Imprinted genes exhibit a parent-of-origin specific pattern of expression. Such genes have been shown to be targets of molecular defects in particular genetic syndromes such as Beckwith-Wiedemann and Angelman syndromes. Recent reports have raised concern about the possibility that assisted reproduction techniques, such as in vitro fertilization or intracytoplasmic sperm injection, might cause genomic imprinting disorders. The number of reported cases of those disorders is still too small to draw firm conclusions and the safety of these widely used assisted reproduction techniques needs to be further evaluated. The first in vitro fertilization (IVF) baby was born in 1978 and intracytoplasmic sperm injection (ICSI) was introduced in 1992 for the treatment of male infertility. Both these techniques have been continually amended and access to them improved for infertile couples. Indeed, assisted reproduction now accounts for 1% to 3% of births in developed countries [Epigenetic modifications are reversible changes of the DNA methylation pattern and chromatin structure that can affect gene expression. In many instances, epigenetic changes governing gene expression can be passed from cell to cell or from parent to offspring. Epigenetic modifications themselves might therefore explain how environmental factors modulate gene expression without affecting the genetic code. The most researched epigenetic phenomenon is DNA methylation .DNA methylation is a covalent modification in which methyl groups are added to cytosine bases located 5' of guanosines (within cytosine-phospho-guanine (CpG) dinucleotides sequences). Methylation is catalyzed by the DNA cytosine-5-methyltransferase (DNA-MTase) enzyme family. Methylation induces changes in chromatin structure and is generally associated with silencing of gene expression, thus providing a way to control gene expression . Indeed,Genomic imprinting is an epigenetic phenomenon by which the expression of a gene is determined by its parental origin. Only one allele of an imprinted gene is expressed. Imprinting is controlled by DNA methylation in such a way that a difference in methylation between the maternal and paternal alleles correlates with the different expression of the two parental alleles.It is estimated that the total number of imprinted genes in the human and mouse genomes ranges between 100 and 200 . ImprintIn order to ensure that every generation receives the appropriate sex-specific imprint, the genome undergoes reprogramming. Epigenetic reprogramming has been shown to occur during gametogenesis and during preimplantation development . During Further genome reprogramming occurs during the preimplantation embryonic stage with epigenetic changes taking place through demethylation in non-imprinted genes in maternal and paternal genomes. This is followed by a genome-wide methylation at the time of implantation. The different stages of imprint establishment, maintenance and manipulations possibly disturbing them are illustrated in Figure The main consequence of the sex-specific establishment and maintenance of imprinted methylation patterns is the creation of maternal- and paternal-allele methylation differences  in or around imprinted genes. A primary DMD is established during gametogenesis and secondary DMDs develop during embryogenesis, most likely due to a direct influence of a nearby primary DMD .IGF2R gene [Imprinted genes are implicated in the regulation of embryonic and fetal growth, as well as many aspects of placental function, including placental growth and the activity of transplacental transport systems . Indeed,F2R gene . ImprintSeveral human syndromes are known to be associated with defects in gene imprinting, including Prader-Willi, Angelman, Beckwith-Wiedemann, Silver-Russell and Albright hereditary oseodystrophy syndromes . AberranIGF2 and KCNQ1OT1, and the maternally expressed genes H19, CDKN1C and KCNQ1 [IGF2 gene, and some of these cases exhibit loss of imprinting (LOI) of IGF2 which is dependent on hypermethylation changes of H19 [KCNQ1OT1, an untranslated RNA within the KCNQ1 gene [KCNQ1OT1 as well as LOI for IGF2 [KCNQ1OT1 is specifically associated with overgrowth and congenital defects, whereas aberrant methylation of H19 is specifically associated with an increased risk of developing tumors [The Beckwith-Wiedemann syndrome (BWS), whose frequency in the general population is about 1/14,000, is characterized by somatic overgrowth, congenital malformations and a predisposition to embryonic neoplasia. The majority of cases occur sporadically. In up to 60% of sporadic cases, the epigenetic changes occur at differentially methylated regions within 11p15.5 in a region of approximately 1 Mb. This region contains an imprinted cluster of at least 12 genes, including the paternally expressed genes g tumors .SNRPN, UBE3A, ZNF127, IPW and NDN. The small percentage of AS cases (<5%) associated with methylation defect involves loss of methylation within the SNRPN imprinting center (IC) and defective expression or silencing of maternally expressed genes within this region. However, the methylation defect associated with PWS involves methylation within the SNRPN IC and a defective expression or silencing of paternally expressed genes within the same region. The IC comprises 2 regulatory regions: the PWS-shortest region of overlap (SRO) and the AS-SRO [The Prader-Willi and Angelman syndromes (PWS/AS) are typical examples of imprinting dysregulations leading to severe neuro-behavioral disturbances. Their frequencies in the general population are approximately 1/10,000 and 1/15,000, respectively. The domain involved in these two pathologies is a 2 Mb domain on the 15q11–13 chromosomal region, including genes as e AS-SRO . PWS-SROe AS-SRO . Indeed,e AS-SRO .In addition, imprinting may have a wider impact on neurological development and behavior. Some reports suggest parent-specific imprinting defect in common neuro-behavioral disorders. Autism, bipolar affective disorder, schizophrenia  and othePrior to the establishment of sex-specific imprints in male and female germ cell lineages, imprints are erased. After erasure of the pre-existing imprints, the timing of acquisition of imprints is significantly different between the two germ lines . In the The first baby conceived by IVF was born 26 years ago. Intracytoplasmic sperm injection (ICSI), developed approximately 10 years ago, was seen to be the reproductive solution for severe male infertility. Several studies have established the general safety of both IVF and ICSI . NeverthKCNQ1OT1 or H19. By comparing this rate of ART-conceived BWS to the rate of ART in the general population during the same time period, sporadic cases of BWS were approximately six times more likely to have been conceived by ART than by natural conception. The authors suggested that causative factors may include the in-vitro culture conditions or the exposure of the gametes or embryos to specific media or growth factors.DeBaun et al.  recentlyKCNQ1OT1 loss of imprinting as the causative molecular defect. Indeed, when compared to the incidence in the general population, ART had a four-fold greater likelihood of being associated with BWS than natural conception. The cases reported by DeBaun et al. [Maher et al. reviewed a different set of sporadic BWS cases and looked for an association with ART . Six outn et al.  and Mahen et al.  were recRecently, a case-control study analyzed the frequency of BWS in 1'316'500 live births and 14'894 babies born after an IVF procedure . The risSNRPN gene at 15q11–13. Considering that the occurrence of AS in the general population is about 1/15,000 and that <5% of cases are due to epigenetic imprinting defects, these reports suggest that the predominant abnormalities seen in ART are epigenetic rather than genetic.Cox et al.  and OrstHowever, no evidence of abnormal methylation patterns at 15q11–13, the locus linked to the pathogenesis of AS and PWS, was found in 92 children conceived by ICSI .MEST and H19) in spermatozoon DNA from normozoospermic and oligozoospermic patients. The data presented suggest an association between abnormal genomic imprinting and hypospermatogenesis [For assisted reproduction by intracytoplasmic sperm injection (ICSI), the injection of a spermatozoon into the ovum by micro-manipulation bypasses several of the steps involved in fertilization. However, in male germ cells, it seems that the paternal imprints are well established in the mature, meiotic stages of spermatogenesis. Furthermore, round spermatid microinjections have confirmed that paternal imprints are completely established in primary spermatocytes . This poogenesis . TheoretWomen with a variety of fertility problems, such as ovarian failure and/or hormonal disturbances, may be more prone to produce gametes with inherent imprinting defects because of the establishment of maternal imprints during the final phase of oocyte growth and meiotic maturation.Although biologically plausible, this is purely speculative at the moment.in vitro culture affected imprint establishment and might lead to loss of methylation at certain imprinted loci, such as IGF2R and gain of methylation at other loci, such as H19. However, to our knowledge, no data concerning the possible effects of ovarian hyperstimulation on imprinting in humans is available yet.In addition to the theoretical possibility that there may be innate defects in oocytes used in ART, the in vitro treatment of oocytes and embryos during ART procedures might affect the establishment of imprints in female germ cells. For example, superovulation or in vitro maturation of oocytes might affect the establishment of the complete array of normal maternal imprints. Oocytes used for assisted reproduction usually originate from women who undergo hormonal hyperstimulation protocol followed by fertilization in vitro. It is not clear to date if the clinical use of high doses of gonadotrophins might alter imprint acquisition. Gonadotrophins might cause the premature release of immature oocytes that have not completed the establishment of their imprints, and establishment may not be completed during in vitro maturation. Shi and Haaf  determinH19 imprinted gene. The loss of methylation at H19 gene was associated with culture in Whitten's media, resulting in LOI in the imprinting control domain upstream of the start of H19 transcription. Khosla et al. [IGF2 and H19 genes, was influenced by the addition of fetal calf serum (FCS) in the culture media. The mechanism by which culture media and other gamete or embryo handling might induce defects and lack of maintenance of methylation at imprinted loci is not clear. It may be due to the facilitation of removal of methyl groups on cytosine bases or the disturbance of the gamete development leading to incompleteness of imprint erasure and/or establishment [Potential disruption of normal imprinting could result from the in vitro manipulation of early stage embryos. In vitro culture with the use of slightly different culture media led to decreased fetal viability and imprinting disturbances in mice. Doherty et al.  first rea et al.  examinedlishment . FurtherDisturbances in imprinting could affect the germline cells of the embryo conceived by assisted reproduction and the problems of imprinting might occur in the offspring of the subsequent generation . Follow-MASH2 gene was shown to regulate the development of spongiotrophoblast [ASCL2 is a transcription factor expressed in the spongiotrophoblast and labyrinthine layers [IGF2 and ASCL2 genes showed fetal growth restriction and death during embryonic development [A critical way of regulating intrauterine development is through placental function and growth. Most imprinted genes are expressed in fetal and placental tissues, and are involved in fetal growth . In genephoblast . Igf2 trphoblast , and ASCe layers . Indeed,elopment ,42.In humans, several imprinting disorders are associated with intrauterine growth restriction (IUGR) . StudiesConcern has been raised about the possible increased incidence of genetic syndromes due to imprinting defects in children conceived by assisted reproduction. In particular, experimental reports in mice have raised the question that some of the steps involved in these techniques, such as ovarian hyperstimulation or certain culture media for in vitro culture of embryos might be detrimental to the formation of genomic imprints. In order to be able to adequately counsel infertile couples enquiring about ART, solid evidence from large, well-designed studies as well as cautious long-term evaluation of the safety of these techniques need to be available. Although the unraveling of the mechanisms underlying genomic imprinting is only at the beginning, there is a clear need to investigate and better understand the regulation of this process during fecundation and embryogenesis.The authors declare that they have no competing interests.Both authors contributed to the writing of this review and both read and approved the final manuscript.


