This last effect is potentially mutagenic in vivo. Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes.We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA) on DNA synthesis catalyzed by mammalian DNA polymerase δ(pol δ). In the presence of homologous PCNA, pol δ exhibits 1) increased absolute activity; 2) increased processivity of DNA synthesis; 3) stable binding of synthetic oligonucleotide template-primers , can only substitute poorly for either calf thymus or human PCNA (~10% as well) in affecting calf thymus pol δ. However, by mutating one or only a few amino acids in the region of Drosophila PCNA thought to interact with pol δ, all four effects can be enhanced dramatically.Drosophila offers the potential for immediate in vivo genetic analyses. Although it has proven difficult to obtain sufficient amounts of homologous pol δ for parallel in vitro biochemical studies, by altering Drosophila PCNA using site-directed mutagenesis as suggested by our results, in vitro biochemical studies may now be performed using human and/or calf thymus pol δ preparations.Our results therefore suggest that all four above effects depend at least in part on the PCNA-pol δ interaction. Moreover unlike mammals,  Drosophila melanogaster homologs of the proteins required for both DNA replication and repair have been identified and in several cases purified to apparent homogeneity. These include DNA polymerase α holoenzyme [in vitro using purified SV40 T-antigen and Drosophila cell-free extracts [Many loenzyme ,2, DNA ploenzyme , replicaloenzyme ), replicloenzyme -9) and vloenzyme ,11). Morextracts .Drosophila PCNA was first identified both as a highly purified protein able to substitute, albeit poorly, for human PCNA in a cell-free SV40 DNA replication system reconstituted from purified proteins [Drosophila PCNA cDNA and gene, express the protein in E. coli and deduce its complete amino acid sequence. Further results indicated that in flies, PCNA was encoded by a single gene located at position 56F5-15 on the right arm of chromosome 2. This was subsequently identified as the Drosophila mus209 locus [Drosophila PCNA gene of limited homology to the original and of unknown biological function has also been found [A protein about which much information has been obtained is proliferating cell nuclear antigen (PCNA). proteins  and by Yproteins  who used09 locus . RecentlE. coli [2-terminally his-tagged but otherwise wild-type human PCNA, also engineered for bacterial expression [Drosophila PCNA and strategies for site-directed mutagenesis have been devised and implemented [Protocols have been established for purification of wild-type human PCNA from tissue culture cells ,17, unmoE. coli  and NH2-pression . All werlemented .Drosophila PCNA sequences in this region indicated that of these four amino acids, three (residues 126, 127 and 128) are identical. The fourth, residue 123, is glutamine (Q123) in wild-type Drosophila PCNA. The corresponding residue in human PCNA is valine (V).Recently, Zhang et al.   Drosophila PCNA molecules on purified calf thymus pol δ , conferred upon it, enhanced ability to affect several properties of calf thymus pol δ. More extensive mutagenesis, in which the entire interdomain connector loop of Drosophila PCNA (amino acids 119-133) was replaced by the corresponding human residues, was still more effective at stimulation of calf thymus pol δ, than either wild-type or Q123V Drosophila PCNA. However, it was considerably less effective than wild-type human PCNA at altering the properties of calf thymus pol δ. These results therefore suggest that in addition to the interdomain connnector loop, other regions of PCNA are also important effectors of pol δ activity. They also provide a means to couple operationally, the considerable power of in vivo genetic analyses performed in Drosophila with the sophistication of mammalian biochemistry.To investigate the role of the interdomain connector loop of PCNA on the effects of PCNA on pol δ, we mutagenized residues within this region of orm; see ,24). Calorm; see -27 and cDrosophila homologs. Of the 15 interdomain connector loop residues, nine are identical between the two; identical residues are shaded , we compared human and ded Fig. . Overallans; see . Of thesCNA Fig. . Shown  NH2-terminally his-tagged wild-type Drosophila PCNA (dPCNA) and two dPCNA derivatives; 3) one in which amino acid 123 was mutated from glutamine to valine (Q123V dPCNA); and 4) the other, in which Drosophila amino acids 119-133 were replaced by the corresponding human sequence (dr119-133h dPCNA). Then all four were transformed separately into E. coli (strain M15 [pREP4]) and respective proteins were expressed. Finally bacteria were lysed and his-tagged proteins were purified using various procedures including Ni2+-IDA Sepharose chromatography. The purity of