiate; these mature cells did not resume tumorigenesis after the oncogenes were reactivated. In this study, Felsher and colleagues show that the ability of the MYC overexpression in the liver cells of embryonic, neonatal, and adult mice, the authors used a biotech trick  that controls gene expression dose and timing with a drug. The system relies on the interplay of two elements: a gene  fused to a regulatory enhancer, and a transcription factor that binds to the enhancer and activates the gene. Administering a tetracycline-like drug  prevents the transcriptional activation of the gene.To study the consequences of MYC oncogene in mice during embryonic development or at birth occasioned their demise fairly quickly . In contrast, overexpression of MYC in adult mice resulted in tumorigenesis only after a long latency period. When the authors evaluated the cellular effects of MYC overexpression, they found that hepatocytes from neonatal transgenic mice showed evidence of increased proliferation (replicated DNA content) compared to normal hepatocytes, while transgenic adult hepatocytes showed increased cell and nuclear growth (some nuclei had as many as twelve genome copies instead of two) without dividing. Since these adult cells eventually developed into tumors, some clearly acquired the ability to divide, which the authors show is facilitated, among other events, by the loss of the p53 tumor suppressor.Overexpressing the MYC activation, Felsher and colleagues conclude, depend on the cell's developmental program, which determines whether a cell can grow and divide, or simply grow. In adult hepatocytes—which are normally quiescent—MYC requires additional genetic events to induce cell division and tumorigenesis; in immature hepatocytes—which are already committed to a program of cellular proliferation—MYC activation alone is sufficient. The next step will be to identify the epigenetic developmental factors, both internal and external, that lead to tumor formation, and how to prevent it.Altogether these results suggest that whether oncogene activation can support tumor growth depends on the age of the host, which in turn suggests the role of genetically distinct pathways in young and adult mice. The consequences of


There is accumulated evidence that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality. Both fasting levels of Hcy and post methionine loading levels are used as prognostic markers. The aim of the present study was to investigate the existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration.® formula).Six healthy men aged 23–26 years participated in 4 experiments. In the first and second experiments, the daily rhythm in Hcy as well as in other amino acids was investigated under a normal or an inverse sleep-wake cycle. In the third and fourth, Hcy concentrations were investigated after a morning and evening methionine loading. To standardize food consumption in the first two experiments, subjects received every 3 hours 150 ml of specially designed low-protein liquid food (EnsureIn both the first and second experiments there was a significant daily rhythm in Hcy concentrations with a mid-day nadir and a nocturnal peak. Strikingly different 24-h patterns were observed in methionine, leucine, isoleucine and tyrosine. In all, the 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments, which were not significantly different from each other.There is a daily rhythm in plasma concentration of the amino acid Hcy, and this rhythm is independent of sleep-wake and food consumption. In view of the fact that increased Hcy concentrations may be associated with increased cardiovascular risks, these findings may have clinical implications for the health of rotating shift workers. Experimental results accumulated in recent years have revealed that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality -5. PlasmStudies conducted during the 1960s have demonstrated that plasma levels of several amino acids vary in a daily manner. Feigin, Klainer and Beisel  were theMore recently, plasma Hcy levels were also shown to vary in a daily manner in humans with an evening peak and a morning nadir . SignifiIn the present study, we further investigated the possible existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration.2). They were instructed to avoid alcohol and coffee beverages during the 24 hours that preceded each of the experimental periods. The study was approved by the local Human Ethics Committee, and subjects gave written informed conset before being enrolled in the first experiment. Subjects were paid for their participation.Six healthy men aged 23–26 years participated in 4 experiments. All were students who maintained a normal and regular sleep-wake cycle for at least three months prior to the studies. They were screened to ensure an adequate state of health by physical examination, detailed medical history and blood testing. All had a normal body weight (mean body mass index (BMI) = 23.5 ± 1.6 Kg/mIn the first and second experiments, daily rhythms in Hcy as well as in other amino acids were investigated under a normal or an inverse sleep-wake cycle. In the third and fourth experiments, Hcy concentrations were investigated after morning and evening methionine loading.