R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinumIV (DAP) is a DNA-damaging agent that will be entering clinical trials for its potent cytotoxic effects against cisplatin-resistant tumour cells. This cytotoxicity may reside in its ability to selectively activate G1-phase checkpoint response by inhibiting CDKs via the p53/p21 pathway. We have now evaluated the role of another CDK inhibitor p27 as a contributor to DAP-mediated inhibition of G1-phase CDK2 activity. Our studies in ovarian A2780 tumour cells demonstrate that p27 levels induced by DAP are comparable to or greater than those seen for p21. The induction of p27 is not through a transcriptional mechanism, but rather is due to a four-fold increase in protein stabilisation through a mechanism dependent on p21. Moreover, DAP-induced p21 promoted the selective increase of p27 in the CDK2 complex, but not in CDK4 complex, and this selective increase contributed to inhibition of the CDK2 kinase activity. The inhibited complex contained either p27 or p21, but not both, with the relative levels of cyclin E associated with p27 and p21 indicating that about 25% of the inhibition of CDK2 activity was due to p27 and 75% due to p21. This study provides the first evidence that p27 upregulation is directly attributable to activation of the p53/p21 pathway by a DNA-damaging agent, and promulgates p53/p21/p27 axis as a significant component of checkpoint response.The cisplatin analogue 1 R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinumIV (DAP) will undergo clinical trials as a therapeutically promising DNA-damaging agent owing to its ability to circumvent cisplatin resistance  was assessed from fitting a sigmoidal curve to the data using the SigmaPlot 2000 software program, and the data are presented as mean±s.e. (N=7 separate data sets). For biochemical and molecular assays, cells (1–1.3 × 106) were plated in 100 mm culture dishes and cultured for at least 24 h before the cultures were changed into fresh medium containing 0.6 μM DAP. The cells were further cultured for indicated times before they were extracted for total proteins by using procedures described previously .His-p21 and His-p27 vectors and human p21 or p27 cDNA were kindly provided by Dr Mong-Hong Lee. The cDNA was used as the template for PCR amplification. For the myc-p21 vector, the forward primer was 5′-ccggaattcggatcccgatgtcagaaccggctggg-3′, which contained an μl of LF2000 and 2 μg of plasmids for 5 h, washed and then replenished with normal media. Cells were re-incubated and collected after 24 h to prepare lysates, as described before  from Invitrogen , using the manufacturer's instructions (version: 07/09/2004). Cells were exposed to the transfection mixture containing 4 −1) formaldehyde. RNA was transferred to nylon membranes, hybridised with 32P-labelled p27 or glyceraldehyde-3-phosphate dehydrogenase cDNA at 42°C for 18 h, and washed with 0.1 × standard saline citrate/0.1% sodium dodecyl sulphate (SDS) for at least 30 min. Blots were then exposed to X-ray film for 6–16 h.The procedure has been previously reported by us (M siRNA using LipofectAMINE 2000 (LF2000), as described above. The depletion of p21 protein levels by siRNA is maintained for at least 2 days, and all experiments were completed within this time. Cells were incubated with the transfection mixture for 5 h, washed, re-incubated with complete media for 1 h, and then exposed to DAP . Cells were collected at 30 h after initiating DAP treatment, and lysates prepared, as described above.Both p21 siRNA duplexes , which targets a proprietary segment in Exon 3 of p21 mRNA, and control siRNA  were purchased from Ambion . A2780 cells were transfected with 100 nImmunoprecipitation was performed as described previously (β-actin (A5060) was purchased from Sigma Chemical Company . The horseradish peroxidase-conjugated secondary antibody was acquired from Amersham Pharmacia . The protein bands on immunoblots were visualised by ECL detection (Amersham Pharmacia) using different lengths of exposures, with exposures that gave linear or near-linear range of signals selected. To quantify signals on immunoblots, the developed films were scanned and band densities analysed with NIH Image software (version 1.62).Immunoblotting for individual proteins was performed as described previously , and quantified using bovine serum albumin (Sigma) as the standard. Recombinant p21 and p27 proteins of known molar ratios were then simultaneously separated by SDS–PAGE, transblotted to nitrocellulose membrane and immunoblotted with a combination of p21 and p27 monoclonal antibodies. For maximal primary antibody bindings, the blots were incubated with 2 μg ml−1 of each of the antibodies at 4°C for at least 14 h. This antibody concentration was 5–10 times higher than the concentration used for our standard immunoblots, and the 14-h incubation time was 4–7 times longer than the standard procedure. After exposure to the primary antibody binding, the blots were processed as for the standard immunoblots. Finally, the simultaneously developed p21 and p27 bands on the same immunoblot were quantified and their relative intensities determined. Comparison of the relative protein intensities by immunoblot analysis against relative Coomassie blue staining intensities allowed an assessment of whether it was valid to determine p21 and p27 simultaneously on the same immunoblot.Simultaneous quantification of p21 and p27 on immunoblots was validated using calibrated amounts of purified standard proteins. Recombinant His-tagged p21 and p27 were produced in Kinase assay to assess CDK2 complex activity was performed as described before  and Amersham . For each gel filtration, 0.5 ml of the standards or crude cell lysates supplemented with 0.5 mg of purified glutathione S-transferase were loaded onto the column and chromatographed at a flow rate of ∼0.3 ml min−1 with collection of 1-ml fractions. Immediately after chromatography, 40 μl aliquots were taken from fractions 19 to 35 and separated by 12.5% SDS–PAGE. The membranes were then immunoblotted with anti-p27 antibody using the standard procedure. To normalise the signals from different chromatographies for comparison, the highest signal for each protein from each chromatography was arbitrarily defined as 100 and other signals were normalised to this.All gel filtration chromatographies were performed at 4°C on a 40-ml Ultrogel AcA34 column  with a buffer containing 80 m−1) were prepared from untreated or DAP-treated (0.6 μM × 30 h) A2780 cells as described above, and pre-absorbed for 4 h at 4°C with 200 μl of protein-A agarose (Sigma). To isolate the p27-containing complex, the pre-cleared cell lysates were incubated overnight at 4°C with 20 μg of polyclonal anti-p27 antibodies or rabbit IgG (Sigma) that were covalently linked to 30 μl of Affi-prep protein A beads (Bio-Rad) through dimethylpimelimidate catalysed reaction , and the precipitated proteins were eluted with SDS–PAGE sample buffer and resolved by 12.5% SDS–PAGE. Following electrophoresis, each gel slab was fixed overnight in 40% ethanol and 10% acetic acid and washed first for 10 min with 40% ethanol and then for another 15 min with deionised water. The gel was next sensitised by a 1-min incubation in 0.02% sodium thiosulphate followed by two 1-min rinses with distilled water. After rinsing, the gel was submerged in 0.1% silver nitrate for 30 min and developed in 0.04% formalin in 3% sodium carbonate to desired intensities. The development was stopped by addition of one-tenth volume of 2.3 M citric acid to the developer and incubation of the gel in this solution for 15 min. The gel was then rinsed three times for 10 min each time with distilled water and stored in distilled water at 4°C  in 25 mM ammonium bicarbonate, and the peptide fragments were extracted twice with 0.01% trifluoroacetic acid in 50% acetonitrile and concentrated to less than 10 μl by centrifugation in a Speedvac . The peptides were purified by HPLC on C-18 Ziptip  by using 2% acetonitrile/0.1% formic acid to wash and 70% acetonitrile/0.1% formic acid to elute the peptides. After the acetonitrile was removed from the collected samples by centrifugation in a Speedvac for 2–3 min, peptide mass fingerprinting (PMF) was performed on a Voyager DE-STR MALDI TOF instrument  in positive ion and reflected mode. Dried droplet deposits of α-cyano-4-hydroxycinnamic acid were prepared by mixing the peptide solutions 1 : 1 with 10 mg ml−1 matrix in methanol. Database searching was performed by using Mascot (http://www.matrixscience.com) and Protein Prospector (http://prospector.ucsf.edu). To validate PMF results, peptides were sequenced by using an LCQ Deca XP electrospray quadrupole ion trap mass spectrometer  coupled to an HP 1090 liquid chromatography . The solvent system for the HPLC included solution A (2% acetonitrile/0.01% trifluoroacetic acid) and solution B (98% acetonitrile/0.01% trifluoroacetic acid). A 40-min linear gradient of 5–60% B was run to elute the peptides from a 0.3-mm C-18 column . Data-dependent acquisition was performed for 60 min by using two MS-MS steps after each MS1. Database search with the MS-MS data was performed with Sonar .Polypeptide bands of interest were excised from the silver-stained gels and de-stained with the Invitrogen Silver Quest kit . After de-staining, the bands were washed five times with distilled water and twice with 25 m50 determined with