Entamoeba histolytica and Entamoeba dispar are not available. Therefore, the present study was aimed to assess the in vitro susceptibility of clinical isolates of E. histolytica and E. dispar to metronidazole, chloroquine, emetine and tinidazole.Amoebiasis is a major public health problem in tropical and subtropical countries. Although a number of antiamoebic agents are used for its treatment, yet the susceptibility data on clinical isolates of E. histolytica and 30 E. dispar) were maintained in polyxenic cultures followed by monoxenic cultures. In vitro drug sensitivity (IC50) of clinical isolates and standard reference strain of E. histolytica (HM1: IMSS) was assessed by nitro blue tetrazolium (NBT) reduction assay after exposure to various concentrations of each drug.A total of 45 clinical isolates  13.2,26.3,31.2 and 12.4] compared to E. dispar isolates [IC50(μm) 15.6,28.9,32.8 and 13.2] and the reference strain of E. histolytica [IC50 (μm) 9.5, 15.5, 29.9 and 10.2] to the metronidazole, chloroquine, emetine and tinidazole respectively.The results showed that all clinical isolates had a higher ICEntamoeba isolates in India do not seem to be resistant to the commonly used antiamoebic drugs.The results indicate that till date,  Entamoeba histolytica, is the etiological agent of amoebic dysentery and amoebic liver abscess (ALA). Worldwide, 40–50 million symptomatic cases of amoebiasis occur annually and 70,000 to 100,000 deaths due to this infection [Entamoeba: Entamoeba histolytica, which is pathogenic and Entamoeba dispar, which is non-pathogenic. E. histolytica, has the capacity to invade intestinal mucosa resulting in intestinal amoebiasis and cause extra intestinal amoebiasis [amoebic liver abscess (ALA)] [s (ALA)] .E. histolytica mainly by inhibiting protein synthesis.Infection is primarily treated by instituting antiamoebic therapy. Drugs of choice for invasive amoebiasis are tissue active agents, like metronidazole, tinidazole and chloroquine or the more toxic emetine derivatives, including dehydroemetine. Metronidazole and tinidazole are derived from 5-nitroimdazole which kill the trophozoites by alterations in the protoplasmic organelles of the amoeba, but are ineffective in the treatment of cyst passers. Chloroquine is derived from 4-aminoquinolines, which acts on the vegetative forms of the parasite and kills it by inhibiting DNA synthesis. Emetine, a plant alkaloid, kills the trophozoites of E. histolytica does not appear to be a serious problem, there are occasional reports of failure with metronidazole suggesting that this could probably be heralding the development of drug resistance clinically [E. histolytica have been reported, indicating that there may be a small percentage of amoebae which are either resistant to the drug or may even eventually become resistant due to abuse of antiamoebic agents [E. histolytica [E. histolytica and E. dispar have not been reported. Therefore, in the present study an attempt has been made to assess the in vitro activity of antiamoebic drugs (emetine, chloroquine, metronidazole and tinidazole) against clinical isolates of E. histolytica and E. dispar.Indiscriminate use of drugs has led to an increase in the minimum inhibitory concentration (MIC) of these therapeutic agents . Althouginically . Recurreinically . Howeverc agents . Althougtolytica -9, yet tE. histolytica (15) or E. dispar (30) by hexokinase isoenzyme analysis and by Techlab ELISA were used in the present study. These have been cultured in modified Boeck and Drbohlav (NIH) medium [Forty-five isolates from patients attending the Out Patient Departments of Nehru hospital, attached to the Post Graduate Institute of Medical Education & Research, Chandigarh, India, identified earlier  as eithe) medium  followed) medium .E. histolytica (HM1: IMSS) maintained axenically in TYI-S-33 medium was included as control [Reference strain of  control .The drugs (metronidazole, chloroquine, emetine dihydrochloride and tinidazole) used in the study were procured as pure salt from Sigma-Aldrich Co., St. Louis, MO., 63178 USA. The stock solutions of drugs (each 0.1 M) were prepared in dimethyl sulphoxide (DMSO)  and storE. histolytica strain (HM1: IMSS). Amoebae were harvested from 24 hour old cultures and suspended in medium. The parasite count was adjusted to 3 × 105 parasites/ml in medium by haemocytometer [Drug sensitivity to all the compounds was carried out by nitroblue tetrazolium (NBT) reduction method . Each clytometer .5/well) was added to all the rows (A-H). Each test included control (without drug) and blank wells (medium only). The plates were incubated at 37°C for 4 hrs. The contents of the plates were discarded and washed with pre warmed Hank's balanced salt solution (HBSS pH 7.2). Thereafter, 100 μl of NBT/well in HBSS was added and the plates were incubated at 37°C for 45 min. followed by aspiration of the contents. Plates were then washed with HBSS twice and 200 μl/well of DMSO (100% v/v) was added. Following incubation at 37°C for 10 min, the optical density (OD) was measured in an ELISA recorder at 540 nm.The assay was carried out in microtiter plates (Grenier bio-one, Germany). Briefly, in row A 200 μl of drug and in all other rows (B-H) medium was added and doubling dilutions of the drug were performed down the plate. Final drug concentration in rows A-H was as follows: 100, 50, 25, 12.5, 6.25, 3.12, 1.6 and 0.8 (μM). Further 100 μl of parasite suspension . Standard deviation (SD) was used to indicate the extent of variation around group mean values. The p value was calculated using the student's-t test.The mean IC50 values of emetine, chloroquine, metronidazole and tinidazole for the 45 clinical isolates [15 E. histolytica and 30 E. dispar] and the reference strain HM1: IMSS were determined by the NBT reduction assay. The mean IC50 values were significantly higher (P < 0.001) in E. dispar isolates to all the four antiamoebic drugs as compared to the E. histolytica isolates and the reference E. histolytica strain  was also included in each set of experiments.Treatment failure among amoebiasis patients often raises the possibility of drug resistance . In the 50 values (μm) of the E. histolytica/E. dispar isolates against metronidazole, chloroquine, emetine and tinidazole were 13.2/15.6, 26.3/28.9, 31.2/32.8 and 12.4/13.2 respectively. The IC50 values (μm) of the reference strain against all the four respective drugs were 9.5, 15.5, 29.9 and 10.2. Recently Upcroft & Upcroft [E. histolytica strains. Adagu, et.al. [50 value as 18.47 μm for the most susceptible isolates of E. histolytica with a > 30 μm value as the cut off for resistance. Burchard & Mirelman, studied in vitro sensitivity to metronidazole and emetine of non-pathogenic zymodemes and showed that all were similarly sensitive to both the drugs (1–10 μg/ml) [Results showed a significant difference in drug sensitivity in clinical isolates as compared to the reference strain with all the four drugs. The mean IC Upcroft  have repTrichomonas vaginalis [Giardia lamblia [Leishmania donovani [E. histolytica and E. dispar.Although resistance to metronidazole has been reported against aginalis , Giardia lamblia  and Leisdonovani , yet to 50 value of all four drugs to the clinical isolates as compared to the reference strain. E. dispar isolates showed higher IC50 values when compared to E. histolytica or reference strain. This is the first report of in vitro drug sensitivity pattern to clinical isolates of E. histolytica and E. dispar. There is definitely a need to monitor the random drug susceptibility among clinical isolates especially in context to widespread use of metronidazole and tinidazole, which are available over the counter in many countries. Increased awareness and continued surveillance for the possible emergence of resistance among clinical isolates is necessary for the ultimate prevention and control of amoebiasis.The results of the present study are in agreement with previous findings ,9,14, exDB, carried the practical work mentioned in the manuscript.RS, was responsible for formulation of the project and provided guidance time to time.YC, he is clinician and carried clinical examination and proposed clinical diagnosis of the patient.RCM, he guided the proposed work related to differentiation of Entamoeba histolytica and Entamoeba dispar and in vitro drug sensitivity.NM, proposed the concept for this manuscript and guided the practical work and writing of the manuscript.