® formula) with the following composition: proteins , fat , carbohydrates , vitamins and minerals, in 77 ml water. No other food except for water was allowed.Subjcts were admitted to the laboratory at 1800 for a period of 24 hours, after having a normal day. A catheter was inserted into an antecubital vein and was kept patent by a drip of saline. Electrodes were attached for polysomnographic monitoring to determine sleep stages. These included EEG, EMG, EOG, respiration by respiratpry belt and nasal thermistor, and oximetry. Starting at 1900, 5-ml blood samples were drawn every hour until 1900 on the next day. Thoughout this period subjects were either in a supine or a sitting position in individual rooms where they could read, use their personal computers or watch television. From 2300 to 0800 the room lights were turned off during the sleep period. Blood samples were collected into EDTA treated tubes, immediately centrifuged at 4°C, and plasma was stored at -70°C until assay. Hourly blood sampling during sleep continued with minimal disturbance to subjects' sleep. To standardize food consumption and to provide adequate energy intake, subjects received every 3 hours 150 ml of specially designed liquid food . At the start of each methionine loading test, subjects were administered 100 mg/kg body weight methionine, mixed in fruit juice. Blood samples (5 ml) were taken into EDTA treated tubes before methionine loading, designated as time 0, and then at +2, +4, +6 and +8 hours after methionine administration. Light carbohydrate rich meals were provided at +1 and +6 hours after the methionine loading in each of the test periods.12 were also measured in all samples of all subjects using commercially available kits from Abbott. The assays were performed on an Abbott IMX analyzer that utilizes ion capture technology for folate determination and microparticle enzyme immunoassay (MEIA) technology for B12. The assays were performed according to the manufacturers' instructions and used quality control sera supplied by Abbott.Plasma amino acids levels  were measured in duplicates using a Biochrom 20 Amino-Acid analyzer  as described before . The meaRepeated measurements ANOVA was used to compare the means of the amino acids between the first two experiments. To obtain the average 24-h Hcy curves, each individual data point was replaced by a z-transformation based on the individual 24-h mean and standard deviation, before averaging across subjects. Then, each of the individual time series was subjected to Cosinor analysis to determine its amplitude and acrophase. Since Experiment 1 was perfomed during the summer (August) and Experiment 2 was performed during early winter (late November), approximately 2 months after the change from Summer daylight-saving time to Winter time, during which the clock in Israel was advanced by one hour, the 24-h curves of the first experiment were advanced by 1 hour before the analysis. Then repeated measurements ANOVA was used to determine differences in acrophase between the experiments. In the third and fourth experimens, the concentrations of Hcy at times 0, 2, 4, 6, and 8 hours after methionine loading were analysed by repeated measures ANOVA to determine if there were any significant morning-evening differences in Hcy levels.All subjects successfully completed the four experiments. In experiment 1 when they slept from 2300 to 0700, average sleep latency was 22.2 ± 7.3 min, total sleep time was 407.3 ± 51.8 min, and sleep efficiency was 77.7 ± 9.2%. In experiment 2 when they slept from 0720 to 1500, average sleep latency was 4 ± 3.1 min, total sleep time was 371.5 ± 59.4 min, and sleep efficiency was 83.6 ± 12.2%. In spite of the reversal of the sleep-wake cycle, the 24 h means and coefficients of variation of Hcy in the two experiments were very similar to each other, 8.82 μmol/L and 29.7% and 8.51 μmol/L and 27.7%, in experiments 1 and 2, respectively. None of the subjects had abnormal Hcy levels (>15 μmol/L) at any point across the 24 hours.Figure Strikingly different 24-h patterns were observed for the other amino acids: methionine, leucine, isoleucine and tyrosine. In all, the average z-transformed 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Their level was notably lower during the sleep period, regardless of its timing, and increased every two hours in synchrony with the times of feeding. This pattern is exemplified in Figure 12 and folic acid. While folic acid showed a linear increase throughout the study period, the 24-h pattern of B12 was rather constant with slight elevation during the night time (data not shown).We did not find any evidence for rhythmicity in the concentrations of BAs expected, methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments Figure . AnalysiThe present study demonstrated that under strictly controlled dietary conditions plasma levels of Hcy shows significant daily rhythmicity, which is independent of the 24-h cycle of sleep and wake, with a peak at around 2200 to 2400. Previously, similar rhythmicity in Hcy with an evening peak was reported in obese diabetic patients by Bremner et al  and with12 dependent while the reaction with betaine is not. In the transsulforation pathway, Hcy condenses with serine to form cystationine in an irreversible reaction catalyzed by the pyridoxal-5'-phosphate (PLP)-containing enzyme, cystationine beta synthase. Although we do not have any information as yet on the underlying mechanism responsible for the daily rhythm in plasma Hcy, it is most probably related to the balance between its rates of production and disposal. A high Hcy concentration could be due to an elevated production rate, a decreased rate of transsulforation, a decreased rate of remethylation to methionine, or any combination of these processes.Homocysteine is a non-protein sulfur containing amino acid, and an intermediate in the metabolism of the essential amino acid methionine. The metabolism of Hcy is accomplished by two major pathways, remethylation into methionine and transsulfuration to cystationine . In reme12 deficiencies, or by methylenetetrahydrofolate reductase thermolability. The variations in plasma vitamin concentrations, however, could not provide an explanation for the daily rhythms in Hcy. The 24-pattern of folate levels showed a linear increase from the beginning to the end of the study. Although the plasma concentrations of vitamin B12 varied across the 24 hours – in contrast however to what was expected if B12 were involved in the daily rhythm in Hcy, ie, increasing levels of B12 associated with decreasing levels of Hcy – the 24-h pattern in B12 was parallel to that of Hcy with a daytime nadir and a night time peak. Thus, it is unlikely that a daily rhythm in plasma vitamin concentrations can  explain the daily rhythm in Hcy.The fact that the range of the daily variations in the plasma levels of Hcy is on the same order of magnitude as