each was determined by SDS-PAGE and is shown as indicated  and replacement of the entire fly interdomain connector loop with corresponding human amino acids (dr119-133h dPCNA) had demonstrable effects. Of note, at relatively high concentrations, Drosophila PCNA but with the entire fly interdomain connector loop replaced by corresponding human amino acids (dr119-133h dPCNA) was similarly effective to wild-type human PCNA at stimulating the activity of calf thymus pol δ; however, it was considerably less effective at lower concentrations  was purCNA Fig. . Mutatio acid Q12V dPCNA aDrosophila PCNA had relatively much less effect on the processivity of calf thymus pol δ  was readily detectable but neither gave results as robust as those seen with wild-type human PCNA . PCNA-dependent TLS by pol δ was first reported by O'Day et al.  and subssic site ) used prsic site ). The momer Fig.  lane 3. l δ Fig.  lane 4 aded Fig.  lane 5. ded Fig.  lane 6. Drosophila PCNA are more than 70% identical at the level of primary amino acid sequence, wild-type Drosophila PCNA is only a very poor substitute for human PCNA in cell-free reactions with calf thymus pol δ. This is documented both in this report and previously [Drosophila PCNA amino acid, glutamine at position 123 (Q123) to valine (V), leads to a dramatic enhancement in the abilities of Drosophila PCNA to stimulate calf thymus pol δ. Effects were shown on total activity  with the corresponding residues from human PCNA resulted in additional enhancement  equivalent to wild-type human PCNA in the stimulation of calf thymus pol δ.Although human PCNA and eviously ,32. Howeity Fig. , pol δ•Pity Fig.  and exteity Fig. . Replacint Figs. ,5,6, butDrosophila PCNA amino acid at position 123 (in addition to conserved residues 126–128) is very important for pol δ-stimulation, the further enhancement of stimulation seen when the entire interdomain connector loop of Drosophila PCNA (amino acids 119-133) was replaced with the corresponding residues from human PCNA suggests that other residues in this loop are also involved directly in binding pol δ. Alternatively, it is possible that loop residues other than 123 and 126–128 play a secondary or indirect  role in positioning crucial amino acids so as to optimize their direct binding to pol δ.Our data indicate that although a single in vitro may not accurately predict properties manifest at much different and generally much higher intranuclear concentrations present in vivo. Alternatively, PCNA must be present as a trimer (three-subunit ring) in order to function. Since the equilibrium among monomer, dimer and trimer was shown to depend on PCNA protein concentration [eq for PCNA multimerization. These two possibilities, concerning both complicated pol δ•PCNA interactions and PCNA multimerization, are not mutually exclusive.In this context, we would like to call attention to the fact that at relatively low concentrations, dr119-133h dPCNA is considerably less effective than wild-type human PCNA in stimulating the activity of calf thymus pol δ; at higher concentrations, dr119-133h dPCNA and wild-type human PCNA stimulate calf thymus pol δ similarly. This implies complex protein-protein interactions between PCNA and pol δ such that biochemical properties recorded in dilute solutions Drosophila PCNA (amino acids 119-133) with the corresponding residues from human PCNA did not result in a molecule as effective in stimulating calf thymus pol δ as human PCNA suggests that regions other than the interdomain connector loop are important for pol δ-stimulation. Our data do not address the question of whether these putative "other regions" affect pol δ directly (e.g., like the interdomain loop) or indirectly . Additional mutagenesis studies may shed light on this issue. For example, based on experiments of others, it seems likely that the extreme C-terminus of PCNA also interacts directly with pol δ (see [Drosophila PCNA, rather than the interdomain connector loop.Similarly, the fact that replacement of the entire interdomain connector loop of l δ see ,34-36). -36. DrosS. cerevisiae PCNA. As in our studies, Ola et al. [We think it should also be noted that both Oku et al.  and Ola a et al.  found thDrosophila PCNA, thereby allowing efficient in vitro function with purified calf thymus pol δ, and then elsewhere in the PCNA molecule corresponding to interesting sites defined phenotypically by in vivo genetic studies of others. For example, it might be possible to determine if particular mus209 mutations leading to enhanced mutagen sensitivity among affected organisms  by which point mutations in the Drosophila PCNA gene lead to enhanced mutagen sensitivity among animals bearing these mutations.It may also be of interest to prepare double-mutants, first in the interdomain connector loop of sms  yet revisiae ,46. CleaDrosophila