Investigation of maternal near-miss is a useful complement to the investigation of maternal mortality with the aim of meeting the United Nations' fifth Millennium Development Goal. The present study was conducted to investigate the frequency of near-miss events, to calculate the mortality index for each event and to compare the socio-demographic and obstetrical data  of the near-miss cases with maternal deaths.Near-miss cases and events , maternal deaths and their causes were retrospectively reviewed and the mortality index for each event was calculated in Kassala Hospital, eastern Sudan over a 2-year period, from January 2008 to December 2010. Disease-specific criteria were applied for these events.There were 9578 deliveries, 205 near-miss cases, 228 near-miss events and 40 maternal deaths. Maternal near-miss and maternal mortality ratio were 22.1/1000 live births and 432/100 000 live births, respectively. Hemorrhage accounted for the most common event (40.8%), followed by infection (21.5%), hypertensive disorders (18.0%), anemia (11.8%) and dystocia (7.9%). The mortality index were 22.2%, 10.0%, 10.0%, 8.8% and 2.4% for infection, dystocia, anemia, hemorrhage and hypertensive disorders, respectively.There is a high frequency of maternal morbidity and mortality at the level of this facility. Therefore maternal health policy needs to be concerned not only with averting the loss of life, but also with preventing or ameliorating maternal-near miss events  at all care levels including primary level. Maternal near-miss cases of women who nearly died but survived a complication during pregnancy, childbirth or postpartum  are increasingly recognized as useful means to examine quality of obstetric care since pregnancy complications occur in 15% of women worldwide. The practical implementation of maternal near miss concept should provide an important contribution to improving quality of obstetric care to reduce maternal deaths and improve maternal health . More thAlthough a high maternal mortality ratio (644/100 000 live birth), with septicemia and hemorrhage as the main direct causes, was previously reported in Kassala hospital in eastern Sudan , there aMedical files of pregnant women and who delivered recently at Kassala Maternity Hospital from January 2008 to December 2010 were retrospectively retrieved. Kassala is located in eastern Sudan, 600 km from Khartoum; it is 42,282 square km, with a population of 1.8 million people. Of these, 440,491 women are of reproductive age. Kassala Hospital provides tertiary care for women who receive antenatal care at the hospital, as well as for referrals from other clinics and hospitals, and for women who live close to the hospital facility. All women with risk factors or obstetric complications are referred to the hospital. However, the referral criteria are not strictly adhered to and many patients without any significant complications deliver at the hospital.Maternal near-miss cases were defined as women with at least one near-miss event as follows: acute obstetric complications that immediately threaten a woman's survival but do not result in her death, either by chance or because of hospital care she receives during pregnancy, labor or within 6 weeks after termination of pregnancy or delivery . For theFor each obstetric condition, we calculated the mortality index to determine the standard of care provided for each disease process. The mortality index was expressed as a percentage and calculated as the number of maternal deaths resulting from a particular obstetric condition divided by the sum of the near-miss morbidities and maternal deaths resulting from such obstetric conditions. These near-miss cases were retrospectively retrieved among all pregnant and recently delivered women with pregnancy-related complications and who were admitted to the hospital. The data of cases and maternal deaths were gathered applying the above-mentioned criteria for definition of near-miss cases, and maternal death was defined as the death of a woman while pregnant or within 42 days of termination of pregnancy, irrespective of the duration and site of the pregnancy, from any cause. For each case, information on socio-demographic characteristics, parity, gestational age at the time of the near-miss morbidity, nature of the obstetric complication(s), presence of organ and/or system dysfunction, duration of hospital stay, and source of referral were collected. Socio-demographic data, parity and gestational age were compared between the near-miss cases and maternal deaths.2 test were used to compare means and proportions, respectively. P < 0.05 was considered significant.Data were entered in a computer database using SPSS  for Windows and were double-checked before analysis. The Student's t-test and xThe research approved and received ethical clearance from the Health Research Committee in the ministry of health at Kassala State, eastern Sudan.During the study period, there were 9578 deliveries, 9262 live births, 205 near-miss cases and 40 maternal deaths. A total of 228 near-miss events were identified among the near-miss cases, which implies that 23 women had more than one event, yielding a mean of 1.1 near-miss morbidities per case. This is resulted in a total maternal near-miss and maternal mortality ratio of 22.1/1000 live births and 432/100 000 live births, respectively. The total mortality index for near-miss cases was 19.5% . Hemorrhage accounted for the most common event (40.8%), followed by infection (21.5%), hypertensive disorders (18.0%), anemia (11.8%) and dystocia (7.9%), table There were no cases reported of organ or system dysfunction/failure. The duration of hospital stay ranged from 3 to 15 days. Most of the cases (42.9%) were referred from surrounding rural hospitals.During the period of the study there were 316 (33/1000 birth) and 86 (9/1000 live birth) stillbirths and neonatal deaths, respectively. Likewise, 23.7% and 5.9% stillbirths and early neonatal deaths respectively were observed among 152 women of the near-miss cases that associated with labor.Various causes of maternal deaths were identified, table P < 0.001) in near-miss cases was significantly higher than that in maternal death cases, there was no significant difference in age, parity, educational levels, residence and antenatal care coverage in the near-miss cases compared with maternal deaths, table While gestational age  is 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) . It mighIn the current study, there is a high total mortality index for near-miss cases 19.5%), which mean that for every maternal death there are 5 near miss-cases. This reflects a poor care and unacceptable high maternal mortality in this setting. Septicemia and hemorrhage were the major causes of death and maternal morbidity in this study. A high maternal mortality ratio , with septicemia and hemorrhage as the main direct causes, has been previously reported in the same hospital [.5%, whicIn this setting, the health care providers were faced with a high percentage of life-threatening obstetric situations. Complications resulted in near-miss and maternal deaths with septicemia, dystocia and anemia, with a higher mortality index, which constitute an important and significant threat to the survival of pregnant women. Despite the high morbidity from hemorrhage and hypertensive disorders  their mortality index was lower than that of the other events. This could be due to the availability of blood bank services and the introduction of magnesium sulfate as a preventive measure for these events. An increased level of care and effort are required to deal with near-miss events with a high mortality index, e.g., infection, dystocia and maternal anemia.In the current study, lack of antenatal care services, illiteracy and rural residence might be predictors for maternal mortality since the majority of the near miss cases were illiterate, had no antenatal care and of rural residence.One of the limitations of this study is weakness of retrospective method of data collection with respect to the quality of those records, the availability of all records, the coding of diagnosing and the correctness of diagnosis. For future studies, prospective collection of data using clear definition of near-miss and including audit of the care given is advised. By doing so and introducing of the audit cycle, the quality of the maternal care might be improved.There is a high frequency of maternal morbidity and mortality at the level of this facility. Therefore maternal health policy needs to be concerned not only with averting the loss of life, but also with preventing or ameliorating maternal-near miss events  at all care levels including primary level.The authors declare that they have no competing interests.AAA, AKH and AO carried out the study and participated in the statistical analysis and procedures. GKA and IA coordinated and participated in the study design, statistical analysis and the drafting of the manuscript. All the authors read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/11/48/prepub


Liver function tests (LFTs) can be affected by many factors and the proposed effects of coffee on LFT require a comprehensive evaluation. The aim of this study was to elucidate whether drinking coffee, smoking, or drinking alcohol have independent effects on LFTs in Korean health-check examinees.We used the responses of 500 health-check examinees, who had participated in a self-administered questionnaire survey about coffee, alcohol drinking, and smoking habits.Coffee consumption was closely related to male gender, high body mass index (BMI), alcohol drinking, and smoking. On univariable and multivariable analyses, drinking coffee lowered serum levels of total protein, albumin, and aspartate aminotransferases (AST). On multivariable analyses, smoking raised serum γ-glutamyl transferase (GGT) level and decreased serum protein and albumin levels, while alcohol drinking raised GGT level after adjustment for age, gender, regular medication, BMI, coffee and alcohol drinking amounts, and smoking.Coffee consumption, smoking, and alcohol drinking affect the individual components of LFT in different ways, and the above 3 habits each have an impact on LFTs. Therefore, their effects on LFTs should be carefully interpreted, and further study on the mechanism of the effects is warranted. Liver function tests (LFT) are useful tools in clinical practice to assess potential liver diseases, to monitor treatment responses, and to predict prognosis of the patients with liver diseases. As a battery, LFTs consists most commonly of serum total cholesterol (TC), total protein, albumin, alkaline phosphatase (ALP), total bilirubin (TB), aspartate amino transferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transferase (GGT). However, the interpretation of LFTs should be