Similarities between obsessive-compulsive disorder (OCD) and trichotillomania (TTM) have been widely recognized. Nevertheless, there is evidence of important differences between these two disorders. Some authors have conceptualized the disorders as lying on an OCD spectrum of conditions.Two hundred and seventy eight OCD patients  and 54 TTM patients  of all ages were interviewed. Female patients were compared on select demographic and clinical variables, including comorbid axis I and II disorders, and temperament/character profiles.OCD patients reported significantly more lifetime disability, but fewer TTM patients reported response to treatment. OCD patients reported higher comorbidity, more harm avoidance and less novelty seeking, more maladaptive beliefs, and more sexual abuse. OCD and TTM symptoms were equally likely to worsen during menstruation, but OCD onset or worsening was more likely associated with pregnancy/puerperium.These findings support previous work demonstrating significant differences between OCD and TTM. The classification of TTM as an impulse control disorder is also problematic, and TTM may have more in common with conditions characterized by stereotypical self-injurious symptoms, such as skin-picking. Differences between OCD and TTM may reflect differences in underlying psychobiology, and may necessitate contrasting treatment approaches. Trichotillomania (TTM) is characterized by repetitive stereotypical hair-pulling from different sites resulting in noticeable hair loss . PhenomeHowever, in addition to overlapping phenomenology between OCD and TTM, there are also significant differences. For example, in contrast to compulsions in OCD, hair-pulling in TTM is not in response to obsessive thoughts (such as worry about harm to self or others) but rather because of an irresistible urge and the promise of gratification when pulling out hair ,6. Also,Examination of demographic variables in OCD and TTM supports the argument that these are two distinctive disorders. TTM is much more prevalent in females  . Age of Additional clinical observations further support a distinction between OCD and TTM. Patients with TTM tend to have fewer comorbid obsessive-compulsive symptoms, as well as less depression and anxiety compared to OCD patients . ResponsRelatively few empirical studies have, however, documented the phenomenological similarities and differences between OCD and TTM ,18,19. ATwo hundred and seventy eight OCD patients , and 54 TTM patients , ranging in age between 8 and 75 years, took part in the study  for adult patients (aged 18 or older) , the intThe Yale-Brown Obsessive-Compulsive Severity Scale (Y-BOCS)  was implPatients' level of insight into the senselessness or excessiveness of their symptoms was assessed on the relevant YBOCS item. When an adequate trial of pharmacotherapy with an SRI (i.e. for both OCD and TTM groups, at least 10 weeks on the medication with a minimum of 6 weeks on mid-range dose) had been undertaken, response to pharmacotherapy was assessed using the global improvement item of the Clinical Global Impression (CGI) scale; subjects with CGI scores of 1 ('very much improved') or 2 ('much improved') were defined as responders . SimilarQuestions addressing potential precipitating or exacerbating factors, including the impact of menstrual/reproductive cycle changes, brain trauma and history of autoimmune infections on OCD/TTM symptom fluctuations, were included in the interview.Severity of comorbid depression was evaluated with the Beck Depression Inventory (BDI) . The ChiThe self-report Temperament and Character Inventory (TCI)  was alsoAs there were few males with TTM Table , only clGender distribution of the sample differed significantly, with a marked predominance of female participants with TTM compared to almost equal numbers of male and female participants with OCD. TTM patients had an earlier age of onset of illness compared to patients with OCD Table .A number of disorders were more frequent in females with OCD: major depressive disorder (MDD), dysthymia, panic disorder, hypochondriasis and intermittent explosive disorder. In terms of the selected Axis II disorders, obsessive-compulsive personality disorder (OCPD) was more frequent in females with OCD Table .The severity of OC symptoms in OCD patients, as measured by the YBOCS severity scale, was 20.1 (± 8.0). TTM patients scored 16.1 (± 6.5) on average on the MGHHPS. Compared to TTM patients, females with OCD had significantly higher depressive symptom scores on the BDI  indicated that OCD and TTM patients differed significantly on 5 schemas, i.e. mistrust / abuse, social isolation, shame / defectiveness, subjugation and emotional inhibition Table . More spOCD patients reported more childhood sexual abuse than did TTM patients (p = .04).OCD and TTM patients did not differ significantly in terms of a history of serious head injury associated with the onset of OCD or TTM.Compared to none in the TTM group, 9 OCD patients reported onset of their OCD with an episode of bacterial pharyngitis (p = .06). In terms of other autoimmune infections, OCD and TTM patients did not differ significantly.2 = 6.8; p = .009).Female OCD and TTM patients did not differ significantly in terms of the impact of premenstrual/menstrual/menopausal symptoms on their illness. Compared to 42 (38.5%) of 109 OCD patients who reported OC symptom changes in the premenstrual/menstrual period, 16 of 32 TTM patients (50%) reported regular changes in their symptoms during this time. Seventeen (n = 17) OCD patients were menopausal and 35.3% (n = 6) of these women reported that their OC symptoms only started with menopause. One of the TTM patients had gone through menopause with no effect on her hair-pulling symptoms. However, OCD and TTM patients differed significantly in terms of the temporal association between pregnancy/puerperium and onset of illness: 42.6% (26 of 61) OCD patients reported OCD onset while pregnant or within a month of childbirth, compared to 7.7% (1 of 13) of TTM patients  and more maladaptive schemas. Fewer TTM patients, however, reported having responded to treatment.The gender ratio findings in both OCD and TTM groups were similar to other surveys where a mean female:male ratio of 1.5:1.0 in OCD ,32,33 anOur comorbidity findings are consistent with existing data suggesting that depressive and anxiety disorders are highly prevalent in both OCD and TTM, and significantly more prevalent in OCD ,16,18. IOur findings of increased disability in OCD is consistent with studies on OCD suggesting it is one of the most impairing of all medical disorders . A numbeOCD patients reported significantly more sexual abuse than TTM patients (p = .04). This finding differs from our previous data suggesting similar rates of childhood interpersonal trauma (CIT) in OCD and TTM . HoweverTTM patients had significantly more novelty seeking (NS) than OCD patients, whereas OCD patients scored significantly higher on harm avoidance (HA) compared to TTM. Our findings are consistent with previous work on temperament / character in OCD, showing increased HA and decreased NS -53. Of nOCD patients had more maladaptive cognitive schemas than TTM, i.e. mistrust / abuse, social isolation, shame / defectiveness, subjugation and emotional inhibition. The schemas that OCD and TTM patients differed on are included in 2 of the 4 higher order factors (i.e. "impaired autonomy" and "disconnection") described by Lee and colleagues' YSQ factor model . While sHormonal influences have previously been investigated in OCD  and TTM Although rare, brain injury may play a role in some cases of OCD ,62; in oA number of patients associated the onset of their OCD onset with an infection, possibly bacterial pharyngitis. This finding is consistent with a body of data suggesting post-streptococcal disease is a cause of OCD in children and adolescents , and perIn our study, more OCD patients reported a positive response to treatment (with CBT or SRI's) than TTM patients. These data should be interpreted cautiously given the retrospective assessments. Nevertheless, there is evidence that SRI's in TTM may not be as effective over the long-term as in OCD . About 4Although there is evidence for the usefulness of behaviour therapy in both OCD  and TTM Several limitations of the current study should be acknowledged. First, interviewers were not blind to the patients' psychiatric diagnosis, so potentially biasing clinician's assessments. Nevertheless, a structured diagnostic instrument ensures a reasonable degree of reliability. Second, instruments employed in the current study are intended for use in adults rather than younger subjects. However, in the case of children and adolescents, the SCID-I was supplemented with a clinical interview of parents or guardians, and self-report data was included only when it was clear that questionnaires had been completed meaningfully. Third, source of referral, and the duration of OCD/TTM, were not controlled for in the analysis, so potentially biasing the analyses. However, given the chronicity of both conditions, this is unlikely to have materially affected the findings. Fourth, males, as well as patients with comorbid OCD and TTM were excluded from the investigation; so that the results here may not be generalizable to all OCD or TTM subjects. Given evidence that the phenotype of OCD  and of TIn conclusion, our data suggest that despite some overlap, TTM differs from OCD in terms of demographics (gender distribution), associated clinical variables (e.g. comorbidity, cognitive schemas, temperament/character profiles and disability), precipitating factors (trauma history) and treatment response. It has been suggested that although TTM is not the same as OCD, it lies on a compulsive-impulsive spectrum of disorders . HoweverThe author(s) declare that they have no competing interests.CL:• participated in the design of the study,• was responsible for patient recruitment,• did most of the clinical assessments,• performed the statistical analyses,• helped to obtain funds for the research, and• was responsible for the final writing up of data.SS:• participated in the design of the study,• assisted with recruitment of patients, and• supervised writing and statistical analyses.PLduT:• participated in the design of the study,• assisted with recruitment of patients, and• did some of the clinical assessments.DGN:• was the primary statistical consultant for analyses.DJHN:• participated in the design and coordination of the study,• was responsible for patient recruitment, and• assisted with clinical assessments.RS:• assisted with literature review of cognitive schema data, and• asisted statistical analysis of schema data.DJS:• conceived of the study• supervised coordination, statistical analysis, and writing,• did the final revision of paper before submission, and• assisted with obtaining of funds.