PCNA could be "humanized" and that "humanization" (mutation of key Drosophila residues to human ones) increased effects on mammalian pol δ. The highly purified two-subunit form of pol δ was used for all of our studies. It is possible, though we think it unlikely, that different conclusions would be reached if a different form of pol δ (three-or four-subunit) was used. Nevertheless two of the effects we observed could be considered beneficial. They were enhancement of polymerase activity and processivity. A third effect seems likely to be detrimental, at least over the long term, that is increased synthesis opposite and beyond a chemically defined template base lesion (TLS). Our data suggest that all three of these effects result from enhancement of PCNA-dependent stability of the pol δ•PCNA•template-primer complex. In other words, in the range that we have studied, the more tightly pol δ binds to DNA, the greater its activity, the greater its processivity, but also the more likely it is to catalyze TLS. Our results provide an explicit approach to correlate in vivo genetic studies with rigorous in vitro biochemistry.Through our experiments, we showed that 32P]ATP and [α-32P]dTTP were from Amersham Corp. E. coli DNA polymerase I Klenow fragment without 3'-5' exonuclease activity (exo-), was expressed and purified according to standard protocols [2+-IDA Sepharose was from Pharmacia (Piscataway, NJ). Acrylamide and methylene bis-acrylamide were from Eastman Organic Chemicals and for protein SDS-PAGE, were further purified by adsorption of impurities to activated charcoal. For PAGE of nucleic acids, they were purified by adsorption to an ion exchange resin. All other materials were of reagent grade and were used without additional purification.Unlabeled deoxyribonucleoside triphosphates (dNTPs) were from Boehringer-Mannheim; [α-rotocols . TerminaE. coli extract, also to apparent homogeneity [D. melanogaster PCNA was purified to apparent homogeneity identically after bacterial expression [2-termini of both human and Drosophila PCNA by cDNA insertion into pQE30  using BamH1 and HindIII restriction endonuclease sites.PCNA was purified to apparent homogeneity from calf thymus  as was pogeneity . D. melapression . A his-tTemplates and primers, all of defined sequence, were synthesized conventionally by Dr. F. Johnson and colleagues (Stony Brook). Before use, they were purified by standard denaturing PAGE . All othDrosophila PCNA was with affinity purified polyclonal rabbit anti-Drosophila PCNA antibodies [Much of the methodology was described in detail previously ,41,50,51tibodies . Restrictibodies  using a 2-terminally his-tagged Drosophila PCNA was performed exactly as described [Drosophila PCNA sequence except for amino acids 119-133 which were replaced by the corresponding residues from human PCNA.Site-directed mutagenesis of NHescribed  to generPurification of his-tagged PCNA to apparent homogeneity was performed exactly as previously described . Charact32P]ATP. Afterward, labeled primer was annealed to an unlabeled template. The standard reaction mixture for pol δ contained 40 mM Bis-Tris, pH 6.7, 6 mM MgCl2, 1 mM dithiothreitol, 10% glycerol and 40 μg/ml bovine serum albumin. Additional details are provided in the figure legends. Incubations were terminated by addition of standard stop solution and aliquots were subjected to 12% PAGE in the presence of 7 M urea and 15% formamide. After electrophoresis, gels were subjected to autoradiography and/or Molecular Dynamics 445 SI PhosphorImager analyses.Assays of pol δ on synthetic oligonucleotide template-primers were performed essentially as previously described . Primers~500 annealed to (dT)12–18 (both from Pharmacia) in a final volume of 5 μl containing 6 nmol poly(dA) (nucleotide), 0.2 nmol (dT)12–18 (nucleotide), 10 μM dTTP, 100 μCi [α-32P]dTTP, 40 mM Bis-Tris, pH 6.7, 6 mM MgCl, 1 mM dithiothreitol, 10% glycerol, 40 μg/ml bovine serum albumin, 10 ng of highly purified pol δ and various quantities of different PCNA samples as indicated. Assays were for 5 min at room temperature and were stopped by addition of standard PAGE stop solution and PAGE in the presence of 7 M urea. After electrophoresis, gels were subjected to autoradiography and/or Molecular Dynamics 445 SI PhosphorImager analyses.Processivity was evaluated qualitatively using (dA)2 and otherwise as detailed in the figure legend. EDTA was included in each incubation and in the gel electrophoresis buffer at a final concentration of 3 mM.Nondenaturing PAGE band mobility shift assays were performed essentially as previously described  but withDJuM performed all enzymologic and mobility shift assays with DNA polymerase δ in combination with both wild-type and various mutant PCNA molecules. He also designed, engineered and characterized all recombinant PCNA molecules. DJuM expressed several recombinant proteins in bacteria and purified them. Finally, he participated in DNA polymerase purification and drafted the original manuscript. MM expressed some recombinant proteins in bacteria and purified them. She also purified and characterized most DNA polymerase substrates. HM participated in DNA polymerase purification and manuscript preparation. PAF advised DJuM on execution and interpretation of experiments and assisted both in figure design and all other aspects of manuscript preparation. All authors read and approved the final manuscript.