comprehensive and careful because LFTs can be influenced by many personal and environmental factors, including age, gender , body math highest coffee consuming country in the world, consuming 65 thousand tons of coffee a year. Considering the large amounts of consumed coffee globally and its lifetime consumption in most individuals, the effects of coffee on health merit great attention. Interestingly, coffee has been suggested to have a potential favorable impact on liver diseases. In North America and Europe, studies have shown that coffee drinking reduces the risk of liver cirrhosis and hepatocellular carcinoma (HCC) [Although the influence of alcohol drinking on the liver function has been extensively studied, studies on the effects of coffee drinking or cigarette smoking have been relatively limited. According to the international coffee organization, people drink approximately 2.25 billion cups of coffee everyday across the globe, and Korea is the 11ma (HCC) . Moreovema (HCC) .Recently, it has been suggested that smoking increases GGT and boosts the alcohol-induced GGT elevations . AlthougNevertheless, most previous studies deal with the separate effects of alcohol, coffee, or smoking on one or a few components of LFTs, and there has been a lack of data on the comprehensive effects of alcohol, smoking and coffee drinking on the commonly used all components of LFTs. Moreover, those studies have been reported primarily from Western countries and Japan, while the study of the effects of these behaviors on LFTs for persons living in Asian countries have had limited study. Thus, this study was conducted in a Korean population to evaluate the effects of coffee consumption, smoking, and alcohol drinking on the most commonly used sets of LFTs in order to consider the interactive association with major demographic factors.In this cross-sectional study, all health-check examinees were consecutively enrolled in the health promotion center of Seoul National University Bundang Hospital from August 2007 to October 2007. Of 909 examinees during this study period, 500 (55%) subjects agreed to participate to a comprehensive self-administered questionnaire survey about smoking, alcohol consumption, and coffee drinking habits. Moreover, they were requested to answer the questions about their current diseases undergoing medical care, their past histories of hypertension, of diabetes mellitus, of hypercholesterolemia, or of any other unspecified diseases and regular medications, in order to discern the effects of regular medication on serum LFTs. This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital and all participants provided written informed consent prior to this study.Detailed information on each subject’s demographics, medical history, physical findings, and laboratory results were obtained from medical records. The information on coffee and alcohol consumption, and cigarette smoking habits was collected from the self-reported questionnaire survey , moderate , and heavy lifetime drinkers , according to their lifetime drinking amounts which were calculated based on the number of years that a person had been drinking and the daily amounts of alcohol consumption.Alcohol drinking status was classified as none, past, or current drinker and information on the detailed alcohol drinking duration and daily amount of consumed alcoholic beverages were obtained with similar way to those of coffee consumption. Since an alcoholic consumption of more than 60 g/day in men (20 g/day in women) over more than 10 years has been documented as a major risk factor for alcoholic liver disease , we cateSmoking histories were classified in a similar manner as above, participants were asked about their mean cigarette amounts (packs) smoked per day over the previous 6 months and the number of years having smoked. Then, participants were classified into none, mild (<0.5 pack/day), moderate (0.5-1 pack/day), and heavy daily smokers (> 1 packs/day) and into never, mild (<10 pack-years), moderate (10–20 pack-years), and heavy (≥20 pack-years) lifetime smokers.The height and weight of each participant were measured in order to calculate BMI. Biochemical assays for serum TC, total protein, albumin, ALP, TB, AST, ALT and GGT were performed in all subjects. The serologic status of chronic viral hepatitis was evaluated by assays for hepatitis B surface antigen (HBsAg)/antibody (HBsAb) and anti-hepatitis C virus antibody (anti-HCV). All assays were performed at the laboratory of Seoul National University Bundang Hospital, which complied with the International Federation of Clinical Chemistry and Laboratory Medicine. We performed abdominal sonography in all subjects, which was included in the routine health-check program of our hospital.2 or tests for trend using linear-by linear association for categorized variables.The LFTs were recorded as continuous variables, but serum GGT level was transformed to a natural logarithmic scale to normalize its non-standard distribution. To compare baseline characteristics of subgroups, univariable analyses were performed using Student T tests or analysis of variance (ANOVA) for continuous variables and χEstimated means and 95% confidence intervals(CI) for each LFT result were calculated from multivariable linear regression models after adjustment for age, gender, BMI, regular medication for hypertension/diabetes mellitus/hypercholesterolemia, daily alcohol drinking  < 60 g or female (F) < 20 g, M ≥ 60 g or F ≥ 20 g), and smoking  histories to determine the association between coffee drinking and each LFT test result. To estimate independent effects of alcohol or smoking on LFTs, the same multivariable analyses were performed after adjustment for daily coffee consumption amounts , and smoking or alcohol drinking amounts, separately.P-value of <0.05 was considered statistically significant on all analyses.All statistical analyses were performed using SPSS 17.0  software. A 2 and 144 (28.8%) were obese (BMI ≥ 25 kg/m2) [The mean age of all participants (n=500) was 46.5 years. Mean BMI was 23.5 kg/mA total of 443 (88.6%) participants were coffee drinkers at the time of the study and only 38 (7.6%) were never-coffee drinkers Table , and morThe results of univariable analyses on the association between daily amount of coffee consumption, smoking, or alcohol drinking and each individual test of LFT are summarized in Table Among the tests, serum total protein, albumin and AST levels were significantly affected by coffee consumption according to univariable analysis. Then, we performed multivariable analyses on the association between the coffee consumption and LFT changes. The results are summarized in Table Of 500 participants, 119 (24.2%) were current smokers Table .In the multivariable models adjusted for age, gender, BMI, regular medication, daily amounts of alcohol and coffee drinking, current smokers had significantly lower serum levels of total protein, and albumin, but higher GGT levels compared with never or past smokers Table . In addiOf our study population, 173 34.6%) did not drink alcoholic beverages, 19 (3.8%) had quit drinking, and 308 (61.6%) were current alcohol drinkers , but not with all of these common lifestyle habits were reported.We demonstrated that total protein and albumin levels were decreased by both coffee drinking and smoking. The decrease of serum protein and albumin levels according to increase of daily coffee consumption amount was also confirmed in subjects who had never drunken alcohol nor smoked in the present study (data not shown). Although most previous studies had not mentioned total protein or albumin levels, a study documented that current or past coffee consumption and smoking lower serum albumin, globulin, and all other protein fractions . MoreovePrevious epidemiological studies suggested a hepatoprotective effect of coffee drinking on liver function . FurtherThe present study is the first to describe a detailed effect of smoking on the individual test of LFTs. Previous studies showed that current smokers revealed high GGT compared to non-smokers  and peopIt was interesting to observe the strong correlations between coffee consumption, alcohol drinking, and smoking habits in our study subjects. In addition, these correlations have been documented in most prior studies  suggestiIn the present study, we carried out a survey on health-check examinee volunteers who were eager to be evaluated about their lifestyle and the effects on their health status, and would provide almost complete responses about alcohol drinking, smoking and coffee consumption; this provided the opportunity to perform a comprehensive analysis of the relationship of these behaviors with LFTs. The strengths of our study included an intensive data collection, and we distinguished participants who had quitted coffee, alcohol and smoking (past users) from those with no history and current users, which made it possible to gain information about lifetime consumption amounts. An additional strength was that we presented independent effects of coffee consumption, smoking, and alcohol drinking on the most commonly used comprehensive items of LFTs, adjusted by extensive confounding factors that included age, gender, BMI, and regular medications. Nevertheless, the lack of evidence of the causal-relationship between lifestyle and LFT changes remained a limitation of our study due to its cross-sectional design. In addition, some heavy alcohol drinker or smoker might be excluded from the present study since 45% of eligible patients refused to respond to the questionnaire. Moreover, coffee-consumption patterns, such as high frequency of instant coffee consumption in Korea, might be a barrier to generalize our results. As we mentioned above, 71% of participants usually consumed instant coffee (57.8%, instant coffee only; 13.2%, instant and brewed coffee, Table High coffee consumption and heavy smoking were both associated with low total protein and albumin levels. High coffee consumption lowered serum AST levels, heavy smoking raised GGT levels, and heavy alcohol drinking raised GGT levels, independently. Because smoking, coffee and alcohol drinking habits showed strong interactions among each other, the association of those habits and LFTs should be carefully analyzed and interpreted. Further studies on the mechanisms of these associations are warranted.LFT: Liver function test; TC: Total cholesterol; ALP: Alkaline phosphatase; TB: Total bilirubin; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; GGT: γ-glutamyl transferase; BMI: Body mass index; HCC: Hepatocellular carcinoma; HBsAg: Hepatitis B virus surface antigen; HBsAb: Hepatitis B virus surface antibody; anti-HCV: Anti-hepatitis C virus antibody; ANOVA: Analysis of variance; CI: Confidence interval.The authors have no conflict of interest to declare.ESJ carried out the statistical analysis and drafted the manuscript. S-HJ participated in the study design and coordination and helped to draft the manuscript. SHH and HYK contributed the acquisition of clinical data and the recruitment of subjects. SYA and JL helped the statistical analysis. S-HL, YSP, JHH, J-WK, NK, and DHL contributed the acquisition of data and revised manuscript critically for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/12/145/prepubPersonal habits questionnaire form about coffee, alcohol drinking and smoking.Click here for file