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Caesarean section (CS) has become an increasingly common method of delivery worldwide, rising in the UK from 9% of deliveries in 1980 to over 21% 2001. This increase, and the question of whether CS should be available to women on request, has been the subject of considerable debate, and national reports and guidelines have specifically highlighted the importance of patient choice in the decision making process. For women who have already experienced CS, the UK National Institute of Clinical Excellence recommends that the decision should consider maternal preferences and priorities in addition to general discussion of the overall risks and benefits of CS. Decision aids for many different medical treatment and screening decisions have been developed and evaluated, but there is relatively little evidence for the use of decision aids for choice of mode of delivery among women with a previous CS. The aim of the study is to evaluate two interventions to assist decision making about mode of delivery among pregnant women with one previous CS.Women with one previous CS are recruited to the trial during their booking visit at approximately 12–20 weeks' gestation in participating maternity units in Bristol, Weston and Dundee. Using central randomisation, women are allocated to one of three arms: information programme and website; decision analysis; usual care. Both interventions are computer-based, and are designed to provide women with detailed information about the potential outcomes for both mother and baby of planned vaginal delivery, planned CS and emergency CS. The decision analysis intervention additionally provides a recommended 'preferred option' based on maximised expected utility. There are two primary outcomes , and five secondary outcomes . Primary follow up for the questionnaire measures is at 36–37 weeks' gestation, and a total of 660 women will be recruited to the study. The primary intention-to-treat analyses will comprise three pair-wise comparisons between decision analysis, information and usual care groups, for each of the two primary outcomes. A qualitative study will investigate women's experiences of the decision making in more depth, and an economic evaluation from the perspective of the NHS will be conducted.Provision of information to women facing this decision appears variable. The DiAMOND study aims to inform best practice in this area by evaluating the effectiveness of two interventions designed to aid decision making. Over the last 20 years caesarean section (CS) has become an increasingly common method of delivery. The CS rate in the United Kingdom rose from 9% of deliveries in 1980 to 21% in 2001 .The Changing Childbirth report has emphasised the importance of patient choice when decisions need to be made in relation to the management of pregnancy and childbirth . HoweverIn an attempt to ensure appropriateness of CS in the UK, a set of evidence-based guidelines on indications for CS have recently been published . The guiIt has been proposed that the way in which clinical decisions are made lies on a continuum, from paternalistic (clinician decides) through partnership (clinician and patient share the decision) to informed (patient decides) . AlthougDecision aids are designed to help people select between various treatment strategies by providing information on the options and outcomes relevant to a person's health. Decision aids for many clinical conditions have been developed , and evaThe specific content of decision aids may vary, but in general they aim to present more than one strategy for clinical management, help people understand the probable outcomes of treatment choices and allow people to consider the personal value they place on benefits versus harms. Decision aids can take several formats, such as leaflets, interactive videodisks, individualised decision analysis, personal counselling sessions and audio workbooks. Interventions to assist patient decision making can improve knowledge about treatment options, make patients more realistic in their expectations, reduce decisional conflict and increase active involvement in decision making .As an intervention to aid patient decision making, individualised decision analysis has so far received limited attention. By explicitly combining patients' values regarding treatment outcomes and individual probability information, decision analysis attempts to provide a rational framework to guide patient decision making. The use of individualised decision analysis has been debated , but empThe aim of this paper is to describe the protocol for a randomised controlled trial of two interventions to aid decision making about mode of delivery among pregnant women with one previous CS. The interventions being assessed are (1) Decision analysis, and (2) Information programme and website. Both decision aids will be compared with usual care given by the obstetric team. The interventions will be assessed in terms of decisional conflict, planned and actual mode of delivery, anxiety, knowledge, perception of shared decision making and satisfaction with the decision making process. Development and piloting of the interventions took place in 2003–2004, and the main phase of the study started in May 2004 and will continue until December 2006. Ethical approval for the study was obtained from the UK South West Multi-Centre Research Ethics Committee.The sample will comprise pregnant women with one previous lower segment CS , no current obstetric problems and delivery expected at ≥ 37 weeks. Women are being recruited to the study by research midwives during their initial booking visit at approximately 12–20 weeks' gestation. Recruitment takes place from maternity units in St Michael's and Southmead Hospitals in Bristol, Weston General Hospital, and Ninewells Hospital in Dundee. The current CS rates for these units range between 18 and 24% and are representative of rates for other units throughout England and Scotland. The women are informed that although both vaginal delivery and repeat CS carry their own benefits and risks, the best method of presenting this information in order to assist women in reaching a decision is not known. Women expressing an interest in participating in the trial at the booking visit are given an information sheet, a written consent form and a baseline questionnaire to take home. Following receipt of the baseline questionnaire and written informed consent to enter the trial, women are randomised to one of three arms as detailed below. Allocation is stratified by maternity unit and preferred mode of delivery and blocked (using random permuted blocks of sizes 6, 9, 12 and 15) to ensure reasonable balance between the trial groups through time. The randomisation sequence was generated by a member of the study team (AAM), and allocation of participants is performed by a staff member with no other involvement in the study.Both interventions are computer based. Women allocated to receive an intervention have an appointment with a researcher to allow the decision aid to be delivered using a laptop computer, usually in the woman's own home or workplace.This intervention provides information about the outcomes associated with planned vaginal delivery, planned CS, and emergency CS. This includes descriptions of outcomes for both mother and baby, and the probabilities of these outcomes based on the best available evidence. Both the probabilities of having and not having the event are given, and all probabilities are presented in both numerical and pictorial format . The proThere are generally four main steps involved in a decision analysis. The first is to draw up a decision tree that maps out the likely outcomes of the strategies in question . These oThe decision analysis intervention in the trial proceeds according to the principles described above. First, women are given information about the outcomes associated with planned vaginal delivery, planned CS, and emergency CS. This includes descriptions of outcomes for both mother and baby, but not the probabilities of these events. These are embedded in the decision tree which is not visible to users. Second, women are required to rate  each possible outcome using a visual analogue scale. Finally, the programme combines the elicited utilities and the probabilities of each outcome in a decision tree to produce a recommended 'preferred option' based on maximised expected utility. Each woman is given a computer printout of the outcome of the decision analysis.This comprises care normally given by the obstetric and midwifery team. Women allocated to decision analysis or information programme receive these interventions in addition to usual care.Women in both intervention arms are contacted by letter at 35 weeks' gestation. The purpose is to encourage discussion of the intervention with their obstetrician and/or midwife when they attend the clinic at 36–37 weeks to finalise their planned method of delivery. Participation in the study is recorded in the medical records of all women in the trial.There are two primary outcomes:(1) Decisional Conflict Scale (DCS) ,29. This(2) Actual mode of delivery . Unlike a previous trial , we are There are five secondary outcomes: anxiety;  knowledgThe primary and secondary outcomes are assessed in all three groups at baseline, and approximately two weeks after randomisation. The questionnaire at two weeks will constitute a secondary follow up, and will enable sufficient time for delivery of the appropriate interventions. As part of usual care, women in the trial normally attend the clinic at around 36–37 weeks' gestation to finalise plans for their preferred method of delivery. Questionnaire outcomes are measured again after this visit, and this constitutes the primary follow up for this trial. Actual and attempted mode of delivery  and satisfaction with choice are assessed by a further postal questionnaire at approximately six to eight weeks after giving birth.As noted above, differences in excess of 0.35 standard deviations have been considered as important for the total DCS score, and differences of this magnitude are feasible for interventions of this kind . With reWith two-sided 1% alpha, a total sample size of 600 provides 82–99% power to detect a standardised difference of 0.35–0.5 in total decisional conflict score between the groups, and 84–95% power to detect odds ratios of 2.1–2.4 in women achieving vaginal delivery. A pair-wise alpha of 1%, corrected for multiple comparisons using Tukey's procedure, maintains an overall study-wise alpha of 3.4%. In order to allow for pre-term deliveries, malpresentations, and losses to follow-up, we will therefore recruit 660 women to the trial.Data analysis will proceed according to CONSORT guidelines for randomised controlled trials. The first stage of the analysis will be to use descriptive statistics to describe the group of individuals recruited to the trial in relation to those eligible, and to investigate comparability of the trial arms at baseline. The primary analyses will comprise three pair-wise intention-to-treat comparisons between decision analysis, information and usual care groups, for each of the two primary outcomes. These comparisons will use appropriate (that is, standard or logistic) multivariable regression models, adjusting for maternity unit, initial preference regarding mode of delivery, and value of the outcome variable at baseline. Full attention will be paid to the estimates and confidence intervals for these comparisons as well as the p-values, with the latter adjusted for multiple comparisons using Tukey's procedure. Secondary outcomes will then be analysed in the same way, using appropriate multivariable regression models depending on the