each of these assays were similar, with 0.22±0.05 μM obtained in the MTT assay, 0.26±0.03 μM with trypan blue and 0.31±0.09 μM by Annexin V. Therefore, the 0.6 μM drug concentration used predominantly in the following studies was about 2–3 × IC50 level. This exposure level has been previously demonstrated by us to preferentially induce G1-phase arrest in A2780 cells  for 30 h and determined whether this treatment increased expression of p27. Immunoblotting of crude cell lysates showed that p21 was induced in a dose-dependent manner . Under such conditions, if the monoclonal antibodies recognise p21 and p27 with similar efficiencies, then Coomassie blue staining and immunoblot signals of purified inhibitor proteins should give similar p27 : p21 ratios for the band intensities. The results from duplicate protein loading are presented in β-actin, showed that the half-life of p27 increased from 0.75 to 3 h in response to DAP treatment . Western blot analysis of p27 in eluted fractions no. 19–35 showed that p27 complexes from both control and DAP-treated cell lysates peaked in fractions 26 and 27, between the 158- and 44-kDa gel filtration standards, suggesting that no additional component(s) is associated with p27 complexes after DAP treatment . To confAs Although our data show that DAP-induced p21 is responsible for increasing p27 in CDK2 complex, the functional consequence of this required examination. Therefore, we immunoprecipitated majority of p21- or p27-bound CDK2 complexes with excessive levels of p21 or p27 antibodies and then measured the relative amounts of CDK2 complex to which they were bound in cells exposed to DAP. As expected from In summary, these results demonstrate that p27 levels in the CDK2 complex is regulated by DAP-induced p21, and stabilisation of p27 within the complex inhibits CDK2 activity.Our present study demonstrates that p21 not only inhibits CDK activity directly, but also indirectly through stabilising p27 protein that increases cellular levels of this inhibitor. Moreover, DAP-induced p21 promotes the selective increase of p27 in CDK2 complexes, and not in CDK4 complexes, and this selective increase contributes to the inhibition of CDK2 kinase activity. Thus, our data provide evidence that p27 contributes to DAP-induced G1-phase checkpoint response in a manner similar to that observed with p21. This is the first evidence that p27 regulation is linked directly to activation of the p53/p21 pathway by a DNA-damaging agent.Hyperproliferation of cancer cells is associated with deregulation of cell cycle progression, which is driven by the activities of CDKs. Therefore, inhibition of CDK activity is a realistic goal in the clinical management of cancer . This isRegulation of p27 in human cancers is predominantly through a mechanism involving Skp2-mediated proteosomal degradation of p27 . FurtherUnlike previous reports on the induction of p27 by ionising radiation or antitumour agents (Our study has also brought awareness to the novel observation that p21-dependent increases of p27 are only reflected within the CDK2 complex, and not CDK4 complex. With the data available, it is possible to propose a conceptual model to explain the induction of p27 by DAP. In this model, basal levels of p27, which are regulated independently of p21, are recruited into both CDK4 and CDK2 complexes . HoweverIn conclusion, our data highlight the significance of p21 and p27 expression in DAP-mediated G1-phase cell cycle arrest in order to prevent G1/S transition. More importantly, we have provided evidence that increased association of p27 within the CDK2 complex is mediated through a p21-dependent pathway, which prevents destabilising phosphorylation of p27. We have previously demonstrated the importance of the p53/p21 pathway in cell cycle effects of DAP. Based on our present study, which demonstrates substantial p27 induction and p27-dependent inhibition of CDK2 by DAP, it is appropriate for the p53/p21/p27 axis to assume greater prominence and become a new focus of attention in checkpoint response to DNA damage.


Plasmodium malariae. Rosetting of infected and uninfected erythrocytes, a putative virulence factor in falciparum malaria, was observed in the blood slide. Severe disease caused by P. malariae is extremely rare. The patient made a rapid recovery with intravenous quinine treatment.We describe a 32-year-old Bangladeshi male presenting with severe malaria caused by a mono-infection with  A 32-year-old previously well Bangladeshi male labourer from Fatickchari in Chittagong District, Bangladesh, was admitted to Chittagong Medical College Hospital in July 2008 because of a 10-day history of evening fevers, chills, rigors, headache, anorexia, generalised myalgia, dry cough, palpitations, drowsiness and several generalised convulsions. He complained of abdominal pain and nausea without vomiting or diarrhoea for the preceding 5 days. He was treated initially with paracetamol at his local health complex, which controlled his fever. The day before admission he became disorientated and had generalized convulsions and was referred to Chittagong Medical College Hospital (CMCH) for further management.3.On admission to CMCH, the patient was febrile (temperature 38.9 °C) and disorientated in time and place. His pulse rate was 110 beats per minute, blood pressure 95/55 mmHg, and respiratory rate 28 breaths per minute. Glasgow Coma Score was 14/15 . Physical examination was otherwise unremarkable. Haemoglobin, leukocyte count, blood glucose and blood biochemistry, including serum creatinine, were normal and he had a mild thrombocytopaenia of 93 000 per mmP. malariae with a parasitaemia of 3 per 1000 red cells (14 017/uL), with typical band form trophozoites, schizonts containing 4-6 merozoites, and occasional gametocytes. Rosettes were observed in a frequency of 50 per 100 trophozoite-infected red cells . Polymerase chain reaction (nested PCR based on the 18 s rRNA gene) performed on the same blood sample subsequently confirmed a diagnosis of Plasmodium malariae malaria, whereas the PCRs for P. falciparum, P. vivax, P. ovale and P. knowlesi were negative . The patient received a total of 4 doses of intravenous quinine followed by further oral treatment and made a rapid recovery. He was afebrile after 3 days and discharged himself from hospital.P. malariae malaria has been reported rarely from Bangladesh.P. malariae can be difficult to distinguish from other species by microscopy alone and its prevalence may be underestimated. This is the first PCR-confirmed case of P. malariae infection to be reported from Bangladesh. The patient was remarkable because of his presentation with neurological features (prostration and multiple convulsions) compatible with a diagnosis of severe malaria in accordance with World Health Organization criteria.P. malariae.P. vivax, P. ovale and P. malariae,P. falciparumP. falciparum is thought to be mediated by binding of various erythrocyte surface cell receptors to the surface expressed variant protein PfEMP1. The mechanism in the other three species is unknown. It is highly uncertain whether in P. malariae rosettes significantly obstruct microvascular flow, since parasitaemia is invariably low. Further studies will be needed to establish their resistance to shear forces and the relationship with disease severity in P. malariae. Usual treatment of uncomplicated P. malariae infection is with chloroquine or an artemisinin-based combination therapy.This patient showed rosette formation in the peripheral blood slide, defined as the binding of two or more infected red cells to an uninfected red cell.P. malariae presenting with severe disease and rosetting in the peripheral blood film. The patient made a quick recovery with intravenous quinine.In summary, we report a rare case of a PCR-confirmed monoinfection with All authors contributed to the conception and content of this case report. WR carried out the clinical assessment. RJM took the photographs. NT did the PCR. RJM, WR, KC, KS, NT, AH, MAF and AMD drafted and revised the manuscript. RJM, NT, KC and KS prepared the figures. All authors read and approved the final manuscript.Mahidol-Oxford Tropical Medicine Research Unit is funded by the Wellcome Trust of Great Britain.None declared.Ethical clearance was obtained from the Oxford Tropical Research Ethics Committee (OXTREC) and the Bangladesh Medical Research Council. The patient gave informed written consent.AMD is guarantor of the paper