Intraguild predation (IGP) is common in nature, but its ecological role is still illusive. A number of studies have investigated a three species IGP module that consists of an intraguild predator, intraguild prey, and resource species in which the intraguild predator and the intraguild prey exploitatively compete for the resource while the intraguild predator also consumes the intraguild prey. A common prediction of models of the IGP module is that the coexistence of the species is difficult, which is considered inconsistent to the ubiquity of IGP in nature. This study revisits the IGP module and provides an alternative coexistence mechanism by focusing on a commonly used analysis method  in light of individual variation in adaptive behavior.Invasion analysis underestimates the possibility of coexistence regardless of the presence or absence of adaptive behavior. Coexistence is possible even when invasion analysis predicts otherwise. The underestimation by invasion analysis is pronounced when the intraguild predator forages adaptively, which is even further pronounced when the expression of foraging behavior is variable among intraguild predators.The possibility of coexistence in the IGP module is greater than previously thought, which may have been partly due to how models were analyzed. Inconsistent conclusions may result from the same model depending on how the model is analyzed. Individual variation in adaptive behavior can be an important factor promoting the coexistence of species in IGP modules. Intraguild predation (IGP) is where predation occurs among predators of the same guild and is one of the most frequently observed species interactions ,2. PredaOrganisms exhibit a variety of behavioral expressions that are thought to enhance their fitness. The importance of such adaptive behaviors has been recognized in a variety of ecological food web modules including ones with IGP ,13-16. FMechanisms of coexistence in food web modules (including ones with IGP) are commonly studied using invasion analysis  e.g. ,18. In aTo investigate the coexistence of the species in the IGP module, this study considers two factors: analysis methods and individual variation in adaptive behavior. We show that coexistence is possible even when mutual invasibility does not hold. Subsequently, we show that when adaptive behavior and individual variation are considered, invasion analysis further underestimates the likelihood of coexistence; individual variation in adaptive behavior can substantially enhance the coexistence of the IGpredator and IGprey.P, IGprey density N, and resource density R, are described byWe follow Křivan and Diehl  who consr is the intrinsic growth rate of the resource, K is the carrying capacity, λij is the encounter rate of species j for species ieij is the efficiency of converting energy of species i for species jhij is the handling time of species j for species i, and mi is the density-independent mortality rate of species i. uiP is the probability that the IGpredator attacks species i upon an encounter. The fixed behavior model assumes that uNP = 1 and uRP = 1 .where eNP/hNP > eRP/hRP), the IGpredator always attacks the IGprey  when it encounters an IGprey. The IGpredator also always attacks the resource upon an encounter  when the the IGprey density is below a threshold density NT = eRP/(λNPhNPhRP(eNP/hNPeRP/hRP)) but always ignores the resource  otherwise. Similarly, when the resource is more profitable than the IGprey (eRP/hRP > eNP/hNP), the IGpredator always attacks the resource  and also always attacks the IGprey  only when the resource density is below a threshold density RT = eNP/(λRPhRPhNP(eRP/hRPeNP/hNP)) and entirely reject the IGprey  when the resource density is above the threshold density (R >  RT).Křivan and Diehl  considerWe consider a model which incorporates individual variation in the prey choice behavior where IGpredators have variable perceptions about the densities of the interacting species. In our model, individual IGpredators do not show partial preference ,25,26. SuRP and uNP) depends on the density of the more profitable prey of the two, and thus perceptual variation in the density leads to variable behavioral expressions among IGpredators [αβ) to describe their distribution. By specifying the mean μ and variance σ2 of the distribution, α and β can be described as α =  μ2/σ2 and β =  σ2/μ. We assume that the mean is the true density (e.g. μ =  N if eNP/hNP > eRP/hRP). We also assume that the perceptional variance is the same as the mean (σ2 = μ). Then the dynamics of the IGP module with individual variation can be described by,The optimal behavioral expression  is the gamma distribution discussed above. In other words, qRN is the proportion of IGpredators that perceives the density of the IGprey is less than the threshold density NT. Because individual IGpredators either perceive that the density of the IGprey is greater than the threshold or not , the proportion of IGpredators that perceive that the density of the IGprey is above the threshold (qN) is 1 − qRN. Similarly, when the resource is more profitable (which leads to qN = 0), and the proportion of IGpredators that perceives that the density of the resource is less than the threshold density, RT is,where qR = 1 − qRN.followed by r = 0.3, λRN = 0.037, λRP = 0.025, λNP = 0.025, hRN = 3, hRP = 4, hNP = 4, eRN = 0.6, eRP = 0.36, eNP = 0.6, mN = 0.03, mP = 0.0275.The effect of adaptive behavior and individual variation on coexistence is examined using invasion and stability analyses supplemented with numerical simulations. The parameter values used in the analyses follows a previous study for comparison : r = 0.3In this invasion analysis, we examine whether the IGpredator can invade communities that consist of the IGprey and resource, and whether the IGprey can invade communities that consist of the IGpredator and resource. When both the IGpredator and IGprey can invade each other , coexistence is implied.Suppose when resident communities are at equilibrium, the invasion conditions for the IGprey and IGpredator, respectively, are,where RP and τRN are the periodicity of the cycle for the resource-IGpredator and the resource-IGprey resident communities, assuming resident populations are on the trajectory of the limit cycles.where τThe inclusion of individual variation does not affect results of the invasion analysis. The possibility of invasion of the IGprey is not affected by individual variation because the perceptual variance is the same as the true density . The possibility of invasion of the IGpredator is also unaffected by individual variation if we assume that some invading individuals exhibit optimal behavior.In invasion analysis, when the mutual invasibility condition is not met, coexistence is considered impossible . HoweverK is further extended in the current analysis , there is little difference between the fixed behavior model and the adaptive behavior model. When the IGprey is less profitable (eNP = 0.25), the possibility of coexistence is extended in the adaptive behavior model, though by a small margin [eNP = 0.25.The results of invasion analysis are the same as those shown in Křivan and Diehl  except ts Figure . When thl margin . CoexisteNP = 0.25.Even when invasion analysis predicts that the IGpredator will be excluded Figure , stabilieNP = 0.25, invasion analysis predicts that coexistence is impossible at high productivities  have small domains of attraction, the difference between the analysis methods may not be ecologically significant because the community dynamics can easily move away from the attractors due to inherent ecological stochasticity. In our model, internal attractors usually have wide domains of attraction Figure . When thAlthough IGP is common in nature, models often predict that it is unlikely. This study shows that this discrepancy can be partly due to how models are analyzed. Invasion analysis, though commonly used, may predict that coexistence is impossible when it may be readily possible through locally stable attractors (e.g. stable equilibrium). In fact, locally stable attractors exist in wide parameter regions when individual variation in adaptive behavior is considered. These results suggest that both individual variation and analysis methods are important for examining the dynamics of the IGP module.The result about the analysis methods  is valid no matter which model is considered. In other words, neither adaptive behavior nor individual variation is necessary. For example, the species can coexist even at very high productivity levels without an additional ecological factor . However, the coexistence possibility further expands with inclusions of adaptive behavior and individual variation. This difference between invasibility and coexistence exists due to nonlinear interactions . The nonlinear functions allow the IGP module to have alternative equilibria  ,7,30. WhOne may argue that invasion analysis is a more conservative way to study coexistence. For example, even if an internal attractor exists , a community may not persist if it experiences a strong perturbation. However, the domain of attraction is not negligible Figure . When coThe interpretation of the effect of adaptive foraging in the IGP module is not straightforward. Here we make two comparisons. One comparison is between the fixed behavior model and the adaptive behavior model without individual variation. This comparison shows that the inclusion of optimal foraging enhances the possibility of coexistence Figures  and 3. TAbrams and Fung  studied Individual variation in adaptive behavior can substantially enhance coexistence in the IGP module, and model predictions can be quite different depending on how they are analyzed. The findings reduce the mismatch between the ubiquity of IGP modules in nature and modeled coexistence. Although the inadequacy of invasion analysis has been known for a long time, we do not know how common it may be (e.g. in other food web modules) and suspect that such deviations may be more common especially when adaptive behavior and/or individual variation are considered.The authors declare that they have no competing interests.WSH and TO analyzed the models and wrote the manuscript. Both authors read and approved the final manuscript.