nature of the outcomes.Other secondary analyses will involve investigation of the short-term effects of the interventions using data from the two week follow up, and the effects at 36–37' weeks gestation adjusted for both baseline and two week follow up. Pre-planned subgroup analyses employing appropriate interaction terms in the regression models will be used to ascertain any differential effects of the interventions on the two primary outcomes across the following categories of women: previous caesarean section occurring before or after labour; previous successful vaginal delivery; stated preferred mode of delivery. Since the trial is powered to detect overall differences between the groups rather than interactions of this kind, the results of these essentially exploratory analyses will be presented using confidence intervals as well as p-values, and interpreted with due caution. Finally, we will investigate the effect of differential use of the information intervention via the internet using descriptive statistics and appropriate comparisons with the other groups.Qualitative research methods will be used in order to explore aspects of the interventions and women's experiences of the decision making in more depth. Specifically, semi-structured interviews will be conducted with a sample of women from each of the intervention arms , across the research sites in Bristol and Dundee. These interviews will explore:(1) Women's views and experiences of the intervention and its delivery – for example, the quality and relevance of the decision aid/information, what they felt about the risk information presented to them, and which particular aspects of each intervention were helpful or unhelpful.(2) Which factors women felt had most influenced their preferences regarding method of delivery.(3) Whether the women had prior preferences about method of delivery, whether/how these changed during their pregnancy and in the case of the decision analysis, what they felt about the method of delivery proposed by the intervention compared to any prior preferences.(4) Any other information sources women sought and used to help them make a decision about method of delivery .(5) Women's views and feelings about their actual method of delivery compared with any prior preferences and the method of delivery suggested by the intervention .A small number of interviews may also be conducted with women in the usual care arm of the trial to explore what support and advice was normally provided during pregnancy to those who did not receive an intervention.A sample of approximately 30 women across the two intervention arms will be interviewed in depth to ensure a thorough exploration of emergent themes and concepts. Within each arm, women will be purposefully chosen to include those with different parities/ages/socio-economic backgrounds, and where possible different methods of delivery/outcomes, following a maximum variation sampling strategy . The intInterviews will be conducted in the womens' homes or other suitable setting chosen by them. A check-list of topics will be used to ensure that the primary issues are covered, whilst allowing flexibility for new issues to emerge from each interview. Interviews will be recorded on mini-disc, fully transcribed and anonymised to protect confidentiality. Transcripts will be studied in detail and a list of common themes and concepts drawn up. Data collection and analysis will run in parallel and the coding index added to or refined and coded material regrouped as new themes and categories emerge from subsequent interviews . FurtherIn addition to the interviews with women, a small number of focus groups with health professionals (e.g. obstetricians, midwives) in each research site may be conducted near the end of the trial, resources and time permitting. Focus groups are often used in evaluations of new services/interventions and are useful for exploring group views, concerns and preferences (e.g. consensus or disagreement about an issue) . They maThe aim of the economic evaluation is to compare the costs and benefits of the two interventions with usual care. The analysis will be from an NHS perspective and will be based on the costs incurred during pregnancy, delivery and 6–8 weeks following delivery. Incremental cost effectiveness ratios will be formed comparing (i) the cost per point improvement on the Decisional Conflict Scale (DCS) and (ii) the cost per extra patient able to make a decision (represented by a 0.5 standard deviation change on the DCS). We will also compare the average cost of care per patient in each arm of the trial with patient satisfaction. Differences in the cost of care of women receiving the two interventions will be compared with usual care from the point at which patients are randomised. The analysis will include all resources under the control of the NHS that may differ as a result of the interventions and will include resources used by both mother and baby. The costs identified as being of relevance are: antenatal appointments in addition to routine appointments, including both primary and secondary care; mode of delivery and related hospital stay for mother and baby; follow up care for mother and baby, including primary and secondary care appointments, outpatient appointments, inpatient stays, A&E visits, and prescribed medication.Data on resource use will be collected principally by two questionnaires completed by the women. The first questionnaire, completed at 36–37 weeks' gestation, will provide information on all antenatal appointments, and the proportion of these that involved discussion about mode of delivery. Women will be asked how often mode of delivery was addressed at (i) routine appointments, and (ii) appointments initiated by them to discuss mode of delivery. A sample of 10 hand-held records from each arm at each main centre  will be scrutinised to validate the information given in response to the questionnaire. The second questionnaire, completed six to eight weeks after delivery, will provide information on mode of delivery and all non-routine postnatal health service contacts and prescriptions for both mother and baby.The principle of opportunity cost will underlie the valuation of resource use. In many cases, market rates will act as a proxy for opportunity cost. National data sets such as the Unit Costs of Health and Social Care  and the Costs and outcomes will not be discounted, as the economic evaluation will be limited to a period of 12 months. Sensitivity analysis will be conducted in areas where there is uncertainty around assumptions about resource use measurement and/or valuation. Variation in resource use and the effectiveness of the intervention is not captured by a cost-effectiveness/utility ratio. We will use bootstrapping to address this, and construct a cost effectiveness acceptability curve.Women with an uncomplicated pregnancy and expected term delivery who have previously delivered by CS face a choice between repeat elective CS or attempted trial of labour. Guidelines in the UK emphasise the importance of involving women in the decision making process and taking account of maternal preferences and priorities, but the type and amount of information available to women facing this choice appears variable. The DiAMOND study is a randomised trial that aims to inform best practice in this area, by evaluating the effectiveness of two interventions to assist decision making in terms of decision quality and actual mode of delivery.The author(s) declare that they have no competing interests.Alan Montgomery, Deirdre Murphy, Ali Shaw and Sandra Hollinghurst drafted the manuscript, with input from the other members of the DiAMOND Study Group.The Decision Aids for Mode Of Next Delivery (DiAMOND) Study Group comprises the following members: Clare Emmett, Tom Fahey, Peter Gregor, Sandra Hollinghurst, Claire Jones, Beverley Lovering, Alan Montgomery, Irene Munro, Deirdre Murphy, Roshni Patel, Tim Peters, Ian Ricketts, Anne Schlegelmilch, Ali Shaw, Kav Vedhara, Kate Warren.The pre-publication history for this paper can be accessed here:


Caenorhabditis elegans interrupts its reproductive cycle and forms a specialised larva called dauer (enduring). This process is regulated by TGF-β and insulin-signalling pathways and is connected with the control of life span through the insulin pathway components DAF-2 and DAF-16. We found that replacing cholesterol with its methylated metabolite lophenol induced worms to form dauer larvae in the presence of food and low population density. Our data indicate that methylated sterols do not actively induce the dauer formation but rather that the reproductive growth requires a cholesterol-derived hormone that cannot be produced from methylated sterols. Using the effect of lophenol on growth, we have partially purified activity, named gamravali, which promotes the reproduction. In addition, the effect of lophenol allowed us to determine the role of sterols during dauer larva formation and longevity. In the absence of gamravali, the nuclear hormone receptor DAF-12 is activated and thereby initiates the dauer formation program. Active DAF-12 triggers in neurons the nuclear import of DAF-16, a forkhead domain transcription factor that contributes to dauer differentiation. This hormonal control of DAF-16 activation is, however, independent of insulin signalling and has no influence on life span.Upon starvation or overcrowding,  A sterol-derived activity is partially purified and shown to support reproductive growth under sterol-free conditions that normally induce dauer larva formation in nematodes Caenorhabditis elegans provides a valuable model system to study the orchestration of cholesterol metabolism and function at the level of a whole organism. C. elegans, like other nematodes, cannot synthesise sterols de novo , a C. elegans homologue of Drosophila orphan nuclear receptor (DHR3) that is induced by ecdysone, leads to defective shedding of the old cuticle  or to prevent their formation (Daf-d mutants) . Geneticd daf-12 . It was receptor , 2000, s mammals . The dafC. elegans, we developed a protocol for strict elimination of sterols in the medium and food. Under sterol-free conditions, the first generation of worms developed from eggs to adults without external cholesterol. In the second generation they become dauer-like larvae but molting was incomplete. We found that replacing cholesterol with its natural metabolite lophenol, a methylated sterol, induced all worms to form regular dauer larvae. Using the effect of lophenol on growth, we could partially purify activity supporting the reproduction and determine the role of sterols during dauer larva formation and longevity. In the absence of this hormone, the nuclear hormone receptor DAF-12 is derepressed and thereby activates the dauer formation program. Active DAF-12 triggers in neurons the nuclear import of DAF-16 that contributes to dauer differentiation. Thus, the effect of lophenol allowed us to reveal a novel function of DAF-16 downstream of DAF-12 that is required for the execution of the dauer program but has no effect on life span.As a starting point for our investigations on the role of sterols in Escherichia coli to be fed to worms . It has been previously established that  product A  required for reproductive growth. The