Investigating severe maternal morbidity (near-miss) is a newly recognised tool that identifies women at highest risk of maternal death and helps allocate resources especially in low income countries. This study aims to i. document the frequency and nature of maternal near-miss at hospital level in Damascus, Capital of Syria, ii. evaluate the level of care at maternal life-saving emergency services by comparatively analysing near-misses and maternal mortalities.Retrospective facility-based review of cases of near-miss and maternal mortality that took place in the years 2006-2007 at Damascus Maternity University Hospital, Syria. Near-miss cases were defined based on disease-specific criteria (Filippi 2005) including: haemorrhage, hypertensive disorders in pregnancy, dystocia, infection and anaemia. Main outcomes included maternal mortality ratio (MMR), maternal near miss ratio (MNMR), mortality indices and proportion of near-miss cases and mortality cases to hospital admissions.There were 28 025 deliveries, 15 maternal deaths and 901 near-miss cases. The study showed a MNMR of 32.9/1000 live births, a MMR of 54.8/100 000 live births and a relatively low mortality index of 1.7%. Hypertensive disorders (52%) and haemorrhage (34%) were the top causes of near-misses. Late pregnancy haemorrhage was the leading cause of maternal mortality (60%) while sepsis had the highest mortality index (7.4%). Most cases (93%) were referred in critical conditions from other facilities; namely traditional birth attendants homes (67%), primary (5%) and secondary (10%) healthcare unites and private practices (11%). 26% of near-miss cases were admitted to Intensive Care Unit (ICU).Near-miss analyses provide valuable information on obstetric care. The study highlights the need to improve antenatal care which would help early identification of high risk pregnancies. It also emphasises the importance of both: developing protocols to prevent/manage post-partum haemorrhage and training health care professionals to manage infrequent but fatal conditions like sepsis. An urgent review of the referral system and the emergency obstetric care in Syria is highly recommended. Since the declaration of the Millennium Development Goals (MDGs) in 2000, reproductive health, the 5th goal of the MDGs, has been recognised as fundamental for human development. National governments and the international community have increasingly embraced language which supports reproductive health. However, re-orienting policies and programmes has been more challenging, particularly in the Middle East and North Africa region (MENA) . DecisioEach year approximately 529 000 women die of complications during pregnancy and delivery; almost all of which (99%) occur in low-resource countries . HoweverNear-miss is a serious adverse event that leads to harm and morbidity in the mother, but from which she survives . Since 'Essentially, three different methods have been used for identifying near-miss cases. These different approaches depend on either a set of clinical criteria defining common diagnostic categories; a set of management-based criteria related to specific interventions or on organ(s)/system(s) failure .Although a considerable body of obstetric near-miss analyses have been recently conducted in developing countries -11, suchIn this retrospective study, we aim to document the frequency and nature of maternal near-misses at a hospital level in Damascus, the Capital of Syria. We also aim to evaluate the level of care at the maternal emergency services in Syria by comparatively analysing maternal near-misses and mortalities. This review is expected to serve as a guiding tool for policy-makers by highlighting the most common and the infrequent but serious maternal morbidities to help direct the expenditure of the modest national health budget towards health care priorities.We conducted a facility-based retrospective study between February and July 2008 for all admissions to Damascus Maternity University Hospital (DMUH), the women teaching hospital for Damascus University, in the years 2006 and 2007. DMUH is one of two maternity referral hospitals in Damascus which serve a region populated by over 3 million inhabitants. This 250-bed hospital has approximately 21,000 admissions yearly, 60% of which are accepted in labour and pregnancy units. This study was approved by Damascus Faculty of Medicine (21 Jan 2007).A near-miss event is 'a woman who nearly died but survived a complication that occurred during pregnancy, childbirth or within 42 days of termination of pregnancy' .et al [The disease-specific criteria set by Filippi et al  were empIn 2009, the World Health Organisation (WHO) published a consensus on maternal near-miss definition and set a criteria for cases identification . These cAll maternal near-misses which occurred before or after arrival at the hospital were included in this study. These were further classified into direct (resulting from complications occurring during or after pregnancy) or indirect (resulting from previously existing disease aggravated by pregnancy). Causes of maternal mortality, as defined by the International Classification of Diseases (ICD-10), were alMaternal deaths and near-miss cases were retrospectively identified by searching the Medical Records Register at DMUH. Each patient's discharge summary includes 'primary' and 'secondary' diagnoses sections. It also has a 'complication' section, where all complications which happened as a result of either the primary or the secondary diagnoses are listed. Discharge summaries also include 'investigations'  and 'interventions'  sections.Using the data documented in the admission/discharge sheets of the hospital, we identified cases where any of Filippi's main disease-specific categories was mentioned . The full notes of these cases were then scrutinised and carefully checked against Filippi's criteria for final inclusion. The data collection was done by 3 investigators using a pre-coded form specially developed for this study. All data was then inserted into a Microsoft Excel spreadsheet. Causes of maternal deaths were generally based on clinical or surgical findings. Data on deliveries and live births were derived from statistics reported by the DMUH's Statistical Department.For each case, we collected data on demographic characteristics including patient's age, parity, previous deliveries, and gestational age at delivery. We also collected data on the nature of the obstetric complication(s) responsible and where it developed , type of delivery, foetal outcomes, any ICU admissions including length of stay, and any special procedure carried out during the care of the woman. Special procedures were defined as propaedeutic or therapeutic interventions not normally used during prenatal, intra-partum, or postnatal care e.g. echocardiography, hysterectomy, transfusion ≥ 4 units, electric cardioversion and iliac arteries ligation.SPSS 13.0 software  was used for descriptive statistical analysis. Results are presented as frequencies, percentages and descriptive statistics. MMRs and MNMRs were calculated using live births as the denominator. In order to evaluate the standard of care provided for each disease category, we calculated the mortality index for each obstetric condition. This was defined as the number of maternal deaths due to a particular obstetric condition divided by the sum of near-miss morbidities and maternal deaths which resulted from this condition, expressed as a percentage .During the 2-year period reviewed, there were 28 025 hospital deliveries, 27 350 live births, 901 near-miss cases and 15 maternal deaths. This resulted in a total MMR of 54.8/100 000 live births. The total MNMR was 32.9/1000 live births and the total mortality index for near-miss cases was 1.67% . Most of the cases  were referred to the hospital with the complication while only 7% developed the complication within the hospital.During the study period, 54% of women with near-miss delivered by caesarean section . Foetal outcomes were 82% liveborn; 11% abortions and 6% stillbirths. Other demographic characteristics of women who sustained near-miss complications and those who died are presented in table 14 (93.3%) of the 15 maternal deaths were due to causes directly related to pregnancy, with severe late pregnancy haemorrhage leading the causes (n = 9), followed by hypertensive disorders  and sepsis (n = 2). One additional death followed uterine rupture and one death was due to an exacerbation of a pre-existing cardiac problem. The mean 'stay in hospital to death' period was 2.6 days (SD = 4.2), table The disease profile for near-miss morbidity differed from that of maternal mortality table . The mosThe majority of the near-miss cases (93%) were referred in critical condition from other facilities namely traditional birth attendant homes (67%), primary (5%) and secondary (10%) healthcare units and private practices (11%) within Damascus city and countryside. This figure varied according to categories . All severe haemorrhage in early pregnancy cases (n = 66) were referrals while 13% (n = 32) of late pregnancy haemorrhages developed in the hospital.Two hundreds and forty five women with near-miss (27%) were admitted to ICU, with a mean stay of 3.5 days  while sepsis had the higher mortality index (7.4%). Most of the cases (93%) had near-miss upon arrival at the hospital. Almost quarter of near-miss cases were admitted to ICU.The MNMR which this study describes (32.9 per 1000 live births) is located within the wide range of ratios reported in studies from other developing countries which used similar criteria for near-miss definition (12.3 - 82.3 per 1000 deliveries) . On the Studying cases of women who nearly died but survived a complication during pregnancy, childbirth or postpartum  is increasingly recognised as a useful means to examine the quality of obstetric care. Nevertheless, routine implementation and wider application of this concept in reviewing clinical care has been limited due to the lack of a standard definition and uniform case-identification criteria.The WHO initiated a process aiming to develop a uniform set of criteria for identifying maternal near-miss cases. According to the new WHO criteria, published in 2009, near-miss cases are identified by dysfunctional system  based on a set of clinical criteria, laboratory