The indigenous gut microbiota are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease.We monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in three of the four individuals was quite distinct. In the fourth patient, a community that was much like that seen in a healthy colon was established, and this patient also had the most benign clinical course of the four patients, without the development of pouchitis 2 years after IPAA.The microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by the intact colon, the precise structure and function of the pouch microbiome, relative to a normal colonic microbiota, will determine if there is establishment of a stable, healthy mucosal environment or the reinitiation of the pathogenic cascade that results in intestinal inflammation. The inflammatory bowel diseases (IBD) are a heterogeneous group of chronic, relapsing ailments of unknown origin that afflict over a million people in the US. There are two major forms of IBD. Crohn’s disease is an inflammatory disorder that can involve any part of the gastrointestinal tract, but more often involves the ileo-cecal region. Ulcerative colitis (UC), on the other hand, only involves the colon and, almost without exception, extends from the rectum proximally in a continuous manner. The singular localization of these diseases suggests that topical or regional factors are important in their development. In this regard, indigenous enteric microbes may play an important role, particularly as they have been strongly implicated in the etiopathogenesis of inflammatory bowel diseases -4.The search for typical bacterial pathogens for the etiology of IBD has not been particularly fruitful. More recently, the concept that specific communities of microbes play a key role in the pathogenesis of IBD has been aided by the use of culture-independent surveys of microbial community structure . A growiPouchitis is an inflammatory condition of the surgically created pseudorectum reservoir that develops within 1 year in over half of UC patients who undergo total colectomy with ileal pouch anal anastomosis (IPAA) ,10. The In this study, we monitored the development of the pouch microbiota in a group of UC patients who underwent colectomy with IPAA. Starting from a time just prior to the takedown of the diverting ileostomy, we noted the transition of the pouch microbial community between multiple structural states, many of which are still distinct from the structural and functional characteristics of a healthy colonic microbiota. The driver for these changes appears to be the restoration of the flow of intestinal contents to the pouch and of the conversion of the distal ileum to a reservoir where fecal stasis occurs. It remains to be determined if the ultimate maturation of the pouch microbiome to one that resembles that seen in the healthy colon prevents the eventual development of pouchitis.In this study, four patients Table  with a hThe study design was as follows. The date of ileostomy takedown was considered as the reference point for comparison of subsequent samples. One to 2 weeks prior to takedown of the patient’s loop ileostomy, a pouchoscopy was performed . Cytology brushes were advanced through the endoscope, and mucosal brushings were obtained with the intent to cover a large surface area with each brush. The cytology brush was then covered in a sterile sheath prior to being removed from the endoscope. Patient 200 was on 5 mg of prednisone for 1 week following ostomy takedown. Patient 210 was on a low dose of prednisone following ostomy takedown (5 mg daily), which was slowly tapered off by visit 4. The other two patients were on no medications during the study visits.Biopsy and brush samples were collected during the endoscopy, stored accordingly depending upon the downstream workflow, placed on dry ice at the time of collection (apart from those targeted for histology) and archived at −80°C thereafter. Storage conditions according to workflow were as follows: biopsies targeted for bulk DNA extraction were placed in Fecal Dry Bead Tubes , and brushes targeted for microbial cultivation were stored in cryovials containing 1× anaerobic GBSS buffer  supplemented with DMSO . Bulk DNA was extracted from biopsies using a modified protocol for the Roche MagNA Pure System incorporating mechanical disruption (bead beating) as described previously .Replicate amplicon libraries for bacterial 16S v3 through v5 regions (Bv3v5) were constructed from all samples. The adapter and 16S rRNA-encoding gene-specific sequences are shown in Table Both primer sets were designed to capture over 95% of known eubacterial diversity. They match 16S genes from 50 to 100% of the members of all known phyla in our reference database based on the SILVA 106 release  with the4, 200 uM dNTPs  and 50 nM combined primers. Between 5–25 ng of sample DNA was added to a master mix to a final volume of 100 ul, and this was divided into three replicate 33-ul reactions. Cycling conditions included an initial denaturation at 94°C for 3 min; 30 cycles of 94°C for 30 s; 60°C for 45 s, 72°C for 1 min; and a final extension at 72°C for 2 min using an Applied Biosystems 2720 or 9700 cycler. The three replicates were pooled and analyzed on a Bioanalyzer DNA1000 chip. Reactions were cleaned and products under 300 base pairs removed using Ampure beads at 0.75× volume (Agilent Technologies). The final products were resuspended in 100 ul of 10 mM Tris-EDTA, quantitated using PicoGreen Quant-IT assay (Invitrogen/LifeTechnologies) and assayed again on a DNA1000 chip.The individual oligos were mixed in equal proportions to create F/R primer pools. 16S rRNA-encoding gene amplicons were generated by polymerase chain reaction containing 1X Platinum HiFi Taq polymerase buffer, 1.6 units Platinum HiFi polymerase (Invitrogen/LifeTechnologies), 3.7 mM MgSOUp to 40 amplicon libraries were pooled prior to emulsion PCR. The emPCR, enrichment and sequencing were done according to current Roche Titanium amplicon sequencing protocols . A typical sequencing run generated an average of 80,000 tags per sample . Reads were trimmed after anchor sequence. Chimeras were removed using UChime [de novo and reference database (ChimeraSlayer GOLD) modes. Taxonomy was assigned using GAST [http://vamps.mbl.edu) for analysis. OTU clustering was performed using UClust [http://www.ncbi.nlm.nih.gov/bioproject/46315. The 16S PCoA analyses were performed using the Morisita-Horn, Yue-Clayton and UniFrac inter-community distance metrics at both full sampling depth and with each data set subsampled to 8,461 sequences . All PCoAs were virtually identical.Pyrosequencing reads were quality-filtered by removing reads that did not have exact matches to the MID and the proximal primer (341 F); that contained an ambiguous base (N); that lacked a conserved distal anchor region used for trimming; or that had an average quality score less than 30 . The v3vg UChime , combiniing GAST  and the g UClust  using 97http://microbiomes.msu.edu/resources/) buffer (with 5% dimethyl sulfoxide) until processing. Samples were thawed on ice and vortexed for 30 s. The brush was removed and vortexed for 30 s in a second tube with GBSS buffer, then the brush was discarded. The suspended samples were combined, homogenized by vortexing for an additional 60 s before aliquots were removed for microscopy and cultivation.Brush samples were frozen in 1 ml of GBSS . Plates were incubated at 37°C for as many as 7 days in anoxic  or oxic  atmospheres. The total number of colony-forming units (CFUs) at day 5 was used to compare viable cell density (CFU/ml) for all samples.Viable cell counts were obtained through plating of serial dilutions. Triplicate dilution series for each sample  were performed in GBSS buffer on ice in the same environment used for downstream cultivation, followed by plating onto complex media containing various carbon, nitrogen and sulfate sources common in the human GI tract  at 37°C for 1 h, followed by mixing with 0.1 M hydrogen chloride . Further dilutions were made as necessary in 0.1 M hydrogen chloride; 4 μl of the treated sample was loaded into the counting chamber . Microbes deposited in nine grids (200 μm × 200 μm/grid) were counted for each of triplicate samples load. The average number of cells was used to estimate total cell counts in the original sample. Repeated measures ANOVA was performed to test the statistical significance of changes in direct or viable cell counts over sampling time points. A linear regression model was applied for log-transformed direct cell counts to estimate microbial population doubling time.Clostridia where species discrimination was applied  or butyryl-CoA:acetate CoA-transferase (but) genes, the final genes of the two main microbial butyrate synthesis pathways, to facilitate data comparison with the results from the companion manuscript.The 16S data were harvested for known butyrate producers of the human colon (based on ) at the Changes in the pouch mucosal microbiota after restoration of the fecal stream with the ileal pouch were monitored by sampling before and after takedown of the diverting ileostomy Figure . AmplicoAs a comparison, we obtained samples from the colonic mucosa of healthy subjects without inflammatory bowel disease via colonoscopy. When these samples were subjected to 16S pyrotag analysis and compared to the pouch mucosa samples, three of the four samples obtained prior to ileostomy takedown clustered as one group, one patient clustered alone, and samples from normal colonic mucosa clustered separately Figure . The samWe compared the overall diversity of the mucosal communities at each time point by calculating the Shannon index Figure . For thrp = 0.003). More interestingly, the increase was modeled as exponential (adjusted R2 = 0.73-0.88) over the whole sampling period, suggesting an active proliferation of the microbial community after ileostomy takedown. Model regression led to an estimated doubling time of 9 to 16 days for the pouch microbiota over the sampling time. By the last sampling point, which was 2 months after ileostomy takedown, the overall microbial cell density in all three patients was still at least one order of magnitude lower than that in healthy colon mucosa samples.We also tracked the development of pouch microbiota by direct and viable cell counts. The overall density of microorganisms in the pouch mucosa continuously increased by approximately two orders of magnitude during the sampling time series , whereas abundant communities with this potential were established in all patients shortly after ileostomy takedown (visit 2) and remained consistent during the remaining two sampling points. Profiles were distinct, however, between individuals with abundant buk-carrying candidates (mainly Clostridium butyricum and C. perfringens) in patients 206 and 207, whereas species associated with but were dominant in patients 200 and 210. Only patient 210 exhibited both Faecalibacterium sp. and Roseburia sp., which are considered to be among the most abundant butyrate producers in the healthy colon [but-linked bacteria, namely Acidaminococcus sp. and Coprococcus sp., were present at high concentrations in several samples.The 16S pyrotag data and the cultivation data document significant changes in the community inhabiting the pouch mucosa following ileostomy takedown. To determine if these changes in community structure were associated with potential functional changes, we analyzed the 16S rRNA gene data from biopsy samples at the genus and species level for butyrate-producing candidates  a clearly defined starting point when all patients are free of disease and off medications, (2) the opportunity to observe the development of a nascent pouch microbiota relative to the maturation of the ileal pouch, (3) sampling of mucosa-associated pouch microbiota without colonic lavage  and (4) C. difficile of the ileal pouch 10 months following visit 4. Patients 206 and 207 both had episodes of acute pouchitis at 16 and 17 months, respectively, following closure of the ileostomy. Both patients responded well to antibiotic treatment. Patient 210’s microbial community structure moved closest to the community structure observed in healthy colon samples, and this patient has had the best clinical outcome of the four subjects. Two years into the study, he continues to do well with no episodes of pouchitis.We were able to determine that in all four patients the pouch microbial community evolved over time, but it did so in a manner that was unique to each individual. As expected, the shift in the microbial community in each individual was