rationale of our approach was to rescue the formation of dauer larvae induced in the presence of lophenol by a substance derived from a lipidic extract of worms. Obviously, this substance should differ from cholesterol and its direct metabolites such as 7-dehydrocholesterol and lathosterol. The lipidic extract of worms see  was frac8 column A, and frWe name this activity gamravali  because it is required for reproduction in worms. Currently we are attempting to determine the molecular formula of gamravali using mass spectroscopy. This task, however, is very demanding because of the tiny amounts of the substance in worms. Even more demanding will be the final identification of the structure by nuclear magnetic resonance or X-ray analysis. We estimated that the latter might require scaling of the preparation see  up to moOur data indicate that gamravali is much more hydrophilic than sterols. Remarkably, retention times on the column of many mammalian steroids tested (pregnenolone, β-estradiol, testosterone, etc.) and the insect molting hormone ecdysone are very similar A. Thus, The effect of lophenol on growth also made it possible to identify steps of the dauer formation pathway at which gamravali is required. For this we examined the phenotype of several dauer formation-defective (Daf-d) mutants when grown on lophenol. We assumed that mutants that are defective in metabolism of gamravali and thus act upstream of the hormone receptor should produce dauer larvae on lophenol. Mutants in genes acting downstream of the gamravali action should reproduce normally.daf-12 null mutant with lophenol as the sole source of sterols. DAF-12 as a putative nuclear hormone receptor is a good candidate to be a receptor for gamravali. In contrast to wild-type worms, mutants of daf-12 grown on lophenol produced no dauer larvae and developed normally for more than seven generations. daf-12 could also reproduce normally on lophanol and 4αF-cholestanol . It must be noted, however, that worms grown in the absence of cholesterol and the presence of lophenol do not have a developmental phenotype in the first generation and therefore may have some maternal rescue of adult life span. Because the second generation does not grow to adulthood (forms dauer larvae), the definitive experiment cannot be performed. Growth on lophenol also had no influence on the intrinsic thermotolerance of worms at 39 °C. Thus, the activation of DAF-16 induced by the absence of gamravali might have different physiological consequences than its activation by diminished IGF-1 signalling.pe worms , and in pe worms . This efC. elegans can regulate membrane properties in response to temperature changes by altering fatty acid composition of phospholipids . We propose the following model to explain these results . Gamravaome P450 , could bhenotype . We did 2 strain . However2 strain , differeOur data uncover a dual role for DAF-16, first during the reproductive/dauer decision and second during dauer differentiation. It has been established that DAF-16 acts via the insulin-dependent pathway and is involved in the inhibition of hormone production , therebyA direct physical interaction between nuclear hormone receptors and forkhead domain (FOXO) transcription factors has recently been reported . It is tdaf-16 is a major regulator of the longevity process . Electrophoresis-grade ultraPURE agarose was the product of Life Technologies (Paisley, Scotland, United Kingdom). Dulbecco's medium (DMEM) was from Invitrogen (Karlsruhe, Germany). Sterols, steroids, and antioxidant BTH were from Sigma (Sigma-Aldrich Chemie, Taufkirchen, Germany).daf-12 (rh61rh411) and daf-9 (dh6) daf-12 (rh61rh411) were obtained from the Caenorhabditis Genetics Center. daf-12 and the double mutant daf-9 daf-12 were a kind gift of Adam Antebi (Max Planck Institute for Molecular Genetics, Berlin, Germany). The following mutant strains were used throughout the study: daf-22 (m130)II, daf-6 (e1377)X, daf-3 (mgDf90)X, daf-5 (e1386)II, daf-10 (e1387)IV, daf-12 (rh61rh411)X, daf-2 (e1370ts)III, and daf-16 (mgDf50)I. For imaging studies strains daf-16 (mu86)I; muIs71[pKL99(daf-16a::GFP/bKO)+pRF4(rol-6)]X and daf-16 (mu86)I; muIs61 (daf-16::GFP (pKL78)+rol-6(pRF4) were tested. Since the former, daf-16a::GFP/bKO, gave a brighter signal, as previously reported (All mutants except reported , resultsE. coli was grown on a sterol-free culture medium DMEM. Bacteria were rinsed with M9 medium before use.Wild-type N2 Bristol and mutant strains were routinely propagated on NGM-agar plates as described in For preparation of sterol-containing plates, cholesterol or lophenol was dissolved in methanol to the concentration of 5 mM and mixed 1:1 (v/v) with cholesterol-free bacterial suspension in M9. After evaporation of methanol in SpeedVac, the suspension was mixed with fresh bacteria to obtain the desired end concentration of tested substance. Bacterial suspensions were spread on cholesterol-free agarose plates.Light and confocal fluorescence microscopy were done using Zeiss Axioplan and Axiovert LSM 510 microscopes, respectively. For nuclear labelling, larvae were grown on medium containing 5 μg/ml of Hoechst (Molecular Probes, Eugene, Oregon, United States). One hour before imaging, larvae were transferred to medium without Hoechst, washed briefly with M9 medium, anaesthetised with 40 mM sodium azide in M9, and mounted on agarose pads. For electron-microscopic studies arrested larvae were washed two times with M9, harvested by centrifugation, and mixed with an equal volume of 2×Fixative . Worms were cut at room temperature with a razor blade on a microscopic slide, transferred to a centrifuge tube, and incubated for 2 h in a refrigerator. Afterwards worms were centrifuged and embedded in EmBed-812 (EMS, Ft. Washington, Pennsylvania, United States). Images were acquired by Tecnai 12 (FEI, Eindhoven, The Netherlands) or Phillips 400 electron microscopes.C. elegans, 10-cm NGM agar plates were prepared without cholesterol. A quantity of 300 μl of bacterial suspension containing 13 μM cholesterol was supplemented with 4 μCi of [3H]-cholesterol . Worms were harvested from the plates with M9 medium and subjected to three cycles of freezing-thawing, and lipids were extracted by the Bligh and Dyer method (To investigate cholesterol metabolism in r method . Eggs deTLC was performed on glass-backed plates of silica gel 60 (Merk, Darmstadt, Germany). Solvents used for the separation of cholesterol metabolites were chloroform-methanol (24:1).After chromatography, plates were sprayed with a scintillator (Lumasafe, Lumac LSC B.V., Groningen, The Netherlands) and exposed to a film (Hyperfilm MP, Amersham Biosciences Europe, Freiburg, Germany).We quantified relative amounts of ms and nms by scanning films exposed to radioactivity for short time and using Adobe Photoshop software.The synthesis of 4α-substituted 5α-cholestan-3β-ols is described in the 2 and thawing by sonication in an ultrasound bath at 37 °C. Worm suspension was then transferred into a glass bottle, 19 volumes of methanol containing 10 μg/ml of antioxidant BHT was added, and extraction was performed overnight at room temperature under continuous agitation. Extract was separated from worm remnants by filtration through a Whatman GF/A glass filter and remnants were reextracted with a fresh portion of methanol. Methanol extracts were combined and extracted two times with one volume of hexane. The obtained hexane extract was washed twice with a methanol-water mixture (9:1), dried under N2 flow, and dissolved in 7 ml of hexane.Worms of mixed population from 150 15-cm plates were collected by rinsing with ice-cold water and left overnight at 4 °C to sediment. The final volume of the sediment was about 150 ml. After decantation, aliquots of the worm suspension were transferred into 50-ml Falcon tubes and subjected to three cycles of freezing in liquid N2 flow, and dissolved in 200 μl of methanol. Two preps (400 μl) were subjected to reverse-phase HPLC chromatography on an Alliance 2695 solvent module (Waters GmbH, Eschborn, Germany) linked to a Waters 996 photodiode array detector using an XTerra Prep MS C18 10 μm 10 × 250-mm column (Waters). The elution protocol was as follows: 15% solvent A (20% methanol in water) and 85% solvent B (methanol) for 11 min, a gradient from 85% B to 100% B in 11 min, and 100% B for 18 min. The flow rate was 5 ml/min. Fractions of 2 min were collected, dried, dissolved in 400 μl of isopropanol, and stored at −80 °C until use.In order to dispose very hydrophobic substances, the extract was subjected to a solid-phase separation. A quantity of 200 μl of the hexane fraction was applied to a 20-ml LC-18-SPE cartridge (SUPELCO, Bellefonte, Pennsylvania, United States) equilibrated with methanol. Twenty millilitres of flow-through methanol was collected, dried under NTesting of the biological activity of HPLC fractions was performed in 12-well cell culture plates (Nunc, Roskilde, Denmark). Each well contained 1 ml of sterol-free agarose mixed with 0.1% tergitol. A quantity of 100 μl of HPLC fractions was added per well and dried in the laminar flow cabinet. Before seeding worms, 30 μl of sterol-free bacteria containing 10 μM lophenol was added to plates and left to stay overnight at room temperature.Worms for the bioassay were prepared as follows. The first generation of adult worms grown on lophenol (see above) was bleached, and eggs were placed on sterol-free plates without food and were kept for 3 d to obtain synchronised L1 larvae.About ten starved L1 larvae were placed in each well with HPLC fractions at room temperature. After 4 d worms were scored and the activity of fractions was represented as the percentage of worms that reached L4 or adult stages. Quadruplicates of each fraction per experiment were analysed.3, ecdysone, 20-hydroxyecdysone, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 19-hydroxycholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, 24-hydroxycholesterol, 26-hydroxycholesterol, cholic acid, dehydrocholic acid, deoxycholic acid, litocholic acid, taurodeoxycholic acid, and chenodeoxycholic acid were tested. 7α-hydroxycholesterol, 19-hydroxycholesterol, and 26-hydroxycholesterol were from Steraloids (Newport, Rhode Island, United States); all others were from Sigma.Pregnenolone, testosterone, estrone, β-estradiol, progesterone, androstenol, vitamin Ddaf-16(mu86)I; daf-12 (rh61rh411)X was generated by crossing daf-16 (mu86)I; muIs71[pKL99(daf-16a::GFP/bKO)+pRF4(rol-6)]X hermaphrodites with daf-12 (rh61rh411) males. Progeny displaying no Roller phenotype and fluorescence and able to grow on lophenol were selected. The daf-16(mu86) mutation was identified by PCR. Consequently, the mutations were verified by sequencing.The double mutant daf-16(mu86)I; daf-12 (rh61rh411)X was then used to generate daf-16 (mu86)I; daf-12 (rh61rh411) muIs71[pKL99(daf-16a::GFP/bKO)+pRF4(rol-6)]X worms by backcrossing to the original daf-16 (mu86)I; muIs71[pKL99(daf-16a::GFP/bKO)+pRF4(rol-6)]X. The mutations were identified and verified in a way similar to that used for the double mutant.The double mutant The life span and thermotolerance of worms were investigated according to the method of Figure S1(3.8 MB PDF).Click here for additional data file.Protocol S1(114 KB DOC).Click here for additional data file.