markers, or management-based proxies which were strictly defined . It is hIn 2008, when the data collection of our study commenced, the WHO consensus on maternal near-miss definition was not yet published. The clinical criteria method, which we chose to define our near-miss cases, has many advantages including its easy interpretation, retrospective nature, and ability to assess both complication rates and quality of care of a particular disease .Although case identification using admission to ICU is easier; it would have heavily underestimated severe morbidity, as generally <10% of near-miss cases in low-resource settings receive intensive care ,24  which implies effective management of these complications upon arrival at hospital.Of the various types of life-threatening obstetric haemorrhage, postpartum haemorrhage (PPH) constituted the greatest danger to affected women. While accounting for almost tenth of the maternal morbidities, PPH is responsible of 40% of maternal deaths. This suggests that either women had no access to care or poor care was provided by health professional .Another area that needs improvement is management of sepsis which has the highest mortality index (12:1). This, indeed, constitutes a significant threat to the survival of affected patients although it is the least frequent cause of life-threatening obstetric conditions (0.9/1000 live births).One critical defect this study helped expose is the delay in referrals to higher levels of care. Surprisingly, the majority of women with near-miss morbidity (93%) arrived at the hospital in critical condition having been referred from other hospitals, private practices or homes. This fact underscores the significance of pre-hospital barriers even in this setting with its availability of free maternal health care .One possible barrier is related to transporting patients to hospital. A recent study on maternal mortality in Syria showed that in 31% of maternal deaths where deliveries took place at home, women died on the way to the hospital. Besides, in 18% of hospital deliveries, women died in another hospital as they were referred from hospitals which refused to admit them due to incapability of dealing with such cases. Small proportion of women were transported by ambulance (6%), while almost two thirds of them were brought by private cars or taxis .Another obstacle to timely arrival are to be found in 'the first delay' , which iHowever, even if women arrive late, much can be done to save them and their babies, and a review into the care received in the hospital may lead to positive changes in the procedures and resources available for managing these complications . This faIt is important to mention that our study has limitations. Its retrospective nature carries the possibility of underestimating near-miss cases due to poor documentation . Other lNear miss cases share many characteristics with maternal deaths and can directly inform about obstacles that had to be overcome after the onset of an acute complication, hence providing valuable information on obstetric care. This allows for corrective action to be taken on identified problems to reduce related mortality and long-term morbidity. Based on our findings, we recommend a number of actions to avert future maternal deaths: i. improving antenatal care to help early identification of high risk pregnancies including pre-eclampsia, ii. developing protocols to prevent/manage post-partum haemorrhage including raising the awareness about using active management during third stage, iii. training obstetric health professionals on managing infrequent but fatal conditions like sepsis, iv. urgently reviewing the referral system and emergency obstetric care in Syria.The authors declare that they have no competing interests.YA was responsible for the study design, together with AA, MQA and HEM. YA, AA and YS collected the data. MQA and HEM did the analysis and wrote the first draft in consultation with YA. AA revised the manuscript and all authors agreed on the final draft.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/10/65/prepub


Fistulae between an ileal pouch and the vagina are an uncommon complication of ileal pouch-anal anastomosis following proctocolectomy and mucosectomy in patients with familial adenomatous polyposis coli. Several reports describe the successful use of muscle flaps to close recurrent pouch-vaginal-fistulae (PVF). However, series only contain small numbers and an optimal management has not yet been determined. We report the case of a 26-year old woman with a third recurrence of a PVF after proctocolectomy for treatment of familial adenomatous polyposis in October 2005. Because local approaches failed, definitive closure of the fistula was achieved by interposition of a gracilis muscle flap between the pouch-anal anastomosis and the vagina. The postoperative course was uneventful; the patient was discharged 7 days after surgery and remained free of recurrence and symptomatic complaints for 22 months now. The gracilis muscle flap proved to be an effective method in the treatment of recurrent PVF. Pouch vaginal fistulae (PVF) after restaurative proctocolectomy with an ileoanal pouch are a serious complication for the patient and a frustrating and diffficult problem for the surgeon. The most common etiologic factor is anastomotic leakage , which oSymptoms that are attributed to PVF are discharge of flatus and feces through the vagina, recurrent vaginitis, perianal irritation, and incontinence. Many procedures have been proposed for PVF such as transvaginal repair, fistulectomy, diversion, and transabdominal procedures such as omentum flap and pre-anal repair. However, these procedures are associated with high recurrence rates ranging from 29–86% in the current literature [.According to the high recurrence rate of PVF after conventional surgical intervention, the interposition of muscle flaps was advocated by several groups with encouraging results , 4. The However this treatment offers new perspectives for patients with PVF as well, in particular after previous attempts at repair have failed. Due to the low incidence of PVF and little experience with this procedure in most of the centers, there is only a small series of patients. Therefore, clear indications for a gracilis muscle flap and operative management are not yet determined. We present our experience with this procedure.A 26-year-old female otherwise healthy patient with no evidence of Crohn's disease underwent a proctocolectomy with a stapled ileo-anal-pouch anastomosis and protective ileostomy for FAP. The postoperative course was uneventful and there were no signs of an anastomotic leakage. One year later, after closing the protective ileostomy she presented with the first PVF. For fistula-closure a pre-anal-repair and a protective ileal diversion was performed. Intraoperatively the pouch-anal anastomosis was found to be vital, tension-free, and without any abscess cavities or granulation tissue. Six months later the fistula recurred. Again a pre-anal-repair was performed with augmentation by placement of an omentum-flap between vagina and ileal pouch. Four months later the third recurrence was approached by a transvaginal fistulectomy. Histopathologically the specimen consisted of fibrotic granulation tissue. Microbiological analysis revealed only bacteria of the gut flora. An antimicrobial treatment was initiated with ceftriaxone and metronidazole for three days.Eight months later, contrast enema revealed another fistula in the stapled anastomosis area. In order to seal the fistula with a larger amount of healthy tissue and to reconstruct the rectovaginal septum, a different approach with fistulectomy, and interposition of a gracilis muscle flap was chosen.Under general anesthesia the patient was placed in the lithotomy position. After a perineal incision with left lateral extension the PVF was identified. After preparing the fatty tissue the fistula was excised subsequently. The pouch opening and vaginal opening of the fistula was then closed by using absorbable sutures. Now another incision was made at the left medial thigh, the gracilis muscle was identified and secured with a loop . The lefPVF after restorative proctocolectomy represents a rare complication with tremendous impairment of the patient's quality of life; it is also a challenge for the surgeon. These fistulae occur more often in patients with UC 6.3%) than in patients with FAP (1.2%) , which m.3% than