those seen in mild hyperhomocysteinemia, may suggest that the two phenomena share a common underlying mechanism. Mild hyperhomocystenemia seen under fasting conditions is due to mild impairement in the methylation pathway. This may be caused by folate or BThe methionine loading test has been used to test the individual's ability to dispose of methionine through the transsulforation pathway . The facA different possibility that cannot be ruled out at this point is the involvement of the Hcy cellular export mechanism. The small amount of plasma Hcy is the result of a cellular export mechanism that is essential for keeping intracellular concentrations low to avoid potentially Hcy cytotoxic effects. Thus the daily rhythm in plasma Hcy may reflect variations in the activity of the cellular export mechanism, which result in varying levels of Hcy disposed to the plasma at different phases of the 24 hours rather than in its rate of metabolism. Further studies are needed to test this possibility.Finally, what may be the clinical implications of the present findings? We would like to suggest that the existence of a daily rhythm in Hcy concentration may have possible health-related consequences to shift workers, who were shown to be at an increased cardiovascular risk . FirstlySecondly, we do not know how the desynchronization between the circadian system and the enviornment which occurs in rotating shift workers may affect the rhythm in Hcy concentrations and its overall plasma concentration. Recently, Martins et al  reportedIn view of the fact that Hcy is a risk factor for cardiovascular morbidity, more research is needed on the possible role of hyperhomocysteinemia as a cardiovascular risk factor in shift workers.12 and folic acid. Methionine loading at 9 AM and 9 PM produced a comparable time-dependent increase in Hcy concentrations with a tendency toward a higher increase in the evening during the first 2 hours after loading. In view of the possible involvement of Hcy in cardiovascular morbidity, and of the increased cardiovascular morbidity in shift wokers, these findings may have implications to shift workers health.Our results demonstrated a daily rhythm in plasma concentrations of Hcy with a nocturnal peak that was independent of sleep-wake cycle and food consumption. There were no comparable rhythms in the concentrations of methionine, leucine, isoleucine and tyrosine, nor in the concentrations of BHcy – homocysteineEEG – ElectroencephalographyEMG – electromyographyEOG – electrooculographyEDTA – ethylanediaminetetraacetic acidCV – coefficient of variationANOVA – analysis of varianceNone declared.PL and LL co-designed the study, supervised the data collection and data analysis and wrote the paper.


The past few years have seen an explosion of data on the mouse genome, widely hailed as a guidebook to the genetic origins of human disease. Scientists are particularly interested in charting the location of SNPs—single nucleotide polymorphisms—throughout the genome. SNPs are DNA sequence variations, or mutations, that change a single nucleotide in the genome, replacing a cytosine (C) base, say, with thymine (T). Many SNPs, even those found within genes, have no functional effect. Others, however, can increase risk of specific diseases or alter a person's response to pathogens and drugs. Whether or not they are involved directly in a disease, SNPs are attractive markers for population studies aimed at identifying the multiple genes underlying complex diseases like diabetes and cancer.Extensive data exist on multiple inbred strains of mice linking their genetic makeup (genotype) to physical traits (phenotype), and scientists have used these data to guide investigations of gene function and disease. Many data have been gathered by crossing mouse strains and painstakingly analyzing their progeny, to tease out the relative contributions different genes make to pathogenesis. But these efforts take time. Investigators would greatly benefit from high-throughput methods to scan the mouse genome and flag markers for candidate disease genes. In 2001, Andrew Grupe et al. introduced an “in silico”  approach to do that very thing. The method scanned mouse SNP data to home in on chromosomal areas regulating complex traits and reduce the time needed to analyze mouse disease models from “many months” to “milliseconds.”For a number of reasons, however, it wasn't clear whether in silico mapping could deliver on its promise. For one thing, the density of SNP maps was sufficient to provide meaningful markers for only a few mouse strains, and phenotype information was lacking for many strains. In a new study, Tim Wiltshire and colleagues have addressed these limitations by mapping nearly 11,000 SNP probes to 48 mouse strains. They have also been able to use this dataset for in silico mapping to predict genomic regions with functionally important phenotypes.Wiltshire and colleagues first show that their method can predict the genomic location of a Mendelian trait (controlled by a single gene), in this case coat color, which the authors acknowledge is a “minimum requirement for a viable in silico mapping method.” They go on to map complex “quantitative” traits —gallstone development and plasma levels of high-density lipoprotein cholesterol—and find that their predictions fall in line with loci identified by traditional mouse disease studies. Noting a high correlation between QTLs predicted in silico and those identified experimentally, the authors argue that loci predicted using this method are “very likely to be biologically relevant.”Wiltshire and colleagues are careful to point out that in silico mapping is meant to complement, not replace, traditional gene mapping models. After all, computers are no match for living organisms in modeling the subtleties inherent in biological reactions. But this approach is a good starting point for identifying significant genomic areas in a new strain. And as new strains are genotyped and phenotyped, and refinements are made to the SNP database, the robustness of this method should only get better.