most evident immediately after closure of the diverting ileostomy and reinstitution of the fecal stream through the pouch. While the microbial communities continued to evolve over time, the changes were less pronounced. The microbial community appeared to move toward a more “healthy” colonic community at each subsequent visit in patients 206, 207 and 210. However, in patient 200, while the microbial community initially moved toward a “healthy” colonic community, it drifted away from the healthy colonic community structure 1 and 2 months after ileostomy closure. Interestingly, this patient whose microbial community was less similar over time to the communities of the healthy colons developed chronic pouchitis 7 months after visit 4 and was diagnosed with Although our sample size was small, our findings suggest that the failure to develop a mature microbial community similar to that found in the healthy colon in the months following ostomy closure increases susceptibility to pouchitis. Alternatively, if an individual’s pouch microbiota develop into a diverse, mature community similar to that found in the healthy colon, we propose the risk of pouchitis is decreased. Falk and colleagues also performed a longitudinal study in two patients with a history of UC undergoing colectomy with IPAA . They foFusobacter (phylum Proteobacteria) and a decrease in Streptococci (phylum Firmicutes). In another study examining the mucosal-associated microbiota from ileal pouches, diversity was greater in the UC patients without pouchitis compared to the UC patients with pouchitis. There were no specific phylotypes associated with pouchitis [Others have compared the pouch microbiota in patients with and without pouchitis, although the results from these various studies have varied, and no clear association between particular bacterial profiles has been identified -26. Komaouchitis .Dysbiosis of the intestinal microbiota is commonly found in patients with IBD , but in Several studies have suggested that the overall diversity of the gut microbiota is decreased in the setting of IBD. However, our results indicate that diversity in and of itself (as measured by a metric such as the Shannon diversity) is not necessarily a predictor of disease. While it is true that patients 210 and 200 both developed pouch mucosal communities that were similar in overall diversity to that seen in the normal colon, patient 200 developed severe, refractory pouchitis, while patient 210 remains healthy after 2 years of follow-up. Rather than using overall diversity as a measure of “health” of the microbiome, it is likely that the specific composition and thus function of the community are the key predictors. In this regard, the community in patient 210 is most similar in composition and structure to that seen in the healthy colon, while the community in patient 200 is quite distinct, despite having relatively high overall diversity. This is also reflected in the relative potential of the communities to produce the beneficial SCFA butyrate where the community in patient 210 had a much higher potential of butyrogenesis compared to that in patient 200.Roseburia sp. and Faecalibacterium sp.  and the functional gene-targeted approach were consistent, only the latter could reveal butyrate-producing gene families and their inferred taxa in detail to more directly evaluate the butyrogenic potential. Unfortunately, it was not possible to use the functional gene-targeted approach in this mucosal study because of the low amount of bacterial DNA in most biopsy samples. However, 16S rRNA gene patterns specific for butyrate producers from Vital et al. [but genes linked to Roseburia sp./Eubacterium sp. and F. prausnitzii (data not shown) at abundances similar to luminal aspirates and consistent with the 16S rRNA gene data presented in Figure The importance of specific microbial functions is further indicated in our analysis of the butyrogenic potential of an individual patient’s microbiome. Abundant butyrate-producing communities were established in all patients after ileostomy takedown, but only patient 210 exhibited both taxa . Figure , which al et al.  were ideThis study describes the establishment of the pouch microbiota in a longitudinal manner. This is the first step toward understanding how the intestinal microbiota influence or trigger the development of the inflammatory response seen in IBD. Despite our small sample size, we can confirm that there is an evolution of the microbial community in all individuals. These changes were apparent when the communities were followed by multiple methodologies. This includes the use of culture-independent surveys based on retrieval of 16S rRNA-encoding gene sequences, specific culture-based techniques and targeted analysis of functional genes.Structurally and functionally, we find that the mucosal microbial community of the pouch moves toward a community more similar to a colonic microbial community following ileostomy takedown. Nonetheless, the membership within the community is distinct from the colonic microbial community, likely secondary to the influence of the small intestinal epithelium on the development of the pouch microbiota and the overall anatomic differences inherent to IPAA. The establishment of a pouch microbiota similar to a healthy colonic microbial community is potentially protective against pouchitis. In the future, longitudinal studies, such as this one, may provide the opportunity to identify patterns of the microbial community, both in terms of structure and function, that predict the onset of inflammation or disease in at-risk individuals.The results of this study demonstrate a marked shift in the structure and function of the microbiota that inhabit the mucosa of patients who have ileal-anal pouch anastomosis for ulcerative colitis. This shift is characterized by a transition to a community that is selected for fermentation, preferentially a beneficial one such as butyrate production. It is likely that the community structure and function of the pouch microbiome will influence the likelihood of the development of pouchitis, with the development of a pouch microbiome that resembles that seen in the normal colon being protective against the development of disease. Monitoring of the development of the pouch microbiome may be able to predict which patients require more aggressive monitoring for the development of pouchitis and potential early treatment/prevention using modalities designed to shift the microbiota into a more normal community.http://vamps.mbl.edu), which is supported by NSF grant NSF DBI-0960626 to S. Huse.The data sets supporting the results of this article are available in the VAMPS repository  and IMG (//img.jgi.doe.gov). Table S3 Taxonomic composition for pouch communities at four time points .Click here for file


Objectives. Many techniques were described for ureteroileal anastomosis in orthotopic bladder substitution, ranging from nonrefluxing to refluxing techniques, all aiming at preservation of the upper tract. We describe our technique of dipping the ureter into the ileal pouch, which is simple and had no complications. Patients and Methods. Our technique implies dipping the ureter in the lateral side of the pouch, in right and left corners, with two rows of four sutures fixing the seromuscular layer of the ureter to the seromuscular layer of the ileal pouch. The procedure was applied in both normal ureteric calibre and dilated ureter. Total number of procedures done was 1,340 ureters in 670 patients after radical cystectomy for invasive carcinoma of the bladder of urothelial and nonurothelial cancer. Results. Followup of patients every six months and onward did not show stenosis in the ureteroileal anastomotic site. Filling of the ureter with contrast dye on ascending pouchogram was observed in patients who had considerably dilated ureters at the time of cystectomy. Normal ureter did not show clinical reflux but radiological filling of the ureter when the intravesical pressure exceeded the leak point pressure. Time to perform the dipping technique was 5–7 minutes for each site. Conclusion. Dipping technique for ureteroileal anastomosis in orthotopic ileal neobladder avoids the incidence of stenosis, preserves the upper tract, is a fast procedure, stands the evaluation in long-term followup, and was practiced successfully for twenty years. Since the introduction of orthotopic ileal neobladder in 1985, the procedure gained wide acceptance and has been practiced by most urologist. The turning point in the technique of orthotopic ileal neobladder was the introduction of the detubularised ileal pouch that ensured low pressure reservoir protecting the upper tract from high-pressure reflux. Many techniques addressed the antireflux procedures in the ureteroileal anastomosis (UIA), which were first described by Le duc et al. ; Studer From 1993 to 2013, we applied the dipping technique in the ureteroileal anastomosis in orthotopic detubularised ileal neobladder substitution in 670 patients after radical cystectomy (RC) for invasive carcimoma of the bladder. 560 were men with age ranging from 45 to 75 (mean 57 ∓ 5 years) and 110 women with age range 35–65 (mean 52 ± 4 years). Invasive urothelial carcinoma was in 330 patients while squamous cell carcinoma was in 340 patients. The dipping technique was performed in the 670 patients on both sides for UIA with a total of 1340 procedures. The dipping technique was applied in normal calibre ureters and in dilated ureters with varying degree of dilatation. Followup after RC and orthotopic ileal neobladder was every 6 months and onward in order to assess kidney function and to monitor changes and dilatation of the upper tract. It was done by ultrasonography, intravenous urography, and ascending pouchogram being done simultaneously with urodynamic recording of the intrapouch pressure and the leak point.After RC for invasive carcinoma of the bladder, an isolated ileal segment of 35 cm is selected and detubularised, a pouch is constructed in a spherical or U-shape, and then it is closed except for the 2 cm width opening in its lower part which will receive the urethraileal anastomosis.On either side of the pouch in its lateral aspect, a hole is opened with a diameter equal to the ureteric calibre; the hole is done using a scalpel with haemostasis using bipolar coagulation for the submucosal small vessels in the ileal wall . An 80 cThe ureteral end is introduced into the pouch hole for a length of 1 cm to allow secure anastomosis; the ileal pouch wall of the seromuscular layer is anchored to the serosa muscularis of the outer wall of the ureter; sutures are arranged in two rows each with four sutures with 0/4 polyglycolic acid sutures .Final step is the anastomosis of the pouch to the urethral stump with four 0/3 sutures; the pouch is drained with a ballon silicone catheter anchored to the abdominal wall with Harris suture and with a supra pubic pouch catheter, two kidney drainage catheters, and one pelvic drainage tube . The ope2O; the resting pouch pressure was 15–25 cm H2O. Patients with the radiological finding of ureteral filling did not have severe urinary tract infection or febrile attacks. Deterioration of the upper tract was detected in 10 patients who developed urethral stricture that was treated with visual internal urethrotomy and pouch drainage for two weeks.Followup of the 670 patients, every 6 months and onward, was done with kidney function tests, urine analysis, and imaging. Stenosis was not detected in all the 1,340 ureteroileal anastomosis using the dipping technique ; radioloThere are numerous published series on the need for an antireflux technique for UIA –6, and t2O. Further elevation of intrapouch pressure will reach the leak point which will protect the upper tract from high pressure reflux. Low pressure reflux was observed to have no influence on the upper tract. The finding of ureteral filling during ascending pouchogram should not be considered as reflux, but should be labelled as imaging finding without adverse effects. The detubularized pouch in both shapes, spherical or U-shape, ensures a low pressure reservoir with a pouch pressure lower than 30 cm HDipping technique for ureteroileal anastomosis in ileal neobladder substitution proved to be without complications, and preserved the upper tract from the hazards of stenosis. The radiological finding of ureteral filling in pouchogram was associated with high pressure pouch during artificial filling. In nonartificial situations, this ureteral filling happens in case of high intrapouch pressure as a result of uretheral strictures.


Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4 Cdt2, to target the potent cell cycle regulator p21 for subsequent degradation. The DNA polymerase-δ cofactor PCNA provides a molecular platform for initial substrate recognition by this ligase, and subsequent p21 depletion prevents its continued interaction with PCNA which otherwise inhibits efficient viral replication. Virally-induced p21 degradation represents another way of promoting efficient replication of DNA polymerase-δ-dependent viruses.Many DNA viruses induce and then exploit host cellular DNA damage responses to generate a suitable environment for their continued replication. Parvoviruses, important disease agents in both humans and animals, rely on host DNA polymerases to replicate their genomes in cell-cycle arrested cells. We show that efficient parvovirus replication requires the recruitment to viral replication compartments of a host cellular E3-ubiquitin ligase, CRL4 WAF1/Cip1 (hereafter referred to as p21) expression. Transient expression of the MVM NS1 protein alone also leads to an increase in p21 levels in vitroMinute Virus of Mice (MVM) is an autonomously-replicating parvovirus which induces a DNA damage response resulting in substantial p53 activation which persists throughout the course of viral replication Cdt2 consists of the scaffold protein Cullin 4 and the homo-trimeric protein DDB1 which serves as an adaptor for the putative substrate recognition protein Cdt2. This ligase has been shown to program the ubiquitination and subsequent degradation of p21 in response to DNA damaging agents such as UV treatment in order to ensure low p21 levels during S-phase Cdt2 is recruited to chromatin via PCNA interaction where it targets substrate proteins for degradation Viruses often make use of the ubiquitin conjugation machinery to target for degradation cellular proteins that might otherwise negatively affect viral replication Cdt2 E3-ubiquitin ligase which was re-localized to viral chromatin within active MVM replication centers. PCNA provides a molecular platform that aids substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase, and p21 targeting to this ligase during MVM infection required its interaction with PCNA. PCNA is also an important co-factor for DNA polymerase δ-dependent MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which could no longer interact with PCNA did not. Introduction of a p21-derived peptide that bound to PCNA also substantially decreased viral replication. Our results suggest that interaction with PCNA was important for targeting p21 to the re-localized CRL4Cdt2 ubiquitin ligase, yet subsequent depletion of p21 was required to prevent its sustained interaction with PCNA which otherwise inhibited efficient viral replication.We show here that efficient MVM replication in S/G2 arrested cells required the targeting for proteasomal degradation of p21 by the CRL4Cdt2 E3 ubiquitin ligase has been implicated in targeting p21 for proteasomal degradation upon S-phase entry and after cellular DNA damage via RNAi in the protocol illustrated in The CRL4Cdt2 ligase also present in several other ubiquitin ligases (data not shown). Taken together, these results indicated that the CRL4Cdt2 ligase was responsible for p21 degradation throughout virus infection.For these experiments cells were parasynchronized prior to infection to maximize the number of cells progressing uniformly through S-phase. This both synchronized infection and the characterization of p21 depletion. At the time of release from the synchronization procedure (as the cells progressed from G0 to G1) there were high levels of p21 expression Mock T0, , lanes 1Cdt2 also targets Set8 and Cdt1 for degradation during S-phase and in response to DNA damaging agents CRL4Cdt2 E3 ligase depletion for viral replication using the protocol illustrated in Cdt2 E3 ubiquitin ligase was necessary for efficient viral replication.We next examined the functional consequence of CRL4via BrdU staining, to serve as sites of ongoing viral replication in infected cells and can be visualized by staining for the viral replicator protein NS1 Cdt2 ligase complex to APAR bodies. The recruitment was specific for the CRL4Cdt2 ligase because the APC/CCdc20 E3 ligase, which targets p21 for degradation after mitotic entry MVM replicates in nuclear compartments termed autonomous parvovirus associated replication (APAR) bodies which have been shown to be enriched for cellular proteins such as DNA polymerase δ, RPA, cyclin A, and PCNA, which are also essential for parvovirus replication Cdt2 E3 ubiquitin ligase to cellular chromatin via its interaction with Cdt2 during normal cell division Cdt2 ligase requires Cdt2 interaction with the p21 degron motif, as well as interaction between p21 and PCNA via its PCNA-interaction (PIP) box via interaction with PCNA, and a p21 mutant defective in binding to PCNA was resistant to degradation following UV treatment The DNA polymerase δ cofactor PCNA is known to target the CRL4Cdt2 ligase we generated stable murine cell lines via lentivirus transduction that conditionally expressed FLAG-tagged wild-type or mutant p21  abolished p21 interaction with the CRL4Cdt2 complex following its transient transfection as reflected by loss of interaction with DDB1 , the p21Δdegron protein was resistant to degradation  following infection . As a result, we could not use cell lines expressing this mutant to determine whether stabilized, p21 affected MVM replication via PCNA binding. Thus we generated a murine cell line conditionally expressing a mutant p21 in which all seven lysines in p21 were changed to arginine . Afterwards cells were pre-extracted for in cytoskeletal buffer containing 0.5% Triton X-100, protease and phosphatase inhibitors for 3 minutes on ice, washed, fixed with 4% paraformaldehyde and stained for the indicated proteins. Nuclei were visualized by staining with To-Pro-3 (Invitrogen). The coverslips were mounted in Fluoromount-G (Southern Biotech) and images were acquired using a Zeiss LSM 510 META confocal microscope. All images were captured using an objective of 63×.For IF, A9 cells were grown on glass coverslips in 35 mm dishes and infected with MVMp at an MOI of 10. At 24 hr pi, cells were washed twice with cold phosphate-buffered saline (PBS) solution and then with cytoskeleton buffer [10 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgClSouthern blots were carried out as previously described A9 cells were fixed in 70% ethanol overnight at 4°C. Cells were then pelleted, washed in PBS and resuspended in PBS containing 0.2 mg/ml RNAse A for 1 hr at 37°C, then propidium iodide was added at 40 µg/ml. Flow cytometry was performed using FACScan (BD biosciences). Data were analyzed using Summit software (Beckman Coulter).Figure S1Set8 but not Cdt1 is degraded during MVM infection. Parasynchronous A9 cells were infected with MVM at an MOI of 10. Western blots were performed against the indicated proteins. MVM infection resulted in reduction in Set8 protein levels. Cdt1, whose expression levels were not altered, appeared to run as a doublet upon MVM infection.(TIF)Click here for additional data file.Figure S2Cdt2 knockdown does not significantly affect S-phase entry.A) Schematic illustrating the experimental protocol for S1B. B) Murine A9 cells were treated with siRNA as shown in S1A and were processed for flow cytometry as described in Experimental Procedures. Mock T0 cells were processed at time of release from parasynchronization and show a majority of cells in G0/G1. siRNA-transfected T16 cells were processed at 16 hr post-release and showed the expected reduction in G0/G1 levels compared to mock T0 cells. There was no significant reduction in S-phase accumulation upon Cdt2 knockdown compared to control siRNA treatment; however, the G2/M to S ratio under these conditions varies between experiments. PI stands for propidium iodide.(TIF)Click here for additional data file.Figure S3APC/C E3 ubiquitin ligase is not recruited to APAR bodies. Murine A9 cells were mock infected or infected with MVM at an MOI of 10. At 32 hr pi cells were processed for immunofluorescence as described in experimental procedures, without detergent pre-extraction, using antibodies against NS1 and Cdc20.(TIF)Click here for additional data file.Figure S4Cdt2 -dependent manner following MVM infection.Overexpressed p21 is degraded in a proteasome and CRL4A) Parasynchronized murine A9 cell lines stably expressing FLAG-tagged p21WT were mock infected or infected with MVM at an MOI of 10. At 18 hr pi cells were treated with doxycycline to induce p21 expression and treated with MG132 as indicated. Cells were harvested 6 hrs later and processed for western blotting using the antibodies indicated. B and C) p21WT cell lines were treated with control siRNA or siRNA targeted to Cul4A (B) or DDB1 (C), as indicated, during parasynchronization. Cells were released and mock infected or infected with MVM at an MOI of 10. At 18 hr pi cells were treated with doxycycline to induce p21 expression. Cells were harvested at 24 hr pi and processed for western blotting using the antibodies indicated.(TIF)Click here for additional data file.Figure S5K7RΔPIP does not inhibit MVM replication.p21 p21WT and p21K7RΔPIP cell lines were parasynchronized, released and infected with MVM at an MOI of 0.5. At 16 hr pi cells were treated with doxycycline to induce p21 expression and harvested 8 hrs later. Cells were processed for Southern blotting (top panel), or for western blotting using the indicated antibodies (bottom panels).(TIF)Click here for additional data file.Figure S6p21 mutants are recruited to MVM replication compartments. Murine A9 cell lines stably expressing FLAG-tagged p21PCNA, p21ΔDegron or HA-tagged p21K7R or p21K7RΔPIP were mock infected or infected with MVM at an MOI of 10. At 18 hr pi cells were treated with doxycycline to induce p21 expression. At 24 hr pi cells were processed for IF using antibodies against NS1 and FLAG or HA.(TIF)Click here for additional data file.