CIP1/WAF1 and p27KIP1. Tax increases p27KIP1 protein stability by activating the anaphase promoting complex/cyclosome (APC/C) precociously, causing degradation of Skp2 and inactivation of SCFSkp2, the E3 ligase that targets p27KIP1. The rate of p21CIP1/WAF1 protein turnover, however, is unaffected by Tax. Rather, the mRNA of p21CIP1/WAF1 is greatly up-regulated. Here we show that Tax increases p21 mRNA expression by transcriptional activation and mRNA stabilization. Transcriptional activation of p21CIP1/WAF1 by Tax occurs in a p53-independent manner and requires two tumor growth factor-β-inducible Sp1 binding sites in the -84 to -60 region of the p21CIP1/WAF1 promoter. Tax binds Sp1 directly, and the CBP/p300-binding activity of Tax is required for p21CIP1/WAF1 trans-activation. Tax also increases the stability of p21CIP1/WAF1 transcript. Several Tax mutants trans-activated the p21 promoter, but were attenuated in stabilizing p21CIP1/WAF1 mRNA, and were less proficient in increasing p21CIP1/WAF1 expression. The possible involvement of Tax-mediated APC/C activation in p21CIP1/WAF1 mRNA stabilization is discussed.HTLV-1 Tax can induce senescence by up-regulating the levels of cyclin-dependent kinase inhibitors p21 Human T cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-associated myelopathy. HTLV-1 encodes a 40 kDa trans-activator, Tax, which plays a crucial role in viral replication and cell transformation . Tax actCIP1/WAF1 (p21) and p27KIP1 (p27) [tax gene and infection with HTLV-1 can cause HeLa cells and SupT1 cells to become senescent or cell cycle arrested with greatly elevated levels of p21 and p27 [Skp2, to be degraded during S phase. The loss of Skp2 leads to the inactivation of SCFSkp2 and the dramatic stabilization of p27 [Skp2 [Previous studies have indicated that Tax causes many eukaryotic cells to develop mitotic abnormalities ,22. We hP1 p27) . More re and p27 . The sha and p27 ,25-27. T . More rn of p27 , a key s27 [Skp2 -30.1/S checkpoint [P21 was initially characterized as an important inhibitor of cyclin/Cdk2 complexes . It becoeckpoint -35. Subseckpoint . As a poeckpoint -35. It ceckpoint . Paradoxeckpoint .A wide variety of mechanisms including transcriptional regulation, mRNA degradation, ubiquitin-dependent or ubiquitin-independent proteolysis, and subcellular localization are known to regulate the level and activity of p21 . At the Transcriptional activation of the p21 promoter alone, however, cannot fully explain the dramatic increase in p21 brought about by Tax. We provide evidence to show that p21 mRNA stability is greatly increased in the presence of Tax. Analyses of newly isolated Tax mutants suggest that the ability of Tax to activate APC/C also correlates with p21 mRNA stabilization. These results reveal a hitherto unknown property of Tax in regulating p21 mRNA turnover.cis elements in the p21 promoter responsible for Tax trans-activation have not been well defined, however. To this end, we obtained a full-length p21 promoter-luciferase reporter plasmid (p21) and a derivative (p21Δ1) that contains a truncated p21 promoter, which begins at 298 nucleotides upstream (-298) and ends at 16 nucleotides downstream (+16) of the p21 mRNA start site. We also constructed two p21 promoter deletions with progressively shortened DNA sequences (100 and 60 bps) upstream of the p21 transcriptional start site. These deletions were inserted upstream of the firefly luciferase gene to produce p21Δ2, and p21Δ3 respectively. Each of these reporter plasmids was transfected into HeLa/18 × 21-EGFP cells together with a Tax expression plasmid, pBC12-Tax. The luciferase reporter activities were then measured 48 h after transfection.Previous studies have shown that HTLV-1 Tax can trans-activate the p21 promoter independently of the tumor suppressor, p53 . The ciscis element responsible for the transcriptional induction by Tax resides between the -100 to -60 region. Interestingly, previous studies have shown that two binding sites for the Sp1 family of transcriptional factors reside within the sequence between -83 and -64, and this region mediates up-regulation of p21 transcription in response to tumor growth factor β (TGF-β) and arsenic compounds [As shown in Fig. ompounds .cis element responsible for Tax induction, we analyzed three mutants: p21P93-S-mut-1, mut-2 and mut-3, each containing ten consecutive base substitutions in the -93 to -84, -83 to -74, and -73 to -64 sequences, with the latter two mutations spanning the Sp1-binding sites Sp1–3 and Sp1–4 respectively. As indicated by reporter assays, base substitutions in the sequence between -93 and -84 had no effect on Tax induction. By contrast, p21P93-S-mut-2 completely lost Tax responsiveness, while p21P93-S-mut-3 was partially impaired . The Sp1 level in the transfected cells, however, was unchanged when compared to that in cells transfected with a control plasmid Fig. , indicatlls Fig. , and HTLlls Fig. , but notlls Fig. .S. cerevisiae, we have isolated and characterized tax mutants whose expression in S. cerevisiae cells did not lead to cell cycle arrest. Many of these mutants, including L235F and A108T, remain capable of trans-activating both the HTLV-LTR-Luc and the NF-κB-Luc reporters, but are attenuated in induction of p21 and p27, and cell cycle arrest/senescence in HeLa cells. These mutants were previously found to be deficient in activating the anaphase promoting complex/cyclosome (APC/C) [Taking advantage of the ability of Tax to cause cell cycle arrest in tax mutants: M22, M47, and V89A that had been shown to be impaired, respectively, in NF-κB activation, LTR trans-activation, and CBP/p300-binding and LTR trans-activation [tax, suggesting that CBP/p300 recruitment is critical for the transcriptional activation of the p21 promoter by Tax. Interestingly, the M47 mutant, which remains capable of CBP/p300 binding but is defective in LTR activation, stimulated p21 promoter to a much greater extent than the wild-type Tax. These results support the notion that by directly interacting with Sp1, Tax may facilitate the recruitment of CBP/p300 to the p21 promoter to activate transcription.To further elucidate the mechanism by which Tax up-regulates p21 expression, we analyzed APC/C-deficient mutants L235F and A108T along with three well-characterized tivation . As showActivation of the p21 promoter alone, however, is not ultimately sufficient to account for the Tax-induced increase in p21 level. Tax mutants, M22, L235F, and A108T trans-activated the p21 promoter to levels comparable to that of the wild-type Tax, yet were attenuated in elevating the p21 protein level Fig.  and 4B. Skp2. As a result, p27, a key substrates of SCFSkp2, becomes greatly stabilized when Tax is expressed, causing HeLa cells to enter into a state of irreversible G1 arrest termed Tax-induced rapid senescence (Tax-IRS) [Skp2 [Skp2. To determine the rate of p21 and p27 protein turnover, HeLa/18 × 21-EGFP cells transduced with LV-Tax or a control lentivirus vector, LV-puro, were treated with cycloheximide to inhibit de novo protein synthesis. The abundance of p21 and p27 in transduced cells was then measured after the cessation of protein translation. As indicated and reported previously [Skp2 and becomes stabilized as a consequence of premature activation of APC/C and inactivation of SCFSkp2 by Tax. By contrast, even though the Tax-induced increase in p21 and p27 correlated with APC/C activation, the up-regulation of p21 by Tax is achieved by a mechanism independent of SCFSkp2- and ubiquitin-mediated protein turnover.We have shown recently that the premature activation of the mitotic E3 ubiquitin ligase, the anaphase promoting complex, by Tax leads to the polyubiqutination and degradation of Skp2, and consequently, inactivation of the G1/S E3 ubiquitin ligase, SCFTax-IRS) . Based oS) [Skp2 , we initeviously , the haltax gene transfer into HeLa/18 × 21-EGFP could be assessed by fluorescence microscopy. Care was taken to ensure that greater than 90% and comparable numbers of reporter cells were transduced with the respective lentivirus vectors  and up-regulated p21 mRNA and protein expression to a much greater extent than the wild-type Tax  near the p21 mRNA start site. This region has been shown previously to be critical for TGF-β-mediated trans-activation of p21. Tax does not alter the intracellular level of Sp1. Rather it binds Sp1 directly. Analyses of Tax mutants further suggest that the interaction between Tax and CBP/p300 is important for p21 promoter trans-activation. Our results are consistent with a model in which Tax may serve as a tether between Sp1 and CBP/p300, and possibly help recruit the latter to the p21 promoter to activate transcription. These results are in agreement with a previous report that Tax can interact with Sp1 to activate c-sis/platelet-derived growth factor-B promoter [In this study, we have shown that two independent mechanisms: trans-activation and mRNA stabilization are responsible for the dramatic up-regulation of p21 by Tax. Our data are in agreement with other studies showing that Tax up-regulates p21 expression ,45-49. Wpromoter . Importapromoter . Our datThe level of p21, like that of p27, is cell cycle-regulated. It increases transiently after cellular exit from mitosis and entry into G1, and declines during G1/S transition. The increase in p21 and p27 after mitotic exit serves to maintain cells in G1 until such time when a new round of DNA replication is warranted. P21 also functions as a scaffold/chaperone for Cdk4-Cdk6/cyclin D assembly, thus helps set the stage for a new round of cell cycle. Since APC/C is intimately involved in regulating mitotic progression and mitotic exit, it is not surprising that the transient increase of p21 and p27 during early G1 is linked to the APC/C activity.Skp2 [Cdh1 targets the degradation of Skp2 and Cks1, thereby rendering SCFSkp2 inactive [P27 is a substrate of the E3 ubiquitin ligase, SCFSkp2 -30, whicinactive ,52. Thisinactive , thus cainactive ).Skp2 [Skp2. Contrary to earlier reports, our present data suggest that p21 is not a substrate of SCFSkp2, and the protein stability of p21, unlike that of p27, is not affected by Tax  of the p21 transcript contains AU-rich elements (AREs) that destabilizes p21 mRNA . RNA-binThe HeLa/18 × 21-EGFP cell line was generated previously by transducing the parental HeLa cell line