We have developed a new method that enables agar microstructures to be used to cultivate cardiac myocyte cells in a manner that allows their connection patterns to be controlled. Non-contact three-dimensional photo-thermal etching with a 1064-nm infrared focused laser beam was used to form the shapes of agar microstructures. This wavelength was selected as it is not absorbed by water or agar. Identical rat cardiac myocytes were cultured in adjacent microstructures connected by microchannels and the interactions of asynchronous beating cardiac myocyte cells observed. Two isolated and independently beating cardiac myocytes were shown to form contacts through the narrow microchannels and by 90 minutes had synchronized their oscillations. This occurred by one of the two cells stopping their oscillation and following the pattern of the other cell. In contrast, when two sets of synchronized beating cells came into contact, those two sets synchronized without any observable interruptions to their rhythms. The results indicate that the synchronization process of cardiac myocytes may be dependent on the community size and network pattern of these cells. Single-cell based analysis methods have become more and more important for understanding the cell-group effects such as how information is controlled and recorded in a cell group or a network shape. Early tissue culture studies of cardiac myocyte cells demonstrated that a single beating cell can influence the rate of a neighbouring cell in close contact and that a group of heart cells in a culture, beating synchronously with a rapid rhythm, can act as pacemaker for a contiguous cell sheet . Though This paper reports the practical use of the agar chamber for screening the community size effect of the synchronization process of adjacent cardiac myocyte cells having independent oscillation.Figure 2 HPO4, 1.5 mM KH2PO4, pH 7.4) containing 0.9 mM CaCl2 and 0.5 mM MgCl2. The ventricles were minced in PBS without CaCl2 or MgCl2 and then incubated in PBS containing 0.25% collagenase (Wako, Osaka, Japan) for 30 minutes at 37°C to digest the ventricular tissue. This procedure was repeated twice more and the cell suspension was then transferred to cell culture medium  at 4°C. The cells were filtered through a 40-μm nylon mesh and centrifuged at 180 g for 5 minutes at room temperature. The cell pellet was re-suspended in a HEPES buffer (20 mM HEPES, 110 mM NaCl, 1 mM NaH2PO4, 5 mM glucose, 5 mM KCl, and 1 mM MgSO4, pH 7.4). Cardiac myocytes present in the suspension were separated from other cells  by the density centrifugation method. The cell suspension was then layered onto 40.5% Percoll  diluted in the HEPES buffer, which had previously been layered onto 58.5% Percoll diluted in the same buffer. The cell suspension was then centrifuged at 2200 g for 30 minutes at room temperature. Cardiac myocytes were retrieved from the interface of the 40.5% and 58.5% Percoll layers. Retrieved cells were then re-suspended in the cell culture medium. An aliquot (5- μl) of the suspension was diluted to achieve a final concentration of 3.0 × 105 cells/ml then plated into the chip. Individual cardiac myocytes were picked up by a micropipette and manually introduced into each chip microchamber and incubated on a cell-cultivation microscope system at 37°C in the presence of a humidified atmosphere of 95% air /5% CO2. It should be noted that because the microchamber sidewalls were made of agar, then the cells could not easily pass over the chambers.Neonatal rat cardiac myocytes were isolated and purified as follows. First, the hearts of 1- to 3-day-old Wistar rats (Nippon Bio-supp. Center, Tokyo, Japan) were excised under ether anaesthesia. The ventricles were separated from the atria and then washed with phosphate buffered saline  of cardiac myocytes, which changed considerably with contraction, were also analyzed and recorded every 1/30 s by a personal computer with a video capture board.Figure The same method was also used to make more complicated network patterns of cardiac myocytes. Figure In conclusion, we present a 1064-nm photo-thermal etching technology with which to create agarose microchambers for growing networks of cardiac myocyte cells. Using the system, we first observed the differences of the synchronization process of cardiac myocyte cells and their dependence on community size. This system has great potential for use in the biological/medical fields for cultivating the next stage of single-cell based networks and measuring their properties in laboratories.KK and TK carried out the microchamber design, cell preparation, single cell cultivation and observation, image analysis. Both authors contributed equally to this article. KY conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.two cells. synchronized oscillation of the two cellsClick here for filetwo sets of cells. synchronized oscillation of the two sets of cellsClick here for file