The biological effect of HCA is poorly understood. Among cinnamic acid and its related compounds, HCA has a specific-anabolic effect on bone, being found to stimulate osteoblastogenesis and to inhibit osteoclastogenesis through the suppression of NF-κB signaling, thereby preventing bone loss. Bone marrow mesenchymal stem cells give rise to ostoblasts and adipocytes. HCA might therefore have effects on osteoblastogenesis and adipogenesis in bone marrow culture. This study demonstrates (1) that HCA has stimulatory effects on osteoblastogenesis and mineralization and suppressive effects on adipogenesis in mouse bone marrow culture and (2) that HCA depresses adipogenesis in mouse 3T3-L1 preadipocytes in vitro. Such effects of HCA might be involved in the differentiation of mesenchymal stem cells.The bioactive flavonoid  Bone homeostasis is maintained through a delicate balance between osteoblastic bone formation and osteoclastic bone resorption. Numerous pathological processes have the capacity to disrupt this equilibrium leading to conditions in which the rate of bone resorption outpaces the rate of bone formation leading to osteoporosis  is an intermediate-metabolic substance in plants and fruits and is synthesized from tyrosine. Among cinnamic acid and its related compounds , HCA has been shown to have specific-anabolic effects on bone in vitro  and antibiotics (penicillin and streptomycin) were purchased from Invitrogen . Fetal bovine serum (FBS) was from Hyclone. HCA (100 % pure) was obtained from Wako Pure Chemical  and was dissolved in ethanol for use in experiments. Insulin, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), PD98059, staurosporine and other reagents were purchased from Sigma . Insulin was dissolved in diluted acidic acid solution and other reagents were dissolved in 100 % ethanol.6 cells/wells per 1 ml), which were isolated from mice  purchased from the Jackson Laboratory (USA), were cultured in 12-well plates in DMEM containing 10 % FBS, 1 % penicillin-streptomycin  and mineralization medium (MM) containing ascorbic acid (100 ng/ml) and 4 mM β-glycerophosphate in the presence of either vehicle or HCA  or its absence, after which the medium was replaced by medium without HCA and the cells were cultured for an additional 14 days. The medium was changed every 3 days. After culture, the cells were washed with phosphate-buffered saline and stained with Alizarin red stain. For quantitation, 10 % cetylpyridinium chloride solution was added to each well to elute the dye. After complete elution, absorbance of the eluted solution was measured at 570 nm on a microtiter plate reader.Bone marrow cells (1 × 106 cells/well per 1 ml in 12-well plates), which were obtained from the femoral tissues of wild-type mice , were cultured for 48 h in DMEM (10 % FBS and 1 % P/S) containing either vehicle or HCA (1–1000 nM) in the presence or absence of the differentiation medium (DM) consisting of dexamethasone (1 μM/ml medium) and IBMX . In other experiments, the cells were cultured in DM for 48 h with HCA (1–1000 nM) or without HCA and then the medium was replaced and the cells were cultured in medium containing insulin (10 μg/ml medium) for 4 days without HCA. After culture, the medium was removed and the adipocytes were stained with Oil Red O. Adipocytes were counted by light microscopy. For quantification, the dye was extracted with 0.2 ml isopropanol for 1 min and the absorbance (490 nm) was read by using a Spectra Count microplate photometer.Bone marrow cells  were cultured in DMEM containing 10 % bovine calf serum and 1 % P/S in 96-well plates in order to obtain subconfluent undifferentiated cells. After preculture (day 0), the undifferentiated cells were cultured in DM consisting of DMEM (with 10 % FBS and 1 % P/S) containing 0.5 mM IBMX and 1 μM dexamethasone for 2 days and then DM was replaced with 10 % DMEM containing insulin (5 μg/ml) with HCA (0.1–10 μM) or without HCA and the cells were cultured for 2 additional days.3T3-L1 mouse embryo fibroblasts were obtained from the American Type Culture Collection and were cultured as described elsewhere  content in the cells after cell culture was quantified by using the commercially available AdipoRed assay reagent according to the manufacturer’s instructions  was performed according to the manufacturer’s instructions. Fluorescence was measured at 560–590 nm in a 96-well plate reader  to determine the formazan concentration, which is proportional to the number of cells . Multiple comparisons were performed by one-way analysis of variance (ANOVA) with the Tukey-Kramer multiple comparisons post test for parametric data as indicated. To determine the effects of HCA on osteoblastogenesis and mineralization, mouse bone marrow cells were cultured in medium containing either vehicle or mineralization medium in the presence of HCA 1–1000 nM) or its absence for 21 days. Culture with HCA stimulated osteoblastogenesis and mineralization  or its absence for 6 days. Culture with HCA suppressed adipogenesis Fig. a, b. SucTo determine the effect of HCA on adipogenesis in preadipocytes, mouse 3T3-L1 preadipocytes were cultured in DM for 2 days with HCA (1–100 nM) or without HCA. The medium was then replaced with DMEM containing insulin without HCA  or their absence and were then cultured for 2 days in medium containing insulin in the presence of either the vehicle or HCA (0.1–100 nM) or their absence. The cell viability of mature adipocytes was not significantly changed after culture with HCA (data not shown), indicating that culture with HCA did not cause cell death.To characterize the effect of HCA on the insulin-stimulated lipid accumulation, 3T3-L1 preadipocytes were cultured for 2 days in DM without HCA followed by 2 days in DMEM with HCA (0.1 to 100 nM) or without HCA in the presence or absence of insulin. Culture with HCA did not cause a significant change in lipid content in mature adipocytes cultured in the absence of insulin Fig. . This reTo determine whether suppressive effects of HCA on insulin-stimulated adipogenesis are mediated through insulin signaling, mature adipocytes were cultured in the presence of staurosporine, an inhibitor of general protein kinases including protein kinase C  alpha and PPARγ (Prusty et al. In conclusion, this study demonstrates that HCA has stimulatory effects on osteoblastogenesis and mineralization and suppressive effects on adipogenesis in mouse bone marrow cell culture in vitro and that it has suppressive effects on insulin-stimulated adipogenesis in preadipocytes. Adipocyte hypertrophy and hyperplasia are important processes in the development of obesity, which induces osteoporosis. The flavonoid HCA might therefore have preventive effects on osteoporosis and obesity.


The achievement of an adequate nutritional status is an important goal in the treatment of HIV infection and requires a proper assessment. We studied the nutritional status of a group of HIV-infected patients using biochemical and anthropometric measures.We assessed 91 outpatients followed in an urban infectious disease clinic in south Madrid (Spain). In each case age, gender, race, HIV risk group, hepatitis C virus (HCV) infection status, smoking status, degree of immune depression, viral load, presence or absence of an AIDS diagnosis, time since diagnosis of HIV infection, antiretroviral therapy (ART), type of ART, anthropometric measures , and blood and biochemical measures .Median age was 46 years; 69.2% were males, 81.3% Caucasian. Viral load was below 20 copies/ml and mean CD4 count 468 cells/µL Using Changi's mini nutritional assessment, which involves immunological, biochemical and anthropometric parameters, we found malnutrition in 81.1%; it was severe degree in 16.7%. Most patients were classified as caloric malnutrition (67.8%). Serum cholesterol was increased in 24.7% and triglyceride in 33.3%. Serum albumin was normal in 96.6%.Anthropometric measurements showed diminished fat compartment  whereas muscular compartment was preserved . Tricipital fold 50th percentile was 8 mm in males and 19.9 mm in women; corresponding values for arm circumference were 26 cm and 22 cm, respectively. Of males, 6.6% were underweight, as were 23.1% of women. Malnutrition was associated with BMI (p=.0001), waist circumference (p=.029) and with HCV infection (p=.037). Caloric malnutrition was associated with BMI (p=.0001), waist circumference (p=.046) and chronic HCV infection (p=.049). It was also related with the degree of immune suppression .Despite HAART we found malnutrition, at least of small degree, in more than 80% of patients, mainly caloric malnutrition. Arm fat compartment was most frequently involved. Chronic HCV infection, degree of immune suppression, low BMI and waist circumference are related with the presence of malnutrition.