with a reporter cassette containing the EGFP gene under the control of 18 copies of the Tax-responsive 21-bp repeat element . Cells wThe full-length human p21 promoter construct, WWP-luc, was kindly provided by Dr. Wafik S EL-Deiry. It contains a DNA fragment encompassing 2.4 kb of the sequence upstream of the p21 mRNA start site. The p21Δ1 (REP-p21) construct was a generous gift of Dr. KJ. Zhao's at the NIH. It contains a truncated p21 promoter that begins at 298 nucleotides upstream (-298) and ends at 16 nucleotides downstream (+16) of the p21 mRNA start site. To construct p21Δ2, primers were designed to amplify the sequence between -100 to +16. The primer sequences were: 5'-AGTGCTAGCCGCGGTGGGCCGAGCGCG-3', and 5'-CCGAAGCTTAAGGAAC TGACTTCGGCA-3'. Nhe I and Hind III sites were incorporated at the ends of the PCR product, and the restriction endonucleases-digested DNA fragment was inserted into the REP-Luc construct. The p21Δ3 (-60 to +16) construct was made similarly except the PCR product was digested with Sma I and Hind III, and inserted into the Pvu II and Hind III sites of REP-Luc. Mutants of p21p93S mut1, 2, and 3 were generously provided by Dr. XF Wang.5 cells/well in six-well plates one day before transfection. For each well, 5 μg of a plasmid encoding wild-type or mutant Tax and 0.5 μg of each of the p21 promoter constructs were used. To control for the variability in transfection, 0.5 μg of pRL-TK containing an HSV thymidine kinase (TK) promoter-driven Renilla luciferase reporter cassette (Promega) was included as an internal control. Cells were harvested 48 h after DNA transfection, and the firefly luciferase activity was assayed and normalized against Renilla luciferase activity. Luciferase assays were performed as previously reported [The production of lentivirus vectors (LV-Tax and LV-puro) was as described previously . For lucreported .g for 5 min at 4°C. The cell pellet was washed once with 5 ml of cold PBS and resuspended for 15 minutes in 1 ml of lysis buffer  supplemented with a protease inhibitor cocktail (Roche Diagnostics). The cell lysates were cleared by spinning at 14,000 × g for 5 min at 4°C. The supernatant was pre-absorbed with 50 μl of a 50% slurry solution of immobilized rProtein G-Agarose (Invitrogen) for 1 h at 4°C. Thereupon, 400 μl of the supernatant was mixed with 2 μg of either a monoclonal anti-Tax (4C5) or a rabbit polyclonal anti-Sp1  antibody. The samples were incubated for 1 h at 4°C with constant rotation, and 30 μl of a 50% slurry solution of rProtein G-Agarose was then added to each sample. The reaction mixtures were incubated for 2–16 h at 4°C with rotation. The beads were then gently washed 4–5 times with 500 μl of the lysis buffer. The bound proteins were eluted in SDS sample loading buffer, subjected to electrophoresis in a 10% denaturing polyacrylamide gel, transferred to nitrocellulose membrane, and immunoblotted for Tax and Sp1. Sp1-Tax co-immunoprecipitation from HTLV-1 transformed MT4 T cells, was carried out similarly as above except that 3 × 106 cells were used for each sample, and an HTLV-1-unrelated T cell line, Jurkat, was used as a control.HEK 293 T cells grown to 40–50% confluence in a 100-mm tissue culture dish was transfected with 10 μg of a control plasmid, pcDNA3, or the Tax expression plasmid, pBC12-Tax, using the calcium phosphate method. Forty-eight hours post-transfection, cells were harvested by trypsinization and centrifugation at 14,000 × Approximately 200 ng of purified Sp1 (Promega) was mixed with 30 μl of Ni-NTA agarose beads in absence or presence of purified hexa-histidine-tagged Tax (TaxH6) (~2 μg) in 500 μl of binding buffer containing 20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% Triton-X100, 100 mM imidazole, and 0.5 mg/ml BSA for 2 h at 4°C. The beads were collected by centrifugation and washed 3–4 times with 500 μl of the binding buffer without BSA. The bound proteins were eluted in SDS sample buffer and analyzed by immunoblotting (Sp1) or Coomassie blue staining (Tax).5 HeLa/18 × 21-EGFP cells were seeded into each well of a six-well plate the day before treatment. LV-Tax and LV-puro were added at a titer of 5 × 105 infectious units/well for 48 h. For protein stability measurement, the transduced cells were treated with 100 μg/ml of cycloheximide for different times . The cells were then harvested, dissolved in SDS-sample buffer, and resolved with SDS-PAGE for immunoblottig with p21 and p27 antibodies. The intensities of the bands were determined by the Scion Image software and plotted using the Prism program.Around 1.5 × 10Invitrogen). To minimize genomic DNA contamination, about 20 μg of total cellular RNA was treated with 10 units of RNase-free DNase I at 37°C for 1 h. RNA was precipitated by adding one tenth volume of 3 M sodium acetate (pH 5.2), followed by the addition of 2 volumes of 100% ethanol, and kept frozen at -80°C for 1 h. The precipitated RNA was collected by centrifugation at 16,000 g for 20 min. Reverse transcription was performed using 1 μg total RNA in 10 μl of a reaction buffer from the Superscript First-strand Synthesis system (Invitrogen). For real-time PCR, the MicroAmp optical 96-well plates (Applied Biosystems) were used. One microliter cDNA template was added to each well, followed by the addition of 24 μl of a master mix containing 12.5 μl SYBER green (Applied Biosystems), 6.5 μl water, 2.5 μl (0.5 μM) each of the forward and reverse primer set. The 18S ribosomal RNA was used as an internal control to standardize the RT-PCR. Three replications were performed to ensure reproducibility of the measurements. The primers designed by the Primer Express software and are listed as follows: 18S rRNA forward primer: 5'-CGGCTACCACATCCAAGGAA-3' and 18S rRNA reverse primer: 5'-GCTGGAATTACCGCGGCT-3'; and p21 forward primer: 5'-CCATGTGGACCTGTCACTGT-3' and p21 reverse primer: 5'-TGGTAGAAATCTGTCATGCTGGTC-3'.For p21 mRNA half-life determination, the transduced cells were treated with actinomycin D (2 μg/ml) for different times  and harvested for RNA preparation. Total RNA was isolated using the TRIZOL Reagent kit (The authors declare that they have no competing interests.LZ and C-ZG were responsible for the design of the study and the draft of the manuscript. LZ performed most of experiments. H-JZ performed Tax-Sp1 co-immunoprecipitation. M-HL and Y-LK provided reagents and cell lines, and assisted in interpretation of results. All authors read and approved the final manuscript.


PLoS Medicine Editors discuss what role food aid should play in addressing the global childhood malnutrition crisis.The The Economist made the case that childhood hunger and malnutrition have a far greater impact upon child health than was previously thought  emergency” . The rea


Congenital pouch colon is one of rare congenital anomalies. We report a 3-day-old male child with congenital pouch colon who underwent a window colostomy but died because of overwhelming sepsis. Due to its rarity, many surgeons in our part of the world may not be aware of it, hence increasing the potential to its mismanagement. However, with simple keen observations, we can safely come to its diagnosis. The aim of this report is to bring attention to congenital pouch colon associated with anorectal malformation in our country, with a brief emphasis on an approach to its diagnosis and initial management. Congenital pouch colon is a rare anorectal malformation in which the distal bowel is shortened forming a pouch-like dilatation, the pouch usually terminating in a fistula communicating with the genitourinary tract. There is a wide anatomical variation, ranging from a complete absence of normal colon with the distal ileum opening into the colonic pouch, to the presence of a near complete length of normal proximal colon, with only the rectum or rectosigmoid colon being affected. CPC has been described in very few literatures, mostly from India . Here weA 3-day-old male child, delivered vaginally at term, was brought to our hospital with complaints of failing to pass stool since birth and abdominal distension. There was no history of meconuria. On examination, his weight was 2.5 kg, was dehydrated, lethargic, had gross abdominal distension, and was found to have no anal opening Figure . A nasogCongenital pouch colon (CPC) was first reported by Trusler et al in 1954 . It has CPC is a rare variant of ARM, and it is distinctly different from other ARMs. Preoperative diagnosis of CPC requires a high degree of suspicion, especially to pick up the dilated terminal bowel loop and an air-fluid level that occupies more than half of the diameter of the abdomen on an abdominal radiograph, which is so nearly forgotten in evaluating a child of imperforate anus.There are different options in the management of CPC . It rangEtiology of pouch colon points to an early vascular insult in the form of intra-uterine obliteration of inferior mesenteric artery . ClusterCPC: Congenital pouch colon; ARM: Anorectal malformation.Written informed consent was obtained from the patient's parents for publication of this case report and accompanying image. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.VCS, SK and PP made substantial contributions to concept and design of the article and acquisition of materials. CSA, RK and SA contributed significantly in critical revision and drafting the manuscript. All authors read and approved the final version of the manuscript.