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 (acute, intermittent crisis, chronic), the tasks assigned to the bereaved , or the population targeted for the intervention (children, adults, seniors). 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 [children . The resKleber and Brom (1987) conducted a comparative outcome study of three forms of short-term psychotherapy . They copsychodynamic therapy, which strives for the patient to understand and cope better with feelings by re-experiencing them and talking them through with the aid of the therapist, was found to be quite prevalent in the bereavement care literature. Overall, the results are mixed, with more support found in the group format of psychodynamic therapy than in individual therapy. Of the studies we evaluated as psychodynamic therapy, six were individual in format, seven had a group format, and eight employed control groups; five of these claimed random allocation .Studies of Psychoanalysis, as exemplified by Freud, proceeds with an inward investigation of unconscious mental processes and childhood experiences as the principal therapeutic procedure. Problematic measurement methodology beset the one study that utilized a group format to provide a psychoanalytic-based intervention (with no details regarding the tasks assigned to the patients)[patients). This stBehavioral therapy uses learning principles (such as behavior modification, systematic desensitization, and aversion) to eliminate or reduce unwanted reactions to either external situations, one's thoughts and feelings, and bodily sensations or functions. Behavioral therapy was used in only one study, which compared traumatic desensitization to hypnotherapy and psychodynamic therapies [herapies . As descInterpersonal therapy aims to improve communication skills and increase self-esteem during a short time period by focusing on a patient's behaviors and social interactions with family and friends, directly teaching how better to relate to others. Only one study used interpersonal therapy as a bereavement care intervention, and this study found no effect on grief as the only measured outcome [ outcome .Seven studies featured interventions that altered the manner in which the healthcare system interacted with patients, family, and friends prior to death, guided by an underlying (yet not fully explicated) notion that interactions experienced by loved ones prior to death can influence the subsequent bereavement process Table  73-79].-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 (Difco Laboratories, Detroit, MI) 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 (8 × 103 transformants per μg of DNA, [The expression of  of DNA, ).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 (bands β, γ2, γ3, δ) 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 [Pseudomonas sp. strain CBS-3 [Arthrobacter sp. strain SU [Bacillus halodurans [Escherichia coli thioesterase II [We have found the HotDog domain, as we suggest calling the Hotdog fold, to be widespread in eukaryotes, bacteria, and archaea and to be involved in a range of cellular processes, from thioester hydrolysis, to phenylacetic acid degradation and transcriptional regulation of fatty acid biosynthesis. We present the superfamily and its functional subfamilies here. The Hotdog fold was first observed in the structure of og' fold . They dein CBS-3  and Arthlodurans  and in Eerase II .Although several proteins are now known to contain a Hotdog fold from structural analysis it has not to our knowledge been demonstrated that these proteins can be related to each other by sequence similarity. We have attempted to unify these structurally related proteins using a sequence analysis approach. Using sequence analysis means that we will identify additional proteins that are likely to contain a Hotdog fold. We have used the PSI-BLAST program  and usedThe Pfam database  containsOur searches have found a total of 1357 proteins see  to be rThe largest subfamily represents over a hundred acyl-CoA thioesterases that are widespread throughout the prokaryotic kingdom, with members also found in eukaryotes. This group of enzymes catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and coenzyme A (CoA-SH.) . The subE. coli the products of the fab operon catalyze the four sequential reactions necessary for each round of fatty acid elongation [trans-2-decenoyl-ACP. FabZ is effective at dehydrating both short-chain and long chain saturated and unsaturated pathway intermediates.Members of this subfamily are found in a wide range of bacteria and sporadically in eukaryotes. In ongation . The thiPseudomonas syringae, a plant pathogen that causes disease in many agriculturally important plants [This subfamily also contains a dehydratase component of the coronafacic acid (CFA) biosynthetic cluster encoded by the cfa2 gene ,21. CFA t plants .maoC gene exists as an operon with the maoA gene in E. coli and is an enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the fatty acid oxidation pathway to polyhydroxyalkanoate (PHA) biosynthetic pathways in fadB mutant E. coli strains. It was identified through its homology to P. aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1) [Aeromonas caviae has shown that this enzyme also contains a Hotdog fold/domain [E. coli MaoC C-terminal HotDog domain is most likely responsible for its enoyl-CoA hydratase actvity. MaoC also contains an N-terminal short-chain dehydrogenase domain, involved in catalysing dehydrogenation of a variety of aliphatic and aromatic aldehydes using NADP as a cofactor. This subfamily also includes the human 17 β-hydroxysteroid dehydrogenase (17 β HSD) type 4, one of four different human 17 β HSDs that catalyze the redox reactions at position C17 of steroid molecules, one of the final steps in androgen and estrogen biosynthesis [Rhizobium and related species form nodules on the roots of their legume hosts, a symbiotic process that requires production of Nod factors, which are signal molecules involved in root hair deformation and meristematic cell division [The  (PhaJ1) . PHAs ar (PhaJ1) , with in (PhaJ1) . The cryd/domain . The E. ynthesis ,25. We adivision . The nodtol-pal cluster in the gamma-proteobacterium Haemophilus influenzae [tol-pal cluster is present in many Gram-negative bacteria and is important for the maintenance of cell envelope integrity [This subfamily contains a large number of proteins about which very little is known except for the YbgC protein. The YbgC protein of the fluenzae  has beenntegrity  and this17 residue is conserved in YbgC from Haemophilus influenzae and Pseudomonas aeruginosa, along with the backbone amide NH of Tyr24, suggestive of a nucleophilic attack mechanism very similar to the Pseudomonas sp. strain CBS-3 thioesterase mechanism discussed below in the 4HBT class I section.The Asptrans-2-decenoyl-ACP. FabA is uniquely able to isomerise trans-2-decenoyl-ACP to cis-3-decenoyl ACP, initiating unsaturated fatty acid biosynthesis [The dehydration of the β-hydroxyacyl-ACP protein intermediate during the third step in each cycle of fatty acid elongation can be catalyzed by β-hydroxydecanoyl-ACP dehydratase/isomerase (FabA), as well as by FabZ, to give ynthesis  and is sPseudomonas spp. and Streptomyces spp. They include clinically useful drugs such as the antibiotic erythromycin A and the immunosuppressants FK506 and rapamycin. The biosythesis of polyketides is very similar to that of fatty acids [Shewanella sp. SCRC-2738, Moritella marina strain MP-1 and Photobacterium profundum strain SS9 contain an eicosapentaenoic acid (EPA) biosynthetic cluster (pfaA-D), responsible for the synthesis of this omega-3 polunsaturated fatty acid (PUFA), [P. profundum), which are also found in the eukaryotic marine protist, Schizochytrium, suggesting that the PUFA synthetic cluster has undergone lateral gene transfer [Polyketides are a large and structurally diverse class of natural products, produced mainly by soil-dwelling bacteria such as ty acids  and polyty acids ,30. The ty acids  such as  (PUFA), ,33. The transfer .Mycobacterium bovis fatty acid synthase. This multifunctional protein is capable of catalysing de novo synthesis and chain elongation of fatty acids [This subfamily also includes several fatty acid synthase proteins from bacteria, such as ty acids  and has 70 and Asp84, His70 is conserved in FabZ dehydratase, but Asp84 is replaced with Glutamate. This replacement may be responsible for FabZ's inability to catalyze the isomerization reaction [The catalytic residues of FabA's bifunctional active site are Hisreaction .fatA and fatB, based on sequence similarity and substrate specificities [Arabidopsis FatA displays highest activity towards oleoyl-ACP whereas Arabidopsis FatB is most active towards palmitoyl-ACP [In plants, fatty acid synthesis occurs in the stroma of plastids, where the acyl chains are bound to the acyl carrier protein (ACP) during extension cycles . Acyl-ACficities ,38. Arabtoyl-ACP . This sutoyl-ACP . The