Playing video games in childhood may help achieve advanced laparoscopic skills later in life. The virtual operating room will soon become a reality, as “doctor games 2.0” will doubtlessly begin to incorporate virtual laparoscopic techniques.To teach surgical skills to schoolchildren in order to attract them to urology as a professional choice later in life.As part of EAU Urology Week 2010, 108 school children aged 15–19 attended a seminar with lectures and simulators  at the 62nd Congress of the German Society of Urology in Düsseldorf. A Pub-Med and Google Scholar search was also performed in order to review the beneficial effects of early virtual surgical training. MeSh terms used were “video games,” “children,” and “surgical skills.” Searches were performed without restriction for a certain period of time.In terms of publicity for urology, EAU Urology Week, and the German Society of Urology, the event was immensely successful. Regarding the literature search, four relevant publications were found involving children. An additional three articles evaluated the usefulness of video gaming in medical students and residents.Making use of virtual reality to attract and educate a new generation of urologists is an important step in designing the future of urology. Each year in September, the European Association of Urology (EAU) encourages urological societies in Europe to create public awareness about urological diseases. This can be done through small local events with lectures in a small community hospital or through a large, centrally organized meeting that may draw media attention. As an example, the 2008 Urology Week in oslo had numerous lectures and an enormous walk-through prostate model, and was broadcasted to approximately 3 million Norwegians through television and radio. All urologists are invited to participate in Urology Week, and more information can be found on www.urologyweek.org. Since its introduction in 2008, approximately 20 countries regularly take part in EAU Urology Week, informing people about prostate cancer, urinary incontinence, and erectile dysfunction. In 2010, the German Society of Urology (DGU) took the event a step further by presenting urology to possible future colleagues—schoolchildren aged 15 to 19. Under the motto “Become a urologist for one day,” they were invited to attend a special session at the DGU Congress in Düsseldorf including hands-on training in surgical techniques.owing to the rapid progress in the development of minimally invasive techniques, urology has changed. however, even more so than today, future urologists will depend on well-developed eye-hand coordination along with advanced spatial skills. To provide training on these skills during childhood, it has been suggested that playing video games may have positive effects on laparoscopic performance later in life. owing to major advances in virtual reality (VR), video games simulating laparoscopic surgery are not only widely accessible for aspiring young urologists but are also on the verge of entering childhood bedrooms as “doctor games 2.0” for video consoles. The technical aspects of surgery require well-developed psychomotor abilities that should be learned as early as possible. Furthermore, it has been shown that video games enhance spatial skills in children . Video gour initiative basically aimed to encourage the future generation of doctors to develop their basic skills in laparoscopy as early as possible, preferably with a career in urology in mind. This was because hardly any undergraduates entering medical school consider urology as their first career choice. The prejudiced prospect of a profession connected to urine and older men prevents many aspiring doctors from considering urology. Moreover, undergraduate urological education is rudimentary in quite a few European countries, with a quarter of all European medical students not receiving any formal training .Recruitment was carried out through active communication with schools in the wider Düsseldorf area and by issuing several press releases prior to the event. Every 15-year-old and above considering a career in medicine was eligible to participate. The course was a one-day class at the venue of the congress and was held on two separate days to maximize the number of participants. The program started with two lectures that introduced aspects of urology. Then, small groups of 5–6 participants moved for approximately two hours between different workstations, where they were introduced to a variety of practical urological tasks. There were five stations: laparoscopy, transurethral resection (TUR), cystoscopy, ultrasound, and tying knots/suturing. The equipment used was made available by Karl Storz Gmbh, hitachi Medical Systems Gmbh, and B. Braun Melsungen AG. TUR was the only station that used a VR environment. Authentic equipment was used to simulate laparoscopy and cystoscopy. To make this exercise more enjoyable, we used jelly bears as training objects that had to be removed out of groups of red peppers. For suturing, bananas and bacon were used. Residents from the German Society of Residents in Urology (GESRU) oversaw each group. The session concluded with a lecture on science and the purpose of scientific congresses.In order to evaluate the usefulness of childhood spatial and psychomotor training, a literature search through PubMed and Google Scholar was performed, using the MeSh terms “video games,” “children,” and “surgical skills.”A total of 108 schoolchildren, aged 15 to 19, attended the seminar. Media attention was extensive, with three major TV stations and two regional and national newspapers covering the event. Feedback given by the participants themselves was very positive.A relatively short time after video games became widely accessible, a study showed that they could improve spatial skills in young girls and boys . The besResidents in surgical specialties and medical students have been studied quite frequently in terms of a correlation between video game experience and laparoscopic skills. Boyle et al.(2012) looked at physical and virtual laparoscopic performance in medical students with no experience in laparoscopy, no experience with the Nintendo Wii™ and no periods of regular video gaming . In thisThere has been little interest in the question of whether video gaming enhances operating room performance in established surgeons. however, it has been shown that a preoperative warm-up video game session significantly enhances laparoscopic surgery performance on basic tasks in novices . DespitePlaying video games  on a regular basis trains cognitive skills essential for laparoscopic surgery. It is quite possible that medical games that incorporate aspects of laparoscopy will be put on the market. It is clear that schoolchildren thinking about a career in medicine stand only to gain from playing video games frequently, in terms of spatial, sensorimotor, and attention skills. Introducing the PlayStation generation to urology at an early age (similar to our initiative “Become a urologist for one day”) is likely to bring new talent to the specialty within a few years. however, it remains somewhat unclear whether today’s computer-literate children, who will eventually become surgeons, will actually outperform all others. Future research should focus more on this topic.


Arteriovenous malformations (AVM) are high-flow lesions having a direct connection between an artery and vein, with bypass of the capillary bed.Treatment of such lesions remains a surgical challenge. Despite multiple procedures, total eradication is often not achieved. For some patients, limb amputation becomes necessary and carries significant risk.We report a case of congenital AVM of the upper limb causing cardiac decompensation. The patient was safely and successfully treated by performing a 2-stage upper-limb amputation.A 32-year-old man presented with a congenital high-flow AVM involving the left upper limb and anterior chest Fig . In receIn addition, there was a chronically infected wound on the dorsum of the left hand from which he suffered a massive bleed 6 years previously. On that occasion, he lost 3500 mL of blood. Amputation of the digits through the metacarpophalangeal joints had been previously performed at another hospital. At presentation, the left upper limb was warm and hypertrophic with pulsating vessels. Bruits were audible and a thrill palpable over the axillary artery. A dry eschar covered the chronic wound on the dorsum of the hand. The vascular malformation over the anterior chest manifested as an uncomplicated reddened patch.Magnetic resonance imaging (MRI) was performed to determine extent of involved tissues. This revealed an extensive diffuse AVM involving all compartments of the left upper limb. There was no skeletal hypertrophy Fig . SelectiThe first stage comprised disarticulation through the elbow. We intentionally avoided amputation through bone to avoid the risk of uncontrollable bleeding from the bone end. To minimize intraoperative bleeding, a tourniquet was inflated above the elbow and 1/0 PDS  transcutaneous sutures placed proximal to the incision. The temporary sutures served to compress cutaneous and subcutaneous vessels thereby reducing bleeding. Consequently, it became easier to formally transfix these vessels during the amputation.The second stage was performed 1 week later. Cardiovascular surgeons performed ligation of left subclavian vessels Fig , immediaA fasciocutaneous flap overlying the deltoid was raised and used for wound coverage. A suction drain was inserted and a soft, bulky dressing applied. Postoperative recovery, including wound healing, was unremarkable Fig . The pat-5The management of AVM remains a significant challenge, particularly those of the upper limb. Intervention should only be contemplated when the lesion becomes symptomatic. A variety of treatment strategies have been reported with varying success.2We believe that plastic surgeons should be an essential member of the multidisciplinary team treating such lesions. In some instances, surgical resection of the AVM with maximal preservation of upper-limb function may be required.Clinical presentation of upper-limb AVMs can be diverse. Patients may suffer pain, nerve compression, ulceration, hand dysfunction, and spontaneous bleeding.9Rarely, extensive arteriovenous shunting may lead to cardiac decompensation.9Management of AVM varies, with a conservative approach adopted for patients that are asymptomatic or have minor symptoms. If treatment is required, techniques that may be used include catheter embolization or direct percutaneous sclerotherapy. Embolization and sclerotherapy may be performed even after major surgery.Surgical ligation of the main feeding vessels to the AVM often yields a poor result, as a collateral circulation subsequently develops.With the advent of highly selective embolization techniques, a number of authors have reported safe and successful outcomes when treating AVM with this modality.8Regardless of the embolic agent used, the underlying aim is to obliterate the nidus. However, embolotherapy must not be considered a definitive cure and can unintentionally worsen symptoms. Furthermore, repeat embolization may be necessary to deal with recurrences.In our patient, embolotherapy would not have been appropriate. The diffuse nature of the AVM with multiple nidi wound render embolotherapy ineffective. Our patient had a life-threatening, symptomatic, nonfunctional upper-limb AVM. In this situation, we believe that amputation or disarticulation should be explained to the patient at the outset. Amputation should be viewed as the “reconstructive” first step in the patient's rehabilitation.We favored glenohumeral disarticulation rather than amputation through the neck of the humerus for a number of reasons. First, the latter provides no functional advantage, as the patient cannot grip or stabilize large objects between the stump and thorax. Second, even though the MRI indicated no apparent involvement of the humerus, amputation through the bone may result in significant bleeding. Third, the patient was not keen for preservation of proximal humerus with associated soft tissue reconstruction. His concern was a protracted postoperative stay and rehabilitation. The cosmetic deformity with glenohumeral disarticulation, however, is greater. Shoulder width and axillary contour are both lost.Our rationale for a 2-stage amputation was based on the following: the aim of the first stage was to control the risk of bleeding and infection at the hand. Elbow disarticulation could be easily performed as a tourniquet could be applied to the arm. After control of infection, the second stage could be performed with the reduced risk of wound infection and breakdown.In one series of 33 patients with high-flow AVM of the upper limb, the majority of patients were treated by surgical excision.A multidisciplinary approach is central to the effective management of AVMs. These lesions are complex and difficult to treat. Despite advances in embolic agents and microcatheter techniques, outcomes for high-flow upper-limb AVM is far from satisfactory. For diffuse lesions that are life-threatening, amputation as the first step in the patient's rehabilitation may be appropriate. Indeed, it has been previously stated that amputation was unnecessarily delayed in such patients.5Surgical excision must be performed in a systematic manner, involving steps to minimize the risk of uncontrollable intraoperative bleeding and postoperative complications. In this case, successful amputation was achieved using temporary transcutaneous sutures to control bleeding from skin edges, ligation of the subclavian vessels, and glenohumeral disarticulation.