This study, using in vitro strips of human uterine smooth muscle, aimed to investigate the direct non-genomic effects of 17P on spontaneous and induced contractions in tissues obtained during pregnancy, and in the non-pregnant state.Biopsies of human myometrium were obtained at elective cesarean section, and from hysterectomy specimens, and dissected strips suspended for isometric recordings. The effects of 17P (1 nmol/L -10 micro mol/L) on spontaneous and agonist-induced (oxytocin 0.5 nmol/L for pregnant, phenylephrine 10 μmol/L for non-pregnant) contractions were measured. Integrals of contractile activity, including the mean maximal inhibition values (MMI) observed at the maximal concentration, were compared with those from simultaneously run control strips.There was no significant direct effect exerted by 17P on pregnant or non-pregnant human myometrial contractility. The MMI ± SEM for spontaneous contractions in pregnant myometrium was 4.9% ± 7.2  and for oxytocin-induced contractions was 2.2% ± 1.3 . For non-pregnant myometrium, the MMI ± SEM for spontaneous contractions was 8.8% ± 11.0  and for phenylephrine induced contractions was -7.9% ± 6.5 .The putative benefits of 17P for preterm labor prevention are not achieved, even partially, by a direct utero-relaxant effect. These findings outline the possibility that genomic effects of 17P, achieved over long periods of administration, are required for its reported therapeutic benefits. Preterm delivery constitutes a major problem in obstetric practice because of the large associated contribution to perinatal mortality and morbidity ,2. A sigIt has however been recently reported that weekly injections of 17-alpha-hydroxyprogesterone caproate (17P), in women who have had a previous spontaneous preterm delivery, significantly reduces the risk of preterm delivery before 37, 35 and 32 week's gestation . In addi2SO4, 1.2 mmol/L CaCl2, 1.2 mmol/L KPO4, 25 mmol/L NaHCO3, and 11 mmol/L glucose. Tissues were stored at 4° C and used within 12 hours of collection.Patient recruitment took place in the Department of Obstetrics and Gynaecology, University College Hospital Galway. Ethical committee approval for tissue collection was obtained from the Research Ethics Committee at University College Hospital Galway and recruitment was by written informed consent. The biopsies were excised from the upper lip of the lower uterine segment incision in the midline i.e. upper portion of lower uterine segment. Women undergoing induction of labor were excluded from the study. For hysterectomy specimens, all women were pre-menopausal and undergoing abdominal hysterectomy without evidence of malignant uterine disease. Biopsies of myometrial tissue from hysterectomy specimens were obtained from the fundus. The biopsies were immediately placed in Krebs-Henseleit physiological salt solution (PSS), pH 7.4, containing: 4.7 mmol/L KCl, 118 mmol/L NaCl, 1.2 mmol/L Mg2/5%CO2. Myometrial strips were allowed to equilibrate for at least 60 minutes, during which time the Krebs-Henseleit physiologic salt solution was changed every 15 minutes. After the equilibration period, regular spontaneous myometrial contractions were allowed to develop. In separate experiments contractions were induced by the addition of oxytocin (0.5 nmol/L) to pregnant myometrial strips, and phenylephrine (10 μmol/L) to strips obtained from non-pregnant myometrium.Longitudinal myometrial strips were dissected measuring approximately 2 × 2 × 10 mm. The strips were mounted for isometric recording under 2 grams of tension in organ tissue baths, as previously described . The tis-9 - 1 × 10-5M). Control strips (i.e. without exposure to 17P) were run simultaneously and for a similar duration, consisting of two separate groups of experiments as follows: 1. exposure of strips to PSS only; 2. exposure of strips to PSS and the vehicle for 17P. The overall duration of experiments was therefore 3 hours which is in accordance with standard in vitro myometrial experiments, allowing for an accurate drug exposure period of 20 minutes. The effects of 17P were assessed by subtracting the integrals of contractility measured after each bath exposure of 17P, from those obtained in control experiments (vehicle only). This allowed for calculation of the net effect of 17P on myometrial contractility. Potential effects of the vehicle were obtained by subtraction of the mean integrals measured after vehicle exposure, from those obtained without addition of vehicle i.e. spontaneous or agonist-induced contractions in PSS only. Percentage contractility was calculated by expressing the net integral measured after each 17P concentration addition, as a percentage of the integral calculated in the 20 minute period prior to any 17P addition. The mean maximal inhibition (MMI) refers to the mean percentage relaxation [i.e. 100% - mean contractility measured for each separate n sample of non-pregnant and pregnant myometrium] observed at the maximal bath concentration of 17P (i.e. 10 μmol/L), or corresponding concentration of vehicle for control strips.Once regular phasic contractions had developed, the integrated tension for the first 20 minutes was calculated, and this value served as a control since no significant spontaneous reduction in myometrial contractility was observed over the duration of experiments in control strips (without addition of vehicle or 17P). The mechanical response of tissues was then measured by calculation of the integral of selected areas for 20 minute periods, corresponding to the cumulative exposure to 17P or vehicle, using the PowerLab hardware data acquisition system and Chart v3.6 software (AD Instruments, Hastings, UK). At 20 minute intervals 17P was added in a cumulative manner at concentrations of 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 μmol/L, and 10 μmol/L (i.e., 1 × 10-3M) stock solution of 17P. The resultant final tissue bath concentrations of DMSO and ethanol, at the maximum concentration of 17P investigated (i.e. 10 μmol/L or 10-5M), were 0.83% and 0.28% respectively, allowing for the cumulative effect at each bath addition. DMSO and ethanol were purchased from Sigma-Aldrich, Dublin, Ireland. Fresh Krebs-Henseleit physiological salt solution was made daily. Fresh 17P solutions were prepared on the day of experimentation and were maintained at room temperature for the duration of the experiment.Oxytocin, phenylephrine and 17P were purchased from Sigma-Aldrich, Dublin, Ireland. Because 17P is a lipid soluble compound it was necessary to dissolve it in a lipophilic vehicle. The vehicle used consisted of 75% Dimethyl sulfoxide (DMSO): 25% ethanol, to make a 1 mmol/L . The integrals of contractile activity were compared using a one-way ANOVA, which was followed by a Tukey HSD post-hoc. A value of P < 0.05 was accepted as statistical significance.There were 6 women recruited for the study at the time of elective cesarean section. The reasons for cesarean section included previous cesarean section (n = 2), breech presentation (n = 2), unstable lie (n = 1) and postmaturity with poor cervical Bishop score (n = 1). The mean age, ± standard error of the mean (SEM), was 36.3 ± 2.8 years (range 26–46). The median gestation at the time of cesarean section was 39 weeks (range 38–41). In relation to parity, 2 of the women were para 0, 1 was para 1, and 3 were of parity greater than 1, at the time of recruitment. There were 6 women recruited at the time of abdominal hysterectomy. The reasons for hysterectomy included menorrhagia (n = 4), irregular vaginal bleeding (n = 1), and fibroids with an ovarian cyst (n = 1). The mean age ± SEM was 42.2 ± 2.4 years (range 36–50).For non-pregnant myometrial tissue representative recordings are shown in Figure The results obtained from myometrium during pregnancy are similarly shown in Figure The vehicle for 17P independently exerted a uterorelaxant effect on spontaneous and agonist-induced contractions in pregnant myometrial tissue only , and this was not observed in non-pregnant myometrial tissue  (Table These results demonstrate that 17P does not appear to exert a direct relaxant effect on human myometrial contractions in vitro, in tissue obtained during the third trimester of pregnancy, or in the non-pregnant state. These findings are in contrast to the inhibitory effect of progesterone derivatives on spontaneous contractions in animal uterine tissues ,14, and Previous studies, using various progestins, have yielded conflicting results in terms of the direct effects of these metabolites on human myometrial contractility in vitro. Progesterone metabolites, in some studies, have been reported to decrease both the frequency and amplitude of contractions -17, whil17P is a naturally occurring progesterone which has been isolated from corpus luteum and adrenal gland. The synthetic caproate ester, like the naturally occurring compound, is a steroid, is highly lipophilic and is inactive when administered orally. To achieve solubility for clinical studies, the vehicle used for injection was castor-oil . This leThere are some limitations to the methodology used in our study. The tissue biopsies from the non-pregnant uterus were obtained from the body of the uterus, while those obtained at the time of caesarean section were excised from the lower uterine segment. The results were similar for both tissue types, and there is reasonable evidence to suggest that the functional characteristics of lower and upper uterine segment myometrium are similar . At presWhether 17P injections become incorporated for routine use in the management of preterm labor remains to be seen. Questions surrounding its true benefits, the associated difficulties in terms of vehicle solubility and placebo, and the mechanism of action, remain. Our findings highlight the solubility problems for scientific evaluation as occurred in clinical studies. Whatever the possible mechanism of action of 17P in reducing the incidence and adverse sequelae of preterm labor, this study demonstrates that it does not exert a direct relaxant effect, unlike other conventional methods of tocolysis investigated, and raised the likeliehood of a genomic effect secondary to long term administration during pregnancy.DJS performed the experiments and wrote the manuscript. MWO'R performed the experiments. AMF analysed the data and wrote the manuscript. JJM designed, supervised the study and wrote the manuscript. All authors read and approved the final manuscript.