proE. coli medium chain length acyl-CoA thioesterase II [tesB gene [This subfamily contains the erase II  encoded esB gene , which iesB gene . hTE couesB gene . HoweveresB gene ,41, depeE. coli thioesterase II was identified by site directed mutagenesis and involves a hydrogen-bonded triad of Asp204, Thr 228, and Gln 278, which synergistically activate a water molecule for nucleophilic attack of the carbonyl thioester carbon of medium chain length acyl-CoA substrates [Pseudomonas and Arthrobacter discussed below. This subfamily is found in bacteria and eukaryotes.The catalytic site of bstrates . This isArthrobacter sp. strains SU and TM1 encoded by the fcbC gene [Pseudomonas thioesterase uses the Asp17 residue to mediate the hydrolysis reaction as discussed below in the 4HBT class I section. Gln58 from Arthrobacter corresponds to the Asp17 residue in Pseudomonas but inspection of the Arthrobacter strain SU active site has revealed the catalytic base (or nucleophile) to be Glu73, on the opposite side of the substrate binding pocket to Asp17[Pseudomonas thioesterase dimers form a tetramer with their long α-helices facing inwards, in contrast to Arthrobacter thioesterase where the dimers form a tetramer with their long α-helices facing outwards [Pseudomonas and Arthrobacter thioesterases, the 4-hydroxyphenacyl moieties are positioned in such an orientation that the thioester C = O interacts with the α-helical N-terminus by means of hydrogen bonding to a backbone amide NH, on Tyr24 in Pseudomonas and Gly65 in Arthrobacter, and it is this contact that results in polarization of the C = O for nucleophilic attack [Arthrobacter sp. strain SU thioesterase displays a similar Hotdog-fold topology to the 4HBT class I Pseudomonas enzyme, the enzymes differ at the level of catalytic platform, CoA binding site and quaternary structure [et al. have found that 12 of the 31 superfamilies they analyzed displayed positional variation for residues playing equivalent catalytic roles [This subfamily includes 4-hydroxybenzoyl CoA thioesterase (4HBT) from cbC gene . The Psetructure . This isic roles .Bacillus subtilis. ComA is a response regulator and transcription factor [comA locus is composed of two ORFs. ComA2 is cotranscribed with ComA1, which is required for competence while ComA2 is not [A surprising inclusion in this subfamily is the ComA2 protein from n factor  that tog2 is not , and so E. coli has been characterised and found to contain 14 genes, allowing catabolism of this aromatic compound into likely Krebs cycle intermediates [paa operon in E. coli encodes PaaI, which is probably a thioesterase involved in the catabolism of PA. The catabolism of phenylacetic acid (PA) in E. coli begins with an activation step where Phenylacetyl-CoA ligase, PaaK, converts phenylacetate into Phenylacetyl-CoA. 4-chlorobenzoate-CoA ligase catalyzes a similar reaction at the first step of the 4-chlorobenzoate-degradation pathway. The thioesterase, PaaI, may be involved in a reaction similar to the last step in the degradation of 4-chlorobenzoate (see 4HBT class I below), however this remains to be demonstrated.The phenylacetic acid (PA) catabolic pathway in mediates . The paaBacillus [This small subfamily is restricted to firmicutes. FapR is a highly conserved transcriptional regulator found in many gram-positive organisms, including all species of Bacillus . It contPseudomonas sp. strain CBS-3 [Pseudomonas may be used as a bioremediation agent for degrading 4CBA. Pseudomonas sp. strain CBS-3 contains an fcb operon responsible for hydrolytic dechlorination of 4CBA, with 4CBA-CoA ligase (FcbA), 4CBA-CoA dehalogenase (FcbB), and 4HBT (FcbC) catalyzing sequential reactions that result in the degradation of 4CBA to 4-hydroxybenzoate. The thioesterase catalyzes the third step in the degradation pathway, which is the hydrolysis of the 4-hydroxybenzoyl-CoA thioester moiety to give 4-hydroxybenzoate and CoA [The crystal structure of 4HBT from the soil-inhabiting bacterium in CBS-3  has helpin CBS-3 . 4CBA isin CBS-3  and this and CoA .Pseudomonas sp. strain DJ-12 [fcb operon in strain DJ-12 is different from that observed in strain CBS-3. The fcb genes are organised as B-A-C in both strains but strain DJ-12 has three ORFs between A and C called T1, T2, and T3 that are unique to this strain. These three genes are similar to the C4-dicarboxylate transport system in Rhodobacter capsulatus, suggesting that they may encode membrane proteins involved in the uptake of 4CBA [Arthrobacter sp. strain SU and strain TM1 have an A-B-C order [4HBT from in DJ-12  is also  of 4CBA . This is-C order . There iBacillus halodurans C-125 contains a gene called BH1999, encoding a novel gentisyl-CoA thioesterase, which catalyzes the hydrolysis of gentisyl-CoA (2,5-dihydroxybenzoyl-CoA)[B. halodurans. Gentisate has been implicated as an