Using a simplified slot machine task, we measured behavioral and neural responses to gambling outcomes. Compared to “full-misses,” near-misses were experienced as less pleasant, but increased desire to play. This effect was restricted to trials where the subject had personal control over arranging their gamble. Near-miss outcomes recruited striatal and insula circuitry that also responded to monetary wins; in addition, near-miss-related activity in the rostral anterior cingulate cortex varied as a function of personal control. Insula activity to near-misses correlated with self-report ratings as well as a questionnaire measure of gambling propensity. These data indicate that near-misses invigorate gambling through the anomalous recruitment of reward circuitry, despite the objective lack of monetary reinforcement on these trials. Gambling is a prevalent and culturally ubiquitous form of entertainment that becomes dysfunctional in a small but significant minority (1%–5%), in whom it resembles a substance addiction in several core respects . The popIn this study, we focus on two common characteristics of gambling games that can be modeled in the laboratory and are known to promote gambling tendencies: the impact of near-misses and the influence of personal control. Near-misses occur when an unsuccessful outcome is proximal to the designated win, such as when a chosen horse finishes in second place or when two cherries are displayed on the slot machine payline. Their significance to the gambler has long been recognized e.g., , to the The second factor that was modeled in our task was personal control, which refers to the gambler's level of involvement in arranging their gamble. On games of pure chance like the lottery, craps, and roulette, gamblers have an equal chance of winning regardless of whether they, or another agent, places the gamble. However, it is repeatedly observed that gamblers have inflated confidence  when given the opportunity to choose their lottery ticket or throw the dice or roulette ball themselves, compared to conditions where the action is performed by another . Craps pThe aims of the present study were threefold: first, to devise a task to elicit near-miss and control phenomena in the laboratory, as measured by self-report ratings administered on a trial-by-trial basis. Second, we explored the neural mechanisms underlying these cognitive distortions, using functional magnetic resonance imaging (fMRI). We were specifically interested in a neural system comprising the ventral striatum and medial frontal cortex, which previous research has robustly implicated in processing unexpected monetary wins , as wellWe developed a gambling task see  resembli39 = 5.09, p < 0.001; see 39 = 2.50, p = 0.017; see In a behavioral experiment in healthy volunteers (n = 40), three sets of self-report ratings were taken on each trial. Immediately after the selection phase, participants were asked “How do you rate your chances of winning?” After the outcome phase, two further ratings were taken: “How pleased are you with the result?” and “How much do you want to continue to play the game?” Each subjects' ratings were z transformed to their own mean and standard deviation for statistical analysis. Personal control over the gamble was associated with significantly higher ratings of “chances of winning,” compared to trials where the computer selected the play icon . Compared to computer-chosen near-misses, participant-chosen near-misses were significantly less pleasant  but significantly more motivating . Compared to participant-chosen full-misses, participant-chosen near-misses were significantly less pleasant  and significantly more motivating .The subjective response to the near-miss outcomes was striking: near-misses were experienced as aversive according to the ratings of “pleased with result,” but simultaneously increased ratings of “continue to play.” Both effects varied as a function of personal control , and subjective ratings were only taken intermittently . An event-related analysis was used to identify neural responses at the receipt of the outcome, with the selection and anticipation phases entered as covariates in the design matrix see . The conFWE-corr = 0.005; 12, 2, −2, Z = 4.25, pFWE-corr = 0.006) and the right anterior insula , despite the lack of objective monetary gain on those trials . When this contrast was confined to near-misses that had passed through the payline and stopped in the next position , these voxels in the rACC were fully significant despite the restricted number of events . An analysis of extracted signal change from these voxels showed greater activity on near-misses compared to full-misses on participant-chosen trials , with a marginally significant effect in the opposite direction on computer-chosen trials . This correlation indicates that subjects who reported greater subjective effects of the near-misses on their ratings of “continue to play” showed a greater BOLD response to near-miss outcomes in the anterior insula/caudolateral orbitofrontal cortex  and a mean near-miss effect for each subject (ratings on available near-miss outcomes minus ratings on available full-miss outcomes). There were no significant voxels within the win mask when the subjective win effect was regressed against win-related brain activity. When the subjective near-miss effect was regressed against near-miss related brain activity, adjacent voxels in the anterior insula (Brodmann Area 13) and caudolateral orbitofrontal cortex (Brodmann Area 47) were the only significant effects at a reduced threshold of p < 0.001 uncorrected for multiple comparisons , there were significant differences between the patterns of neural response to the near-misses and full-misses. Near-misses were associated with significantly greater BOLD signal in the ventral striatum and anterior insula; areas that were also activated by unpredictable monetary wins on the task. This win contrast detected additional responses in the rACC, midbrain, and thalamus, confirming a well-established circuit of areas linked to reinforcement processing , often rThe anterior insula was recruited during both monetary wins and near-miss outcomes, and in addition, the BOLD response to near-misses in this region was associated with two sets of psychological variables. There was a significant positive correlation between insula activity to near-misses and the GRCS, a questionnaire measure of the susceptibility to gambling biases. We also observed a significant positive correlation between insula activity to near-misses and the subjective ratings of the near-misses on “How much do you want to continue to play the game?” In each case, the insula was the only area in the win-related circuit to show these predictive relationships. Thus, the neural response to near-miss outcomes in the anterior insula was associated with both the subjective impact of those events during scanning and a trait-related index of gambling propensity that is significantly elevated in problem gamblers . While tOur insula data are congruent with accumulating evidence for insula involvement in drug craving and extend these findings to gambling as a behavior that can become addictive . FunctioThese findings provide a number of further insights into the psychological basis of the near-miss effect. By one account, the near-miss may be conceptualized as the omission of an expected reward and considered within the context of fMRI studies of reinforcement learning by prediction error e.g., . By thisSeveral aspects of the current data present problems for an account of the near-miss effect solely in terms of reward expectation and omission. First, why should unpleasant reward omission invigorate behavior? It is unclear how this account would explain our finding that near-misses increased self-report ratings of desire to play the game. In many real-world situations , near-miss outcomes are indicative of skill acquisition and, as such, constitute useful signals of imminent success. In these environments, it is advantageous for reinforcement learning algorithms to compute a value function that can assign some value to near-misses, despite the objective absence of reinforcement on these trials e.g., . In manyA second issue is that the reward expectation and omission components were matched across the participant-chosen and computer-chosen conditions, but we saw differences between the near-miss outcomes as a function of personal control, in both the subjective ratings and the rACC response. These effects are more consistent with cognitive formulations of gambling that invoke an “illusion of control,” where the gambler interprets the near-miss as evidence that he has acquired skill at the game. Crucially, this appraisal of skill is most likely to occur when the player has control over their gamble selection. Thus, by interpreting the near-miss event as evidence of skill acquisition, the subject is motivated to continue gambling in order to exploit this (perceived) knowledge. Our finding that the rACC is sensitive to this manipulation is consistent with much that is known about ACC involvement in reward processing and decision-making. Lesion studies in experimental animals have shown a critical role for ACC in deciding how much effort to invest in obtaining a reward  and in uAn alternative, lower-level account of these near-miss phenomena is that they are an effect of perceptual generalization. By virtue of their spatial proximity to the goal state, near-misses may engender some goal-related neural processing. Such an account seems unlikely for two reasons. First, spatial proximity to the payline is equally present across the participant-chosen and computer-chosen conditions, but the impact of the near-misses on ratings of “continue to play” and rACC signal diverged as a function of personal control. Second, both the subjective ratings and the rACC signal were predominantly modulated by near-misses that passed through the payline before stopping; the near-misses that stopped one position short of the payline were much less effective. A perceptual generalization account would predict comparable effects for near-misses either side of the payline.The effects of near-misses and personal control in the present study were observed in healthy volunteers who did not gamble regularly. When nine subjects with moderate gambling involvement (South Oaks Gambling Screen scores 2–5) were excluded from the behavioral analysis (n = 31), the potency of the near-miss and personal control manipulations were unaffected see . HoweverThese data demonstrate that two cognitive distortions associated with gambling behavior can be elicited in a laboratory setting, in healthy subjects who do not gamble with any regularity. Gambling near-misses were associated with significant recruitment of brain win-related circuitry and acted to increase desire to gamble when the subject had personal control over selecting the gamble. These neural responses may be described as anomalous, in the sense that they occur in the absence of objective reinforcement on near-miss trials. In this sense, these findings are congruent with data showing that striatal activity is tied to the subjective utility rather than the objective value of the outcome; for example, the ventral striatal response to monetary wins is also sensitive to framing effects  and indiForty undergraduate volunteers  were recruited through university advertisements that asked “Do you enjoy gambling?” Subjects attended a single test session, where they completed the slot machine task (30 min), the South Oaks Gambling Screen  and