In 2002 the Australian National Competitive Grants System was opened to the Institute of Advanced Studies at the Australian National University as part of a commitment to transparency, competitiveness, and collaboration in national research funding.The block grant to the John Curtin School of Medical Research had progressively eroded over many years. Access to the National Competitive Grants Schemes and associated infrastructure (through an agreed 'buy-in' price of 20% of block funding) has succeeded in its aims and in reversing this progressive effective decrease in funding.Access to the National Competitive Grant Scheme has allowed the John Curtin School of Medical Research to contribute more broadly to Australia's health and medical research effort through increased collaboration, in a transparent and competitive funding environment. Australia has developed a unique blend of medical research institutions, from universities, teaching hospitals, independent medical research institutes to a range of smaller centres. Universities with medical schools have been the primary forces in medical research and many of the university teaching hospitals fostered the development of research institutes within their grounds . The speThe Australian National University (ANU) was established as a research university  with fouIt can be argued that JCSMR's position as one of the original block-funded research schools of the Institute of Advances Studies (IAS) has given it the opportunity to pursue long-term, independent medical research. Many staff and alumni of JCSMR, including its present Director, argue that the Nobel Prizes, the Albert Einstein World Award, the Japan Prize, the Copley Medal, and a host of other international awards attest to the success of this funding strategy over the past 50 years, and that the scientific achievements that those prizes signify could only have been made by research that could be conducted over long (10 to 20 year) time frames.But the funding climate has changed dramatically over the last few decades. Twenty five years ago many tenured Australian university or teaching hospital staff were able to undertake research even without external funding. Infrastructure was relatively generous, there was still funding for university technical research staff, hospitals provided drugs and consumables and even beds at no cost to the researcher, and research activities merged relatively seamlessly into those of teaching and patient care. Today research is increasingly separated from teaching and care, and the days of in-kind support are long gone. Research is an investment for the nation and the future, but in contemporary funding climates within universities and hospitals it is increasingly becoming an optional extra, undertaken only when researchers are able to attract sufficient external resources.The change in environment and the disappearance of cross subsidisation from patient care and/or teaching has both advantages and disadvantages. Today's research (and research funding) is more transparent: what is spent on research as opposed to care and teaching, at least at the micro level, is much better defined. And, in parallel, the loss of capacity to undertake research that is not peer reviewed has almost certainly raised research standards. The negatives are equally obvious. The University of Melbourne submission to the Wills review stated 'The danger is that if the importance of the nexus between research and learning is not visible to tertiary students because University research is allowed to run down through poor infrastructure, equipment or lack of opportunity of research scientists then research institutes and teaching hospitals will inevitably suffer from a lack of quality of graduates available to them'.The JCSMR has not been exempted from these pressures, and over the last 20 years the value of the block grant has been progressively eroded. The JCSMR grant was a one-line grant to the Director, which provided flexibility, but inhibited collaboration because of restrictive rules around the National Competitive Grants Scheme. Accordingly, a desire for transparency, competitiveness and collaboration led to a decision in 2000 by the Commonwealth Government that the IAS of the ANU could enter the National Competitive Grants Scheme. The negotiations that led to partial entry of JCSMR into the NHMRC schemes in 2001 (for 2002) were long and complex. However the negotiations between JCSMR and NHMRC were paralleled by those between the ARC and ANU and led to a jointly agreed 'buy in' price of 20% of 2000 funding for the IAS to gain access to the various national funding, research training and related schemes. This was $1.7 million per annum for JCSMR access to the National Competitive Grant Schemes and a similar amount for access to the training and infrastructure schemes. It was determined initially that entry would be phased, but after one year the ARC determined that the phase-in was unnecessary and gave the IAS full entry from 2002 (for 2003), and the NHMRC followed suit.The JCSMR felt that, at last, it was able to redress the competitive disadvantage the School faced because of its essentially fixed funding over the last 10 to 15 years, at a time when government funding for the NHMRC system had doubled and then redoubled, from around $65 million in the late eighties to $176 million in 1999 to $381 million in 2004. The rest of the system waited, not without apprehension, for the outcome, but there had long been a wish within the research community that JCSMR be subject to the same forms of peer review as the broader medical research community.st century approaches to improving health. The World Health Organisation has stated that the likely trends in global health in the 21st century include widespread absolute and relative poverty, demographic changes, ageing, growth of cities, epidemiological changes, continuing high influence of infectious diseases, increasing incidence of non-communicable diseases, injuries and violence, global environmental threats to human survival, new technologies, information and telemedicine services, advances in biotechnology, evolving partnerships for health that include private and public sectors and civil society, and globalisation of trade, travel and the spread of values and ideas. Research to deal with global health problems will therefore necessarily be multidisciplinary, involving biomedical, clinical, public health and health services research, and include the social sciences, information sciences and engineering, physics, chemistry, ecology and environmental sciences and economics. As part of the IAS, the JCSMR is accordingly strongly positioned. Not that this need for a multidisciplinary approach is really a new concept – in 1902 Osler stated that the remit of medical research was 'to wrest from nature the secrets which have perplexed philosophers in all ages, to trace to their sources the causes of disease, to correlate the vast stores of knowledge, that they may be quickly available for the prevention and the cure of disease – these are our ambitions' [Removing barriers to collaboration, the outcome of these changes, is important for 21bitions' .In the first three years that JCSMR has been eligible to apply for research funding, their researchers have been awarded a total of $18 million (88% NHMRC, 12% ARC) in competitive funding for periods of up to five years. All three NHMRC program grants held by researchers at the JCSMR are collaborative with other Australian institutions and School researchers hold four ARC linkage grants. This collaboration bodes well for Australian health and for biotechnology growth. In a highly competitive world, Australian researchers need every opportunity to succeed.


Obesity, in particular visceral adiposity, is positively correlated with insulin resistance and type 2 diabetes. Although the link is well established in humans and in rodent models, the mechanisms involved in obesity-related insulin resistance are not clear. One possibility is that hormones secreted by adipocytes compromise peripheral insulin sensitivity, and a number of candidates for such adipocyte signals have been identified. One of them, resistin, was discovered a few years ago by Mitchell Lazar and colleagues, who showed that the protein is expressed by mouse adipocytes and regulated by a group of anti-diabetic drugs called thiazolidinediones. Several lines of evidence from functional studies in rodents suggested that resistin could be the missing mechanistic link between obesity and diabetes.The human homolog of resistin has subsequently been under intense investigation, but initial studies revealed more differences than similarities between the human and rodent proteins: human resistin is mostly expressed in macrophages, not in adipocytes, and its serum levels do not correlate as clearly with obesity, insulin resistance, or diabetes. Similarly, genetic association studies between allelic variants of the resistin gene and metabolic abnormalities have so far been inconclusive. These results prompted some of the scientists in the field who had jumped on the resistin bandwagon after the initial results in rodents to jump off again. Others, including the resistin discoverers, continue their quest to uncover resistin's role in humans, and have started to think outside the framework defined by the mouse data.Starting with the role of macrophages in inflammation and encouraged by the fact that obesity and insulin resistance are associated with markers of systemic inflammation, Lazar and colleagues examined the resistin response to inflammatory stimulators. As they report in this issue, resistin production in macrophages and serum levels in patients are significantly increased by these stimulators. This response can be blocked by the thiazolidinedione rosiglitazone and by aspirin, two drugs that have dual anti-inflammatory and insulin-sensitizing actions and antagonize the immune regulator NF-kappaB. The researchers go on to show that activation of NF-kappaB is sufficient to induce resistin expression. And NF-kappaB is necessary for the resistin response to inflammatory stimuli.Lazar and colleagues now view obesity as a state of chronic inflammation and speculate that in obese individuals inflammatory cytokines lead to elevated production of resistin by macrophages and elevated serum resistin levels, which in turn contribute to insulin resistance and diabetes. This is consistent with some studies that have found higher resistin levels in obese individuals and patients with insulin resistance and/or diabetes, but not all studies have found such differences.Jeffrey Flier, an obesity researcher who was not involved in the study, calls the article “an excellent and timely paper that demonstrates the fact that inflammatory pathways induce resistin expression and levels in human monocytes ex vivo, and in intact humans. The work appears to provide a novel link between inflammation and insulin resistance, through monocyte derived resistin.” He points out, however, that “several other factors also appear to contribute directly to insulin resistance in inflammation (e.g., cytokines themselves, without invoking resistin) so the full biologic implications of the high resistin levels for insulin resistance in humans cannot be determined from this study.” Resistin, it seems, continues to resist easy interpretations.