intermediate in the degradation of several industrial aromatic compounds [zoyl-CoA),52 to yiompounds .Pseudomonas perform different physiological functions but remain in the same subfamily because they are highly related. The active site residues Asp16 and Asp31 of gentisyl-CoA thioesterase align with Asp17 and Asp32 of 4HBT. These are crucial residues that are proposed to function in nucleophilic catalysis and substrate binding respectively. Loss of Asp17 in the Pseudomonas enzyme effectively halts catalysis, while loss of the corresponding Asp16 residue to the Bacillus halodurans enzyme only reduces its catalytic rate by 230-fold, perhaps indicating that the hydrolysis reaction does not proceed through an Asp16-mediated nucleophilic attack mechanism previously proposed for Asp17 [17 in Pseudomonas strain CBS-3 has been suggested to participate in nucleophilic catalysis rather than general base catalysis based on the following observations. The Asp17 carboxylate is located at a distance of 3.2 Å from the substrate C = O thioester bond, its aligned trajectory and the absence of a water molecule near the reaction centre are all suggestive of a role for Asp17 as a catalytic nucleophile [32 in Pseudomonas interacts with the benzoyl OH of 4-hydroxybenzoyl-CoA [31 plays a similar role.Gentisyl-CoA thioesterase and 4HBT from or Asp17 ,4. Asp17leophile ,53. Asp3zoyl-CoA  and perhIn the above sections we have described the 11 subfamilies that have some functional characterization. In this section we describe the other 6 subfamilies that have no functional characterization, except they are associated with other domains or have been structurally characterized.Bacillus halodurans. The BH3175 protein contains two homologous copies of the CBS domain [et al. have recently shown that tandem pairs of CBS domains act as sensors of cellular energy status by binding AMP, ATP, or S-adenosyl methionine and mutations in CBS domains impair this binding in several hereditary disorders [The CBS associated subfamily contains the hypothetical protein BH3175 from S domain . Scott eisorders . AlthougAgrobacterium tumefaciens strain C58, which contains a HotDog domain at its C-terminus and the two domains (3HCDH_N and 3HCDH) associated with 3-hydroxyacyl-CoA dehydrogenase activity are located at the N-terminus and central portion of this protein. The combination of activities may allow substrate to be passed from one domain to the next.3-hydroxyacyl-CoA dehydrogenase is an enzyme involved in fatty acid metabolism, catalyzing the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA . The hydE. coli, where an acetyltransferase domain is fused. The human mesenchymal stem cell protein DSCD75 and its counterpart in mouse also contain a HotDog domain. A Structural proteomics project has shown that the conserved hypothetical E. coli protein YbaW contains a Hotdog fold [Ralstonia solanacearum hypothetical protein RSp0367, containing a HotDog domain and two AMP-binding domains, found in proteins involved in ATP-dependent covalent binding of AMP to their substrate, is a member of another subfamily.Other subfamilies in the superfamily include the YiiD protein from dog fold . FinallyIt has been shown that proteins that are functionally linked are occasionally found to be fused in various genomes. These fusion proteins have been termed Rosetta proteins ,58 and cChlorobium tepidium  is fused to the FabZ-like HotDog domain in Bacteroides thetaiotaomicron there is a Rosetta protein that contans a haloacid dehalogenase-like hydrolase domain  domain and a HotDog domain -dehalogenase(B)-thioesterase(C). In Agrobacterium tumefaciens strain C58, which possesses the HotDog domain, 3HCDH_N domain (3-hydroxyacyl-CoA dehydrogenase, NAD binding) and 3HCDH  domain -G [IV]-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) [Gene context/operon analysis was carried out with the GeConT tool (xt Tool)  availablxt Tool) .Protein domain analysis was carried out using Pfam  .Click here for fileA complete list of the 1357 HotDog domain containing proteins.Click here for fileThe HotDog domain superfamily. This xml-like file contains 1293 (95%) of the HotDog domain containing sequences, grouped into 85 clusters, which permits investigators to immediately identify HotDog domain(s) in their 'unknown' protein of interest and allow them to infer some functionality.Click here for fileA HotDog domain HMM library. This library can be used in conjunction with the HMMER program to search for HotDog domains in any protein sequence.Click here for fileA list of motifs identified in each subfamily. Motifs were identified using the MASIA program [62]. 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. (That is, the genes evolve different functions after the species diverge from their common ancestor.) 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