the The task was programmed in Microsoft Visual Basic 6, with responses registered on three adjacent keyboard keys. Trial structure and display screen are displayed in selection phase, an anticipation phase, and an outcome phase. The selection phase lasted a fixed 5 s duration, where one shape on the left reel was selected. The anticipation phase lasted a variable duration (2.8–6 s), where the right reel was spun, and decelerated to a standstill. The outcome phase was initiated as the right reel stopped moving: if the right reel stopped on the icon that was selected on the left reel , the subject was awarded £0.50; all other outcomes won nothing. Outcomes were presented in a fully balanced pseudorandom order to ensure a proportionate number of wins over the 60 trials , near-misses  and full-misses . The outcome phase lasted a fixed 4 s duration. At the end of each trial, there was an intertrial interval of variable duration (2–7 s).Each trial consisted of a Two trial types were presented in a pseudorandom order: on 30 trials, the play icon was selected by the participant, and on the other 30 trials, the play icon was selected by the computer. Trial type was indicated by screen background color and a message on the left side of the screen. On participant-chosen trials (white background), the subject chose the play icon using keys 1 and 2 to rotate the reel up and down, and key 3 to select the icon currently displayed in the payline. On computer-chosen trials (black background), the computer selected the icon on the left reel, but the subject was required to confirm the selection by pressing key 3, in order to better equate attentional and motor demands across the two conditions. In both conditions, if selection/confirmation was not completed within the 5 s window, a “Too late” message was displayed and the next trial commenced after the intertrial interval.On each trial, subjective ratings were also acquired using onscreen visual analog scales. After the selection phase, subjects rated “How do you rate your chances of winning?” and after the outcome phase, two further ratings were taken: “How pleased are you with the result?” and “How much do you want to continue to play the game?” Subjects indicated their response on a 21 point scale using keys 1 and 2 to move left and right and key 3 to confirm. No time limit was imposed for the subjective ratings.Subjective ratings for each subject were standardized to a z score, based on the individual's mean and standard deviation for that rating, given the variability in anchoring across subjects. Data were inspected for normality and parametric statistics were applied only where normality was met. Subjective ratings were analyzed using paired t tests (for “chances of winning”) and repeated-measures analysis of variance (for “pleased with outcome” and “continue to play”) with outcome  and control  as factors. The Greenhouse-Geisser correction was applied where sphericity was violated. Two-tailed statistics were thresholded at p < 0.05.Seventeen right-handed subjects with no history of psychiatric or neurological disorder were recruited from advertisements around the University. One subject withdrew due to claustrophobia, and one subject was subsequently excluded from all analysis due to excessive movement , leaving 15 subjects  in the reported analysis. Subjects reported minimal to modest involvement in gambling, indexed by scores on the South Oaks Gambling Screen of 0–3 . Subjects attended a single fMRI session at the Wolfson Brain Imaging Centre in Cambridge, UK. The protocol was approved by the Norfolk & Norwich Research Ethics Committee (COREC 06/Q0101/69) and all volunteers provided written informed consent. Volunteers were reimbursed £20 for participation “with the opportunity to win further money on the task” .Several minor modifications were required for fMRI. First, the auditory feedback was not delivered given the noise of the scanner. Second, more trials were acquired  to enable sufficient power for fMRI analysis. Third, the number of ratings was reduced given the longer task duration: the “pleased with outcome” rating was dropped altogether, and the ratings of “chances of winning” and “continue to play” were acquired, at random, on 1 in 3 trials. Responses were recorded using the first three buttons on a four-button box, resting on the subject's stomach under the dominant hand. Subjects performed 10 practice trials on the task  before entering the fMRI scanner.Scanning was performed on a Siemens TimTrio 3 Tesla magnet using a 32 slice axial oblique sequence, with a repetition time of 2 s . At the start of each run, six dummy scans were discarded to allow for equilibrium effects. Each 60 trial EPI run lasted a maximum of 630 repetitions (21 min), but was terminated early on block completion. A high-resolution T1-weighted three-dimensional magnetization-prepared rapid acquisition gradient-echo sequence (MP-RAGE) structural image was also acquired for use in spatial normalization of the EPI series.fMRI data analysis was performed using SPM5 . Data preprocessing consisted of slice timing correction, within-subject realignment, spatial normalization, and spatial smoothing using a 10 mm Gaussian kernel. Time series were high pass filtered (128 s). Volumes were normalized to the International Consortium for Brain Mapping (ICBM) templates that approximate to (1)Win-related activity: Winning outcomes on participant- and computer- chosen trials minus all nonwinning outcomes on participant- and computer-chosen trials.(2)Near-miss activity: near-miss outcomes on participant- and computer-chosen trials minus full-miss outcomes on participant- and computer-chosen trials. This contrast was restricted to areas showing win-related activity .(3)Near-miss by choice interaction: areas differentially recruited by near-misses compared to full-misses as a factor of participant versus computer control . This contrast was restricted to areas showing win-related activity .(4)Win activity as a function of personal control: winning outcomes on participant-chosen trials minus winning outcomes on computer-chosen trials. This contrast was restricted to areas showing win-related activity .A canonical hemodynamic response function (HRF) was modeled to the onsets of the selection phase, the anticipation phase and the outcome phase on each trial. At the selection onset, two trial types were distinguished: participant-chosen trials and computer-chosen trials. At anticipation and outcome, eight trial types were distinguished, comprising a 2  by 4  factorial design. The design matrix thus comprised 18 (2 + 8 + 8) columns per session. In addition, the movement parameters from realignment were included as covariates of no interest. The HRF was used a covariate in a general linear model, and a parameter estimate was obtained for each voxel, for each event type, reflecting the strength of covariance between the data and the canonical HRF. Contrast images were calculated between parameter estimates from different trial types. The following outcome-related contrasts were computed:http://marsbar.sourceforge.net/) for the purposes of plotting the data.Based on the data from experiment 1, contrasts 2 and 3 were repeated restricted to near-miss outcomes where the play icon passed through the payline. Contrasts 1 and 2 were also calculated for the onset of the anticipation phase. For the selection phase onsets, a single contrast of participant-chosen minus computer-chosen trials was calculated see . Individ


Three cases of type IV congenital pouch colon associated with low anorectal anomaly are reported here. Pouch colon may be a cause of intractable constipation in children operated for low anorectal anomaly. Excellent results can be obtained by exicision of the pouch. The radiological and pathological features of this condition are discussed. Congenital pouch colon (CPC) is a condition associated with anorectal malformations (ARM). Ninety percent of CPC are reported from the Indian subcontinent and predominantly from the northern provinces.–6 Earlie3A 20-day-old female neonate presented with severe diarrhea. She was moderately dehydrated with a distended abdomen and was diagnosed to have diarrhea with an anovestibular fistula and associated UTI. The vestibular opening admitted an 8 Fr dilator. Skiagram of the abdomen showed a dilated rectosigmoid (gas shadow occupying >50 % of the width of the abdomen). After stabilization, barium enema showed dilated rectum and sigmoid with abrupt transition to normal proximal colon ,b. ThereA male neonate was referred with multiple congenital anomalies. Physical examination revealed absent pinna on the right side, tuft of hair on the back, absent anal opening, and abdominal distension. The child was evaluated for possible associated anomalies and diagnosed to have Goldenhar's syndrome. Prone cross-table lateral X-ray was suggestive of low anorectal anomaly and a moderately dilated rectum, and this was attributed to the obstruction. Anoplasty was done. Renal ultrasound was normal. He had persistent constipation without any anal stenosis. A contrast was done in order to rule out pouch colon as he had a moderately dilated rectum in the newborn period. Contrast X-ray was diagnostic of rectal pouch and hence excision of pouch and pull through was done. Post-operatively the constipation resolved. HPE of the pouch revealed mucosal lymphoid aggregates, muscle hypertrophy, and disarray in certain areas with normal ganglion cells.A 1-year-old male child was referred with a mass in the abdomen with constipation and failure to thrive. He had undergone anoplasty for imperforate anus soon after birth at another hospital and was on laxatives for the constipation. Examination revealed normal external genitalia and a loaded colon. Per rectal examination revealed loaded rectum with hard stools. There was no anal stenosis or stricture. Barium enema revealed dilated sigmoid and rectum with abrupt transition to normal colon. Laparotomy revealed a typical type IV pouch involving the sigmoid and rectum. The pouch was supplied by a leash of vessels arising from inferior mesenteric artery. There were no appendices epiploicae, haustrations, and taenia coli, and there was abrupt transition to normal colon proximally . ExcisioCPC is an extremely rare variant of anorectal malformation in which all or part of the colon is replaced by a pouch-like dilatation that communicates distally with the urogenital tract by a large fistula. They are divided into four types.Type I – Normal colon is absent and the ileum opens directly into the colonic pouch.Type II – The ileum opens into a short segment of cecum, which then opens into the pouch.Type III – Presence of a significant length of normal colon between the ileum and the colonic pouch.Type IV – Presence of near normal colon with only the terminal portion of colon (sigmoid and rectum) converted into a pouch.et al. in his article reports only one case of type IV CPC out of a total of 56 cases of pouch colon between 1968 and 1984, and the figure rose to 46 out of a total of 81 cases between 1985 and 1999.[Over the years, there has been a trend of reducing severity of pouch colon, that is, from Type I to Type IV. Pathak eand 1999. CPC occuet al., 3/10 cases of CPC were type IV and found to be associated with colovestibular fistula.[611We treated three cases of type IV CPC  within a period of 2 years. All three cases were associated with low ARM. This supports the literature of increasing incidence of type IV CPC. In a study by Chadha  fistula.61112 desConstipation is commonly associated with low ARM.14 Mega r